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ISSN:0365-6217    

DOI:10.1039/AR95047FP001

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

vi CONTENTS.ERRATA.VOL. 45, 1948.25 for Me* + Ph*CHMe, _3 CH, + PhSCMe,Tead Me* + Ph*CHMea + CH, + Ph*kMe,Lineformula (I.)Page141142143154Page2217332015 for (a) Ph*COa + RH--+Ph*COzH + R*read (a) PhGO,* + RH --+ Ph*CO,H + R-Equations (ai) and (uii) should read(ai) R-S- + CH,:CH*CO,Me --+ [R*S*CH,*CH.CO2Me]-(aii) [R*S*CH,*CH*CO,Me]- + RSH --++R.S.CH,.CH,*CO,Me + R*S-VOL. 46, 1949.Linereference 198 for J. H. Thorn read J. H. Thorp.1631for reference 61 read (O, and insert footnote, 6o Annalen,column 3, for J. H. Thorn read J. H. Thorp.1948, 561, 16

ISSN:0365-6217    

DOI:10.1039/AR9504700006

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

ANNUAL REPORTSON THEPROGRESS OF CHEMISTRY.GENERAL AND PHYSICAL CHEMISTRY.1. MOLECULAR STRUCTURE.Electronic Spectra.-In the period under review a considerable number ofpapers has been published on molecular spectra in the visible and ultra-violetregions.The sharpening of absorption bands and the increase in intensity a t lowtemperatures have been discussed by Keilin and Hartree who have alsomade some interesting new observations on the absorption spectrum ofliquid oxygen in the visible region. Bands in the visible region similar tothose which were primarily observed with H20 vapour have been obtainedwith D,O vapour and assigned to OD.3 The absorption of fluorine has beenre-studied by Nathans who reports that there is no absorption in the 7000-9600-~. region.In the case of bromine monofluoride absorption is observedwith rotational fine structure from 18,500 to 21,100 cm.-l and a pre-dissociation is said to occur a t 21,800 cm.-l. If the molecule dissociates intounexcited atoms a t this frequency then Do = 59.6 -+ 0.2 kcals.has calculated the relative vibrational transition probabilities for the firstfour vibrational levels in the 2C+ - 211 system of OH.Very interesting emission spectra have recently been observed withfluorocarbon vapours under the action of electric discharges.' A system ofviolet-degraded double double-headed bands of which the most prominentis at 2240 A., and a system of red-degraded bands, with marked sequenceslying in the region 1970-2210 A., have been shown to be due to a diatomicmolecule.Vibrational analysis shows that these bands have a lower state incommon, which is probably the ground state, 21T, of CF. Venkateswarlu hasassigned the bands at 2240 A . to the transition 2C - 21T. Laird, Andrews, andBarrow have shown that the bands between 2340 and 500 A . are due to theSchulerKeilin and Hartree, Nature, 1950,165, 504.Idem, ibid., p. 543.Schuler and Reinebeck, 2. Naturforsch., 1949, 5a, 560.Nathans, J . Chem. Phys., 1950,18, 1122.Brodersen and Schumacher, Anal. Asoc. Q d m . Argentina, 1950,38,52.Schuler, J . Chem. Phys., 1950, 18, 1221.Venkateswarlu, Phys. Review, 1950, 77, 676.Laird, Andrews, and Barrow, Trans. Paraday SOC., 1950, 46, 8038 GENERAL AND PHYSICAL CHEMISTRY.CF, radical. They have studied these bands in absorption and preliminarymeasurements show that this radical has a life of about 1 sec.in electrodelessdischarge. Interesting new observations have been made on the absorptionspectrum of the CS,+ molecule-ion.Q Schmitz and Schumacher lo have foundthat ClP, shows absorption beginning a t 4700 A. and rising to a maximumabout 2200 A.Edse l1 has re-examined the absorption spectrum of hydrogen peroxidevapour and obtained results which agree well with those previously reportedby Holt, McLane, and Oldenberg.12 The absorption spectrum of iodine inacetone solution has been studied and the strong band a t 363 mp. is attributedto the tri-iodideCoriolis coupling between two fundamental vibrations of the formaldehydemolecule in its ground state has been shown l4 to perturb the rotationalstructure of the band in the ultra-violet spectrum a t 3600 A.Voden and Astoin l6 have described a new light source for use in thevacuum ultra-violet.It is claimed that with copper electrodes usefulemission is obtained between 100 and 2000 A. The absorption spectra ofmethane, carbon dioxide, water, and ethylene have been investigated in thevacuum ultra-violet .I6 One outstanding requirement of vacuum ultra-violetspectroscopy is an accurate method of determining extinction coefficients.Harrison, Gaddis, and Coffin l7 have developed a technique for determiningthe molar extinction coefficients of compounds in the vapour state and claima precision of 3--5%. The method has been applied to the spectrum ofdivinyl ether.Ethylene oxide has been studied by Liu and Duncan l8 whofind two Rydberg series, one beginning at 1935 and the other a t 1382 A.Both these converge to the same limit, giving an ionization potential of10.81 ev. Two non-Rydberg transitions are observed with origins a t 1713.4and 1572.4 A . These authors assume that the Rydberg transitions arise bythe excitation of an electron from a molecular bonding orbital very similar tothe one responsible for the Rydberg series in ethylene and related compounds.Intensity measurements on the vacuum ultra-violet spectrum of ethylene inthe gaseous state, and octenes, cyclohexane, octynes, and dihydropyran inhexane solution have been made by Platt, Klevens, and Price.lg Quinol,resorcinol, and catechol have been studied in the vapour state,,O and theorigin of main bands has been discussed and some vibrational assignmentshave been made.The hydrides of sulphur, selenium, and tellurium and theLaird and Barrow, Proc. Phy8. SOC., 1950,63, A , 412.10 Schmitz and Schumacher, Anal. Asoc. Quim. Argentina, 1950, 38, 61.l 1 Edse, J . Chm. Physics, 1950,18, 244.1* Holt, McLane, and Oldenberg, ibid., 1948, 16, 255, 638.1s Benesi and Hildebrand, J . Amer. Chm. SOC., 1950,72, 2273.l4 Brand, Trans. Faraday SOC., 1950,46, 805.l5 Voden and Astoin, Nature, 1950, 166, 1029.l6 Wilkinson and Johmon, J . Chem. Phys., 1950,18, 190.l7 Harrison, Gaddia, and Coffin, ibid., p. 221.Liu and Duncan, ibid., 1949,17, 241.l9 Platt, Klevens, and Price, ibid., p.466. ao Beck, ibid., 1950,18, 1135MCDOWELL : MOLEOULAR STRUCTURE. 9methyl derivatives of hydrogen sulphide have been studied in the far ultra-violet .zOaThe assignments of the singlet-triplet emission spectrum in benzene havebeen discussed by Craig, 21 who has also given an interesting theoreticaldiscussion 22 of the perturbation of the forbidden &,-Bern transition in ben-zene by the Egt vibrational frequencies. It is shown that the 606-cm.-lvibration is about 100 times more effective than the 1596-cm.-l vibration inproducing the forbidden transition. Experimental observations agree withthese theoretical results. Schull 23 has made a very complete study of thevibrational fine structure of the 3400-A. triplet-singlet emission band inbenzene as observed by the rigid glass technique of Lewis and K a ~ h a .~ *This 3400-~. band is identified as a 1A1g-3B8n transition. The bands between2000 and 4000 A. observed in absorption in liquid ethylene have been inter-preted by Reidz5 as also being due to a singlet-triplet transition. Con-siderable discussion has recently centred on the interpretation of the ultra-violet spectra of aromatic hydrocarbons, and much progress has beenmade.26* 27 Klevens 28 has indicated that there is a complete correspondencebetween the spectra of azulenes and their six-membered ring isomers withregard to intensity, vibrational structure, and sequence of the five bandsobserved. A simple theoretical treatment which is extremely successful inpredicting maximum possible extinction coefficients of polymeric andpolycyclic benzenoid hydrocarbons has been given by Bra~de.~g Platt 30has shown that the simple L.C.A.O.molecular-orbital treatment includingoverlap is quite successful in helping one to understand the spectra of complexconjugated molecules. In an interesting series of papers a similar theoreticaltreatment has been given 31 for monosubstituted benzenes, thiophenol, com-pounds of type (C6H5),X, and various substituted derivatives of benzene, andnew experimental results have been produced which are discussed in terms ofthis theory.Numerous publications have appeared on the ultra-violet absorption oforganic compounds in solution, and a selection of those of physico-chemicalinterest is given below.Bayliss32 has discussed the effect of electrostaticpolarization of the solvent on the electronic absorption spectra in solution.An interesting consideration of the technique of absorption spectrophoto-metry has shown that fluorescence is often a cause of deviations from the200 Price, Teegan, and Walsh, Proc. Roy. SOC., 1950, A , 201, 600.21 Craig, J. Chem. Phys., 1950, 18, 236.23 Schull, J. Chem. Phys., 1949,17, 295.24 Lewis and Kasha, J. Amer. Chem. SOC., 1944,66, 2100; 1945, 61,997.25 Reid, J . Chem. Phys., 1950,18, 1299.26 Platt and Klevens, Chem. Reviews, 1947,41, 301 ; J . Chem. Phys., 1948,16, 832 ;28 Klevens, J. Chm. Phys., 1950,18, 1063.Platt, J . Chem. Phys., 1950, 18, 1168.31 Matsen, J. Amer. Chem. SOC., 1950, 72, 5243; Robertson and Matsen, ibid., pp.8p Bayliss, J .Chem. Phye., 1950,18, 292.a2 Idem, J., 1950, 59.1949, 17, 470. 27 Platt, ibid., p. 454.2B Braude, J., 1950, 379.6248, 5250, 5252, 5256 ; Robertson, Music, and Matsen, ibid., p. 526010 GENERAL AND PHYSICAL CHEMISTRY.Beer-Lambert law.33 Amongst- the compounds which have been studied insolution have been substituted aromatic nitro-compounds,34 substitutedaniline~,~5 phenols,36 diphenylalkane~,~' various ketones,38 d i t o l y l ~ , ~ ~ cyclicdienes,4O phenolic compounds,P1 azlactones,42 a n t h r a ~ e n e , ~ ~ o-substitutedanilines,& arylmethylallyl alcohols,45 hydroxydiphenylmethane~,~~ hydroxy-naphthoic a ~ i d s , ~ 7 compounds containing the C-I and sulph0xides.4~Grubb and Kistiakowsky have attempted an explanation of thermo-chromism.Fluorescence spectra are often of great assistance in the interpretation ofultra-violet spectra.Bass and Spooner 51 have photographed the fluores-cence spectra of fluorobenzene and chlorobenzene and shown that theiranalysis yields results in agreement with their known ultra-violet spectra.These molecules belong to the symmetry group Cay and it is shown that thefirst electronic transition is A, --+ B,.Raman Spectra.-The experimental development of photoelectric Ramanspectrometers 62--54 has continued and new instruments have been de-scribed.65* 56 Lord and Nielsen 57 have described a simple apparatus whichenables Raman spectra to be obtained at temperatures down to -1150".Careful distillation has been shown to produce samples in which the back-ground scattering is considerably reduced.58 Bender and Lyons 69 havedescribed a modified version of the very satisfactory technique originated byReitz 6o for the determination of depolarization factors, while Rank andss Braude, Fawcett, and Timmons, J., 1950, 1019.34 Fielding and Le FBvre, J., 1950, 2812.36 Robertson and Matsen, J.Amer. Chem. SOC., 1950,72, 1543.36 Idem, ibid., p. 1539.38 Day, Robinson, Bellis, and Till, ibid., p. 1379.39 Pickett, Groth, Duckworth, and Cunliffe, ibid., p. 44.4~ Pullmann and Berthier, Bull. SOC. chim., 1950, 81.41 Coggeshall and Glessner, J. Amer. Chem. SOC., 1950, 72, 2275.4p Schueler and Wang, ibid., p. 2220.4s Gorinda Rau and Venkataraman, Current Sci., 1950,19, 9.44 Grammaticakis, Bull.SOC. chim., 1950, 158.4 5 Braude, Fawcett, and Newmann, J., 1950, 793.4 6 Hunter, Morton, and Carpenter, J., 1950, 441.4 7 Bergmann, Hirshberg, and Pinchas, J., 1950, 2351.Durie, Iredale, and Jarvie, J., 1950, 1181.I9 Koch, J., 1950,2892; Felmel and Carrnack, J. Amer. Chern. SOC., 1950, 72, 1292.Grubb and Kistiakowsky, J. Amer. Chem. SOC., 1950, 72,419.61 Bass and Spooner, J. Opt. SOC. America, 1950, 40, 389.6* Rank, Pfister, and Coleman, ibid., 1942, 32, 390;6s Chien and Bender, J. Chem. Phys., 1947.15, 376.54 Kinell and Traynard, Actu Chem. Scand., 1948,2, 193.5 6 Miller, Long, Woodward, and Thompson, Proc. Phys. SOC., 1949,62, A , 401.66 Sushchinsker, J. Exp. Thew. Phys., U.S.S.R., 1950, 20, 403.6 7 Lord and Nielsen, J.Opt. SOC. America, 1950, 40, 653.5 * Mallory, J. Chem. Phys., 1950,18, 898.69 Bender and Lyons, ibid., p. 348.6o Reitx, 2. physikal. Chem., 1936,3$, B, 368.37 Coggeshall, ibid., p. 2836.Rank and Wiegand, ibid.,1946,36, 325MCDOWELL : MOLECULAR STRUOTURE. 11Kagarise 61 have shown that though their new apparatus appears geometric-ally unsuitable for the accurate determination of p, nevertheless, the resultsare in good agreement with those obtained by more accurate methods whenconvergence corrections are applied.Interest has recently been shown in diatomic molecules and the Ramanspectra of bromine,62 chlorine monoflu~ride,~~ and fluorine have beenobtained. Cyanogen has been re-investigated.C6 Low-frequency Ramanspectra have been observed in single crystals of benzene,66 di-iodoben~enes,~~potassium hydrogen fluoride,68 and potassium chloride.69 Hydrogen bondinghas been shown to be present in crystals of KHC03.70The aluminium hydride ion has been shown to have a tetrahedral structureby Lippincott 7 1 who studied its Raman spectrum in ethereal solution.Inaqueous solution spectra have been reported for nitrates,72 S203-- ion,73di-, tri-, and tetra-thionate ions,74 and the AuC1,- Polarizationmeasurements in the case of S,O,-- have indicated that it is probable thatthis ion has symmetry C3v. The S , 0 6 - - ion would be expected to resembleethane and have either an eclipsed structure ( D 3 h ) or a staggered configura-tion with symmetry D S d .The Raman spectrum, taken in conjunctionwith earlier infra-red data,76 indicates that the structure probably is DSd.No definite decision has been reached concerning the structure of thetrithionate ion S306--. The AuC1,- ion has been shown to have a squarestructure.76Raman-spectrographic studies of nitric acid solutions have led tointeresting and important discoveries concerning the structure of thesesolutions and have shown the existence of a new ion, viz., NO2+, in nitrating78 This work has given a great stimulus to the study of the ion invarious concentrated acid solvents and has led to a most important develop-ment in inorganic chemistry, namely, the preparation and isolation of a hostof new compounds, the nitronium salts of the general formula (NO,+)(X-)61 Rank and Kagarise, J .Opt. SOC. America, 1950, 40, 89.63 Jones, Parkinson, and Burke, J . Chem. Phye., 1950,18,236.64 Andrychuk, ibid., p. 233.65 Langseth and Meller, Acta Chem. Scand., 1950,4, 725.6 7 Korshimov and Sel’ Kin, J . Exp. Theor. Phys., U.S.S.R., 1950, 20, 292.O 8 Mathieu and Conture-Mathieu, Compt. rend., 1950, 230, 1054.6D Stekhanow, J. Exp. Theor. Phys. U.S.S.R., 1950,20, 330.70 Couture-Mathieu, Compt. r e d . , 1949,229, 1215.71 Lippincott, J . Chm. Phys., 1949, 17, 1351.72 Mathieu and Lounsbury, Compt. rend., 1949, 229, 1315.73 Gerding and Eriks, Rec. Trau. chim., 1950, 89, 659.7 6 Goulden, Maccoll, and Millen, J., 1950, 1635.76 Duval and Lecomte, Compt. rend., 1943, 217, 42.7 7 ChBdin, ibid., 1936,292, 220; Ann.Chim., 1937, 8, 243.78 Goddard, Hughes, and Ingold, J . , 1950, 2589 ; Ingold, Millen, and Pooh, J., 1960,2576; Millen, J., 1950, 2589, 2600, 2606; Ingold and Millen, J . , 1950, 2612; Gouldenand Millen, J . , 1950, 2620.Stammereich, Phy8. Review, 1950, 78, 79.Fruhling, J. Chem. Phys., 1950, 18, 1119.l4 Idem, ibid., p. 72412 GENERAL AND PHYSICAL CHEMISTRY.where X- = ClO,-, HS207-, S20,--, FSO,-, S3OlO--, NO,-, e t ~ . ~ ~ This workhas now been described in Further studies 8o of the Raman spectrumof mixtures of nitric acid and acetic anhydride indicate that in this solutionN205 exists as [NO,+][NO,-]. The Raman spectrum of solutions of sulphurtrioxide in nitric acid indicate 81 the existence of the nitronium saltCompounds of which the Raman spectra have been studied include:aromatic nitro-compounds,82 buta-1 : 3-diene,s3 hexachl~rodisilane,~~ phenyl-b ~ t e n e s , ~ ~ amino-acids,86 GeC1Br3,87 SiHBr3,88 trichloromethane deri~atives,~galdehydes,g0 fluoromethane derivatives,gl difl~oroethylene,~2 deuterio-acetylacetone, deuterioacetoacetic ester,93 ethylchlorosilanes,94 methyl-chloro~ilanes,~~ trichlor~alkylsilanes,~~ linoleic monodeuteriatedtoluenes,98 and unsaturated alcohols.99 Binary and ternary mixtures ofacetone, methyl alcohol, and carbon disulphide have been investigated, looand also the effect of increasing acid character on the OH frequency in alcoholsand phenols.lo1Recently there has been a considerableadvance in the design of infra-red prism spectrometers, and many single-and double-beam instruments have been described.lo2 I n America a numberof double-beam instruments are now on the market.A very full account ofthe Perkin-Elmer double-beam spectrometer has recently been given,lo3 andcertain problems which arise in the design of double-beam instruments have“O2+1 CHS207 - 1Infra-red Spectra.-Technique.79 Millen, J., 1950, 2589.Ch6din and Feneant, Compt. rend., 1949, 229, 115.Cerding, Steeman, and Ravallier, Rec. Trau. chim., 1950,69, 944.Richards and J. R. Nielsen, J. Opt. SOC. America, 1950, 40, 438.82 Bolorich and Vol’kenstein, Doklady Akad. Nauk., S.S.S.R., 1950,71, 1045.84 Katayama, Simanonti, Inorino, and Mizushima, J. Chem. Phys., 1950,18,506.85 Golse, Compt.rend., 1950, 230, 1762.a6 Edsall, Otros, and Rich, J. Amer. Chem. SOC., 1950, 72, 474.88 Franqois and Buisset, ibid., p. 1946.90 Harrand, Compt. rend., 1949, 229, 1217.91 Rank, Shull, and Pace, J. Chem. Phya., 1950,18, 885.** Edge11 and Byrd, ibid., p. 892.93 Shirogin and Syrkin, Doklady Akad. Nauk., S.S.S.R., 1950, 70, 1033.95 Shimanouchi, Tsuchiya, and Mikawa, ibid., p. 1306.Delwaulle, Compt. rend., 1950, 230, 1945.Zietlow, Cleveland, and Meister, J. Chem. Phys., 1950,18, 1076.Murata, Okawara, and Watase, J. Chem. Phys., 1950, 18, 1308.Gonbeau and Siebert, 2. anorg. Chem., 1950, 261, 62.Pigulevskii and Naidenova, Doklady Akad. Nauk., S.S.S.R., 1950, 72, 717.98 Smith, Choppin, and Nance, J. Amer. Chem. SOC., 1950,72, 3260.99 Malyshev and Shishkina, J.Exp. Theor. Phys., U.S.S.R., 1950,20,297.loo Joerges and Nikuradse, 2. Naturforech., 1950, 5a, 25.101 Batuev, Merhcheryakov, and Matveeva, J. Exp. Thew. Phys., U.S.S.R., 1950, 20,318.102 Wright and IIerscher, J. Opt. Soc. America, 1047, 37, 211; Baird, O’Bryan,Ogden, and Lee, ibid., p. 754; Brownlie and Cumming, Nature, 1948, 164, 105; Elliott,Ambrose, and Temple, J. Sci. Inetr., 1950, 27, 21.103 ‘White and Liston, J. Opt. SOC. America, 1950, 40, 29, 36, 93MCDOWELL : MOLECULAR STRUCTURE. 13been discussed by Kivenson.lOQ Other technical developments which maybe noted are the suggested use of a carbon arc as an infra-red source,1o6 andthe continued development of photoconductive detectors. The variationof the long-wave limit of lead sulphide, telluride, and selenide photoconduc-tive cells has been investigated by MOSS,^^^ and Fellqett lo7 has discussed thetheory of the ultimate sensitivity of various radiation detectors.Daly andSutherland lo* have considered how the performance of a spectrometer islimited by the detector characteristics. Watts log has shown how a verysimple device increases the sensitivity of a photoconductive cell. A newgrating spectrometer including a thermopile as a photoconductive cell hasbeen described.Davies ll1 has observed that a synthetic silica prism in the Grubb-Parsons single- beam instrument gives a resolution which is comparable withthat observed with a grating spectrometer.Theoretical.-A rigorous treatment of the intensities of the vibrational-rotational absorption spectra of diatomic molecules has been given byCrawford and Dinsmore.l12 With regard to the theory of molecular vibra-tions a general solution for the secular equation has been given byTorkington 113 which is applicable to any molecule.The same authorhas made a normal co-ordinate analysis of the planar vibrations of varioussubstituted ethylenes 114 and also considered the problem of calculatingvalence force displacement co-ordinates for systems in which the anglesdeviate from the ideal,l16 the calculation of moments of inertia of moleculeswith internal rotation,l16 and the cubic secular equation for molecular~ i b r a t i 0 n s . l ~ ~W. J. Taylor 118 has considered the general form of the force constantmatrix for harmonic vibrations with interesting results.The vibrational-rotational energies of planar symmetrical X,Y,X2 molecules 119 and angularinteraction in pyramidal molecules such as phosphorus trifluoride andarsenic trifluoride have been considered.120 Pace 121 has computed the forceconstants of the fluoromethanes. The very extensive theoretical discussion104 Kivenson, J . Opt. SOC. Amrica, 1950, 40, 113.l o 5 Rupert and Strong, ibid., p. 455.lo6 Moss, Proc. Phys. Soc., 1949, 62, B, 741.'0' Fellqett, J . Opt. SOC. America, 1949, 39, 920.lo8 Daly and Sutherland, Proc. Phys. SOC., 1949, 62, A , 205.l U 9 Watts, ibid.. p. 486.l 1 0 Thompson and Miller, Proc. Roy. SOC., 1950, A , 200, 1.1 1 1 Davies, J . Chem. Phys., 1950,18,398.112 Crawford and Dinsmore, ibid., p.983.113 Torkington, ibid., 1949, 17, 1026; Trans. Faraday SOC., 1050, 46, 27.114 Ibid., p. 1279.l l 8 Ibid., p. 407.118 W. J. Taylor, ibid., p. 1301.l l B Herman and Schaffer, ibid., p. 1207.l * O Burnell and Duchesne, ibid., p. 1300; J . Phys. Radiol., 1950,121 Pace, J . Chem.. Phys., 1950,18, 881.115 Ibid., 1950,18, 93.11' Ibid., p. 773.119.1114 GENERAL AND PHYSICAL CHEMISTRY.of the vibrations of molecules recently given by Linnett and his collaborators 122has been extended by a consideration of the force constants of the non-metallic hydrides of Groups IV, V, VI, and VII.Attention has been drawn by Thomas lZ3 to inconsistencies in variousproposed structures for the carboxyl group, and a new assignment of fre-quencies has been submitted.Sheline has considered the problem of theeffective mass of the methyl group in Group-Iv tetramethyl derivatives.Experimental Observations.--Cole and Thompson 126 have measured theintensities of absorption bands due to the bending vibrations of substitutedbenzenes, and have calculated the dipole moment of the C-H bond.Extinction coefficients of near infra-red bands of C-H, N-H, C-C, (3x0, CzN,C-Cl bonds have been recorded.126 Francis lZ7 has given results ofmeasurements of the intensities of absorption bands for aliphatic hydro-carbons. Kaplan 128 has calculated the intensities of the 15-p. carbon dioxideband.Since infra-red absorption is only possible when there is a change in thedipole moment during the transition, homonuclear diatomic molecules shouldnot exhibit infra-red vibrational spectra.Quadrupole absorption has howeverbeen observed 1z9 for hydrogen, and a complete analysis of the quadrupolevibrational-rotational spectrum of this molecule has been given by Herz-berg.130 Using the long-path technique it has also been possible to obtainthe vibrational-rotational absorption spectrum of HD.131 Smith, Keller,and Johnson 132 have also observed infra-red absorption in liquid oxygensimilar to that previously reported by Crawford, Welsh, and Locke 133 fornitrogen, oxygen, and hydrogen at high pressures. They have also observedabsorption in liquid nitrogen. Similar observations in liquid oxygen andnitrogen have been made by Oxholm and Williams 134 and by Van Asseltand Williams.13sOzone has been re-studied by Gutowsky and Petersen 136 who state thatthe new observations do not allow a decision to be made between the acute-angled 13' and the obtuse-angled m0de1.l~~ Badger and Wilson 139 howeverlZ3 Heath and Linnett, Trans.Faraday SOC., 1948, 44, 556, 873, 878, 884; Linnettand Wheatley, ibid., 1949, 45, 33, 39; Heath, Linnett, and Wheatley, ibid., 1950, 46,137.lZ3 Thomas, J . Chern. Phys., 1950,18,76.lZ6 Cole a.nd Thompson, Trans. Faraday SOC., 1950, 46, 103.12a Suhrmann, Angew. Chem., 1950,62,507.lZ7 Francis, J . Chem. Phys., 1950, 18, 861.lZ9 Herzberg, Nature, 1949, 163, 170.Idem, Canad. J . Res., 1950, 28, A , 144.la2 Smith, Keller, and Johnson, Phys. Review, 1950,79, 728.133 Crawford, Welsh, and Locke, ibid., 1949, 75, 1607.lS4 Oxholm and Williams, ibid., 1949, 76, 151.136 Van Asselt and Williams, ibid., 1950, 79, 1016.13* Gutowsky and Petersen, J .Chem. Phys., 1950,18, 564.lS7 Adel and Dennison, ibid., 1946,14, 379.lBB M. K. Wilson and Badger, ibid., 1948,16, 741.1~ Badger and M. K. Wilson, ibid., 1950, 18, 998.ler Sheline, ibid., p. 602.128 Kaplan, ibid., p. 186.lS1 Idem, Naure, 1960, 166, 562MCDOWELL : MOLECULAR STRUCTURE. 16point out that the acute-angled model fails to account for the band observedat 1110 cm.-l. Interesting and important new observations on this problemhave been made by Wilson and Ogg 140 who studied pressure broadeningeffects in the infra-red spectrum. Using pure ozone they observed a Qbranch in the 1043-cm.-l band.An acute-angled model would require twoof the fundamental vibrations to have Q branches whereas the obtuse-angled model would only require one of the fundamentals to have a Q branch.It has previously been shown 137 that the 705-cm.-l vibration lacks a Q branch,so Wilson and Ogg conclude that their observations support the obtuae-angled model.Other simple inorganic molecules which have recently been investigatedare F20,141 S8,142 S2C12,142 and P4,142 16N14N0 and 14N14N0,143 H 2 ’ S 145sulphur monoxide,146 H 2 0 2, 14’* 148 and D202,147 COC1F,149 C1F,160 andCICN.lS1 Perhaps the most interesting observation has been the discoveryby Jones 146 that sulphur monoxide as prepared by Schank’s method cannot beSO but is probably S,02.Iodine pentafluoride has beenshown by Lord, Lynch,Schumb, and Slowinski 152 to have a tetragonal pyramidal structure (C4v)and iodine heptafluoride a pentagonal bipyramidal structure (Dbh). Shelineand Pitzer 153 have studied the infra-red spectra of Fe(CO), and Fe,(CO),.High-resolution measurements ls4 with diborane are consistent with theconclusion that this molecule has the bridge structure with symmetry V , inagreement with Price’s earlier work.156 In passing one may note that Webb,New, and Pitzer 166 have re-studied the Raman spectrum of diborane andalso investigated the infra-red spectrum of deuteriodiborane. Their resultsare in agreement with the bridge structure. Price 157 has studied the infra-redspectra of aluminium, lithium and sodium borohydrides and proposes abridge structure for aluminium borohydride and a tetrahedral structure forthe other two.High-resolution spectra obtained with polarized infra-red radiationsuggest that the urea molecule is planar in the crystalline state.158The vibrational-rotational bands of allene,15, vinyl chloride and fluoride,l 4 0 M. K.Wilson and Ogg, J . Ghem. Phys., 1950,18, 766.141 H. J. Bernstein and Powling, ibid., p. 685.143 Richardson and E. B. Wilson, jun., ibid., p. 694.144 Allen, Cross, and M. K. Wilson, ibid., p. 691.us Noble and Nielsen, ibid., p. 667.147 GiguBre, ibid., p. 88.149 E. A. Jones and Burke, ibid., p. 1308.l 6 0 E. A. Jones, Parkinson, and Burke, ibid., p. 235.151 Richardson and E.B. Wilson, jun., ibid., p. 155.lSa Lord, Lynch, Schumb, and Slowinski, J . Amer. Chern. Soc., 1950, 72, 522.163 Sheline and Pitzer, ibid., p. 1107.lS4 Anderson and Badger, J , Chem. Phys., 1950, 18, 698.156 ?rice, ibz’d., 1948,16, 894,lo@ Webb, New, and Pitzer, ibid,, 1949,17, 1007.157 Price, ibid., pt 1044.IL8 Waldron and Badger, ibid., 1950, 18, 566.lsg Miller and Thompson, Proc. Roy, SOC., 1949, A , 200, 1.142 Idem, ibid., p. 1018.146 A. V. Jones, ibid., p. 1263.148 R. C. Taylor, ibid., p. 89816 OEINERAL AND PHYSIOAL CHEMISTRY.vinylidene fluoride, glyoxal, 160 and methanethiol have been investigatedvery completely. In the case of allene, a type of Coriolis coupling originallypredicted by Nielsen 162 has been observed. cycEoPropane 163 has beenstudied by the long absorbing path technique.164Saksena and Narain 1e5have recently shown a preference for the D4d structure but it has beenpointed out 166 that this is certainly in conflict with much of the experi-mental evidence.167* lB8 Lippincott, Lord, and McDonald 166 believe thatthe D, structure is probably correct, i.e., the " crown " structure.Further studies 169* 170 have led to a new assignment of frequencies inethylene, but Torkington 171 has pointed out several difficulties which thisoccasions.etc. etcThermodynamically the osmotic pressure may be quite generallyexpressed in virial form (Zoc.cit.), and all reported measurements can befitted to this equation using not more than three terms. In many instances,34 Zirnm, J .Chem. Phys., 1948, 18, 1093; Blaker, Badger, and Gilmar, J . Phys.Colloid Chem., 1949, 53, 794; Hadow, Sheffer, and Hyde, Canad. J . Res., 1949, 27, B,791; Schulz and Harborth, Makromol. Chem., 1948, 2, 187; Jullander, Acta Chem.Scand., 1949, 3, 1359.Doty and Steiner34a Doty and Steiner, J . Chem. Phys., 1950, 18, 1211.35 Stuart, Makromol. Chem., 1949, 3, 176; Chem. Weekblad, 1949, 46, 293; Oster,Rec. Trav. chim., 1949,68, 1123; Rousset and Lochet, Rev. Sci., 1948,86,291; Hermans,Plastica, 1950, 3, 187, 222." Sands and Johnson, Ind. Eng. Chem. Anal., 1947, 19, 261 ; Sirianni, Wise, andMcIntosh, Canad. J . Res., 1947,25, B, 301 ; Browning and Ferry, J . Chem. Phys., 1949,17,1107 ; Bawn, Freeman, and Kamaliddin, Trans. Paraday SOC., 1950,47,862 ; Platzek,Chem.Weekblad, 1950,46, 193.37 Masson and Melville, J . Polymer Sci., 1949,4, 323, 337.38 Enoksson, ibid., 1948, 3, 31492 GENERAL AND PHYSICAL CHEMISTRY.linear relationships have been found between x/c, and c,,~~ and this impliesthat A , = 0 Unfortunately, contrary results have been reported by differentworkers for the same polymer in the same solvent, the X / C , against c, plot.being linear in some cases whilst in others it shows a very marked curvature.40Whether this is due to branching or to a different internal architecture of themolecule is not clear. Recent osmotic measurements include poly(viny1acetate)41 and polystyrene42 in a wide range of solvents and at differenttemperatures, polythene in xylene at 72.7" and 9 1 ~ 6 " , ~ ~ poly( isobutyl vinylether) in toluene,44 poly( butyl acrylate) in and poly(methy1methacrylate) in benzene.46The determination of the virial coefficient, A,, is important, since it is ameasure of the mutuaI interaction of the molecules in solution.When A ,is large the segments, through their excluded volumes, repel one another sothat the molecule is swollen, but when A , is made smaller the moleculeshrinks. When A , = 0 the average repulsive and attractive forces balanceand the extension of the molecule will be little affected by the ~ o l v e n t . ~ ~ * Contrary to previously held opinion recent theoretical and experimentalwork shows that A , depends strikingly on molecular ~ e i g h t . l l * ~ ~ * 48 Zimm l1observed a threefold variation of A , for polystyrene in dichloroethane overthe molecular weight range 1,78,3,000-23,700. A slight decrease in A , withmolecular weight was observed for poly(viny1 acetate) in ethyl methylketone.41 The dependence of A , and A , on molecular weight was measuredin toluene by Bawn et aZ.,36 who observe that the plot of A , against 1/Mwas linear although there was considerable scatter of the points at highmolecular weight, and the relationship may need modification when moreaccurate data is available.As would be expected the value of A , decreases inpoor solvents, and this is found in all the cases so far Forpolystyrene, A , has a negative temperature coefficient in good solvents anda positive coefficient in poor solvents.51 Both Zimm l1 and Iiunst 50observed a close correlation between the root mean square length, R, and A ,for single fractions of polystyrene in a range of solvents.Similar, althoughless extensive, results are observed with polyi~obutene.~l Zimm noted that38 Goldberg, Hohenstein, and Mark, J. Polyirier Sci., 1947, 2, 503; Melville and40 Cf. refs. 39 and 42.41 Browning and Ferry, see ref. 36.4 2 Bawn, Freeman, and Kamaliddin, see ref. 36; Breitenbach and Frank, Monatsh.,1949, 79, 445; 1950, 81, 455; Schick, Doty, and Zimm, J. Amer. Chem. SOC., 1950,72,630.Valentine, Trans. Faraday SOC., 1950,46, 210.43 Muthana and Mark, Rec. Trav. chim., 1949, 68, 758.44 Idem, ibid., p. 754.4 5 Bickel and Melville, Trans. Faraday SOC., 1949, 45. 10.19.4 6 Mackay and Melville, ibid., p.323.4 7 McMillan and Mayer, J. Chem. Php., 1945,13, 276.4 8 Benoit, Compt. rend., 1950, 230, 2024.49 Refs. 36, 41, 48.Kunst, Rec. Trav. chim., 1950, 69, 125; h'alure, 1949, 164, 535.51 See refs. 11 and 12BAU" : SOLUTIONS OF HIGH POLYMERS. 93the osmotic effect with polystyrene was able to change the mean extensionof the molecule by a factor of two.Viscosity.-The considerable interest in the experimental and theoreticalinvestigations of the viscosity of solutions of macromolecules arises from theinformation which may be derived about the size and weight of the molecules.The intensity with which this work is pursued may be judged from the factthat many of the new ideas referred to below arose simultaneously in differentplaces.It was early realised that the Einstein model of the compact spherewas not consistent with our ideas of chemical structure, and later calculationswith asymmetric molecules permitted the evaluation of the dimensions ofmolecules of simple shape, such as ellipsoids or rods, once the molecularweight was known. The extensions of these ideas to chain-type polymersshowed the intimate connection between viscosity and molecular weight , andfrom numerous measurements the familiar rule between the intrinsic viscosity,[ r ) ] , and molecular weight, M , viz. [q] = KM", where the exponent a assumesvalues between 0-60 and 1.00, was deduced. The magnitudes of K and ahave been determined for a large number of polymer-solvent systems, andnew work includes measurements of polyi~obutene,~~ p ~ l y s t y r e n e , ~ ~ poly-b~tadiene,~* and cellulose nitrate 55 in a wide range of pure and mixedsolvents.A single relationship has been found to exist between a and K forpolystyrene in pure and mixed s0lvents.~6Striking advances have recently been made in the theoretical under-standing of the relationship between viscosit,y and molecular weight, and ithas been shown that the modified Staudinger law may be derived for theflexible-coil model of a polymer chain. If the molecule is considered as anecklace in which the flow around each bead is not modified by the presenceof the other ones, then the intrinsic viscosity is proportional to R2, where Ris the distance between the chain ends.57 Combination of this result withthe theoretical relationship for R leads to Staudinger's equation.57The assumption that negligible interaction occurs between individualsegments is not true for a long-chain coiled molecule, and recent theories 57include the effects of hydrodynamic interaction. Brinkman and Deb ye andBueche 58 consider the polymer particle as a porous sphere in which thereare 2 beads distributed with uniform density, and evaluate the disturbanceof flow through the particle produced by its independent segments.These52 Baldwin, J . Anter. Cheni. (soc., 1950, 72, 1833; Fox and Flory, ref. 24.53 Bawn, Freeman, and Kamaliddin, Trans. Faraday SOC., 1950, 46, 11 07 ; Schulz,Makromol. Chem., 1949, 3, 146; Breitenbach, ref. 42; Vallet, Rec.Trac. chim., 1950, 69,325.54 Johnson and Wolfangel, Ind. Eng. Chem., 1949, 41, 1580.5 5 Zapf, Makromol. Chem., 1949, 3, 164.Bawn, Grimley, and Wajid, Trans. Faraday SOC., 1950, 46, 1112.Debye, J . Chem. Phys., 1946,14, 636; Hermans, Rec. Trav. chim., 1944, 63, 219;J . Polymer Sci., 1946, 1, 233 ; Kramers, J . Chem. Phys., 1946, 14, 415.5 8 Brinkman, A p p . Sci. Res., A , 1947, 1, 27; Physica, 1947, 13, 565; Debye andBueche, J . Chem. Phys., 1948,16,573 ; Kirkwood and Riseman, ibid., p. 565 ; Kuhn andKuhn, Helr. Chim. Acta, 1945, 28, 98, 1533; 1946, 29, 609. 830; J . Colloid Sci.. 1948,3, 1 1 : J . Polymer S c i . , 1950, 5, 51994 GENERAL AND PHYSICAL UHEMISTRY.segments interfere with one another hydrodynamically with the result thatthe inner parts of the polymer molecule are shielded from the solvent byoutlying portions.This shielding effect is greater for large molecules thanfor small ones. Kuhn and Kuhn and Kirkwood and Riseman s8 employed adifferent mathematical approach, using the statistics of the polymer chainwith hindered rotation rather than the hydrodynamically equivalent sphere.Both theories predict that the exponent o! should decrease from unity forlow-molecular-weight polymers to 0.5 for very high molecular weights andallow of the calculation of R from the measured [q] once the intrinsicviscosity/molecular weight relationship has been determined. The theoryhas also been worked out for rods made up of monomer molecules separatedby rigid bonds.59It has been repeatedly reported that chain polymers exhibit higherintrinsic viscosities in good solvents than in poor solvents, and these effectshave been interpreted in terms of the nature of the interaction betweensolvent and polymer molecules. The above theory may be used to determinethe influence of solvent on the molecular dimensions, and results have beenreported on polystyrene in pure and mixed solvents by Outer, Carr, andZimm 6o and by Bawn, Grimley, and Wajid.S6 The former authors, and alsoK u n ~ t , ~ ' show that the variation of R with solvent is similar to that deter-mined by the light-scattering method (Zoc. cit.).A close correlation between theory and experiment has also been noted byPeterlin 62 for polystyrene and amylose, but with the more rigid cellulosederivatives the theory is not so satisfactory.Both the rotary-diffusionconstant and the intrinsic viscosity depend on the same parameter describingthe shape of the particle and, by a combination of these measurements, offeran additional method of determining molecular weights.58* 61The numerous relationships which have been proposed to represent thedependence of viscosity on concentration 63 may be expressed in the expandedformthe coefficients a2 and a3 are empirically related by the equations a2 =k1[qI2 and a3 = k2[ql3. This equation is obeyed for many systems with k,in the range 0-3-0.6 for dilute solutions. By using certain reasonableapproximations and including hydrodynamic interaction and aggregation,Simha has shown that the above equations may be derived theoreticallyand that the calculated vaIues of k, (0.77 for coils) are of the right order ofmagnitude.Furthermore, the parameter k, is shown to depend in acharacteristic manner on the shape of the particle as determined by flexible?.P/C = [?I + a2c + a3c2;Kirkwood and Riseman, J . Chem. Phys., 1950,18, 512.Outer, Carr, and Zimm, ibid., 1950,18, 836.61 Newman, Riseman, and Eirich, Rec. Truv. chim., 1949,68,921.62 Peterlin, J. Polymer Sci., 1950, 5 , 473.6s Alfrey, " Mechanical behaviour of High Polymers," Interscience, 1948, pp. 459-64 Simha, J. Res. Not. Bur. Stand.. 1949, 42, 409; ,J. ColloidSci., 1950, 5, 386,461BAWN : SOLUTIONS OF HIGH POLYMERS. 95molecules by the solvent environment and the molecular weight.Similarcalculations by Saito 65 lead to the same relationship, with k, = 0.43 forchain molecules, in very close agreement with experiment. At present thetheories do not specifically include polymer-solvent interaction.Fractions of polystyrene prepared a t different temperatures obey theseequations with k, = 0.38 (toluene),66 but when the polymer is made frommonomer containing small quantities of divinylbenzene the resulting k,values are changed t o 0-50--0-84. This is ascribed to cross linkage orbranching. I n ethyl methyl ketone k, has been found to increase from 0.42to 0.63 as the molecular weight increases, and this may be due to progressivecoiling in the bad solvent.Polgelectrowes.-The ionisation of macroelectrolytes containing largenumbers of ionisable groups of different types has attracted much attentionboth experimentally and theoretically.The difference of the titrationbehaviour of a polymeric acid from that of monobasic acid is due to the workexpended in removal of the hydrogen ion from the field of the ionised groups,as well as the work performed by electrostatic forces on stretching the polymermolecule. Several calculations of the field effect have been made and usedto develop theories of titration and dissociation of polymeric acids.67Overbeek, by treating the polymer molecule as a sphere containing a con-tinuous charge, expresses the change of dissociation constant as a functionof the radius of the molecular coil and the charge on the particle. Measure-ments 6* with solutions of poly(methacry1ic acid) agree with theory for lowdegrees of dissociation but for higher extents of dissociation the treatmentof the polymer coil as a sphere is no longer satisfactory.Expressions havebeen developed for the electrical free energy of a statistically coiled electricallycharged chain molecule.67* 69 I n these calculations the end-to-end length Rof the polymer molecule is used as a characteristic parameter €or the shapeand the expansion of the molecule caused by electric charges. The effect ofconcentration and added salts thereon may be expressed as a function of R.The configuration of ionised polymer molecules has also been discussed byGuinard et aL70In a macromolecule of the poly(viny1pyridine) type, in the presence of,for example, an alkyl halide, every carbon atom of the long chain carries apositive pyridinium ion; the charges on the polycation cannot diffuse apartany farther than that corresponding to the maximum extension of the coil.Therefore, in a very dilute solution where the molecules are separate, thereexists a small volume of high positive-charge density localised in the poly-6 5 Saito, J .Phy. SOC., Japan, 1950, 5, 4.6 6 Walker and Winkler, Canad. J . Res., 1950, 28, ;B, 298.6 7 Katchalsky and Gillis, Rec. Trav. chim., 1949, 68, 879; Kuhn, Kunzle, andKatchalsky, Helv. Chim. Acta, 1948, 31, 1994; J. Polymer Sci., 1950, 5 , 283; Overbeekand Hermans, Rec. Trav. chim., 1948, 67, 761 ; Overbeek, Bull. SOC. chim. Belg., 1948,57, 252.Arnold and Overbeek, Rec.TTUV. chitn., 1950,69, 192.Guinard. Boyer, Kawenoki. Dobry, and Tonnelat. Compt. rend., 1949, 229, 143.** Kunzle, ibid., 1949, 68, 69996 GENERAL AND PHYSICAL CHEMISTRY.cation, and we should expect a large number of anions to accompany thepolymeric ion and exist within the molecular coil. Outside, in the body ofthe solution, only anions will be found. When the coils overlap the solutionwill resemble a concentrated solution of a one-one electrolyte. This view-point has been confirmed by Edelson and Fuoss 71 from measurements of theconductivities and viscosities of poly( N-n- butyl-4-vinylpyridinium bromide)and poly(sodium acrylate) at concentrations up to 0 . 3 ~ . These authors alsoshow that, although aqueous solutions of these two polymers at the sameequivalent concentration have approximately the same conductivity, therelative viscosity of the polyacrylate was much greater than that of thepolybromide.The difference is ascribed to cross linking 73 through hydrogenbonds with water molecules and not to differences in molecular weight. Theconsiderable evidence that in aqueous mixtures of poly(acry1ic acid) andsodium hydroxide the sodium and polyacrylate ions are associated to animportant extent has been established by transference measurements,radio-sodium being used as a tracer.74from theoretical calculations of the dependence of pHand equivalent conductivities as a function of concentration, conclude thatthe conductivity of a polyelectrolyte is due almost entirely to hydrogen ions.Theoretical expressions for the intrinsic viscosity of polyelectrolytes havebeen derived by Kuhn 67*75 and Hermans and O ~ e r b e e k , ~ ~ and these havebeen used to evaluate the dimension of the polymer coil in a mannersimilar to that described for un-ionisable polymer .The viscosity behaviourof strong polyelectrolytes has been the subject of research by Puoss and hiscollaborator^.^^ Empirical functions have been deduced for the viscosity ofsolutions which besides containing a factor dependent on molecular weight,as with ordinary polymers, includes terms determined by electrostaticinteractions.The effect of salts on the viscosity of poly(acry1ic acid) solutions has beenshown to be in agreement with Smoluchowski’s electrical theory of viscosityat low concentrations and that of the folding chain at higher concentrationsof added salt.77 The theory of light scattering from solutions and chargedmolecules has been worked out by Hermans 78 and by Doty and Steiner.79The latter authors report measurements on various polyelectrolytes, and havefound that there is a deficiency of light in the forward direction instead of anexcess.The investigation of the properties and behaviour of polyelectrolytes as aWall andi 1 Edelson and FUOSS, J . Amer. Chem. SOC., 1950, 72, 306.72 Wall and Butts, J. Chem. Phys., 1949,17, 1330.73 Edelson and FUOSS, J . Amer. Chem. SOC., 1950,72, 1838.74 Huizenga, Grieger, and Wall, ibid., p. 263.7 5 Kuhn, Kunzle, and Katchalsky, Bull. SOC. chim. Belg., 1948, 57, 421.i0 Fuoss, J . Polymer Sci., 1948, 3, 603; 1949, 4, 47; Fuoss and Strauss, Ann. N . Y .7 7 Markoritz and Kimball, J . Colloid Sci., 1950, 5, 115.7 8 Hermans, Rec. Trav. chim., 1949,68, 859.Acad. Sci., 1949, 51, 836.Doty and Steiner, J . Chem. Phys., 1949, 17, 743BAWN : SOLUTIONS OF HIGH POLYMERS. 97model substance for understanding the more complicated behaviour ofbiological systems has been stressed recently. Methacrylic acid polymerscontaining o.07y0 dipinylbenzene as a cross-linking agent absorb up to 326times their weight 80 of water, giving jellies which are weak although stable inform. Jellies swollen 81 -fold by water shrink to 6-fold in 0-Oh-hydrochloricacid, whereas in water containing 7 yo equivalent weight of potassium hydrox-ide a 202-fold swelling occurred.The molecular expansion and contraction of ionisable polymers may bemade evident on a macroscopic scale as shown by Kuhn et u Z . , ~ ~ who foundthat filaments of poly(acry1ic acid), bearing a load, expanded and contractedreversibly in the presence of alkali or acid, respectively. The degree ofexpansion or contraction was determined by the extent of cross linking ofthe polymer.

ISSN:0365-6217    

DOI:10.1039/AR9504700007

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

INORGANIC CHEMISTRY.IN the present Report the aim has been to continue to present a representativeaccount of new work in the many fields of inorganic chemistry which, asshown by the number of original papers published, continues to attract anincreasing amount of attention. Owing to the time-lag between the publisheddate of some foreign journals and the actual time of their appearance in thiscountry, it has not been possible to include all the work published near theend of 1950; for the same reason reference has been made to a few paperspublished near the end of 1949.Thepresent classification under groups of the Periodic Table is convenient thoughit has its limitations ; inevitably some investigations are concerned withelements in more than one group. In such cases the work is described underthe group heading associated either with the first-mentioned element or withthe most important feature.The chemistry of some elements has receivedmuch attention during 1950; of others, virtually none. The two featureswhich are perhaps the most outstanding are the continued rapid increase inthe interest in the chemistry of fluorine and the wide range of applications ofion-exchange resins, not only for the separation of chemically very similarelements but also for the study of their physicochemical properties and evenfor preparative purposes.Of the reviews which have been published during the year reference maybe made to a collection of papers by a number of authors on a variety ofaspects of complex-compound formation which has been published in honourof the 75th birthday of Paul Pfeiffer, the pioneer in this field,l and to reviewsby Lister 2 on the Chemistry of the Transuranic Elements and by Sharpeon Interhalogen Compounds and Polyhalides.A review, by C ~ a t e s , ~ hasalso been published on the Organometallic Compounds of the First ThreePeriodic Groups which includes a discussion of some uncommon structuraland valency problems provided by these compounds. The centre of interestin organometallic compounds, apart from their use as preparative reagents,has seemed in recent years to belong more and more to the realm of InorganicChemistry, and mention is made in the present report of a number of neworganometallic compounds.Group 0.-A most interesting series of clathrate or cage-like compoundsof the inert-gases argon,5 krypton,6 and xenon has been described byPowell.These are prepared by slowly cooling an aqueous solution of quinolunder a high pressure of inert gas (necessary because of its low solubility).No single system of classification of topics is likely to be ideal.1 Angew. Chem., 1950, 62, 201.2 Lister, Quart. Reviews, 1950, 4, 20.Ibid., p. 217.6 Powell and Guter, Nature, 1949, 164, 240; Powell, J . , 1950, 298.Ibid., p. 115.Powell,ibid., p. 300. Idem, ibid., p. 468FAIRBROTHER. 99The general series of clathrate compounds of ideal formula SC,H,(OH),,Mformed by quinol with a, variety of substances M, which in their ordinarystates at room temperature may be volatile liquids or gases, consist of cage-like structures formed by the quinol on crystallisation in the presence of thesecond component, the atoms or molecules of which are trapped inside thecage.There is no chemical bonding in the usual sense between the inert-gasmolecules and the quinol cage. Nevertheless, these " compounds " areremarkably stable at room temperature and pressure although the inertgas may be effectively under a high pressure [more than 70 atm. for3C6H4( OH),,O.88 ; the fully saturated 3C,H4( OH),,A would contain argonat an effective pressure of 91 atm.]. Since the inert gas may be liberated bywarming or by dissolution of the quinol framework in some solvent, these com-pounds constitute a highly concentrated and portable source of inert gas.Similar compounds may be formed with other organic molecules whichare capable by hydrogen- bond formation of providing the necessary cage-likestructure.The inability of helium to form a clathrate compound withquinol is believed to be due to its small diameter, which permits it to escapethrough holes in the cage walls.Traces of nitrogen and oxygen present in commercial argon or helium canbe effectively removed by passing the gas over titanium powder at 850'.8Group 1.-A simple method of extraction of metallic czesium from theSwedish mineral pollucite, due to Hackspill and tho ma^,^ consists in heatingthe dried ore directly with calcium in vacuo at 900' : the czesium distils offand can be purified by redistillation in vacuo at 350400'.Lithium hydride can also be used lo as a reducing agent for the prepar-ation of other alkali metals by heating it with the corresponding chlorideunder reduced pressure.Superoxide formation, which occurs easily only with the large alkalimetals, has been extended by Stephanou, Schechter, Argersinger, andKleinberg l1 to include sodium superoxide, NaO,.The crystal structure ofthis has now been measured by Templeton and Dauben l2 and differs fromthat of the potassium, rubidium, and caesium superoxides, which are iso-morphous with CaC, and in which each pair of 0-0 atoms is oriented withits axis parallel to the tetragonal c-axis, in that it possesses a rock-saltstructure with the 0,- ions occupying positions like the chlorine ions butwith a random orientation of the 0-0 axis.The effects of the nature of the anion, the basicity of the amine, and thenature of the solvent on the properties of complexes between cupric and cuproussalts and long-chain amines have been investigated by Burkin.13 Cuprouschloride, bromide, and iodide complexes with monoamines l4 polymeriseMallett, Ind. Eng.Chem., 1950, 42, 2095.Compt. rend., 1950, 230, 1119.lo Pearce, Burns, and Gantz, Proc. Indiana Acad. Sci., 1949, 58, 99.l1 J . Amer. Chem. Xoc., 1949, 71, 1819.l2 Ibid., 1950, 72, 2251.l3 J., 1950, 122.Wilkins and Burkin, J . , 1950, 127100 INORGANIC CHEMISTRY.to a tetrameric form, whilst cuprous bromide also forms at the temperatureof freezing benzene bisamine complexes with a dimeric bridge structurewhich dissociate reversibly on warminginto the tetrameric monoamine complexesand free amine.The cuprous halideRH,NRH,N~~ k ~ ~ / \NH,R complexes of empirical formula CuX,NH,Rare oxidised in the air and in presence ofamine salts HX,NH,R form the corresponding cupric halide complexesCuX,,2NH,R.16 I n both cupric and cuprous complexes the usual co-ordination number of 4 is maintained. On the other hand, polarographicstudies of copper complexes with dipyridyl, o-phenanthroline, and thioureaby Onstott and Laitinen l6 indicate that in the presence of considerableexcess (50- to 200-fold) of complexing agent (dipyridyl) the cupric ion canco-ordinate three molecules, i.e., exhibit a co-ordination number of 6, whilstunder the same conditions the cuprous ion only co-ordinates two molecules.Other work, by Pfeiffer and Werdelmann,17 also indicates that in phenan-throline complexes with univalent central atoms (Ag,TI) the central atom hasa co-ordination number of 4.Bivalent perchlorates of Cu, Sn, Fe, Co,Be, Mg, Zn, Cd, and Hg on the other hand are reported to co-ordinate 3phenanthroline molecules with a co-ordination number of 6.Some light has been thrown by X-ray investigations hy Rundle andGoring of the crystal structure of the salt AgC1O4,C,H6 on the mechanismby which the argentous ion can become attached to the benzene ring. Itis found that each silver ion lies between two obliquely inclined benzenerings, equally bonded to two carbons in each, a structure which suggeststhe formation of x bonds between the silver and the aromatic ring.The compounds 5Ag,0,1,07, 2Ag,0,1,07 ,3H,O, and Ag,O,I,O, ,4H,Ohave been isolated by Gyani l 9 from the ternary system Ag20-H10,-H,O.Group II.-Hartford, Lane, and Meyer 2o have prepared and examinedthe two crystalline hydrates of calcium dichromate, CaCr2O7,3H,O, and thepreviously unknown CaCr,07,4.5H,0. When heated, the hydrates undergopartial decomposition by hydrolysis to CaCrO, and CrO, : there is no evidenceof the existence of anhydrous CaCr,O, as a separate crystalline phase.Onfurther heating, oxygen is lost to give a mixture of‘ CaCrO, and Cr203 whichat 11OO--l20Oo loses oxygen to form CaCr,04.Quinoline complexes of zinc and cadmium sulphates have been studied byBattacharya and Sinha.2l Polarographic studies by Douglas, Laitinen, andBailar 2, have demonstrated the existence of complexes, [Cd(en)3] + + ,[Cd( pn),] + + , [ Cd( dien),] + + , [ Cd( trien)] + + , [ Cd( dipy ),I + + , and [Cd(o -phen),] + + ,in the presence of considerable (100-2000-fold) excess of the respectivecomplexing agent.Mehta and Kabadi 2, have isolated sodium zincate, 2NaOH,Zn(OH),,H,O1 5 Wilkins and Burkin, J ., 1950, 132.l6 J . Amer. Chem. SOC., 1950, 72, 4724.l8 J . Amer. Chem. SOC., 1950, 72, 5337.20 J . Amer. Chem. SOC., 1950,72, 3353.s2 J . Amer. Chem. SOC., 1950, 72, 2484.NH2R Br Yli cu’ li c ul7 2. anorg. Chem., 1950,261, 197.lo J . Indian Chem. SOC., 1950, 27, 5.a1 J . Indian Chem. SOC., 1950,27, 21.23 J . Univ. Bombay, Sect.A, 1949,18, 54FAIRBROTHER. 101by slow evaporation in a vacuum of solutions of Zn(OH), in concentratedaqueous sodium hydroxide.Jander 24 has extended his work on acid-base systems in non-aqueousmedia to a study of molten mercuric bromide (m. p. 236") as an electrolyticsolvent in which many reactions can be carried out. Mercuric oxide, titratedconductimetrically with thallous bromide or mercuric sulphate, behaves as aweak base in HgBr,, dissolving in it much as NH, dissolves in water. Assuch it can be displaced from basic salts, e.g., BH,O,HgSO,, by the strongbase TlBr. In a study by Lamure Z5 of the system HgC1,-Cu(OH),-H20between 17" and loo", two definite compounds were found : 2HgC12,3Cu( OH),and HgC1,,3Cu(OH),. The paramagnetism of CU++ is much diminished inthese compounds, which with other observations suggests that they aresalts of complex CU++ ions.Group III.-Mass-spectrometric examination by Norton ,* of residuesfrom B,Hll which had been stored for a long time at -78" showed, in additionto traces of B10H14, B6HI0, and B5H,, species around mass 105-108.It isbelieved that these represent a new hydride B,H,, belonging to the stableB,H, + 27 series, although a formula BgH15 belonging to the less stableBnHn+6 series is not excluded.Stone and Emelkus g8 have examined the reaction of diborane with somealkene oxides and vinyl compounds. Diborane reacts rapidly with ethyleneor propylene oxide at -80" to give respectively diethoxyborine or diiso-propoxyborine and polymers of the type H*[CHR*CH,*O],*BH,, whereR = H or CH,, respectively.Boron trifluoride and ethylene oxide similarlyreact vigorously to give dioxan and a liquid boron-containing polymer.It may be noted that this polymerisation, unlike the polymerisation ofisobutene by boron triflu~ride,~~ does not require the presence of a hydroxylicco-catalyst : it is suggested that possibly the ethylene oxide itself may actas co-catalyst.28The systems BF,-PH,, BF,-ASH,, and BF3-HBr have been investigatedby Martin and Dial.30 The absence of compound formation in the last twosystems has been ascribed on the one hand to the greater radius of arsenicthan of phosphorus, and on the other hand to the small ionic character ofHBr. ASH, co-ordinates with both BCl, and BBr, in which the arsenicatom can approach more closely to the boron than in BF, : also the moreionic HF alone among the hydrogen halides forms co-ordination compoundswith BF,.Brown and Johannsen 31 have investigated the 1 : 1-addition compounds24 Jander andBrodersen, 2.anorg. Chem., 1950, 262, 3 3 ; Jander, Angew. Chem.,25 Compt. rend., 1949, 228, 1731.26 J . Amer. Chem. SOC., 1950, 72, 1849.27 Wiberg, Ber., 1936,69, 2816.28 J . , 1950, 2755.29 Evans and Meadows, J . Polymer Sci., 1949, 4, 359; Symposium on Friedel-Crafts30 J . Amer. Chem. Soc., 1950, 72, 852.1950, 62, 264.Catalysts, Nature, 1949, 164, 655.31 Ibid., p. 2934102 INORGANIC CHEMISTRY.which boron trifluoride forms with benzonitrile, o-, m-, and p-tolunitrile, andmesitonitrile.The dissociation pressure of boron trifluoride over thebenzonitrile addition compound is sufficiently low at room temperature topermit of purification by sublimation, whereas at 80" it is about 55 mm. ofHg : it has been suggested as a method for the purification of boron tri-fluoride.The specific electrical conductivity, electrolysis, and kinematic viscosityof liquid boron trifluoride-ethyl ether BF,,(C,H5),0 in the temperature range-10" to +45" have been the subject of an investigation by Greenwood,Martin, and E m e l k ~ s . ~ ~ The results indicate that considerable ionic characteris introduced into ethyl ether when co-ordinated t o BF, and that the doublecompound is ethyl ethoxyfluoroborate C,H5*[BF,*O*C,H,]. Freezing-pointdata for the COC1,-BF3 system 33 indicate the existence of two compoundsCOCl,,BF, (f.p.- 134.3") and (COC1,),,BF3 (f.p. - 137.0").A phase-rule investigation 34 of the system Al,O,-SO,-H,O at 60" in thebasic region between sulphur trioxide concentrations of 5.10-22.02% hasgiven evidence that only two stable solid phases, Al,O3,SO,,6H,O andAl,O3,2SO,,l1H,O, exist in this range, and that many basic aluminiumsulphates which exist at lower temperatures 36 are not stable chemicalindividuals at 60".An examination of the system MgS04-Al,(S04)3-H,0 by Bassett andWatt 36 has indicated the existence of one double salt MgAl,(S04)4,22H,0,which is the essential constituent of pickeringite, although specimens of thismineral usually contain also excess of MgS0,,7H20.Bassett 37 has also prepared a lead alunite which contains 44% ofPb[Al,(OH),( H,0),][S04], mixed with H,0[A1,(OH)5,H20][S04]2 and alittle H,O[AI,(OH),( H,0),][Al(OH)4],, which supports the view that in thealunite lattice one-third of the aluminium positions can be vacant.The equilibrium diagram of the system As,05-A1,03-H,0, studied byGuerin and Martin,38 indicates the existence of two aluminium arsenates-an acid salt 2As,05,Al,0,,3H,0 which exists in equilibrium with solutions ofhigh (43.2-71 -5 yo) As,05 content, and an orthoarsenate As,05,Al,03,2H,0at lower As,O, concentrations.Wallace and Willard 39 have studied the rate of exchange of radio-chlorinebetween solid aluminium chloride and carbon tetrachloride.The resultssuggest that the exchange takes place by the surface-ionisation of the carbon-halogen bond on charged centres of the ionic AlCl, lattice rather than throughthe formation of soluble molecular or ionic complexes such as occur in ahomogeneous system.Davidson and Jirik40 have found that the anodic oxidation of thallium33 J., 1950, 3030.33 Martin and Faust, J .Phys. CoZZ. Chem., 1949, 53, 1255.34 Henry and King, J . Amer. Chem. SOC., 1950,72, 1282.35 Bassett and Goodwin, J . , 1949, 2239.86 J . , 1950, 1408. 5 7 J . , 1950, 1460.313 Compt. rend., 1950, 230, 2025.39 J . Amer. Chem. SOC., 1950,72, 5275. 'O Ibid., p. 1700FAIRBROTHER. 103in anhydrous acetic acid yields exclusively thallous ion Tl+. The anodicoxidation of aluminium, gallium, and indium leads to a loss of metal from theelectrode considerably greater than that corresponding to Paraday's law onthe assumption that a triply charged metal ion is formed in each case.Itis suggested that the primary anodic products may be mixtures of singly andtriply charged metal ions.It has been shown by Glazer, McRoberts, and Schulman 41 that aluminiumdodecanoate (trilaurate), prepared by reaction between lauric acid andtrimethylaluminium in dry benzene, has no gelling properties in hydrocarbonsolution. This result may be ascribed to the fact that this aluminiumtri-soap does not contain any hydroxy-bonds by which hydrogen- bondedcross-linking can take place. Anhydrous aluminium triacetate and tripro-pionate have also been prepared.42Some aluminium selenates have been prepared and examined byBassett 43 and shown to be very similar to the corresponding sulphates-aresult that may be helpful in elucidating the structures of the sulphates,since the selenium atoms are much more easily distinguished from thealuminium atoms by X-ray diffraction than are sulphur atoms.It is becoming increasingly evident that the frequently postulatedchloro-acids of the type HAlC1, or HGaCl, do not exist as such.I n thisconnection the observations of Brown, Pearsall, and Eddy are significant :no reaction could be observed between hydrogen chloride and aluminium orgallium chloride under a variety of conditions from -120" to 300". Itwould appear that the simultaneous presence of a third, slightly basicprotophilic molecule may be necessary to bring about the ionisation of thehydrogen-chlorine bond.Clusius and Hitzig 46 have studied the halogenation of gallium metal byheating it with a number of other metal halides, and the reduction of thetrichloride by a number of metals.The reaction between silver and GaC1,is reversible : Ag + GaCl, AgCl + GaC1,. GaBr,,(CH3)20 andGaCI,,(CH,),O have been prepared, and their vapour-pressure curvesmeasured, by Van Dyke and Crawford : 46 trimethylamine forms compoundswith gallium chloride or bromide of the type GaC1,,2(CHJ3N. Van Dyke4' hasalso measured the conductance of these two halides in nitrobenzene, whichis increased by the addition of dimethyl ether. The gallium halides and theirether complexes are very weak electrolytes in nitrobenzene; the bromide ismonomeric in nitrobenzene solution.A study48 of some chlorogallates and related co~pounds of the typeM'M"'Cl,, where M' = NH,+, Li+, K+, or Cs+, has yielded information, notonly in respect of the preparation and properties of these compounds, butalso of the relative acidities of the several Lewis acids (Ga+++, Fe+++, A,+++)involved.The same authors have also found 48* 49 that KH,GaCl,, NH,FeCl,,41 J., 1950, 2082.4a Hood and Ihde, J . Amer. Chem. SOC., 1950, 72, 2094.44 J . Amer. Chem. SOC., 1950,72, 5347.46 J . Amer. Chem. SOC., 1950, 72, 2829.48 Friedman and Taube, ibid., p. 2236.43 J., 1950, 1191.4 5 Helv. Chim. Acto, 1950, 33, 506.4 7 Ibid., p. 2823.4~4 I b i d . , p. 3362104 INORGANIC CHEMISTRY.and NH,AlCl, when treated with dimethyl ether distribute themselvesbet-ween the solid phases and the saturated solutions without change incomposition, but that some other compounds of this type including LiGaC1,undergo a change in composition, the saturated ether phase being enrichedin M”’Cl, relative to M‘C1.The conditions under which hydrous gallium oxide is precipitated havebeen reinvestigated electrometrically by Moeller and King.” In thepresence of SO,- ion precipitation begins at a mole ratio of [OH-] : [Ga+++]of approximately unity and is complete a t a ratio of 3 : ’1. In the presenceof C1-, Br-, and NO,-, however, precipitation does not begin until the ratioreaches 3 : 1, whereupon complete flocculation takes place.Measurements of the magnetic susceptibility of TICl, and TI,CI, made byMeir and Garner 51 show that both these compounds are diamagnetic like thegallium and indium dihalide~.~, These compounds therefore contain themetals in 1 and 3 oxidation states or metal-metal bonds.The preparation of milligram quantities of pure actinium by the trans-mutation of radium in a chain-reacting pile, 226Ra (n ; y ) + 227Ra &227Ac, has made possible for the first time a direct observation of the proper-ties of pure actinium since this isotope of mass 227 has theconveniently long half-life of 21.7 years,% in contrast to the short-livednaturally occurring isotopes.Nine pure actinium compounds, all isomor-phous with the corresponding lanthanum compounds, have been preparedon a microgram scale and examined by X-ray diffra~tion.~~ The crystaldata provide additional evidence that actinium can be regarded as the firstmember of the “ actinon ’’ series.Considerable attention has been paid to the study of thechemistry of the lanthanons (rare-earth elements).Methods of separationand purification which have been studied include synthetic-resin ion-exchangemethods 56 and fractional precipitation of complexes with ammoniumacetate,67 ethylenediamine-NNN’N’-tetra-acetic acid,5* and other amino-acids.59 Lutetium may be purified 6o by the fractionation of hexa-antipyrinelutetium iodide [ LU(C,,H,,N,O)~]I,.The unusual dark brown Pr6011 which results when any other oxide, orsalts such as the nitrate or oxalate, of praseodymium are ignited in air, hasbeen examined by X-ray and other methods.The constancy of the latticedimensions of Pr6011 under widely different methods of preparation foundLunthanom.50 J . Phys. Coll. Chek., 1950, 54, 999.51 J . Chem. Physics, 1950, 18, 237.52 Klemm and Tilk, 2. anorg. Chem., 1932,207, 175.53 Hagemann, J . Amer. Chem. SOC., 1950,72,768.64 Curie and Bouissihres, Cahiers Phys., 1944,26, 1.6 5 Fried, Hagemann, and Zacharisen, J . Amer. Chem. SOC., 1950,72, 771.56 Spedding, Fulmer, Butler, and Powell, ibid., pp. 2349, 2354 ; Spedding and Dye,57 Vickery, J . , 1950, 1101 ; Perey, J . Chim. physique, 1949,46, 485.6 8 Marsh, J., 1950, 1819.ibid., p. 5350; Huffman and Ostwalt, ibid., p. 3323.59 Vickery, J . , 1950, 2058.8o Marsh, J . , 1950, 577FAIRBROTHER. 105by McCullogh 61 indicates that Pr601, is a separate and distinct phase inthe Pr-0 system, and a fluorite type of structure is suggested similar to thatassumed by Pro,, but with the random omission of one-twelfth of the anionsfrom the structure. Tensimetric measurements in conjunction with X-rayexamination and thermoelectric measurements 62 confirm that the structureis of a fluorite-minus-oxygen type but indicate that Pr forms a series ofnon-stoicheiometric oxides between Pro,., and Pro,., and that the air-ignited oxide is properly represented by Pr01.83. Pure Pro, can be readilyprepared by heating any lower oxide in oxygen at 50 atm. and about 300",but there is no evidence of oxidation beyond the 4+ state.61The hydrated normal lanthanon carbonates can be very convenientlyprepared by hydrolysis of aqueous solutions of the soluble trichlor~acetates.~~The solubility of ytterbium oxalate in buffered sodium oxalate solutions hasbeen studied by Crouthamel and Martin by a radio-chemical method.The results indicate the formation of Yb(C,O,)+ and Yb(C,O,),- but notof Yb(C,O,):-.In a search for new refractories, the sulphides of cerium have beenstudied.66 In addition to the known red Ce,S,, two new compounds Ce3S,and CeS, which are respectively black and brass-coloured, have been pre-pared.All three are very refractory, especially CeS (m. p. 2450" & 100")which has a v.p. of only lo3 mm. of Hg at 1900". Magnetic-susceptibilitydata show all three compounds to have one free electron, the additionalelectrons available for compositions with less sulphur than Ce,S, presumablybeing used for additional bonding between czsium atoms.The availability of milligram quantities of pure promethium (At.no. 61)from uranium fission products has made it possible for Parker and Lantz 66to make a detailed examination of its absorption spectrum. The solutions,which are rose or pink, show bands in the visible spectrum similar to those ofits neighbour neodymium but separable from them by at least 8 mp.Group N.-It has been shown by MacNevin and Carson 67 that carbonmonoxide is oxidised by iodine in acid aqueous solution : CO + I, +H,O = CO, + ZHI, the reaction being strongly catalysed by palladouschloride, and Katz and Halpern 68 have showed that it can be removedfrom a stream of air at ordinary temperatures by silver permanganatedeposited on a variety of metallic oxides, although dry silver permanganatealone is virtually unreactive towards carbon monoxide.The density, viscosity, and surface tension of anhydrous liquid hydrogencyanide have been determined by Coates and Davies 69 in the temperaturerange - 13.3 to + 25".The variation of these properties has been dis-cussed in relation to the linear association of hydrogen cyanide moleculesdue to hydrogen bonding.61 J . Arner. Chem. SOC., 1950, 72, 1386.62 Martin, Nature, 1950, 165, 202.64 Ibid., p. 1382.6 6 Ibid., p. 2834.8 8 Ind. Eng. Chem., 1950, 42, 345.Salutsky and Quill, J .Amer. Chem. SOC., 1950, 72, 3306.6 5 Eastman, Brewer, Bromley, Gilles, and Lofgren, ibid., p. 2248.e7 Ibid., p. 42.68 J., 1950, 1194106 INORGANIC CHEMISTRY.An examination of the isotherms at 10" and 31" for the system Na,O-Si0,-H,O has shown 70 that the following 6 compounds may occur as solidphases : Na,SiO, with 9, 8, 6 and 5H20; Na,HSiO, with 5 and 2H,O.Strickland 71 has shown that heteropolysilicomolybic acid exists in twoforms with the same empirical formula but different structures and formedaccording to the proportions of acid and MO," ion.When silicon tetrachloride is hydrolysed, even in moist air, the hydrolysisto silica is usually complete, but by partial hydrolysis in ethereal solution a t-78" a series of chlorosiloxanes can be prepared.72The rearrangement reactions previously reported by Anderson betweenmixed inorganic halides have now 73 been extended to include some fluoro-silanes.The redistribution reaction is an efficient method of preparationof bromofluorosilanes, isocyanatofluorosilanes, and especially of iodo-fluorosilanes. Three new iodofluorosilanes have been prepared by thismethod, vix., SiFI,, SiF,I,, and SiF,I, from the redistribution of SiF, andSiI,.SiC1,Br and SiCI,Br, react with Grignard reagents '4 with replacementof both chlorine and bromine, the proportion of bromine replaced beinghigher the larger the alkyl group ; ethyl bromodichlorosilane and ethyl-dibromochlorosilane have been prepared by this means ; SiClBr, and SiBr,are unreactive towards Grignard reagents.Eaborn 75 has examined theinteractions of triethyl- and trimethyl-silicon halides and pseudohalideswith silver salts and has shown that in the series R,SiI+ (R,Si),S+R,SiBr + R,SiNC --+ R,SiCI + R,SiNCS + R,SiNCO a compoundmay be converted into any other ou its right by boiling with the appro-priate silver salt, and has discussed this and other possible similar series interms of the solubilities of the salts and of the energy necessary to breakthe several Si-X bonds into Si+ and X-.A number of new alkylsilicon isocyanates and isothiocyanates have beenprepared 76 by reaction of the appropriate alkylchlorosilanes with silverisocyanate or isothiocyanate.Burg and Kuljian 77 have prepared a number of new silylamines andsilylaminoboron compounds.Trisilylamine (SiH,),N, prepared by thereactions SiCl, -+ 78 SiH, -+ 79 SiH,C1 -+ 8o (SiH,),N, reacts a tLIAIH, HC1 NH,-78" with BCl, to give (SiH3),NBCl, : (SiH,),N + BCI, -+ SiH,Cl +(SiH,),NBCl,. This new silylaminoboron compound is similar in severalAlCl,70 Baker and Jue, J . Phys. Goll. Chem., 1950,54, 299; Wills, ibid., p. 304.7 1 Chem. and Ind., 1950, 393.72 Goubeau and Warncke, 2. anorg. Chem., 1949, 259, 109; Schumb and Stevens,J . Amer. Chem. Soc., 1950, 72, 3178.73 Anderson, ibid., p. 2091.74 Wilkins, Brown, and Stevens, J., 1950, 163.7 5 J . , 1950, 3077.76 Anderson, J . Amer. Chem. SOC., 1950, 72, 196.7 7 Ibid., p. 3103.7 8 Finholt, Bond, Wilzbach, and Schlesinger, ibid., 1947, 69, 2694.79 Stock and Somieski, Ber., 1919, 52, 695.*O Idem, ibid., 1921, 54, 740FAIRBROTEER. 107respects to its carbon analogue (CH,),NBCI,.81 In a similar mannerB,H,Br reacts a t -78" with (SiH,),N with the quantitative formation ofB,H,,SiH,Br and the monomer and dimer of (SiH,),$BH, : this monomerreadily adds diborane to give (SiH,),NB,H,. (SiH,),N also reacts at roomtemperature with (CH,),BBr to give a number of products, including thenew and difficultly volatile, (CH,),BN(SiH,Br),. CH3N( SiH& reacts withBCI, a t -78" to give (CH,NSiH,)BCI,, which in turn decomposes quanti-tatively into SiH,CI and (CH,NBCl),.Milligan and Kraus 82 have prepared tristriphenylgermanylsilane byreaction of sodium triphenylgermanide with SiHCl, in ethereal solution.The hydrogen atom in this silane can be replaced by lithium in ethylaminesolution and by bromine in ethyl bromide solution.Tristriphenylgermanyl-silyl chloride, amine, and the corresponding silo1 have also been prepared.On treatment of the lithium salt of the silane with ethyl bromide in liquidammonia solution, the metal is replaced by the ethyl radical with theformation of ethyltristriphenylgermanylsilane.Bistriethylgermanium sulphate and diethylgermanium sulphate may beprepared by the action of concentrated sulphuric acid on the correspondingoxide.8,Everest and Terrey84 have shown that germanous hydroxide can beobtained in a form which may be either white or coloured according to theparticle size and degree of hydration. Conductivity measurements giveno indication of any reaction between Ge( OH), and NaOH ; no dissolution ofthe germanous hydroxide occurs even when ten equivalents of alkali areadded, and the evidence of the potential of the germanate-germanite couplesuggests that the fact that bivalent germanium can exist at all in alkalinemedia is due to the insolubility of &(OH),.Germanylsodium (NaGeH,), prepared by the quantitative reactionbetween sodium and monogermane in liquid ammonia, reacts with methyliodide and ethyl and propyl bromides to give the corresponding monoalkyl-germanes.Teal and Kraus 85 also find that when a solution of germanyl-sodium in liquid ammonia is electrolysed with a platinum anode and mercurycatahode, the anode reaction is not, as might be expected, the formation ofdigermane but of monogermane and nitrogen : 6GeH,- + 2NH, = GGeH, +N, + 6e-.A new and simplified method for the synthesis of ethylgermaniumtrichloride and diethylgermanium dichloride has been devised by Rochow 86which involves the direct reaction between ethyl chloride and elementarygermanium in the presence of a copper catalyst.The nature and conditions of formation of germanyl ferrocyanide,[Ge(OH),]Fe(CN), or (GeO),Fe(CN),,2H20, formed as an insoluble whiteprecipitate when ferrocyanide ion is added to an acid solution of germanium81 Wiberg and Schuster, 2. anorg. Chem., 1933,213, 77.82 J . Amer. Chem. SOC., 1950, 72, 5297.83 Anderson, ibid., p. 194.04 J., 1950, 2282.a 5 J. Amer. Chem. Soc., 1950,72,4706.B 6 Ibid., p. 198108 INORGANIC CHEMISTRY.dioxide, have been examined by Peisach, Pugh, and Sebba.87 This is thefirst time such a compound has been described.Experiments on tetraethyltin 88 have shown that it can crystallise in atleast ten forms, the m.p.s of which all lie within the range 137" H. to 148" K.Tetraethyl-lead also crystallises in many forms. Tetramethyl-tin and -leadand tetraethylgermanium do not show this unusual kind of polymorphismwhich, it is tentatively suggested, results from the molecular dimensionsenabling the molecules to pack into different lattices in which they havedifferent configurations, that is, to exhibit a form of rotational isomerism inthe solid state.Although previous studies have indicated that the only sodium stannate isNa2H8Sn0,, Grillot 89 has now shown that there exists, in addition to thecorresponding potassium compound, also K2Sn( OH), , which is formed whenthe solution contains excess of potassium hydroxide.The difficult separation of zirconium and hafnium has been attacked bya number of authors and by several methods.The observation by Hansenand Gunnar that silica gel will adsorb hafnium in strong preference tozirconium from a methanol solution of the tetrachloride has now been used 91for the purification of zirconium and, by differential extraction of the usedsilica gel, to the preparation of a concentrated solution of hafnium. Huffmanand Lilly 92 have found that adsorption of zirconium and hafnium as negativetluoro-ions on a strongly basic anion-exchange resin followed by elution witha mixture of O-~M-HC~ and O-O~M-HF gives an excellent separation on amilligram scale, the progress of elution being studied by a tracer method.Kraus and using larger quantities of the two elements and undersomewhat different conditions, have also achieved a partial separation bythis method.A good separation has also been accomplished by Gruen andK a t ~ , 9 ~ by the fractionation of 3ZrC1,,2POC13 (b. p. 360") and 3HfC1,,2POC13(b. p. 355"), by Huffman and Beaufait 95 by solvent extraction of the per-chlorates with thenoyltrifluoroacetone, and by Schultz and Larsen t~ byextraction of a hydrochloric acid solution of the ions with trifluoroacetyl-acetone.Wardlaw and Bradleyg7 have prepared a number of alkyl ortho-estersof zirconium by reaction of zirconium tetrachloride with a primary alcoholand anhydrous ammonia. When secondary alcohols are used in the reactionthe products are hydrolysed esters, the pure esters in this case being obtainedby ester interchange between the ethyl ortho-ester and the secondary alcohol.The ortho-esters of zirconium are much less volatile than those of eithersilicon or titanium.A series of new chloride ethoxides of zirconium has alsobeen ~ r e p a r e d . ~ ' ~87 J . , 1950,949. Staveley, Paget., Goulby, and Warren, J . , 1950, 2290.Compt. rend., 1950, 230, 1179.J . Amer. Chem. SOC., 1949,71,4158.9 1 Hansen, Gunnar, Jacobs, and Simmons, ibid., 1950, 72, 5043.92 Ibid., 1949, 71, 4147.9 j Ibid., p .3179.974 Bradley, Abd-el Halim, and Wsrdlaw, J., 1950, 3450.93 Ibid., p . 3263.96 Ibid., 1950,72, 3610.Ibid., p . 3843.97 Nature, 1950, 165, 75FAIRBROTHER. 109Solvent extraction, with pentan- 1-01 or any of a number of higher alcoholsand ketones, has been usedQ8 as a method of extracting thorium fromaqueous solutions of its naturally occurring mixtures with the lanthanons.D'Eye Qg has made an examination of the crystal structure of thorium bro-mide, with results which indicate that, as in the chloride, the thorium-halogen bonds are partly covalent in character.A series of refractory sulphides of thorium and uranium has beenprepared, loo viz. :Compound. Colour. M. p. d ( g . / c . c . ) .ThS ........................... Silvery > 2200" 9.57Th,S, ........................Brown 1950" f 50" 7.88Th,S,, ........................ Black 1770" f 30" 7.78ThS, ........................ Purple 1905" f 30" 7.36US ........................... Grey metallic >2000" 10.9 u,s, - -USJ, ........................... BIack 1850" f 100" 7.908.8 1 ...........................The compound Th,S, is of particular interest since it is the only definitelyestablished thorium compound which contains the element in the +3 oxid-ation state. None of these sulphides is paramagnetic, which means that allthe electrons even in the lower valency states are paired, and it is suggestedthat the electrons in the first three not used in bonding with the sulphur areused in bonding between the thorium ions in the (rock-salt) lattice just asbonding occurs in a metal lattice.Group V.-The electrolysis of a solution of aluminium iodide in liquidammonia with a platinum cathode and aluminium anode gives an intenselyblue colour similar to those of the alkali metals : lol the colour first appearson the platinum cathode and spreads through the solution.The need for simple compounds lo2 of thorium and uranium that areappreciably soluble in liquid ammonia without reaction more extensive thansolvation has led to the study of the behaviour of a number of compounds ofthese elements in liquid ammonia.A number of ammonolyic reactions havebeen observed.Although many investigations have been carried out on the gradual transi-tions which occur in some pure solids, relatively little is known of thephenomenon in binary mixed crystals of which one or both componentsexhibit such a transition.Mandleber and Staveley lo3 have now investigatedthe volume-temperature relationships of mixed crystals of ammoniumchloride and ammonium bromide.A comprehensive series of investigations into the chemistry of thenitronium ion NO,+ and its compounds has been carried out by Ingold andAsselin, Audrieth, and Comings, J. Phys. Coll. Chem., 1950, 54, 640; Templetonand Hall, ibid., pp. 954, 958.O9 J., 1950, 2764.loo Eastman, Brewer, Bromley, Gilles, and Lofgren, J. Amer. Chem. SOC., 1950, 72,l01 Davidson, Kleinberg, Bennett, and McElroy, ibid., 1949, 71, 377 ; McElroy,lo* Watt, Jenkins, and McCuiston, ibid., p. 2260. lo3 J., 1950, 2736.4019.Kleinberg, and Davidson, ibid., 1950, 72, 5178110 INORCIANIU OHEMISTRY.his ~ o l l a b o r a t o r s .~ ~ ~ 1 ~ Cryoscopic measurements show that NO,+ ions areformed when HNO,, N205, or N204 is dissolved in concentrated sulphuricacid.lM The freezing-point diagram of concentrated nitric acid, over therange of N205-H20 compositions in the neighbourhood of " pure " HNO,,indicates that N20, in nitric acid solution is completely dissociated intosolvated NO,+ and NO3- ions, and that in analytically anhydrous nitric acidat -40" some 3.4% is dissociated to give 102% of NO,+, 1.7% of NO,-, and06% of water.lo6 A study of its Raman spectra 112 has shown that whenN204 is dissolved in dilute nitric acid it is almost completely dissociated,partly homolytically, but chiefly heterolytically : 2N0, N204NO+ 3- NO,-.The nitrosonium ion NOf forms a molecular compound with nitrogendioxide: &0*&02, which may also be produced from nitronium ion andnitric oxide &O2-I$0, and it is believed that this compound arises throughthe formation of a single-electron bond by resonance between the structureskO,*kO and & 0 2 * & 0 .1 1 2 p 113A number of pure crystalline nitronium salts have been prepared,ll* zfiz.,the perchlorate (N02+)(C104-), hydrogen disulphate (N02+)(HS20,-),disulphate (NO,+),(S,O,"), trisulphate (NO,+),( S3010-), and fluorosulphonate(NO,+)(F*SO,-), the structural lattice components of some of which have beenidentified by means of the Raman spectra of the crystalline compounds, whichhas also given experimental confirmation that solid dinitrogen pentoxideis ionic nitronium nitrate (NO,+)(NO,-).Schomaker and Chia-Si Lu 115 have shown by electron-diffraction measure-ments that the molecule of nitrogen trifluoride is a symmetrical pyramid,with a F-N-F bond angle of 102.5' and not a nearly planar molecule as hadbeen conjectured earlier from its very small dipole moment.A study has been made 116 of the direct oxidation of phosphorus by steam,to give phosphoric acid and hydrogen, P, + 16H20 = 4H3P0, + 10H2,by passage of the vapours over a suitable catalyst a t 650-800". Bothplatinum and palladium supported on aluminium metaphosphate orzirconium pyrophosphate are found to be active catalysts, as are also theless stable, but cheaper, supported copper catalysts.104 Gillespie, Graham, Hughes, Ingold, and Peeling, J., 1950, 2504.lo6 Gillespie and Graham, J ., 1950, 2532.lo6 Gillespie, Hughes, and Ingold, J . , 1950, 2552.lo' Millen, J., 1950, 2606.lo8 Ingold, Millen, and Poole, J . , 1950, 2576.log Millen, J . , 1950, 2589.110 Idem, J . , 1950,2600.111 Ingold and Millen, J . , 1950, 2612.112 Goulden and Millen, J . , 1950, 2620.lla Goulden, Ingold, and Millen, Nature, 1950,165, 565.114 Goddard, Hughes, and Ingold, J., 1950, 2559.115 J . Amer. Chem. SOC., 1950, 73, 1182.118 Shultz, Tarbutton, Jones, Deming, Smith, and Cantelou, Ind. Eng. Chem.,1950,42, 1608FAIRBROTHER. I11Much work has recently been published on the chemistry of the condensedp01yphosphates.l~~ The study of the appropriate phase-diagrams 118 hasindicated the existence of two new phosphates, an unstable zinc phosphateZn(H2P0,),,2H3P0,, and an anhydrous manganous phosphate Mn(H,PO,),.Lange and Livingstone 119 have prepared anhydrous difluorophosphonic acid,HP0,F2, by the action of gaseous POF, on anhydrous monofluorophosphonicacid, POF( OH),.A new method has been described by Geach, Jeffery, and Shelton 120 forthe preparation of pure oxide-free arsenic, which involves the reduction in asealed tube of arsenious oxide by zirconium metal powder, mixed with 15 yoby weight of zirconium oxide to mitigate the violence of the reaction.The absorption spectra of vanadium(n1) and vanadium(1v) ions inaqueous chloride and perchlorate solutions have been examined by Furmanand Garner.121 The blue colour of vanadium(1rr) perchlorate solution is dueto the hydrated V3+ ion : on warming to 70" the colour changes to green,and reversibly to blue on cooling, owing to increased hydrolysis a t the highertemperature by the endothermic reaction V+++ + H20 VOH++ + H+.The spectra of aqueous vanadium chloride solutions indicate the formationof complex ions.Jantsch, Bergmann, and Rupp 122 have studied the am-moniates of vanadium(n1) chloride, vix., VCl,(NH,),, where x = 2, 3, 5.6, 7, or 12.Both the English (columbium) and the German (niobium) name forelement41 continue to appear in the literature.* There is still some question 123as to whether the latter will find general acceptance.There is no doubt thatthe oxide of' this element was extracted and recognised as new, and a numberof its properties correctly described, by Charles Hatchett in London in 1801 124and named by him columbium, before Ekeberg's discovery of tantalum 125(1 802) and more than 40 years before Heinrich Rose 126 extracted it and namedit Niobe, under the then current erroneous impression that Hatchett'scolumbium and Ekeberg's tantalum were one and the same.by use of the respectiveradio-isotopes, that columbium (niobium) and tantalum can be efficientlyseparated by fractional adsorption on an anion-exchange resin from solutionin a mixture of ~M-HCI and O-OSM-HF.11' Thilo and Riitz, 2. anorg. Chem., 1949, 260, 255; Andress, Gehring, and Fischer,ibid., p.331 ; Thilo and Hauschild, ibid., 1950,261,324; Van Wazer and Holst, J . Amer.Chem. Soc., 1950,72,639; Van Wazer, ibid., pp. 644,647; Van Wazer and Campanella,ibid., p. 655; Van Wazer, ibid., p. 906; Mehrotra and Dhar, Proc. Nat. Inst. Sci. India,1950, 16, 59; Jones and Monk, J., 1950, 3475; For further references see also Topley,Quart. Reviews, 1949,3, 345.It has been demonstrated by Kraus and11* Salmon and Terrey, J., 1950, 2813.120 J., 1950, 1207.122 Z.anorg. Chem., 1950,262,223.lZ4 Phil. Trans., 1802, 49.126 Pogg. Ann., 1844, 63, 317.* The Commission of Nomenclature of Inorganic Chemistry at the AmsterdamMeeting of the International Union of Pure and Applied Chemistry in 1949 recom-mended the adoption of the name niobium for element 41.llg J .Amer. Chem. SOC., 1950,72, 1280.121 J . Amer. Chem. SOC., 1950,72, 1785.125 Nicholson's J., 1802, 3, 251.12' J . Amer. Chem. Soc., 1949, 71, 3855.las Wichers, J . Amer. Chem.Soc., 1950,72,1431112 INORGANIC CHEMISTRY.Tantalum pentaiodide has been prepared by Alexander and Fairbrother 128by heating the metal in iodine vapour by means of high-frequency inductioncurrents. It forms shiny black crystals which can be sublimed in a vacuumwithout decomposition and have m. p. 496", b. p. 543". Measurements havebeen made of its vapour pressure from about 300" to near the boiling point.The reaction between columbium metal and an excess of iodine undersimilar conditions leads to the formation of well-defined lustrous crystals,essentially CbI,, with a brass colour, which are instantly attacked on exposureto air and lose iodine on gentle heating.2-Tartrato-2-columbic (-niobic) acid, Cb205( C4H405)2, 10H20, has beenprepared by dissolution of the freshly precipitated hydrous oxide in tartaricacid and precipitation in a micro-crystalline form by addition of alcohol.A heteropoly-acid structure of the type H,[Cb '4 ],2-5H20 is sug-ge~ted.l,~ The alkali-metal, alkaline-earth, and other metal salts have alsobeen investigated.130Group VI.-A clarification of the state of knowledge regarding the saltsof some of the simpler peroxy-acids has been made by Partington andFathallah.131 They have shown in the first place that the alkali peroxy-borates are true peroxy-acid salts and not simply addition compounds ofhydrogen peroxide and metaborates.Peroxyborates have also beenprepared, containing a greater proportion of active oxygen than previouslyreported, and corresponding to empirical formule LiBO,,H,O, KBO,,H,O,RbB04,0.5H20, and CsB04,0-5H,0. The previous contradictory experimentalwork on the alkali peroxycarbonates has also been reinvestigated by the sameauthors and, in addition to the confirmation of a number of compoundspreviously reported, the new compounds CsOOH,H,O,, KHCO,, RbHCO,,Rb,C,O,, and Li,C04,H20 have been prepared.Sulphuryl fluoride has been prepared by Woyski 133 by passing sulphurylchloride vapour or an equimolar mixture of sulphur dioxide and chlorineover sodium or potassium fluoride a t 400".Trifluoromethylsulphur penta-fluoride CF,*SF, has been prepared by Silvey and cad^.',^ The fluorin-ation of methanol gives trifluoromethyl hypofluorite CF,*OF. The fluorin-ation of methanethiol, however, either by cobalt(II1) fluoride at 250" orby dilute fluorine and a copper-supported silver fluoride catalyst at ZOO",instead of yielding the sulphur analogue, gives CF3*SF, as one of the products.This compound (b. p. -20.4", m. p. --81"), which is also obtained byfluorination of carbon disulphide, greatly resembles sulphur hexafluoride inits inertness.A new investigation of the solid-liquid equilibria in the system SO,-H,Ohas been carried out by Gable, Betz, and Maron 135 over a range of 0-93.7%of SO,. Eight solid phases, vix., H,S04 with 6, 4, 3, 2, or 1H20 : H,SO,,H2S20,, and ice, are found within this range. All of these except( c ,H 4 0 6 1Is* J., 1949, 2472.1*0 Idem, ibid., pp. 300, 381.las J .Amer. Chem. SOC., 1950,72, 919.laS Srinivasan, PTOC. Indian Acad. Sci., 1950, 31, 194.131 J., 1949, 3420.la' Ibid., p. 3624.13a J . , 1950, 1934.lS6 Ibid., p. 1445FAIRBROTHER. 113H2S04,6H20 and H2S04,3H20 exhibit congruent melting points. Thehydrate H,S04,3H20 has not been reported previously.The behaviour of sulphuric acid as an ionising solvent has beenexamined by Gillespie, Hughes, and Ingold. 136 The experimental methodsof cryoscopy in this solvent have been developed to a high degreeof precision. The cryoscopic constant of sulphuric acid (kf = 5-98 deg.g.-mol.-l kg.) has been obtained by using three types of solute, (a) sulphurylchloride and chlorosulphonic acid, which are un-ionised, ( b ) potassiumsulphate and ammonium sulphate, which are ionised, e.g., K2S04 + H2S04 =2K+ + 2HS04-, and ( c ) acetone and acetic acid, which behave as strongbases, e.g., B + H2S04 = BH+ + HS04-.Results with the three types ofsolute are in good agreement.From a study of the freezing points in the H,O-SO, system at composi-tions near that of sulphuric acid 13’ it is concluded that the ionisation of wateras a base in solvent sulphuric acid is appreciably incomplete, and that onthe other hand, disulphuric acid (H,S207) present in solutions with a higherSO, content, is moderately ionised. This cryoscopic method has also beenused to investigate the nature of the species present in sulphuric acid and indilute 01eum.l~~ In addition to the presence of H2S207 in oleum, evidencehas been obtained of the presence of higher polysulphuric acids, H3S3010and H2S4013 being definitely recognised.Freezing-point depressions of nitronium perchlorate and ammoniumperchlorate in solvent sulphuric acid 139 show that the perchlorate ion inthese salts is largely solvolysed to free perchloric acid ; which means thereforethat perchloric acid is a very weak acid when dissolved in sulphuric acid.On the other hand nitro-compounds e.g., nitromethane, nitrobenzene, andderivatives of nitrobenzene, behave as fairly strong bases in anhydroussulphuric acid, 140 the percentage ionisation in 0.1 M-SOlUtiOn varying from21 yo for nitromethane to 73% for p-nitrotoluene.Cations of high positive valency in aqueous solution usually combinewith the oxygen ions present to form oxy-cations of lower valency beforesalt formation occurs, so that the salts obtained from such solutions areoxy-salts.A method has now been developed by Hayek and Engelbrecht 141for the preparation of sulphates of higher-valency cations, which consistsin treating the chloride of the ion in question, in the absence of water, withsulphur trioxide in sulphuryl chloride solution. In this manner Ti(SO,),,Sn(SO4)2, v2°(s04)& Sb,O(SO4)4, CrO(SO4)2, MoO(SO4)2, WO(SOJ2, andU(SO,), have been prepared.The green solution of chromic sulphate, familiar to anyone who hasheated a solution of chrome alum, and the corresponding heated solutions ofthe chloride, have received considerable attention in the past.On the otherhand, little work has been done on the nitrate, probably because there islittle colour difference between a fresh solution and one that has been boiled136 J., 1950, 2473.138 Idem, J . , 1950, 2516.140 Idem, J . , 1950, 2542.la’ Gillespie, J., 1950, 2493.13@ Idem, J., 1950, 2537.lol Momtsh., 1949, 80, 640114 INORGANIU CHEMISTRY.and cooled. Hall and Eyring 142 have now attacked the problem of chromiccomplexes by a new method, using the so-called ammonium paramolybdate,(NH,),Mo,O~~,~H,O. Conductimetric titration with solutions of this reagentpermits the determination of the average number of oxygen bridges betweenchromium atoms in boiled solutions of chromic salts and has made it possibleto show that a structural change also accompanies the heating or ageing of asolution of chromic nitrate.The formation of oxalate complexes of chromium has been studied bySh~tt1eworth.l~~ Pure anhydrous chromic iodide has been prepared forthe first time by Handy and Gregory by heating CrI, in iodine vapour at1 atm.pressure and 500". The equilibrium Cr13(s) Cr12(s) +has been studied between 309" and 373". A rather long extrapolation leadsto an approximate value of atm. for the dissociation pressure a t 25",the compound being very stable at room temperatures.Hartford and Lane lg5 have described the preparation of a number ofnew compounds belonging t o the class of double chromates of some of' thetransition elements.The constitution of the peroxychromates has beenexamined by G 1 a ~ n e r . l ~ ~ It is suggested that the familiar ether-solubleblue perchromate is formed by the addition of an HO, radical to chromiumtrioxide : CrO, + HO, HCrO,. At a pH above 4.5 or on dilution bythe addition of an excess of hydrogen peroxide, which raises the pH, anothermolecule of hydrogen peroxide is added forming the violet perchromate :HCrO, + H,O, _T H,CrO,. The quinquevalency of the chromium in theperchromates and the reduction of chromic acid by hydrogen peroxide in acidsolution are considered to be due to the odd electron in the HO, radical.Molybdenum blues have been prepared by Sacconi and Cini14' with aMO(VI) : Mo(v) ratio from 9.0 : 1 to 0.56 : 1 and a water content of from17.96% to 19.38%.On heating at 195", almost all the water is lost to givea typical semi-conductor which decomposes at 350" to give a mixture ofMOO, and a more reduced blue. These compounds have a small para-magnetism, which is interpreted by the authors as indicating the presenceof Mo(v)-Mo(v) covalent bonds. The formula of sodium paratungstate hasbeen discussed by Saddington and Cahn.148 As a result of some new chemicaland crystallographic analyses they conclude that the crystalline salt has thecomposition Na,oWl,O,, ,Z8H20.Mair 149 has examined the reaction between ferric salts and sodiumparatungstate in boiling aqueous solution and has isolated 11 -tungstoferric(III)acid, the first member of a new series of 11 -heteropoly-tungstic acids withmainly positive central ions.Mair and Waugh 150 have also prepared threefurther 11-heteropoly-tungstic acids with tervalent central ions, vix., 11-tungstoaluminic, 11 -tungstochromic(IIr), and 1 1 -tungstomanganic(r) acids.1 4 2 J . Amer. Chern. SOC., 1950, 72, 782.143 J . Amer. Leather Chem. ASSOC., 1950, 45, 41.144 J . Amer. Chem. SOC., 1950,72,5049.1Q6 J . , 1950, 2795.145 Ibid., p. 1286.J., 1950, 3526. J., 1950, 2364. 150 J . , 1950,2372.1 4 7 J . Chem. Phys., 1950,18,1124FAIRBROTHER. 115From all three, salts have been prepared. A new and convenient method ofpreparation of free heteropoly-acids, and thence of other salts, has beendevised by using cation-exchange re~ins.1~1 The method is applicable evento the preparation of the free acid from an almost insoluble salt.A series of lithium tungsten '' bronzes " similar in composition to thewell-known sodium t,ungsten " bronzes " has been prepared.ls2Uranium and the Transuranic Elements.This year has seen the public-ation of an account of that part of the immense amount of research workcarried out during the War under the Manhattan Project and the AtomicEnergy Commission of the United States which was known as the PlutoniumProject. The account, which consists of a collection of some 162 originalresearch papers by 114 authors, edited by Seaborg, Katz, and Manning, iscontained in two volumes under the title of " The Transuranic Elements ".ls3The papers deal principally.with the chemistry and physics of neptuniumand plutonium but include also many relating to special techniques and toother radioactive elements such as actinium, thorium, radium, americium,and curium and to the general problem of the transuranic elements.Evidence is steadily increasing, as new elements are made, that thisseries of elements does in fact constitute a new series, variously referred to inthe literature as " actinide ", " actinon ", or " uranide " elements, in whichthe 5f electron shell is in process of being filled, somewhat in a similar mannerto that in which the 4f shell is filled in the rare-earth or lanthanon series.There is, however, still some diversity of opinion 154 as to where this seriesactually starts, that is, whether actinium or uranium should be regarded as thefirst member of the series and which ions contain f electrons and how many.The evidence, of the presence o f f electrons in any particular ion, fromabsorption or magnetic measurements, however, may not only depend on theelectronic configuration of the neutral gaseous atom and the oxidation stateof the ion, but may also depend on its environment, in particular if themultiplicity is reduced by covalent-bond formation.The problem is madeless simple by the fact that the 5f electrons are apparently less firmly boundthan the 4f electrons in the lanthanons. A review of the general lines ofevidence regarding the electronic structure of the heaviest elements fromchemical and physical data has been given by S e a b ~ r g .l ~ ~The hydrolysis of the uranyl and other uranium ions in aqueous solution,and the various acidic and basic compounds that are precipitated on theaddition of bases, have received further attention. By determining thechanges in pH and conductivity of solutions of uranyl nitrate or acetate, onaddition of measured amounts of NH,*OH, NaOH, or KOH, Tridst 156 hasfound that a basic salt is first produced and eventually a hydrated trioxideU03,xH20 at 1.5 mols. of NH,*OH or 1.6 mols. of NaOH or KOH per mol.151 Baker, Loev, and McCutcheon, J . Amer. Chem. SOC., 1950,72,2374.152 Straumanis and Hsu, ibid., p. 4027.153 " The Transuranic Elements," Edited by Sesborg, Katz, and Manning, McGraw15* Cf.Ann. Reports, 1949, 46, 88.165 Ref. 153, p. 1492.Hill Co., New York, 1949.lS6 Ann. Chim., 1950,5,368116 INORGANIC CHEMISTRY.of UO,++. Uranyl nitrate yields a diuranate, M,0,2U03, with an excess ofbase, whereas uranyl acetate first gives an unstable monouranate which isconverted into diuranate in the presence of water or uranyl salts.The existence of the ion U4+ in acid aqueous solution has been confirmedby Kraus and Nelson,ls7 and the hydrolysis of the U4+ and the Pu4+ ionstudied in further detail. The striking similarity between the sharp- bandedabsorption spectra of unhydrolysed Pu4+ and U4+ suggests that both ele-ments are members of the actinon series, but that Pu4+ has only two 5felectrons instead of the expected four.A comparative potentiometric and photometric absorption investigation 15*of uranyl monochloroacetate indicates that three complexes are formed :(UO,)*A+, (UO,)A,, and (U02)A3-, where A = CH,Cl*CO*O-.The solubility of some inorganic nitrates in ether has been examined inrelation to the now familiar purification of uranyl nitrate by ether-extrac-t 3 0 n .l ~ ~ The solubility of bismuth nitrate and ferric nitrate in ethyl etheris considerably greater than that of 23 other common nitrates. The presenceof uranyl nitrate in the ether lowers the solubility of other nitrates, and thepartition coefficients are such that one or two aqueous extractions of anethereal solution will remove any likely impurities from uranyl nitrate. Thethermal instability of the uranyl nitrate-ether complex forme din this extractionhas been examined.160 The concentrated solution, from which the complexUO,(N0,),,2H,0,2(C,H5),0 can be isolated, deflagrates at 85-90" owingto oxidation of the ether by the high concentration of nitrate ion.Glueckauf and McKay 161 have pointed out that the chemical propertiesof uranium and other actinon elements suggest that f-electron orbitals maybe used in bond formation in covalent complexes.The magnetic susceptibilities of elements 92 to 95 (uranium to curium)in most of their oxidation states have been measured in aqueous solutionby Howland and Calvin : 162 the results could only be interpreted on the basisof electronic configurations involving 5f electrons even though the suscepti-bilities were in general lower than the theoretical and experimental values forlanthanon ions with the same number of 4f electrons.Dawson and Lister 163have measured the magnetic susceptibilities of UO, and intermediate oxidesup to U,O, over a range approximately 90-570"~. The susceptibilitiesare consistent with the view that these oxides contain quadri- and sexi-valentbut no quinquevalent uranium.Measurements have also been made 164 of the magnetic susceptibilities ofsolid solutions of uranium dioxide in thorium dioxide. The results indicatethat the magnetic moment of U4+ shows little dependence on the concen-tration of UO, in the solid, and at the lowest concentration (2% UO,) is inagreement with the " spin only " formula for two unpaired electrons.This is15' J . Amer. Chem. Soc., 1950,72, 3901.158 Ahrland, Acta Chem. Scand., 1949, 3, 783.159 Bachelet, Cheylan, and Bris, J. Chim. phys., 1950,47, 62.160 Cheylan, Bull. SOC. chim., 1949, 641.162 J. Chem. Phys., 1950, 18, 239.lS1 Nature, 1950,165, 594.lea J . , 1950, 2181.Trzebistowski and Selwood, J . Amer. Chem. SOC., 1950, 72, 4504FAIRBROTHER. 117interpreted as an indication that these two unpaired electrons should beassigned to the 6d rather than to the 5f shell, on the assumption that quench-ing of the orbital contribution only occurs for the d electrons.Sheft, Fried, and Davidson 165 have prepared pure uranium trioxideby heating U,O, at 600-700" in pure dry oxygen a t a pressure of' about 25atm. This procedure gives a lOOyo yield of UO, over a wide range oftemperature. Above 750°, however, a higher pressure of oxygen is requiredto prevent the formation of oxides intermediate in composition betweenU,O, and UO,.When uranyl oxalate (U0,C,04,3H,0) is heated in theair,166 it loses 2H,O at 120" and the third a t 210". The anhydrous salt beginssuddenly to decompose at 310" to form UO, and U,O,. Continued heatingin the air gives a mixture of U,O, and UO,.The precipitation of uranium as U0,,2H20 from uranyl nitrate andhydrogen peroxide is substantially quantitative at pH 2 . 5 - 3 ~ 5 . ~ ~ ~ Twodifferent crystalline forms of this peroxide are formed, according to whetherthe uranyl nitrate or the hydrogen peroxide is in excess : the U-U distanceis the same in both types of crystal.U04,2H,0 dissolves in aqueous solutionsof sulphites without gas evolution to give solutions which contain, in additionto sulphate, also uranyl sulphite, an intensely yellow compound notpreviously reported.The system uranyl sulphate-water, investigated by Secoy,168 shows atwo-liquid phase region around 300-375" at all concentrations up to 72% ofUO,SO,. The lower critical solution temperature is 295" a t a weightconcentration of about 28% of UO,SO,. The solubility curve of solidU0,S0,,H20, which has been followed up to 363", does not intersect thetwo-liquid phase region.It is reported 169 that intermediate fluorides of uranium, includingU4F1,,U2F9, and ct- and p-forms of UF,, are formed by treatment of finelydivided UP, with gaseous UF, a t suitable temperatures and pressures.Dawson, Ingram, and Bircumshaw 170 have studied the reduction ofuranium hexafluoride with hydrogen.A need for some simple compounds of thorium and uranium that areappreciably soluble in liquid ammonia without other reaction than solvationhas led to the examination of the behaviour of a number of these compoundsin liquid ammonia.171 Thorium(rv) nitrate and sulphate, uranyl nitrate, anduranium(r1r and IV) chlorides and bromides react with the solvent, butthorium(rv) iodate and oxalate and uranium peroxide dihydrate are unreactive.Polarographic measurements of the N~(III)-N~(Iv) couple in perchlorica.nd hydrochloric acid have been made by Hindman and K r i t ~ h e v s k y .~ ~ ~185 J .Amer. Chem. SOC., 1950, 72, 2172.Boulle, Jary, and Dominb-Berg&, Compt. rend., 1950, 230, 300.16' Watt, Achorn, and Marley, J . Amer. Chem. SOC., 1950,72, 3341.168 Ibid., p. 3343.16s Agron and Weller, U.S.P. 2,510,850; Chem. Abs., 1950,44, 71649.170 J . , 1950, 1421.171 Watt, Jenkins, and McCuiston, J . Arner. Chem, SOC,, 1950, 72, 2260,Ibid., p. 953118 INORGANIO CHEMISTRY.The couple is polarographically reversible in hydrochloric acid with a redoxpotential Np4+ + e- Np3+ = +0.142 & 0-005 volt, but is irreversiblein perchloric acid.Some interesting facts have emerged in connection with the separationof americium and curium from rare-earth fission p r 0 d ~ c t s . l ~ ~ As is the casefor the lanthanon elements, the only thermodynamically stable oxidationstate of americium and curium in aqueous solution is the 3+ state, and sincetheir crystal radii overlap those of the lighter lanthanons, being about thesame as that of neodymium, the solubilities of the salts of these elements arevery similar to those of the lanthanon salts.A satisfactory separation hasbeen achieved by the now familiar method of adsorption on a syntheticion-exchange resin and fractional elution with ammonium citrate solution.Whereas, however, the eluent usually removes the elements in reverse orderof their atomic number, Le., the heaviest first, and with 6~-hydrochloric acidamericium and curium are eluted in their normal order, yet elution with13.3~-hydrockloric acid not only separates the americium and curium fromall the rare earths but the expected order of their elution is reversed. This,it is suggested may be due to weak covalent co-ordination of additionalC1- ions in concentrated hydrochloric acid, using the 5f electrons of theseheavy elements, available as a consequence of the smaller separation instability of the 5f and 6d orbitals as compared with the 4f and 5d of thelanthanon elements, and would indicate that the covalent complex ofamericium is slightly more stable than that of curium.Although Am3+ is the stable oxidation state in aqueous solution, it isreported 174 that it can be completely oxidised by ammonium persulphate in0*2~-nitric or -chloric acid to a formal valency of 6+.The colour changesfrom the characteristic pink to a strong yellow, and on addition of sodiumacetate a compound, presumably sodium americyl acetate, was obtained,which was isomorphous with the corresponding sodium uranyl and plutonylacetates, Na(UO,)(OAc), and Na(PuO)( Oh),.The production by Thompson, Ghiorso, and Seaborg 175 of a radioactiveisotope of element 97, which has been named Berkelium (Bk), has madepossible the examination of some of the properties of this element by tracertechnique.176 This isotope (half-life 4.6 hours, decaying by electron capturewith about 0.1% branching decay by a-particle emission) was prepared bythe bombardment of 241Am with about 35 MeV.helium ions and is believed tobe 243Bk or possibly 244Bk. The sequence of elution by ammonium citrate ofthe group berkelium-curium-americium from a synthetic ion-exchange resinindicates the same kind of break in ionic radius a t the point of half filling ofthe 5f electron shell (curium in the actinon series) as is known to occur in thelanthanon series a t gadolinium.Consequently, just as terbium can beoxidised in the solid state to TbO, (though not in aqueous solution), it was173 Street and Seaborg, J . Amer. Chem. Soc., 1950,72, 2790.Asprey, Stephanou, and Penneman, ibid., p. 1425.Phys. Review, 1950, 77, 838.176 Thompson, Cunningham, and Seaborg, J . Amer. Chem. SOC., 1950,72,2798FAIRBROTHER. 119expected that Bk3+ should be capable of oxidation to Bk4+ and that this mightbe achieved even in aqueous solution. This has now been confirmed bytracer experiments, which consisted of the measurement of the activitycarried down by a zirconium phosphate precipitate from solutions of knownoxidation potentials.I n this way a formal oxidation potential for thecouple Bk4+ + e- = Bk3+ of about + 1.6 V. has been indicated.The latest element to be prepared,17' element 98, obtained by bombard-ment of M2Cm with 35-Mev. helium ions, and probably 24498, has been giventhe name Californium (Cf). This isotope has a half-life of about 45 minutesand decays at least partially by ct-particle emission. An examination ofsome of its chemical properties has been made by Street, Thompson, andSeaborg,17* by methods similar to those used for berkelium. Its propertiesindicate that it fits in well as the ninth member of the actinon series, especi-ally as shown by the relative rates of elution by ammonium citrate from acation exchange-resin in the series Cf-Bk-Cm-Am as compared with theirlanthanon analogues, Dy-Tb-Gd-Eu. Only the 3+ oxidation state has sofar been examined, and the existence of a higher oxidation state in the formof an oxygenated ion (CfO,)+, stabilised by the two oxygen atoms, is stilluncertain.Group VII.-An account has now been published 179 of the Symposiumon the Chemistry of Fluorine held at the Royal Institution in November 1949.A new estimation of the dissociation energy of the fluorine molecule, forwhich the values in the literature range between about 64 and 33 kcals.per g.-mol., has been made by Evans, Warhurst, and Whittle; 180 it isconcluded that all the experimental evidence a t present available stronglyindicates a value for D(F2) of about 37 kcals.g.-mol.-l and certainly notgreater than about 45 kcals. g.-mo1.-1. This in turn leads inescapably to avalue for the electron affinity of a gaseous fluorine atom which is less thanthat of chlorine although in solution the total electron affinity of fluorine(i.e., including the heat of solvation of the ion) is still found to be greaterthan that of chlorine.The chemistry of the inter-halogen compounds and especially of thehalogen fluorides has been extensively studied by Emelhus and his collabor-ators. From this work has emerged the striking versatility of brominetrifluoride as an electrolytic solvent and fluorinating agent.The electrical conductivities of several inter-halogen compounds havebeen measured.The specific conductivities of ClF,, BrF,, and IF, 181 arerespectively <1eS (at O"), 8.0 x lo3 (at 25"), and 2-3 x ohm-1cm.-l The conductivity of BrF,, which obeys Ohm's law, decreases withtemperature in the range 15-60", but for IF, there is a positive temperaturecoefficient. The conductivity of molten IClYfe2 in which the degree ofionisation is of the order of 1%, passes through a maximum a t 40". Theconductivity of solid IC1, increases rapidly with temperature and passesThompson, Street, Ghiorso, and Seaborg, Phys. Review, 1950, 78, 298.17* J. Amer. Chem. SOC., 1950, 72,4832.180 J., 1950, 1524. J., 1949, 2861.17g Emelkus, Nature, 1950, 165, 224.lS2 Greenwood and Emelbus, J., 1950, 987120 INORGANIC OHEMISTRY.without marked discontinuity into that of the molten compound, whichshows a maximum at 111".To explain the phenomena in bromine trifluoride, Banks, Emelkis, andWoolf 181 postulated the existence of BrF,+ and BrF,- ions, 'uz'x., ZBrF,BrF,+BrF,- , an assumption amply supported by succeeding investigationswhich have also demonstrated the existence of an " acid-base " system inthis s01vent.l~~ Compounds such as (BrF,)SbF, and (BrF,)2SnF, behaveas acids, and KBrF,, AgBrF,, and Ba(BrF,), as bases, and neutralisationreactions have been shown, by conductivity measurements, to occurbetween them to give salts of SbF,- and SnF,-.Bromine trifluoride has also been found to be a valuable fluorinating agent.Sharp has prepared a compound, AuBrF,, by dissolving gold in BrF,.This compound also behaves as an " acid " in BrF, and with bromotetra-fluorides (" bases ") forms fluoroaurates such as AgAuF,.Similarly, salts of a wide variety of complex fluoro-acids of metals and ofthe nitronium ion have been prepared by Woolf and Emelhus 385 either bytreating an oxy-salt, such as a borate or metaphosphate, directly withBrP,, to give a tetrafluoroborate or hexafluorophosphate, respectively, or by" neutralisation '' reactions in BrF,.Nitronium salts were prepared bydissolving N,O, and an " acid " forming substance, e.g., Sb,O, or SnF,, inBrF, ; in this manner (NO,)BF,, (NO,),SnF,, (NO,)PF,, (NO,)AsF,,(NO,)SbF,, and (NO,)AuF, have been prepared.A variety of nitrosyl complex fluorides capable of functioning as " acids "in BrF, solution, and nitrosyl fluorosulphonate which can undergo reactionswith both " acids " and " bases " to give nitrosyl salts and fluorosulphonates,respectively, have been prepared including the following new salts :AgSO,F, (NO)SO,F, (NO,)SO,F, (NO)AuF,, (NO)PF,, (NO),GeF,,(NO),SnF,.186 Bromine trifluoride has also been used to prepare PtF,,RhF,, and PdF, from the halides of these metals.Niobium (columbium), tantalum, or their pentoxides react with BrF,to give fluorobromonium hexafluorotantalate (BrF,)NbF, and (BrF,)TaF,which act as " acids " in bromine trifluoride and are neutralised by bromo-fluorides. Bismuth pentoxide [70% Bi(v)] reacts with BrF, to give BiOF,containing BiF,, and BiF, forms (BrF,)BiF, which behaves as an " acid "in BrF,.laaThe only simple anhydrous fluoride of molybdenum known hitherto is thehexafluoride. The compound MoF, has now been prepared lag by heatingthe tribromide to 600" in hydrogen fluoride.Reactions of BrF, with mixturesof VCl, and potassium, silver, or barium chlorides give the correspondinghexafluorovanadates. The reaction between BrF, and a number of oxidesand some oxy-acid salts has been studied by Emelbus and W00lf.~~OComplex fluorides prepared by " acid-base " reactions in BrF, are183 Woolf and Emelkus, J., 1949, 2865.185 J., 1950, 1050.188 Gutmann and EmelBus, J . , 1950, 1046.18Q Emel6us and Gutmann, J., 1950, 2979.18' J., 1949, 2901.18' Sharpe, J., 1950, 3444.loo J ., 1950, 164.ls6 Woolf, J., 1950, 1053FAIRBROTHER. 121often impure and contain bromine. It has now been shown by Sharpe lglthat this result is due to solvolysis by the BrF, or incomplete reaction betweenacid and base.Further types of neutralisation reactions in hydrogen fluoride and iodinepentafluoride have been studied by W00lf.l~~ These lead to the formationof complex fluorides by such reactions as :K+€€F,- + H,F+BF,- = KBF, + 3HFKfIF,- + IF,+SbF,- = KSbF, + ZIP,Potassium iodohexafluoride KIF,, has been prepared by Emelbus andSharpe by dissolving K F in excess of IF,, the surplus IF, being removedby evaporation in vacuo. The preparation of iodine heptafluoride from itselements and a study of some of its properties have been described bySchumb and Lynch.lg4 The Raman spectra of IF, and IF, indicate that theIF, molecule is a tetragonal bipyramid with four F atoms at the corners ofa square base, and the iodine and fifth fluorine atom a t the ends of the four-fold axis normal to the base.I n IF, the iodine appears to be located at thecentre of a pentagon of five F atoms, the remaining F atoms being situatedabove and below the iodine, forming a pentagonal bipyramid.lg5The formation of some fluoride complexes of bivalent and tervalentmetals in aqueous solution has also been examined, and the existence of MnF,3-and CrF2+ dem0n~trated.l~~ Woolf and Greenwood lg7 have studied theformation of complex fluorides by addition and neutralisation reactions, inrelation to the conductivities of the non-aqueous liquid fluorides used assolvents.Tantalum trifluoride has been prepared by Emelkus and Gutmann lg8 bythe action of hydrogen fluoride on “tantalum hydride” at 250-300”:some pentafluoride was formed simultaneously. Niobium (columbium), its“ hydride ”, or the impure trichloride, on the other hand, gave only thepentafluoride under similar conditions.Pure tungsten dibromide wasprepared by reducing the tribromide in hydrogen. A number of non-metallic oxyfluorides, wiz., SOF,, SO,F,, H*SO,F, POF,, and SeOF,, havebeen prepared by Wiechert lg9 by treating the corresponding acid chlorideswith hydrogen fluoride without the use of a catalyst.The system NH,F-KF-H,O a t 25” is a simple one; no double salts areformed, the solid phases in equilibrium with the solutions being NH,F, KF,and KF,2H,0.200 Banks and Rudge ,01 have measured the density of liquidchlorine trifluoride under its own vapour pressure from -5” to +as0.Dunitz and Hedberg ,02 have re-investigated the structures of C10 andC10, by electron-diffraction measurements.lg2 J., 1950, 3678.lS1 J., 1950, 2907.lQ4 Ind. Eng. Chem., 1950, 42, 1383.lg5 Lord, Lynch, Schumb, and Slowinski, J. Amer. Chem. Soc., 1950,72, 522.lg6 Schaffer and Hammaker, ibid., p. 2575.leg 2. anorg. Chem., 1950, 261, 310.201 J., 1950, 191.lo3 J., 1949, 2206.lQ7 J . , 1950, 2200. lS8 J., 1950, 2115.Haendler and Jacke, J . Amer. Chem. SOC., 1950, 72, 4137.202 Dunitz and Hedberg, J . Amer. Chem. SOC., 1950,72, 3108122 INORGANIC CHEMISTRY.Lead monoxide is reported to give good yields as a, reducing agent in thepreparation of sodium chlorite from chlorine di~xide.~O~GufmannZM has shown that iodine monochloride also behaves as anionising solvent.A saturated solution of antimony pentachloride (1 mol. yo)possesses a conductivity which is greater than that of the solvent and believedto be due to the ionisation : I S b C l , ~ I + + SbC1,-. Addition of potas-sium chloride reduces the conductivity to a minimum at [KCl] = [SbCl,],followed by a rise caused by the formation of KICI,. Typical neutralisationreactions are observed.The course of the reaction between iodine monochloride and phenolshas been studied by Bennett and Sharp205 under different experimentalconditions : in the absence of a solvent the main reaction is chlorination;in solution it is iodination.This is accord with the increase of dipole momenton dissolution and the prediction that in a solvent of sufficiently highdielectric constant, iodine monochloride would undergo electrolytic dissoci-ation into I+ and C1-.,06A new determination of the acid dissociation constant of hypoiodous acidby Josien and Sourisseau 207 leads to the value 2 x for [H+][IO-]/[IOH]which is greater than the previous estimate by Furth 208 (2-3 x 10-ll) andabout the same as the basic dissociation constant obtained by Murrayzo9(3.2 x 10-l').Further evidence of the existence of iodine, and also to some extentbromine, as unipositive cations has been obtained by several workers.Hildebrand, Benesi, and Mower 210 have shown that the increase of solubilityof iodine in p-xylene and mesitylene, owing to complex formation betweenthe solvent and the iodine, is in good agreement with equilibrium data fromearlier spectroscopic evidence.211 Cromwell and Scott 212 have made are-determination of the equilibrium constant of the formation of the benzenecomplex, C6H6 + I, C,H6,1,, and estimate the heat of formation( A H ) of the complex to vary from -1317 -J= 50 to 1452 80 cals.accordingto the concentration of the solutions. Complex formation also betweeniodine monochloride and benzene and certain of its derivatives has beendemonstrated by Keefer and Andrews by measurement of the absorptionspectra.Several new salts of positive iodine, stabilised by pyridine or a-picoline,with halogenobenzoic acids have been prepared by Zingaro, VanderWerf,and Kleinbe~-g,~l~ and a very labile needle-shaped compound of iodine withdioxan has been obtained by Kortum-Seiler and K ~ r t U r n .~ ~ ~ The heat offormation in cyclohexane solution from dioxan and solid iodine is estimatedto be -4.3 kcals./mole.203 Holst, Ind. Eng. Chem., 1950, 42, 2359.205 J . , 1950, 1383.20' Bull. SOC. chim., 1950, 255.209 J . , 1925, 127, 885.211 Benesi and Hildebrand, ibid., 1949, 71, 2703.212 Ibid., 1950, 72, 3825.914 Ibid., p. 5341.*04 Research, 1950, 3, 337.206 Fairbrother, J., 1936, 847.208 2. Elektrochem., 1922, 28, 57.210 J . Amer. Chem. SOC., 1950, 72, 1017.213 ]bid., p .5170.215 2. Elektrochem., 1950, 54, 70FAIRBROTHER. 123Gonda-Hunwald, Graf, and Korosy 216 have obtained further evidenceof the existence of Br+ from some electrodialysis experiments, and Keefer andAndrews217 have shown that bromine displays a major absorption band inthe neighbourhood of 300 mp. in aromatic solvents similar to that shown byiodine.Pairbrother 2 l 8 has shown that iodine cyanide undergoes a polarisationin an electron-donor solvent which can stabilise the structure I+CN- similarto that shown by molecular iodine.219 It has been shown that the behaviourof the cyanohalogens as halides or as cyanides in their reactions (e.g., ClCNusually behaves as a chloride of cyanogen, and ICN as a cyanide of unipositiveiodine) is due to solvent-solute interaction in a basic solvent and to theenergy relationships involved in the particular reactions.A crystallographicexamination 220 of a series of solid solutions of iodine and bromine over therange 0-38 rnol.% of bromine suggests that the crystals contain orientedIBr molecules.Potassium permanganate and the oxides of manganese, when gentlyheated in fluorine, yield MnF, contaminated with a little MnF, but no oxy-fluoride. On the other hand, as Aynsley, Peacock, and Robinson 221 haveshown, rhenium dioxide and potassium per-rhenate by the same treatmentgive the two new oxyfluorides ReOF, and Re02F,.A spectrophotometric study of Mn(m), which is present in the brown-redsolution obtained when &(II) in hydrochloric acid is oxidised or when MnO,or KMnO, are dissolved in hydrochloric acid has been made by Ibers andDavidson.222 A blue sodium pentamanganate has been prepared by Levi.223Accurately characterised specimens of MnO, &(OH),, Mn,O,, a-Mn203,y-Mi20,, MnO-OH, and MnO, have been prepared, and their chemical and.physical properties Compounds of manganous chloride withpyridine have been studied by F ~ f e .~ ~ ~The thermal explosion in air and the decomposition of ammoniumpermanganate have been studied by Bircumshaw and Tayler.2Z6The technique of ion-exchange separation has been extended to the separ-ation of a number of seventh-group anions.227 Halide ions may be separatedfrom one another as well as TcO,- from Re0,- : permanganate cannot beseparated, as it reacts with the resin.The absorption spectra of rhenium(m), rhenium(v), and variousrhenium(rv) species and their reactivities towards common oxidising andreducing agents have been investigated by Maun and Davidson.228 Theyellow- brown solution of Re@) in hydrochloric acid or reduced per-rhenateis much more easily oxidised than the light green hexachlororhenate.21u Nature, 1950, 166, 68.217 J .Amer. Chem. SOC., 1950, 72, 4677.21g Fairbrother, J., 1948, 1051.220 Heavens and Cheesman, Acta Cryst., 1950,3, 197.222 J . Amer. Chem. SOC., 1950,72,4744.124 Moore, Ellis, and Selwood, J. Amer. Chem. SOC., 1950, 72, 856.225 J., 1950, 790.227 Attenbury and Boyd, J. Amer. Chem. SOC., 1950,72,4804.Z * 8 J., 1950, 180.221 J., 1950, 1622.223 Gazzetta, 1949, 79, 630.aa6 J ., 1950, 3674.228 Ibid., p. 2254124 INORGANIC CHEMISTRY.Aynsley, Peacock, and Robinson 221 have confirmed the observations ofearlier workers that the highest fluoride of rhenium that can be prepared isthe hexafluoride.Group VIII.-The formation of a thin film of y-Fe203 on iron immersedin 0-1N-sodium hydroxide and its relation to the inhibition of corrosion insodium hydroxide solution have been studied by Mayne, Menter, andP r y ~ r . ~ ~ ~ The thermal dissociation of the anhydrous ferric halides has beeninvestigated by Gregory and T h a ~ k r e y , ~ ~ ~ and by Kangro and Petersen.231Feitknecht and Keller 232 have shown that the dark green oxidation productof Fe(OH), does not correspond to a single chemical compound.Crystals of nickel ferrite, NiFe204, of up to 2 mm.side, have been preparedby slowly cooling a mixture of Fe203 and NiO dissolved in borax glass from1330°.233Nyholm has prepared and studied complexes of ferrous and ferrichalides,234 bivalent and tervalentand tervalent rhodium 237 halides with the di(tertiary arsine) chelate groupo-phen ylene bisdime th ylarsine.Azidopentamminocobalt (1x1) complexes have been prepared by Linhardand Flygare 238 by the substitution of N3- for H20 in [Co(NH3),,H20j3+ orby the synthesis of [Co(NH3),N3I2+ from Co2+, N3-, NH,, and NH,+ byatmospheric oxidation.Measurements of the diamagnetic susceptibilities of the cobalt(II1)complex of 3 : 6-dithia-1 : 8-di(salicy1ideneamino)octane and of thecorresponding p-hydroxy- a-naphthylidene complex 239 have confirmed theview, deduced earlier2& from a resolution of the former compound intooptical antipodes, that these chelate molecules can attach themselves at sixpoints to the central metal atom.The corresponding ferrous complex of thefirst-named chelating agent is paramagnetic, with a moment correspondingto two unpaired electrons, which has been interpreted as meaning that theFe(I1) is bound to the sexadentate in a similar manner to that in which Fe(II1)is bound in ferriheme hydroxide2,1 by four bonds resonating among sixpositions.A comparison of the absorption spectra of geometric isomers of cobalt(111)complex compounds of known configuration indicates that absorptionspectra may be used to establish the configuration in other cases wheneverthe positions of the absorption maxima of the second and third bands aresignificantly shifted.242Bassett and Henshall 243 have examined the systems Na,SO,-CoSO,-H20 and have shown that the double salt Na2S04,CoS0,,H20 has a definite229 J ., 1950, 3229.231 2. anorg. Chem., 1950, 261, 157.233 Matthias and Remeika, Phys. Review, 1950, 79,391.234 J., 1950, 851.237 J . , 1950, 857.239 Dwyer, Lions, and Mellor, J. Amer. Chem. SOC., 1950, 72, 5037.240 Dwyer and Lions, ibid., 1947, 89, 2917.241 Pauling and Coryell, ibid., 1937, 59, 633.242 Basolo, ibid., 1950, 72, 4393.bivalent and tervalent230 J . Amer. Chem. SOC., 1950,72,3176.233 Ibid., 1950, 262, 61.238 J . , 1950, 2061; Nature, 1950, 165, 154.238 2. anorg. Chem., 1950,262, 328.235 J., 1950, 2071.24s J., 1950, 1970FAIRBROTHER. 125composition and that isomorphous replacement of Co(H,O), by Na,(H,O),does not occur.that theonly solid phases in equilibrium with their solutions are the hexa- and mono-hydrates.Hume and Kolthoff 245 have shown by polarographic studies of the re-duction of the Ni(CN,)" ion that the dissociation constant of this ion is ofthe order of and that the solid nickel cyanide in equilibrium with asaturated solution is actually Ni[Ni(CN),]. Nickel complex compoundscontaining triethylenetetramine have been studied by Jonassen and Douglas.246Spectrophotometric measurements indicate the presence of [Ni trien],+and [Ni2trien,]4+ in solution. The same complexing agent has beenused 247 to prepare the compounds [Pt trien][PtCl,], [Pd trien][PdCl,], and[trien H,(PtC14)2],2H,0.The lower oxidation states of ruthenium, RU(III) and Ru(Iv), have beenstudied in acid perchlorate solution by Wehner and hind ma^^.^,^ Drychlorine a t 700" converts metallic ruthenium completely into the trichloride,which sublimes in two different polymorphic ~ a r i e t i e s . ~ ~The complex compounds PtBr2,3AsR, and PdBr2,3AsR, (where AsR,is methyldiphenylarsine) have been isolated and studied by N y h ~ l r n . ~ ~ Conductivity measurements in acetone of the palladium compound supportthe view that they are triarsine salts, e.g., [Pd(AsR,),Br]+Br-.UbbelohdeZ5l has studied the effect of adsorption of hydrogen on thewetting of palladium by mercury and has discussed the results in terms ofpseudo-metallic bonding in hydrides.Burstall, Dwyer, and Gyarfas 252 have prepared [ OsC12,2dipy]C1,3H,0and [ Os,3dipy]Cl,,GH,O and the corresponding bromides by the reactionbetween potassium osmichloride K,[ OsCl,] or ammonium osmibromide(NH,J,[ OsBr,] and excess of 2 : 2'-dipyridyl. Optically active enantiomorphsof' the antimony1 tartrate and iodide of the bivalent complex have beenobtained.Several new platinum(I1) and platinum(1v) complex compounds havebeen prepared, vix., C,H4PtC1 and (C,H4PtC12),,253 trans-[ Pt en2C12]C1,,254and a series of octahedral complexes of quadrivalent platinum with thegeneral formula Pt( Hal),2AsR3, various halides and tertiary arsines beingChatt 256 has studied the oxidation of the non-ionic simple com-plexes c~~-[(C,H~)~P],P~C~, and trans-[ (C,H7),P],PtC12 and the bridgedcomplex [ (C,H, ),P,,PtCl,], to the corresponding platinic compounds, and hasdiscussed the role of the d orbitals of the platinum atom in stabilising theplatinous bridge. F. FALRBROTHER.A study of the system NiSe0,-H,Se04-H20 at 30" has shown844 Rohren and Froning, J . Amer. Chem. SOC., 1950,72,4656.246 Ibid., 1949, 71, 4094.248 Ibid., 1950, 72, 3911.250 J . , 1950, 848.253 Chatt and Wilkins, Nature, 1950,165, 859.p54 Basolo and Tam, J . Amer. Chem.. SOC., 1950,72, 2433.255 Nyholm, J . , 1950, 843.245 Ibid., p. 4423.247 Jonassen and Cull, ibid., p. 4097.2*9 Hill and Beamish, ibid., p. 4865.251 J . , 1950, 1143. J . , 1950, 953.256 J . , 1950, 2301

ISSN:0365-6217    

DOI:10.1039/AR9504700098

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

ORGANIC CHEMISTRY.1. INTRODUCTION.A FURTHER step forward has been taken this year towards the objective ofmaking this section a true Annual Report. Some two-thirds of the spaceavailable is devoted to this purpose under the headings : Stereochemistry,General Methods, Aliphatic Compounds, Homocyclic Compounds, Hetero-cyclic Compounds, and Macromolecules ; the section on Theoretical OrganicChemistry deals with addition and elimination reactions and molecularrearrangements which are topics which have not been discussed fully in theseReports in recent years. It has beeh thought necessary to include specialisedarticles on Nucleic Acids and Porphins to cover obvious gaps. With the verylarge number of publications appearing in the space of one year, it is inevit-able that each aspect of organic chemistry cannot be dealt with every year asthere are still severe limitations on space.For this reason treatment of, forexample, sugars and many of the heterocyclic nitrogenous compounds hasbeen postponed for a later Report.We propose next year to devote the whole of our space to an account ofthe progress made during the year which will be organised under the generalheadings we have just mentioned; of these that devoted to Macromoleculesis a new departure which we think necessary in view of the ever increasinginterest of such substances from the organic chemical, ke., structural, pointof view. It seems inevitable, however, that occasional essay articles willbe required even under the new policy.A. W.J.H. N. R.2. THEORETICAL ORGANIC CHEMISTRY.A. Addition Rmctions.-Homolytic additions, particularly of halogenacids to olefinic systems, have recently been considered in these Reports,lbut heterolytic addition processes have received little attention. In 1931,Ingold and Ingold,2* cfs3 examining by a competition method the effects ofsubstituents on the rate of addition of bromine to ethylene derivatives, madeit clear that halogens are usually electrophilic in their attack on unsaturatedcompounds. These authors also adduced evidence that powerful electron-withdrawal from the olefinic centre might polarise the double link sufficientlyto make bromine a nucleophilic reagent. Knowledge of the kinetics of halogenaddition reactions has since been extended, partly by Anantakrishnan’sSmith, Ann.Reports, 1939,36, 219 ; Hey, ibid., 1948,45, 149.Ingold and Ingold, J., 1931, 2354.Anantakrishnan and Ingold, J . , 1935,984,1396DE LA MARE : THEORETICAL ORGANIC CHEMISTRY. 127school 4 and by Nozaki and Ogg 5 but most extensively by P. W. Robertsonand his co-workers,6 who have examined the reactivities of many ethyleniccompounds under various conditions of medium and of catalysis. Theresults obtained in acetic acid (in this solvent complications due to homolytic,heterogeneous, and photochemical reactions may be avoided) have recentlybeen surveyed,’ and they are summarised in the following section.Chromones, Flavones and Flavono1s.-Schmid and his colleagues 98 havecarried out an extensive study of the chromone constituents of carnations,Eugenia aromaticu and E .mryophyllata. Among the products isolated wereeugenin (XXXVIII ; R = R” = H, R’ = Me), eugenitin (XXXVIII ;R = R’ = Me, R” = H), isoeugenitin (XXXVIII; R = H, R’ = R” =Me), and isoeugenitol (XXXVIII; R = R’ = H, R” = Me). The simul-taneous occurrence of eugenitin and the iso-structures suggests the presenceof a monocyclic intermediate, e.g., (XXXIX), which can cyclise in two ways.Syntheses of eugenitin, isoeugenitin, and isoeugenitol were described.The Allan-Robinson synthesis of flavones, by heating of an o-hydroxy- oran o-aroyloxy-acetophenone with an aromatic anhydride and the salt of thecorresponding acid, suffers from the disadvantage that 3-acylflavones areobtained as by-products, often in appreciable amount, and the hydrolyticfission of the acyl group may result in considerable Baker andGlocking loo have discussed the mechanism of this formation of acylflavonesand have devised a method depending on a condensation with benzylamine93 Johnson, Robertson, and Whalley, J., 1950, 2971.94 Brown, Cartwright, Robertson, and Whalley, J., 1949, 859.95 Cram, J .Amer. Chem. SOC., 1948,70,440,4244; Frye, Wallis, and Dougherty, J .Org. Chem., 1949,14, 397.Cartwright, Robertson, and Whalley, J., 1949, 1563.@’ Cram, J . Amer. Chem. Soc., 1950,72, 1001.98 Schmid et al., Helv. Chim. Acta, 1048, 31, 1603; 1949, 32, 813, 1358; 1950, 33,917, 1770.Baker and Butt, J . , 1949, 2142. loo J., 1050, 2759230 ORGANIC CHEMISTRY.for distinguishing between the two possible isomeric acylflavones.If theo-substituted acetophenones are heated alone in glycerol a t 250°, the flavonesthemselves are obtained,lol a method which is also applicable to the synthesisof flavonols.Paper chromatography has been applied to the separation of mixtures offlavones and related compounds.lo2 Briggs and Locker lo3 have examinedthe flavonol content of the bark of certain Melicope spp. and have described avariety of new compounds, some of which are ethers of quercetin(3 : 5 : 7 : 3’ : 4’-pentahydroxyflavone) and others also have substituents inthe 6- or the 8-position. Methods of synthesis were evolved, e.g., of ternatin(XL) consisting of a combination of the Allan-Robinson flavonol method andSeshadri’s nuclear hydroxylation method. lo4 The structure of ginketin, thecolouring matter of the autumn leaves of the maidenhair tree, is still underconsideration but it is probably more complex than the simple flavoneformulation originally put forward.lo5 Moewus lo6 has reviewed the partplayed by flavonols in the sexual processes of certain flowering plants, e.g.,Forsythia.Lactones.-The occurrence of the unsaturated lactone structure 1°7 inmany natural products had led to a great deal of work on the synthesis andproperties of these compounds, Methods of synthesis in the heart-poison logand toad-venom 109 fields have been reviewed. An important syntheticroute for the +unsaturated lactones is that depending on acetylenic inter-mediates,l1° e.g.:The addition of methanol to the triple bond yields the unsaturated methoxy-lactones, as exemplified by Raphael’s synthesis of penicillic acid ;ll1 similarOMe /\, r‘C:CH*CO,H 1 \ ,,CH*CH,-CO,Hi / IPh*CH:CH(O,CO co-o co-0 -(XLI.) (XLII.) (XLIII.)additions to the Py-acetylenic alcohols 112 lead to six-membered unsaturatedlactones, as in the synthesis of (&)-kawain (XLI).l13101 Dunne, Gowan, Keane, O’Kelly, O’Sullivan, Roche, Ryan, and Wheeler, J.,1950, 1252.102 Bate-Smith et al., Biochim. Biophys. Actu, 1950, 4, 427, 441 ; Lindstedt, ActuChem. Scand., 1950,4,448, 1042.lo3 J., 1949, 2157, 2162; 1950,864,2376,2379.104 Proc. Indian Acad. Sci., 1949,3Q, A , 333.106 Baker, Flemons, and Winter, J . , 1949, 1560.106 Angew. Chem., 1950, 62, 496.108 Turner, Chem.Reviews, 1948, 43, 2 3 ; Heusser, “ Fortschritte der Chemie109 Deulofeu, ibid., 1948, 5, 241.11s Fowler and Henbest, i b d . , 1950, 3642.Haynes, Quart. Reviews, 1948,2, 46.Organischer Naturstoffe,” Vienna, 1950, Vol. VII, p. 87.110 Jones, J . , 1950, 756.112 Henbest, Jones, and Walls, ibid., 1949, 2696. J., 1948, 1508JOHNSON AND RYDON : HETEROCYCLIC COMPOUNDS. 23 1Similar principles have been used by Jones et ul.ll* to synthesise analoguesof the plant growth hormone, auxin b, as formulated by Kogl :116[CH,],>XH R*CH( OH)*CH,*CiC*CO,Me ____ -+ R*CH( OH)*CH2*COCH,*C0,MeR-yH-CH,*C( OH) :CH (R = A / ) o------co ! I! --+In contrast to the reported properties of auxin b,l15 the product could not behydrolysed to the corresponding acid and attempts to prepare the acidinvariably led to the formation of the lactone.Further work on the isolationand properties of the natural compound is necessary to remove theseanomalies. Other analogues containing the same side chain were alsodescribed (R = Me, Ph, propenyl, spirocyclohexyl) and some progress hasbeen made towards building of the auxin-a side-chain .l15a Meanwhile Kogland de Bruin 116 have also synthesised the cyclopentenyl analogue of auxin bby means of a Reformatsky reaction of cyclopentenealdehyde with y-bromo-p-ethoxycrotonic ester. No comparisons of the product with auxin b itselfwere made other than the ultra-violet absorption spectrum.The bearing of Bredt's rule on the enol lactonisation of y- and 8-keto-acidshas been discussed by Fawcett,l17 and Linstead and his colleagues havestudied the formation of lactonic acids by cyclodehydration of keto-dicarboxylic acids.Thus treatment of p-ketoadipic acid with acetylchloride-hydrogen chloride gave the lactone (XLII), the properties of whichresembled those of the &-unsaturated lactones (see below). Richter ll9prepared the ester of (XLII) by cyclisation of the monoester of p-ketoadipicacid, but his claim to have prepared the free acid is considered doubtfulby the English workers. Lactonisation of cis-cis-muconic acid gave the+unsaturated lactone (XLIII) ,121 the corresponding ester of which, ontreatment with sodium methoxide,122 gave the cis-truns-isomer of the mono-methyl ester of muconic acid, from which the corresponding acid, not formerlydescribed, was obtained.Johnson et ~ 1 . l ~ ~ have studied the decarboxylationof y-substituted paraconic acids.with N-bromosuccinimide and then treated with silver acetate, to give adiacetate with an extra acetoxy-group in the 2-position of the pyrone ring.Removal of acetic acid as before gave patulin itself although in small yield.Hydrogenation and hydrolysis of the above diacetate gave deoxypatulinicacid (LXII), which was an intermediate in the synthesis of allopatulin(LVIII), as well as a second patulin synthesis via 5-chlorodeoxypatulinicacid.147 The new patulin formula (LVII) was used 144 to interpret theextensive degradative work described by earlier workers.lP1 J., 1944, 415.l a 3 Woodward and Singh, J .Amer. Chem. SOC., 1950,72, 5351.144 Idem, Experientia, 1950, 6, 238.la6 Woodward and Singh, ibid., 1950, 72, 1428.147 Idem, Nature, 1950, 165, 928.la8 Naute, Oosterhuis, van der Linden, van Duyn, and Dienske, Rec. Trau. chim.,142* 148Ira Helv. Chim. Acta, 1949,32, 1166, 1752.Dauben and Weisenborn, J . Amer. Chem. SOC., 1949, 71, 3853,1946,65, 865; Cohen, Chem. and Ind., 1949, 640230 ORGANIC UHEMISTRY.Coumarins.-The fluorescent behaviour of coumarins and its variationwith pH is recommended as a means of identification in this series.149 Therecognition of the blood-anticoagulant and rodenticide properties of a number(LXII.) (LXIII.) (LXIV.)of 3-substituted 4-hydroxycoumarins 150 has intensified studies on thereactions of the 4-hydroxycoumarins, e.g., acylation,151 Michael addition,152and aldol ~0ndensation.l~~ Koelsch and Sundet lM have described Michaeladditions with 4-acetylcoumarin.The coumarin photodimers probablyhave a cycbbutane structure as 3-phenylcoumarin gives a product analogousto that obtained from coumarin i t ~ e 1 f . l ~ ~ Parker and Robertson 156 havesynthesised a chromono(2’ : 3’-3 : 4)coumarin having the nucleus ofrotenonone (LXIII).Methods for the synthesis of isocoumarins (LXIV) have been surveyed.15’Dioxans.-1 : 3-Dioxans, formed by the Prins reaction 15* with substitutedethylenes, may be hydrogenolysed to 1 : 3-diols lSQ or 3-substituted primaryalcohols. 160Sulphur ring systems.Ethylene Sulphides.-The preparation and properties of the ethylenesulphide ring system have been studied by Davies and his co-workers inMelbourne.lG1 They have reviewed the methods of preparation and havereported favourably on the reaction of epoxides with thiourea or relatedcompounds, except in those cases where a strongly polar group was adjacentto the oxide ring.In many respects, although not all, the ring-openingreactions are similar to those of the epoxides, but there is a much greaterGoodwin and Kavanagh, Arch. Biochem., 1950,27, 152.l 5 0 E.g., FuEik, Prochbzka, LQbler, and Strof, Nature, 1950, 166, 830.161 Ukita, Nojima, and Matsurnoto, J . Amer. Chem. Soc., 1950, 72, 5143; Badcock,Dean, Robertson, and Whalley, J . , 1950,903 ; Muller, Syrovatka, and Wlasak, Monatsh.,1950,81, 174.154 Seidman, Robertson, and Link, J.Amer. Chem. SOC., 1950, 72, 5193.153 Ikaws and Link, ibid., p. 4373.154 Ibid., pp. 1681, 1844.166 Schiinberg, Lrttif, Moubaaher, and Awad, J., 1950, 374.1 5 8 Ibid., p. 1121.1 5 7 Kamal, Robertson, and Tittensor, ibid., 1950, 3376 ; Johnston, Kadow, Langs-160 Cf. Johnson, Ann. Reports, 1949,46,151.159 Price and Krishnamurti, J . Amer. Chem. SOC., 1950,72,5335.1‘0 Emerson, Heider, Longley, and Shafer, ibid., p. 5314.161 J., 1949, 278, 282; 1950, 317, 892.See also J . , 1949, 2049.joen, and Shriner, J . Org. Chem., 1948,18,477JOHNSON AND RYDON : HETEROCYCLIC COMPOUNDS. 237tendency of the cyclic sulphides to polymerise. Unlike that in thiophen,the sulphur in a three-membered ring cannot exist in a higher valency state.mophen.-Several papers have been devoted to the substitution of thethiophen ring for aromatic or other heterocyclic rings in natural productsand their prototypes, as well as in the chemotherapeutic field, especiallyantihistamines,162 but this work generally follows the standard reactions ofthiophen chemistry and its main interest is biological.to occur to the extent of 20--30%in the residual " tar " from the preparation of thiophen by reaction of buta-diene with s u 1 p h ~ r .l ~ ~ Liquid thiophen polymers of low molecular weighthave been described, containing predominantly the trimer (LXV).165Osmium tetroxide oxidation of protoporphyrin can be made to yieldspirographis porphyrin (oxidation of only one vinyl group to formyl) or2 : 4-diformyldeuteroporphyrin according to the conditions.Improvementsin the yield of the latter substance have recently been achieved.*7 Fischer and Deilmann, ibid., 1944, 280, 186.2. physiol. Chm., 1936, 242, 133.Lemberg and Falk, Biochem. J . , in the press.Ibid., 1941, 272, 1FALK AND RIMINQTON : PORPHYRINS. 273The glycol which is an intermediate stage in this oxidation, -CH:CH, --+-CH(OH)CH,*OH -+ -CHO --+ -CO,H, can also be ~btained.~The elegant synthesis (I1 + I11 --+ IV) of aetioporphyrin I in excel-lent yield under physiological conditions due to Siedel and Winkler lo has notpreviously been reported. Andrews, Corwin, and Sharp l1 have also reporteda new porphyrin synthesis which proceeds smoothly at room temperature,leading to a porphyrin with four carbethoxy-groups, although such groupshad previously been held to inhibit porphyrin formation.The methoddepends on the fact that the l-methylpyrrole-%aldehyde (V) can supply thecarbon atom for condensation of 2 molecules of (VI) to give a tripyrryl-methane (VII), which is specifically split to the dipyrromethene (VIII).When, instead of (VI), 2 molecules of the dipyrrylmethane (IX) are used, thedihydroporphyrin (X) is formed and is autoxidised to the porphyrin (XI)which crystallises from the reaction mixture in 40 yo yield.Me-C02Et Me -CO,Et Et0,C -Me II II Et0,C ----Me \g’ II IIMe II II CH2-\N/ \I$ Me11 [ICHOMe H\N/(V.1 (VI -1 (IX.)Et0,C -Me Me=CO,EtMel(N!J-CH=(N/MeH (VIII.)-1-I1 IIMe‘N/Me-C0,EtMeEt0,C H, C0,EtMe,(\()OMe/-NH N-\/ \ \--N HN //MJ I T M eEt0,C C0,Et\/\/\/Aluminium isopropoxide under mild conditions reduces the formylside-chain without reducing the isocyclic ring carbonyl group of mesopyro-ph%ophorbide-b.l2 The vinyl group, when present, is not affected by thisreduction.Fischer’s school clung long to the idea of “ Kekulb ” isomers, dependingon the exact position of the double bonds in both the porphyrin and thechlorophyll series.Not a little work has centred around the related question* Fischer and Pfeiffer, Annalen, 1944,556, 131.lo Ibid., 1943, 554, 162.l2 Fischer, Mittenzwei, and Hevkr, Annalen, 1940, 545, 154; “ Organic Reactions,”l1 J . Amer. Chem. SOC., 1950, 72,491.Vol.11, p. 184274 ORGANIC CHEMISTRY.of hydrogen bonding between pyrrole nitrogens.13 The evidence now pointsstrongly to complete resonance in these molecules, and has been well sum-marised by Lemberg and Legge.3aExtra ” Hydrogen Atoms.-The isolationby Fischer and Wendroth in 1940 l4 of the optically active acid (XII) insteadof haematinic acid (XIII) on degradative oxidation of chlorins confirmedthe previous amignment of the ( ( extra ” hydrogen atoms to positions 7 and8, affording also, for the first time, a degradation product which, like theoriginal chlorophyll, was optically active. Since it was already known thatchlorophyll-b differed from chlorophyll-a only in having CHO instead ofCH, a t position 3 (conversion of a phaeophorbide-b derivative, by reduction,into a phaophorbide-a derivative had been achieved), this fixed the ‘( extra ”hydrogens of chlorophyll-b also a t positions 7 and 8.CH,*CH2*C0,H HO,C*CH, CH,*CH,*CO,HH-TY1-H (iNiCH2*C02H O=’ ‘ 1-0The Chlorophyll Series : The“/HO = w = O(XII.) O = w = O H (XIII.) (XIV.)HBromination of chlorins leads to substitution of one of these hydrogensby bromine.15 Chlorination of chlorins leads to substitution of hydrogen atpositions 7 and 8 by hydroxyl groups,16 and of porphyrins to substitution ofmethene-bridge hydrogen by chlorine,17 though in porphyrins like phzeopor-phyrin-a, or phylloerythrin substitution by chlorine occurs first a t position 10.Removal of Iron from Haems.-Fischer and his school devised severaldifferent methods for the removal of iron from haemin.These all dependedon the action of a reducing agent in an acid medium and yielded either proto-porphyrin or mesoporphyrin in which the two vinyl groups of the formerhave also suffered hydrogenation. Side reactions made the purification ofthe porphyrins difficult.A very convenient technique, leading directly to protoporphyrin dimethylester in good yield and applicable to haemoproteins such as haemoglobin, isdue to Grinstein;l9 for work on a micro-scale, the use of pyruvic acid forremoval of iron is excellent.Z0 For splitting the thio-ether linkage whichunites the porphyrin moiety to the protein in cytochrome-c, Paul 21 employssalts of Ag, Hg, Pb, Cu, or Cd, preferably the first mentioned. The porphyrinrelease is a first-order reaction.Improvements have been suggested.18l3 Vestling and Downing, J.Amer. Chem. SOC., 1939, 61, 3511 ; McEwen, iCid., 1936,l4 Annalen, 1940, 545, 140.l5 Fischer and BalQz, ibid., 1943, 555, 81 ; Fischer, Kellermann, and Balaz, Ber.,l7 Fischer and Klendauer, ibid., p. 123.lo Ibid., 1947, 167, 515.21 Acta Chem. Scand., 1950, 4, 239.58, 1124; Ellingson and Corwin, ibid., 1946, 68, 1112.1942,75, 1778. l6 Fischer and Diet], Annalen, 1941, 547, 234.Grinstein and Watson, J. Biol. Chem., 1943,147, 667.20 Paul, personal communication, 1950FALK AND RIMINBTON : PORPHYBINS. 275The relatively inefficient Fischer-Kdgl method for transformation ofprotoporphyrin into mesoporphyrin, modified 22 with improvement of yield,has been further studied by Grinstein and Watson23 who have raised theyield to 60%.The necessity for obtaining maximum yield in isotope erxperi-ments involving step-wise degradation of the porphyrin ring has encouragedfurther study of this transformation. Catalytic hydrogenation, with methylmethacrylate as the supporting colloid, affords yields ofThe biological conversion of one porphyrin into another has never beensatisfactorily demonstrated either in the whole animal or by uae of survivingtissues (excepting the bacterial degradation of haemin into meso- and deutero-porphyrin in the gut), although Fischer and others assumed that this necess-arily took place. Van den Bergh, Grotepass, and Reever's 25 claim that theliver transformed protoporphyrin into coproporphyrin has not beensubstantiated.2sThe administration of protoporphyrin intramuscularly or by mouth failsto raise the blood protoporphyrin, but a small increase in this level is said tofollow intramuscular injection of haernat~porphyrin.~~ Further evidencewill be needed before a biological conversion of haematoporphyrin to proto-porphyrin can be accepted.All modern theories of haem biosynthesis (see below), based on theinterpretation of experiments with isotopically-labelled substances, postulatethe formation first of a highly carboxylated pyrrole derivative which is thenprogressively decarboxylated either before or after cyclisation to the porphyrinring.The occurrence in Nature, under normal or pathological conditions, ofporphyrins representing intermediate stages betweeen the fully carboxylateduroporphyrins (8 C0,H groups) and coproporphyrins (4 C0,H groups) orprotoporphyrin (2 C0,H groups) might thus be expected.Nicholas andRimingtonF8 using paper chromatography, have indeed obtained evidenceof the existence of such porphyrins with seven, six, five, and three carboxylgroups in various materials ; a pentacarboxylic porphyrin has been isolatedin nearly pure condition from a porphyria patient.29The " conchoporphyrin " from pearl mussel shells, described by Fischerand Jordan30 as a pentacarboxylic porphyrin, has on the other hand beenshown by paper chromatography to 'be a mixture of uroporphyrin andc~proporphyrin.~~Chromic acid oxidation of porphyrins causes rupture of the macrocyclicring with production of maleinimide derivatives.The synthesis of ethyl-a2 Schultze, J . Biol. Chem., 1942,142, 89 ; Rimington, Biochem. J., 1938, 32, 460.24 Muir and Neuberger, Biochem. J . , 1949,45, 163. 25 Klin. Woch., 1932, 11, 1534.2o Watson, Pass, and Schwartz, J . Biol. Chem., 1941,139,583 ; Salzburg and Watson,27 Schumm and Beckermann, Arch. exp. Path. Phczrm., 1948,205,98.2s McSwiney, Nicholas, and Prunty, Biochem. J . , 1950, 46, 147.so 2. physiol. Chem., 1930, 190, 75.31 Nicholas and Comfort, Biochern. J . , 1949, 45, 208,J . Biol. Chem., 1943, 147, 671.ibid., p. 593.Scand. J . Clin. Lab. Invest., 1949,1, 12, and personal communication, 1950276 ORGANIC CHEMISTRY.methylmaleimide and of haematinic acid have been improved and theconditions laid down, from a study of their physical characteristics, for theoptimal separation of these substances.24Anmcal.-The distribution of the chromoprofeins, haemoglobin,myoglobin, and cytochrome-c in the tissues of different animals has beenreported by Drabkin 32 who also describes the preparation of crystallinehaemoglobin on a large scale.Directions for the large-scale preparation ofhaemin have been given.33 Determination of porphyrins in body fluids isreviewed by With and by B r ~ g s c h , ~ ~ while Salamanca, Mesorana, et ~ 1 . have studied the determination and characterisation of copro- and uro-porphyrins. Photoelectric and fluorimetric methods are reported for thedetermination of protoporphyrin in blood.36* 36 Attention has been drawnto the necessity for certain preliminary treatments in the determination ofurinary c~proporphyrin,~' and a micro-determination of porphyrins bytitration with a copper salt has been described.38 The use of phosphoricacid in place of hydrochloric acid in the fluorimetric determination of por-phyrins has been re~ommended.~QThe most notable advance in the analysis of mixtures of porphyrins is theapplication to these substances of filter-paper chromatography employinglutidine-water as the solvent system.28* 29 Under the prescribed conditions(requiring 5-10 vg.of porphyrin mixture) individual porphyrins areseparated so that their R, values are linearly related to the number of carboxylgroups in the molecule.The method is applicable to porphyrin-metalcomplexes. A partition-chromatographic method, using columns of silicagel, has also been reported.40 Nicholas 41 has undertaken a thorough studyof adsorption chromatography of porphyrins under carefully standardisedconditions.The molecular extinction coefficients of pure coproporphyrins I andI11 42 have been measured, also those of the uroporphyrin~.~3>~~ An im-proved method for the determination of uroporphyrin in urine has beendescribed which employs a spectrophotometric correction for absorbingimpurities and thus makes it possible to carry out the determination directlyupon diluted porphyria ~rines.~4Separation and quantitative determination of the coproporphyrin32 J . Biol. Chem., 1950,182, 317 ; Arch.Biochem., 1949, 21, 224.33 Org. Synth., 1941, 21, 54.s4 Scand. J . Clin. Lab. Invest., 1949, 1, 164 ; 2. Qes. inn. Med., 1949, 4, 253.35 Arch. Med. Expt. (Madrid), 1949,12, 25, 39.3~ Grinstein and Watson, J. Biol. Chem., 1943, 147, 675; Grinstein and Wintrobe,37 Mmchling, J. Lab. Clin. Med., 1940-41,26, 1676; Raine, Biochem. J . , 1950,47, xiv.38 Oliver and Rawlinson, Biochem. J., in the press.39 Kliewe, 2. Qes. inn. Med., 1948, 3, 543.40 Lucas and Orten, Ped. Proc., 1950,9, 197.Jope and O'Brien, ibid., 1945, 39, 239.43 Nakamiya, Bull. Inst. Phys. Chem. Res., Tokyo, 1942, 21, 252.Sveinsson, Rimington, and Barnes, Scand. J. Clin. Lab. Inveat., 1949,1,2 ; Riming-ibid., 1948, 172, 459.41 Biochem. J., in the press.ton amd Sveinsson, ibid., 1950,2,209FALK AND RIMINGTON : PORPHYRINS. 277isomers, which so often occur together in biological materials, have alwaysbeen a matter of great difficulty.A study of the melting points of mixturesof known composition showed the unreliability of this criterion.42 Separationof the esters by chromatography on alumina, with elution of the type-111isomer by 35% aqueous acetone, has been claimed 45 but not confirmed.42* 46More recently Schwartz et aL4' have employed the quenching a t low temper-ature of fluorescence of coproporphyrin I in 30% aqueous acetone solutionas a means of determining the quantities of the isomers present in a mixture.Figures are published, based upon this method, for the daily coproporphyrinI and I11 excretion in normal urine.4sThe pigment of the malaria parasite has been extracted by a methodavoiding the use of alkali at all stages and has been identified as haematin.49Haem a, The Prosthetic Group of Cytochome-&-Earlier work on thisporphyrin derivative is summarised by Warburg 50 in his book on metallo-porphyrins and enzyme action.Rawlinson and Hale 5 l described a methodfor its isolation from cells of Corynebacterium diphtheriae and from heartmuscle, and studied its spectral and chemical properties, which indicated thepresence of at least one aldehyde group. Simultaneously Lemberg and Falk 52studied the same subject by comparing the absorption spectra of haem andporphyrin-a with the spectra of a series of synthetic haems and porphyrins.Short reports from the two Schools were made to the 1st InternationalCongress of Bio~hemistry.5~ Porphyrin-a resembles in some respects spiro-graphis (ch1orocruoro)porphyrin.Kiese claimed 54 that the latter waspresent among the products of the action of nitrous acid on haemoglobin;the mixture, however, is very complex. Cystalline pigments, which may benitroso-derivatives, are formed, according to Sapir0,~6 by the action of nitrousacid on chlorophyll (mixture of a and b ) .Prodigiosh-The tripyrrylmethene pigment prodigiosin produced byB. prodigiosus (Serratia rnarcescem) is of interest on account of the possibleintervention of a tripyrrylmethene stage in the biosynthesis of porphyrins.Hubbard and Rimington56 have shown by the isotope technique that thenitrogen and the methylene-carbon atom of glycine are specifically utilisedin the bacterial synthesis of prodigiosin, but not the carboxyl-carbon atom.Both carbon atoms of acetic acid are specifically utilised.There is thusconsiderable resemblance between the biosynthesis of this pigment and thatof haem.45 Watson and Schwartz, PTOC. SOC. exp. Biol., 1940, 44, 7.46 Helwig, 2. Ges. inn. Med., 1949, 4, 415.4 7 Schwartz, Hawkinson, Cohen and Watson, Science, 1946,103,338 ; J . Biol. Chem.,** Wat.son, Hawkinson, Schwartz, and Sutherland, J . Clin. Incest., 1949,28,447.50 " Heavy metal prosthetic groups and enzyme action " (trans. Lawson), Clarendon5a Ibid., in the press.53 Rimington, Hale, Rawlinson, Lemberg, and Falk, 1949, Abstracts 1st Inter-1947,168, 133.Rimington, Fulton, and Sheinman, Biochem.J . , 1947, 41, 619.Press, Oxford, 1949.national Congress of Biochemistry, pp. 351, 378, 379.51 Biochem. J . , 1949,45, 247.54 NdtuTWiS8., 1946, 53, 123.5 6 Onderstepoort. J . Vet. Lcci. Animal Ind., 1950, $34,105. 5 6 Biochem. J . , 1950,40,220278 ORGANIC CHEMISTRY.A prodigiosin-like pigment, which may be a higher homologue of prodi-giosin itself, has been des~ribed.~' I n this case the organism was a mould(Actinmycetes), not a bacterium.Bacterial Porphyrins.-The porphyrin, produced by Corynebacteriumdiphdheriae grown in an iron-deficient medium, has been reinvestigated bychromatographic techniques and shown to consist of coproporphyrin IU:together with smaller quantities of uroporphyrin I and porphyrins with fiveand six carboxyl groups 68 Coproporphyrin 111 has alsobeen identified as a cellular constituent of several mycobacteria. 69Uroporphyrins.-The presence of a porphyrin in the urine of Petry, apatient with congenital porphyria, had been noted by Salkowski, Giinther,Schumm, and other early workers but all had confused it with haemato-porphyrin produced chemically from haemin by Nencki.In 1916 Fischer 6oisolated the main porphyrin from Petry's urine and obtained its methyl esterin hair-like crystals, m. p. 293", in quantities of 200-300 mg. per day. Hea t first described it as a heptacarboxylic acid, but later analytical data anddetermination of the carbon dioxide yielded on decarboxylation showed thatthere were eight carboxyl groups in the The most convenientmethod for partial decarboxylation is heating with 1% HCI a t 180-190",624 mols.of carbon dioxide being lost and a coproporphyrin produced. Identi-fication of the isomer type of the latter indicates that of the parent uropor-phyrin also and is thus a valuable aid to structural characterisation. Theuroporphyrin from Petry's urine belonged to the stioporphyrin series I.Of the three possible structures for uroporphyrin I , 'uiz., 1 : 3 : 5 : 7-tetramethylporphin-2 : 4 : 6 : 8-tetrakismethylmalonic acid, 1 : 3 : 5 : 7-tetramethylporphin-2 : 4 : 6 : 8-tetrasuccinic acid, and 1 : 3 : 5 : 7-tetrakis-carboxymethylporphin-2 : 4 : 6 : $-tetrapropionic acid, the last was eventuallyaccepted by Fischer and Hofmann 63 because the carboxylated haematinicacid produced by chromic oxidation of natural uroporphyrin was found to beidentical with a synthetic preparation of the expected material (XIV) .and was stated to bederivable from turacin, the copper-containing pigment of turaco feathers.65Preparations from urines of patients with porphyria exhibited m.p.s (ofthe octamethyl esters) ranging from 255" to about 290". In 1936 Walden-strom 66 and Mertens 67 independently reported the isolation, from urines ofacute porphyria patients, of a new uroporphyrin with ester m. p. 255-260".Since on decarboxylation it yielded coproporphyrin I11 it was claimed thatUroporphyrin I was also found in marine shells6 7 Dietzel, Naturwise., 1948, 35, 345 ; 2.physiol. Chem., 1949, 284, 262.6 8 Gray and Holt, Biochem. J., 1948,43, 191.6 1 Fischer and Hilger, ibid., 1925, 149, 65.62 Fischer and Zerweck, ibid., 1924.137, 242. 6s Ibid., 1937, 246, 15.84 Fischer and Haarer, ibid., 1932, 204, 101; Nicholas and Comfort, Biochem. J . ,1949, 45, 208 ; Comfort, Nature, 1948, 162, 851 ; Science, 1950, 112, 279 ; Tixier, Bull.SOC. Chim. biol., 1946, 28, 394.6 5 Fischer and Hilger, 2. physiol. Chem., 1923,128, 167 ; 1924,138,49.6 8 Deut. Arch. klin. Med., 1935, 178, 38; 2. physiol. Chem., 1936, 239, iii; Walden-etrom, Fink, and Hoerburger, ibid., 1935, 233, 1. 67 Ibid., 1936, 238, i ; 1937, 250, 57.69 Todd, ibid., 1949, 45, 386.2. physiol. Chem., 1915, 95, 34FAT;# AND RIMMQTON : PORPHYRINS. 279this pigment was uroporphyrin 111.In 1945, however, Watson et U Z . ~ ~disputed the homogeneity and nature of such " Waldenstrom esters,"claiming that by chromatography on calcium carbonate they were often (butnot always) separable into a zone regarded as uroporphyrin I (ester m. p.284") since it yielded coproporphyrin I on decarboxylation, and a secondzone (ester m. p. 208') believed from analytical data to be a heptacarboxylicporphyrin (of the isomeric series I11 since it yielded coproporphyrin I11 ondecarboxylation) . Even chromatographically homogeneous Waldenstromesters were stated to yield mixtures of the I and the I11 series coproporphyrinson decarboxylation. Support for these claims has been offered by P r ~ n t y . ~ ~ ~Watson's view is that the I and I11 series porphyrins in the Waldenstromesters form molecular associations which crystallise as an individual material,but the contradictory decarboxylation results obtained by Waldenstrom andMertens on the one hand, and Watson and Prunty on the other, are difficult toreconcile.The identity of the uroporphyrin in turacin has also been called in question.Fischer and Hilger 65 carried out no decarboxylation of their supposeduroporphyrin I ; Rimington 70 found that turacins from eleven differentspecies of Turacos yielded only coproporphyrin 111 when decarboxylated.This has recently been confirmed by Nicholas and Rimington 71 who haveprepared unequivocal uroporphyrin 111 in pure state from turacin.It hasm. p. 264", similar to that of the Waldenstrom ester.Porphobilinogen.-In acute porphyria urines, Waldenstrom 72 detected asubstance, porphobilinogen, a colourless precursor of uroporphyrin.Furtherstudies 73 suggested that porphobilinogen had a molecular weight (by diffusion)of about 350 and thus contained only two pyrrole rings; when this materialwas heated in acid solution, union of two molecules took place. The mainproducts of this reaction were an amorphous pigment, porphobilin (probablyrelated to urobilin) and uroporphyrin 111, together with some of the I isomer.Porphobilinogen can be detected by the formation, on treatment withEhrlich's aldehyde reagent, of a red pigment not extractable by chlor~form.~~Its excretion appears to be characteristic of acute porphyria, Hammond andWelcker 75 having found no false positive reactions in urine of 1000 casesexamined,The Ehrlich reaction affords a method for quantitative determination.76The conversion into urDporphyrin has been the subject of special studies.77* 76That the porphyrin obtained from porphobilinogen-containing urines by6 8 Grinstein, Schwartz, and Watson, J . Biol. Chem., 1945, 157, 323; Watson,Schwartz, and Hawkinson, ibid., p. 345.6B Arch. intern. Med., 1946, 77, 623.7 1 Personal communication, 1950.73 Waldenstrom and Vahlquist, 2. physiol. Chern., 1939, 260, 189.7 5 J . Lab. Clin. Med., 1948,33, 1254.76 Jorgensen and With, Nord. Med., 1945, 27, 1341; Prunty, Biochem. J . , 1945,'I7 Grieg, Askevold, and Sveinsson, Scand. J . Clin. Lab. Invest., 1950,2, 1.70 PTOC.Roy. SOC., 1939, B , 127, 106.72 Acta Med. Scand., 1934, 83, 281.Watson and Schwartz, Proc. SOC. exp. Biol., 1941,47, 393.39, 446280 ORGANIC CHEMISTRY.boiling is not the same as that present in the fresh urine is suggested byGibson and Harrison.78 .Biosynthesis of Porphyrins.-The last ten years have seen a remarkableincrease in knowledge concerning the materials and methods utilised byliving cells in synthesising substances containing the porphin ring. Isotope-labelling techniques have been almost entirely responsible. The N atom ofglycine is specifically utilised for haem production in man 79 and in avian andimmature mammalian erythrocytes in vitro. The rate of disappearanceof labelled haem from the blood stream in the first case leads to the conclusionthat haemoglobin is outside the general metabolic interchange, and alsopermits an estimate of normal red-cell longevity.81 Since rings (I and 11)and (111 and IV) of haem are labelled to an equal extent, it is probable thatglycine supplies the nitrogen of all four pyrrole rings.82 Carbon labellingshowed that the methylene- 83* but not the carboxyl-carbon atom 83* 85 ofglycine is incorporated in haem.Both carbon atoms of acetic acid are alsospecifically utilised,86 making a contribution, most probably, to the pyrroleP-side chains. The methene carbon atoms of the porphin ring are derivedfrom gly~ine.~' By means of a careful, step-wise degradation of the haeminsynthesised by avian erythrocytes in the presence of N- and a-C-labelledglycine, Wittenberg and Shemin 88 have localised the C atoms derived fromglycine as in (XV).The major steps in the degradation were as follows :fro, Haemin -+ Protoporphyrin --+ MesoporphyrinEthylmeth ylmaleinimidefrom rings I & I1E t h ylme th ylmaleinimide --C%Haematinic acidNaClO, E thylmethylmaleinimides (separately) oso,> EthylmethyltartarimideEt*CO,H =MnO4 x-Ketobutyric Pyruvicacid acid78 Biochem. J . , 1950, 46, 154.70 Shemin and Rittenberg, J. BioE. Chem., 1946, 166, 621. 'Shemin, London, and Rittenberg, ibid., 1950, 183, 749, 757.Shemin and Rittenberg, ibid., 1946,166, 627.8a Muir and Neuberger, Biochem. J., 1949,45, 163; Wittenberg and Shemin, J. BioE.83 Radin, Rittenberg, and Shemin, &id., 1950,184, 745.Chem., 1949,178,47.Altman, Casarett, Masters, Noonan, and Salomon, ibid., 1950, 176, 319; Altman,Salomon, and Noonan, ibid., 1949,177,489 ; Altman and Salomon, Science, 1950,111,117.8 5 Grinstein, Kamen, and Moore, J .Biol. Chem., 1948, 174, 767; 1948,179, 359.86 Bloch and Rittenberg, ibid., 1945,159, 45 ; Radin, Rittenberg, and Shemin, ibid.,1950, 184, 755 ; Pontecorvo, Rittenberg, and Bloch, ibid., 1949,179, 839.87 Muir and Neuberger, Biochem. J., 1949, 46, xxxiv; 1950, 47, 97; Wittenbergand Shemin, Fed. Proc., 1950, 9, 247. J . Biol. Chem., 1950,185, 103FALK AND RIMINGTON : PORPHYRINS. 281Radioactivity * was confined to the carboxyl-carbon atom of a-ketobutyricacid and the carbon dioxide arising from the four methene carbon atoms of theCH,:CH MeMe/\\Y+\\/%H:CH, ~LNH NA (0 indicates C derived from h (XV.) methylene of glycine.)HO,C*CH,*CH, CH,*CH,*CO,Hring in the initial chromic acid oxidation.The fact that eight glycinea-carbon atoms are utilised for every four nitrogen atoms 89 indicatesthe occurrence of deamination reactions to provide a C, residue. Neitherformate nor carbon dioxide is utilised for haem synthesis by thissys ternLemberg and Legge 3a and others 91 have reviewed various theories of thebiosynthetic mechanism. Neuberger, Muir, and Gray 92 recently suggestedthat the initial stage is the union of 2 molecules of a-ketoglutaric acid and1 molecule of glycine to give a highly carboxylated pyrrole derivative (XVI)from which the two a-carboxyl groups are then lost, to give (XVII).Addi-tion at one or other a-position of a two carbon fragment (e.g., glyoxylic acid)is then thought to occur, to give, after decarboxylation, either A (XVIII)or B (XIX). Porphyrin formation is postulated by condensation of foursuch units (cf. Siedel and Winkler lo).HO,C*CH, CH,*CH,*C02H I H02C-CH, CH,*CH,*CO,H(XVII .)H02C1Q1C0,H H (XVI.1 \g'If no restriction is placed on this final stage, all four porphyrin isomers couldbe expected. Since only types I and I11 are found in nature, it is postulatedthat in the condensation, the M (potential methene) groups of B but not ofA can be activated enzymically, while both units can act as acceptors of thepotential methene carbon atoms. Such a condition would preclude A-Alinkage and give rise to porphyrin molecules of only the I or the I11 series, thelatter predominating.Isotopic studies have been made of the biosynthesis of porphyrins andRadin, Rittenberg, and Shemin, Fed.Proc., 1949, 8, 240.Bufton, Bentley, and Rimingtoh, Biochem. J . , 1949,44, xlix.01 Shemin, Cold Spring Harbor Symp., 1948, 13, 185; Maitland, Quart. Reuiews,1950,4,45; Rimington, ref. 3b. 9a Nature, 1950, 165, 948282 ORGANIC CHEMISTRY.bile pigments in porphyria and other diseasesQ3 and of bile pigments innormal man.94 The conversion of injected haematin into bile pigment 95has also been demonstrated by this means. The biosynthesis of chlorophyllhas been studied; both glycine and acetate are specifically utilisedjS6 andprotoporphyrin Mg complex, Mg vinylphaeoporphyrin-a5, and protochloro-phyll have been identified as intermediate The relation betweenthe biosynthesis of porphyrins and haems by C .diptheriae has been investi-gated.gs An important role of lactoflavin in regulating the porphyrinproduction of yeast is described by S t i ~ h . ~ ~ Pyridoxine 99a, and folic acid andvitamin B,, s9b may also be important in porphyrin biosynthesis.Physicochemical Aspects.-The infra-red spectra of coproporphyrins I andI11 and stercobilin,lm and of zetioporphyrin I lol have been measured.Vestling and DowninglO1 found a band at 3320 cm.-l in aetioporphyrinin carbon tetrachloride solution which was considered lo1a to be due to a NHvibration lowered by H bonding. This has been confirmed by Willis andFalk,lolb using protoporphyrin; it was found that there is only a smallfrequency shift (from 3320 to 3280-3200 cm.-l) for the NH vibration ongoing from dilute carbon tetrachloride solution to the solid state.Infra-redspectra can distinguish porphyrin isomers of the I and I11 series,100. l o l b andtentative assignments have been made for the frequencies of different typesof carbonyl groups in porphyrin side chains.lolb Rabinovitch lo2 attempteda theoretical interpretation of the absorption spectra of porphyrins andchlorophyll. Kuhn lo3 has calculated porphyrin spectra on the basis of hisuniform-potential, free-electron-gas model ; this has been criticised byDewar.lm Simpson lo5 made a quantum-mechanical interpretation somewhatsimilar to Kuhn's.The molecular-orbital method has been applied byLonguet-Higgins, Rector, and Platt lo6 to porphin and tetrahydroporphin.83 London, West, Shemin, and Rittenberg, J. Biol. Chem., 1950,184,365; Neuberger,Muir, and Gray, Nature, 1950, 165, 948 ; Grinstein, Aldrich, Hawkinson, and Watson,, J . Biol. Chem., 1949, 179, 983 ; Gray and Neuberger, Biochem. J., 1950, 47, 81 ; Gray,Neuberger, and Sneath, ibid., p. 87; Grinstein, Wikoff, de Mello, and Watson, J. Biol.Chew., 1950, 182, 723; London, Shemin, West, and Rittenberg, ibid., 1949, 179, 463;London and West, ibid., 1950,184, 359.84 London, West, Shemin, and Rittenberg, ibid., p. 351.9 5 London, ibid., p. 373.Q6 Salomon, Altman, and Della Rosa, Fed. Proc., 1950,9, 222.Gilder and Granick, J.Gen. Physiol., 1947, 31, 103; Granick, J. Biol. Chem.,Q* Hale, Rawlinson, Gray, Holt, Rimington, and Wilson Smith, Brit. J. Exp. Path.,OD Naturwisa., 1950, 9, 212; Deut. med. Woch., 1950, 37, 1217; Stich and Eisgruber,1948,172, 717; 1948,175,333; 1950,183, 713.1950, 31, 96.Klin. Woch., 1950, 28, 133.Cartwright and Wintrobe, J. Biol. Chem., 1948, 172, 557.BOnard, Gajdos, and Gajdos-Torok, Compt. rend. SOC. Biol., 1950, 144, 38, 350.loo Gray, Neuberger, and Sneath, Biochem. J., 1950, 47, 87.lor Vestling and Downing, J. Amer. Chem. SOC., 1939, 61, 3511.lola Ruswell, Downing, and Rodebush, ibid., p. 3252.lol* Willis and Falk, personal communication, 1950.lo2 Rev. Mod. Physics, 1944,16, 226.lo' J., 1950, 2329.lo9 J .Chem. Physics, 1949,17, 1198.lo6 J . Chem. Physics, 1949, 17, 1218. lo6 Ibdd., 1950, 18, 1174BALK AND RIMINQTON : PORPHYRINS. 283Preliminary X-ray crystallographic analysis lo’ shows that tetramethyl-haematoporphyrin, like the phthalocyanines, is probably planar. A verybrief X-ray examination has also been made of aetioporphyrin I crystals.lo8From X-ray crystallographic studies of haemoglobin, the molecule appearsto be composed of superposed layers of folded polypeptide chains, the haemresidues being attached tangentially ; Perutz’s log* 3* data indicated fourpolypeptide layers in the structural unit, but Dornberger-SchS’s 110calculations of Patterson and Fourier projections suggest a unit of 7layers. Myoglobin 1 1 1 s 3 b appears to be closely analogous in structure tohaemoglobin.In studies 112 of the photo-oxidation of the zinc complex of tetraphenyl-chlorin evidence has been found for an intermediafe triplet state of the chlorinmolecule when p-naphthaquinone is the oxidising agent.This chlorin isoxidised by o- or p-quinones to the corresponding porphyrin, the zinccomplex eight times as fast as the magnesium complex; oxygen also acts asoxidising agent in a similar manner, a secondary reaction with hydrogenperoxide leading, however, to further oxidation. In a study of substanceswhich do and do not quench the fluorescence of chlorophyll-a, it was found 113that those which quench most efficiently are oxidising agents. The complexstructure of the fluorescence spectrum of the magnesium complexes ofphthalocyanine and chlorophyll has been studied.l14Clark and his collaborators have continued their systematic studies of thedissociation constants of ferriporphyrins and of other metalloporphyrins.Values of pK have been determined for the dissociation of ferriproto-porphyrin hydroxide (hydroxyhaemin) 116* 117 and of nicotine ferro-porphyrins (haemochromogens) .l18 Older values for the haemochromogensformed by ferroprotoporphyrin with a number of different bases have beensummarised.llQ A value has been given for the dissociation of hydroxylfrom ferricoproporphyrin hydroxide, and for the dissociation of hydroxylfrom some ferriporphyrin base hydroxides (hydroxymethaemochromo-lZo Lemberg 1-21 has given a pK value for pyridine protomethaemo-chromogen. Because the equilibrium involved is not well understood, pKvalues are not useful as an index of the affinity of bases for ferriporphyrins;it had been found, however, that the linkage of bases to ferroprotoporphyrinlo7 O’Daniel and Damaschke, 2.K r i ~ t . , 1942, 104, 114.lo8 Robertson, Amer. Min., 1942, 27, 219.loo Proc. Roy. SOC., 1949, A , 195, 474.ll1 Kendrew, Proc. Roy. SOC., 1950, A , 201, 62.112 Calvin and Dorough, J . Amer. Chem. SOC., 1948,70,699; Huennekens and Calvin,113 Livingston and Chun-Lin Ke, ibid., 1950, 72, 909.11‘ Gachkovskii, Doklady Akad. Nauk. S.S.S.R., 1950, 71, 509.116 Clark, Cold Spring Harbor Symp., 1939,7, 1.116 Clark and Perkins, J. Biol. Chem., 1940,135, 643.118 Davies, ibid., 1940, 135, 597.120 Vestling, J . Biol. Chem., 1940, 135, 623.121 Lemberg and Foulkes, quoted in ref. 3a, p. 178.1 1 O Acta Cryetall., 1950, 3, 143.ibid., 1949, 71, 4024,4031.Shack and Clark, ibid., 1947, 171, 143.119 Ref. 3a, p. 175284 ORGANIC CHEMISTRY.(haem) is stronger than to ferriprotoporphyrin hydroxide,122 and it has beenpossible 123 to calculate the approximate extent to which the affinity of ferro-porphyrins for bases is greater than that of ferriporphyrins.The (ferrous-ferric) oxidation-reduction potentials of iron-porphyrincompounds have been studied very intensively, the haem-haematin systemand the haemochromogen-methaemochromogen system by Barron lZ4 andby Clark’s school.l18* l2Ov 122,125 Lemberg and Legge have discussed Clark’stheoretical treatment in detai1.126 Systems where the base is protein innature have also been studied, e.g., haem~globin,~~~ myoglobin,12* and cyto-~hr0me-c.l~~The combination of cyanide and alkyl cyanides, and of carboalkylamineswith ferro- and ferri-porphyrins has been further studied.130

ISSN:0365-6217    

DOI:10.1039/AR9504700126

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

BIOCHEMISTRY.1. INTRODUCTION.DURING the past few years these Reports on biochemistry have usuallyconsisted of articles on special topics of current interest. Now that theChemical Society publishes Quartdy Reviews such articles might perhapsappear more conveniently in this new publication. An attempt will thereforebe made to report on larger fields of biochemistry. As space is limited,however, the subjects will not be covered each year but rather on a triennialbasis. Thus the subjects of hormones, nutrition, and microbiology aredivided into three parts and it is hoped that one part of each will appear eachyear. Although the subject will not be covered so completely as in AnnualReviews of Biochemistry, the Reports should give the main themes of develop-ments in different branches of the subject.E..B.2. ANTIBIOTICS.Introduction.-When the antibiotics were first reviewed in these Reportsseven years ago the discovery of the therapeutic power of penicillin hadbegun to stimulate widespread interest in their properties, and since. that timethey have been the subject of an immense number of publications. Theliterature to the end of 1948 has been discussed elsewhere in some detailand many of the publications in 1949 have also been r e ~ i e w e d . ~ * ~ Thepresent article deals mainly with the salient aspects of recent work.The study of an antibiotic may be carried through a number of more orless distinct stages, concerned with : (1) detection, (2) production, (3) isol-ation, (4) chemical structure and synthesis, ( 5 ) antimicrobial and pharma-cological properties, (6) mode of action, and (7) therapeutic use in animals orman.Most of the research is undoubtedly prompted by the hope of findingnew substances of value to medicine. From this point of view it has provedhighly rewarding, for penicillin, streptomycin, chloromycetin, aureomycin,and terramycin are powerful therapeutic agents, and the list of such substancesis likely to grow. Other aspects of the subject, however, should not beneglected. Only a very small proportion of the antibiotics discovered evercome into the hands of the clinician, but among those that for one reason oranother find no place in medicine are many of considerable interest inchemistry or biology.The ability to produce one or more antibiotics is a rather common propertyof mkro-organisms and the mere detection of antagonism between organismseasily grown in vitro often presents little difficulty.By relatively simpleChain and Florey, Ann. Reports, 1943,40, 180.H . W. Florey, Chain, Heathy, Jennings, Sanders, Abraham and M. E. Florey,Carter and Ford, Ann. Rev. Biochem., 1950,19,487.Herrell, Ann. Rev. Microbiol., 1950, 4, 101.“ Antibiotics,” 1949, Oxford Univ. Press286 BIOCHEMISTRY.methods large numbers of fungi,5 actinomycetes,s* and bacteria have beensurveyed in the laboratories of academic institutions and commercial firms.The detection of antibiotics that are active against animal viruses is moredifficult, but a simple technique has been introduced for finding substancesthat affect the action of bacteriophage^.^Before 1944 antibiotics were normally produced by growing organismsin shallow layers of stationary media.The commercial production of peni-cillin was greatly facilitated when it was found possible to use media in deeptanks under conditions of vigorous stirring and aeration. Deep ferment-ation is now the method of choice for antibiotic production and has beenemployed with fungi imperfecti,1° actinomycetes,ll bacteria,l2 and with abasidiomycete,l5 but it does not always prove succe~sful.~~There is so far little information about the biosynthesis of any antibiotic.The way in which the essential features of the penicillin molecule, for example,are put together is still unknown.15 The preferential formation of a partic-ular kind of penicillin that can be induced by addition, to the culture medium,of compounds containing the side chain of the penicillin concerned l6 appearsto be the only case in which biosynthesis has been influenced in a rationalmanner by the addition of a precursor of known structure, although it hasbeen reported that a strain of R.Zicheniformis can be induced to form alicheniformin-like substance instead of ayfivin (bacitracin) by changing thecarbon : nitrogen ratio of the rnedium,l7 and that glycine and acetate areused in the synthesis of prodigiosin.18 In general the best culture media areonly found by trial and error.Work on the production of penicillin emphasised that the yield of anti-biotic may vary not only with the medium but with the strain of micro-organism used, and that valuable new strains may sometimes be obtainedby mutagenic agents.lg A strain of Streptomyces griseus that had lost itsability to form streptomycin yielded a mutant on treatment with X-raysHervey, Bull.Towey Bot. Club, 1947,74,476; Wilkins, Brit. J . Exp. Path., 1948,29, 364; Lacey, J . Qen. Microbiol., 1950, 4, 122.Waksman and Lechevalier, Science, 1949, 109, 305.7 Kane, Finlay, and Sobin, Ann. N . Y . Acad. Sci., 1950, 53, 226; Cerdos andRosemblit, Rev. Argentina Agon., 1950, 17, 98.8 Fredericq, Compt. rend. SOC. Biol., 1950, 144, 986; Gilliver, Brit. J . Exp. Path.,1949, 30, 214; Gardner, ibid., 1950, 31, 102; Sherwood, Russell, Jay, and Bowman, J .Infect.Dis., 1949, 84, 88.O Asheshov, Strelitz, and Hall, Brit. J . Exp. Path., 1949, 30, 175.lo Peterson, Harvey Lectures, 1946/47, 42, 276; Brown and Peterson, Ind. Eng.Chem., 1950,42, 1769.l2 Newton, Brit. J . Exp. Path., 1949, 30, 306; Stansly, Schlosser, Ananenko, andCook, J . Bact., 1948, 55, 573; Garibaldi and Feeney, I n d . Eng. Chem., 1949, 41, 432;Humfeld, J . Bact., 1947, 54, 689.l1 Bartz, J . Biol. Chem., 1948,172, 445.lS Gill-Carey, Brit. J . Exp. Path., 1950,31, 30.l5 Sus, Annalen, 1950, 569, 153. l4 Gardner,ibid., 1949, 30, 130.l6 Behrens in “ The Chemistry of Penicillin,” 1949, Princeton Univ. Press.l 7 Hills, Belton, and Blatchley, Brit. J . Exp. Path., 1949, 80, 427.lo Backus, Stauffer, and Johnson, J . Amcr. Chem.SOC., 1946,88, 152; Foster, 1949,Hubbard and Rimington, Biochem. J., 1950, 46, 220.U.S.P. 2,458,495ABRAHAM AND NEWTON : ANTIBIOTICS. 287which was a good producer of the drug.2o On the other hand the claim thatthe ability of a strain of B. mesentericus to produce an antibiotic was enhancedby animal passage could not be confirmed.21The antibiotics fall into a large variety of chemical types and any newmethod for purifying organic substances may find some application in theirisolation. Partition chromatography was adapted to the purification of thepenicillins soon after its introduction into biochemistry,22 and was later usedwith the polymyxins 23 and with marcescin.M Chromatography on paper,followed by the determination of the position of the active substances bychemical or microbiological techniques, has been increasingly used for thecharacterisation of antibiotics and the analysis of mixtures.25* 26 Counter-current distribution between solvents 27 has proved particularly useful inthe purification of antibacterial polypeptides not yielding readily to othermethods 28-30 and has shown, for example, that crystalline preparations ofgramicidin may be heterogeneous.An important extension of this procedure,using a carrier (a long-chain acid or base) to bring the substance (a water-soluble base or acid) into an organic phase in which it is not normally soluble,arose out of work on the separation of streptomycin and mannosidostrepto-m ycin .3Only a small proportion of the antibiotics detected have been obtained ina homogeneous state or assigned precise chemical structures.Since anti-biotics are often named when little is known of their properties it may some-times happen that different names are given to the same compound. Someof the names in the literature have thus already fallen into disuse and othersare likely to do so.Antibiotics from Fungi.-Penicillin is still the most interesting andimportant of the antibiotics obtained from fungi. Substances such aspenicillic acid, discovered in early work on the metabolic products of moulds,continue to be isolated from different species.32 A new structure has beenintroduced for patulin, and puberulic and puberulonic acids have been shownto contain the tropolone ring system postulated in 1945.33 Several new2o Waksman and Harris, Proc.SOC. Exp. Biol., 1949, 71, 232.21 Savage and H. W. Florey, Brit. J. Exp. Path., 1950, 31, 14.22 Abraham, W. Baker, Chain, and Robinson in “ The Chemistry of Penicillin,”Bell, Bone, English, Fellows, Howard, Rogers, Shepherd, Winterbottom, Dornbush,1949, Chap. 2, Princeton Univ. Press; Boon and Carrington, op. cit., Chap, 5 .Kushner and SubbaRow, Ann. N.Y. Acad. Sci., 1949, 51, 897.24 Fuller and Horton, J. Gen. Microbiol., 1950, 4, 417.25 R e p a and Murphy, J. Amer. Chem. SOC., 1950,72, 1045.26 Kluener, J. Bact., 1949, 57, 101 ; Karnovsky and Johnson, Analyt. Chem., 1949,21, 1125; Peterson and Reineke, J. Amer. Chem. SOC., 1950, 72, 3598; Catch, Jones,and Wilkinson, Ann. N . Y . Acad. Sci., 1949,51, 917.27 L.C. Craig and D. Craig in “ The Technique of Organic Chemistry,” Vol. 111, p.17 1, Interscience Publ., N.Y., 1950.28 L. C. Craig, Gregory, and Barry, Cold Spring Harbor Symp. Quart. Biol., 1949,14,24.sB Work and Callow, 1950, personal communication.Newton and Abraham, Biochem. J., 1950,47, 257.31 Plaut and McCormack, J. Amer. Chem. SOC., 1949,71,2264; O’Keeffe,Dolliver,and32 Burton, Nature, 1950,165,274. 33 Dewar, ibid., 1945,165,50. Stiller, ibid., p. 2452288 BIOCHEMISTRY.antibiotics have been isolated from basidiomycetes, but with one exceptionlittle is yet known of their structure.Penicillin. There appear to be few outstanding publications on theproduction and chemistry of the penicillins that have not recently beenreviewed.29 3* 34 A satisfactory synthesis is still awaited.Butyric, valeric, hexanoic, and hex-3-enoic acids * can be used as pre-cursors to stimulate the biosynthesis of penicillins containing correspondingn-alkyl side chains by P .chrysogenum Q 176. Biosynthetic n-propyl- andn- butyl-penicillins appear identical with two penicillins formed normally ina synthetic medium.35 5-Chloro- and 5- bromo-2-thienylacetic acids havebeen found to stimulate penicillin prod~ction.~~ New chemical proceduresfor estimating penicillin have been described.37It has been reported that penicillin F may be separated from penicillinG by precipitation as its beryllium salt at pH Penicillin K has beenisolated by continuous counter-current solvent fractionation39A unit has been suggested for penicillinase, the enzyme that inactivatespeni~illin.~O Several compounds, including 2- benz ylglyoxaline and penicill-nmine (P-mercaptovaline) have been found to inhibit the action of penicill-inase.While penicillamine increased the action of penicillin against thepenicillinase-producing B. cereus, however, 2- benzylglyoxaline did not .41The surprising report that after treatment with penicillinase penicillin ispartly reactivated by injection into rabbits would appear to need furtherinvestigation.42Patulin. Strong evidence was recently obtained that this antibiotic hasthe structure (I) instead of the previously accepted structure (II).43 Thishas now been confirmed by the synthesis of patulin in small yield from thelnctol acetate (III)?4 aZloPatulin (11) has also been synthesised.434 “ The Chemistry of Penicillin,” 1949, Princeton Univ.Press ; Wintersteiner andDutcher, Ann. Rev. Biochem., 1949,18, 559.35 Thorn and Johnson, J . Amer. Chem. Soc., 1950,72,2052.36 Ford, Prescott, and Colingsworth, ibid., p. 2109.37 Ortenblad, Acta Chem. Xcand., 1950, 4, 518 ; Knoll, 2. Bakt., Abt. I (Orig.), 1950,155,99; Odo and Hirano, J . Agric. Chm. SOC. Japan, 1950,23,237.3839404 14243238 ;44 *C. Pfizer & Co. Inc., B.P. 633,660/1949.Bartels and Dolliver, J . Amer. Chem. SOC., 1950,72, 11.Levy, Nature, 1950, 166, 740.Behrens and Garrison, Arch. Biochem., 1950, 27, 94.Irrgang and Dornbrack, 2. physiol. Chem., 1950,285, 17.Woodward and Singh, J .Amer. Chem. SOC., 1949, 71, 758 ; Experientia, 1950, 6,Engel, Brzerki, and Plattner, Helv. Chim. Acta, 1949,32, 1166.Woodward and Singh, J . Amer. Chem. SOC., 1950, 72, 1428, 5352.Geneva nomenclature, CO,H = 1ABRAHAM AND NEWTON : ANTIBIOTICS. 289Puberulic and Puberuhic Acids. Although these antibiotics were dis-covered in 1932 there has hitherto been no satisfactory structure for thembased on experimental evidence. Puberulic acid has now been shown toyield aconitic acid on oxidation with alkaline hydrogen peroxide, and hasbeen assigned the structure (IV).45 It is a hydroxystipitatic acid.HO 40 HO //o 0yj-C J&-\\ / / \\ od'=" ' d"" I&H &H 06, yoH o f T O H\-(IV-) (V.1 (VI). 0Puberulonic acid, whose molecular formula has been amended to C,H40,,yields puberulic acid and carbon dioxide when heated in aqueous acid.Puberulonic acid was at first thought to have a structure such as (V),4s butthe anhydride structure (VI) has now been proposed to account for itsultra-violet absorption spectrum and behaviour on t i t r a t i ~ n .~ ~ Structure(VI) is supported by the infia-red absorption spectra of puberulonic acid andrelated 47Baccutine A . This substance has been isolated from the mycelium of aspecies of Fusarium (Gibberelk baccuta;). A possible molecular formula isC,,H,,O,N,.Cordycepin. An antibiotic named cordycepin, with the molecularformula C,,H,,O,N,, has been obtained in crystalline form from Cordycepsmizitcaris (Linn) Link.49The culture fluids of the basidiomycetes,Poria corticola, Poria tenuis, and a fungus from white cedar have been foundto contain two similar antibacterial substances named nemotin and nemotinicacid.s0 These substances cannot be handled out of solution since theydecompose in the solid state. Both show similar absorption spectra withfour high peaks between 230 and 290 mp.I n aqueous solution above pH6 nemotin changes into another neutral substance called nemotin A.Nemotin and nemotinic acid inhibit the growth of Staph. aurew in highdilution but are much less active against Bact. coli and Bact. Friedlunderi.Nemotin is much more active than nemotinic acid as an antifungal agent.This antibiotic has been isolated from the culture fluid ofAgrocybe dura, where it occurs partly in an inactive form that can be activatedby boiling.s1 It yields white crystals which blacken in air but are stable at4" in vacuo.It appears to be neutral orweakly acidic and its ultra-violet absorption spectrum is similar to that ofIt is active against Shph. aureus and a number of fungi?*Nemotin and Nemotinic Acid.Agrocybin.Analysis gave : C, 65.6 ; H, 4.3%.Is Corbett, Johnson, and Todd, J., 1950,6, 147.I6 G. Aulin-Erdtman, Acta Chem. Scand., 1950,4, 1326.4 7 Shepard, Johnson, and Todd, personal communication.40 Cunningham, Manson, and Spring, Nature, 1950,166, 949.50 Anchel, Polatnick, and Kavanagh, Arch. Biochem., 1950, 25, 208; Ksvanagh,Guerillot-Vinet, Guyot, MontBgut, and ROUX, Compt. rend., 1950, 230, 1424.Hervey, and Robbins, PTOC.Nat. Acad. Sci., 1950,36, 1. 51 Idem, ibid., p. 102.REP.-VOL. XLVII. 290 BIOCHEMISTRY.nemotin A. Agrocybin is highly aotive against Stuph. aureus, Bact. mli, andMyco. phlei and is also antifungal, but its activity is greatly reduced byhuman blood. It is highly toxic to mice and liable to cause dermatitis inman.Grri,foZin. An antibiotic, C,,H,,O,, obtained in crystalline form from thesporophores of the basidiomycete Grifola conJEuens has been namedgrifolin.62 The results of hydrogenation and degradations with ozone,potassium permanganate, and lead tetra-acetate provide evidence for thestructure (VII). Grifolin inhibits the growth of Xtuph. aureus and some(~11.1 CMe,:CH*CH,-CMe:CH*CH :CHOCK (OH ) CMeE t OHacid-fast bacteria, but is inactive against grambnegative organisms.Itshows low toxicity in mice.IZZudin. Two antibiotics produced by Clitocybe illudens have been iso-lated in the crystalline state and called illudin M and illudin S. They areneutral and have the molecular formula C15H20O3 and C15H,,0, respectively,They both show considerable activity against mycobacteria, but they arehighly toxic to mice and are liable, like agrocybin, to cause dermatitis inman. 53An antibiotic with quinonoid properties has been iso-lated from the culture fluid of the basidiomycete Zenxites trabea (Pers) Fr.(Lenxites thermophila) and named thermophillin. Its probable formula isC,,H605(OMe), and it shows weak activity against Xtaph. uureus.54This antifungal substance has now been separated into isomericCI- and p-viridin.Both compounds are thought to have the molecularformula C,9H,60,.55AZtemrine. A substance called alternarine has been isolated from theculture fluid of AZternaria sohni in the form of white crystalline needles.s6It is active against a number of gram-positive and gram-negative bacteriaand phytopathogens.Miscellaneous. Antibiotics have also been obtained from Pusariumbostry~oides,~~ Aspergillus japonicw,6a and Marasirnus ureus. 69Antibiotics from Actinowcetes.-Following the discovery of strepto-mycin the ability of the actinomycetes to produce antibiotics has probablybeen examined more extensively than that of any other group of micro-organisms. Many thousands of strains have been surveyed, in some caseswith the particular object of detecting substances active against Mpco.tabercuZosis.6 Three antibiotics-chloromycetin, aureomycin, and terra-mycin-that have emerged from these studies in recent years have provedThermphiZZin.Viriddn.58 Hirata and Nakanishi, J.Biol. Chem., 1950,184, 135.53 Anchel, Hervey and Robbins, Proc. Nut. A&. Sci., 1950, 86, 300.54 Burton, Nature, 1950,166, 570.5 5 Vischer, Howland, and Raudnitz, ibid., 1950, 165, 528.66 Darpoux, Farvre-Amiot, and ROUX, Compt. rend., 1960, 230, 993.5 7 Cajori, Hamilton, Urbanish, and Purshottam, Fed. PTOC., 1950, 9, 158.58 Aketsu, J . Agric. Chem. SOC. Japan, 1950,23,343.E)blom, Mdure, 1950,166, 950ABRAHAM AND NEWTON : ANTIBIOTICS. 291effective in man against infections with a number of organisms insensitive topenicillin ; a fourth, neomycin, is undergoing clinical trial.Chloromycetin,which is remarkable in being a natural compound containing a nitro- and adichloroacetyl group, proved to have a simple constitution and is now readilyobtained synthetically. The structure of the other antibiotics is either notyet known or has not been revealed.The production, chemistry, and use of streptomycin hasbeen described in detail in recent review^.^^ go Dihydrostreptomycin has beenisolated in the form of the crystalline free base.61 Streptidine has beensynthesised from D-glucosamine. 62 Residues from the purification ofstreptomycin have been found to contain a streptomycin derivative that ishighly toxic when injected intravenously into mice.63NH[CH(oH)*st12 The derivative is di-( whydroxystreptomycy1)amine(VIII ; St is streptomycin less CHO), and it has beensynthesised by warming a concentrated solution of streptomycin hydro-chloride containing ammonia.Hydroxystreptomycin.Strephyces griseo-carneue, isolated from aJapanese soil, produces a streptomycin-like antibiotic which has been isolatedas a crystalline helianthate and converted into the trihydr~chloride.~* Itwas distinguished from streptomycin only by paper chromatography. Thenew substance, which also appears to have been obtained from a strain ofStreptomyces found in soil a t Illinois,65 contains an additional oxygen atomin the streptose portion of the molecule; this fragment has the structure(IX), instead of (X) as in streptomycin.Streptomycin.(VIII.)-0-CHI 3 T H HC-O-Neomycin. A basic antibacterial substance obtained from a strain ofStreptomyces related to S.fradiue was named neomycin.6 It was later shownby counter-current distribution to contain a t least three active substances andwas called the neomycin complex.6sOne component of the neomycin complex, neomycin A, has been isolatedas a crystalline p(phydroxypheny1azo)benzenesulphonate. The regener-ated hydrochloride shows only end absorption in the ultra-violet region, aWaksman, “ Streptomycin-Its Nature and Practical Application,” 1949,Williams and Wilkins Co., Baltimore, Maryland ; Brink and Folkers, Adu. EnzymoEogy,1950, 10, 145.Rhodehctmel, McCormick, and Kern, Science, 1950,111, 233.62 Wolfrom, Olin, and Polglase, J .Amer. Chem. SOC., 1950, 72, 1724.63 Solomons and Regna, ibid., p. 2974.e5 Grundy, Schenck, Clarke, Hargie, Richards, and Sylvester, Arch. Biochem.,Benedict, Stodola, Shotwell, Borud, and Lindenfelser, Science, 1950, 112, 77.1950,28,160. e6 Swart, Hutchinson, and Waksman, ibid,, 1949,24, 92292 BIOCHEMISTRY.positive ninhydrin test, and negative glucosamine, maltol, and Sakaguchitests. 67 Other workers obtained crude neomycin whose dominant componentwas different from neomycin A and was called neomycin B.26Neomycin is active against many Gram-positive and Gram-negativeorganisms and especially against mycobacteria. It has a strong bactericidalaction. Resistant organisms have been said to develop less readily to neo-mycin than to streptomycin,6* 68 although this has been disputed in the caseof Myco.tubercul~sis.~~ The substance has a relatively low toxicity, butrecent experience indicates that it can damage the kidneys.70* 71 The LD,,,when given subcutaneously to mice, is 450 mg./kg. It is reported to be moreeffective than streptomycin in suppressing infections in mice with Xtaph.aurew, 8. schottmuleri, and S . t y ~ h i . ~ ~Fradicin. This antibiotic, which is also produced by X. fradiae, is activeagainst fungi but not against bacteria.73Chloramphenicol (Ghloromycetin). An antibiotic was isolated from theculture fluid of a species of Streptomyces and given the trade name “ chloro-mycetin.” 11 Degradation and synthesis showed that this substance was( - )-~-threo-2-dichloroacetamido- 1 -p-nitrophenylpropane- 1 : 3-diol (XI).Itis now known by the trivial name chl~ramphenicol.~~ A cylinder-plate assayand a polarographic procedure for estimating chloramphenicol have beendescribed . tiH NH*CO*CHCl,O , R ’ < ~ > - ~ ~ $ - C H ~ * O H (XI.)Chloramphenicol inhibits the growth of a wide range of pathogens at adilution of a t least 1 in lo6, including members of the genera Streptococcus,Corynebacterium, Escherichia, Pasteurella, Salmonella, Shigellu, and Vibrio,and several fungi. Treatment of infected chick embryos showed that it isalso active against Rickettsiae and some of the larger viruses, including theagents responsible for epidemic typhus, scrub typhus, and lymphogranulomavenereum, but that it is ineffective against most of the smaller viruses.7667 Peck, Hoffhine, Gale, and Folkers, J .Amer. Chem. SOC., 1949, 71, 2590.68 Waksman, Katz, and Lechevalier, J. Lab. Clin. Med., 1950, 36, 93; Weiss andWaksman, Proc. Nut. Acud. Sci., 1950, 36, 293; Jen-Yah Hsie and Bryson, Amer. Rev.Tuberc., 1950,62,286; Waisbon and Spink, Proc. SOC. Exp. Biol., 1950,74,35.68 Yegian and Vanderlinde, Amer. Rev. Tuberc., 1950,61, 483 ; Steenken, Wolinsky,and Bolinger, ibid., 1950, 62, 300.7O Waksman, Brit. Med. J., 1950, 11, 595.7l Karlson, Gainer, and Feldman, Amer. Rev. Tuberc., 1950, 62, 345.7% Waksman, Frankel, and Graessle, J . Buct., 1949, 58, 229.73 Swart, Romano, and Waksman, Proc. SOC. Exp. Biol., 1950, 73, 376.’* Rebstock, Crooks, Controulis, and Bartz, J. Amer. Chem. Soc., 1949, 71, 2458;Controulis, Rebstock, and Crooks, ibid., p. 2463; Long and Troutman, ibid., pp.2469, 2473.7 5 Smith, Landers, and Forgacs, J . Lab. Clin. Med., 1950, 36, 154; Hess, Anulyt.Chem., 1950,22,649.v 6 McLean, Schwab, Hillegas, and Schlingman, J . Clin. Invest., 1949,28,953ABRAHAM AND NEWTON : ANTIBIOTICS. 293If, is said to inhibit the multiplication of staphylococcal phage,77 and toeliminate the cytoplasmic " kappa particles " in killer strains of Para-mecium aurelia so that their power to kill sensitive organisms is lost.78Some bacteria sensitive to chloramphenicol contain enzymes able tobring about reduction of the nitro-group, hydrolysis of the amide linkage,oxidation of the secondary hydroxyl group, and a cleavage of the moleculebetween the first and the second carbon atom of the propanediol chain.79Chloramphenicol is readily absorbed into the blood and the body fluid aftereither parenteral or oral administration.A dose of 50 mg./kg. was welltolerated intravenously by dogs and no serious toxic effects resulted fiomcontinued doses of 200 mg./kg. given orally. The LD,, intraperitoneally inmice is 1300 rng./kgeso The drug is not entirely innocuous to animal tissues,however, and in sufficient amount may cause acute respiratory depression anddamage to the kidneys. Concentrations of 10 pg./ml., less than those attainedin the blood of patients, were found to retard the growth of epithelial cellsand fibroblasts.81 When chloramphenicol is given by mouth to man it isexcreted in the urine partly unchanged and partly as the hydrolysis product( -)-~-threo-2-amino-l -p-nitrophenylpropane-1 : 3-diol (XII), but mostlyas the 3-glucuronide of chloramphenicol.The latter has no antibacterialactivity, but chloramphenicol can be liberated from it by the enzyme p-glucuronidase.82Aureomycin. This is a yellow crystalline antibiotic, with amphotericproperties, isolated from the culture fluid of Streptomyces aureofaciens. Likechloramphenicol it contains non-ionic chlorine, analysis giving : C, 54.6 ;H, 5.3 ; N, 5.8 ; C1,7-2. It is unstable in alkali.83 A fluorometric method 84and rapid biological methods 85 for the assay of aureomycin have recentlybeen described.Aureomycin inhibits the growth of a wide range of Gram-positive and Gram-negative bacteria at dilutions of the order of 1 in lo6 86 andin sufficient concentration may kill the majority of organisms in a culture.87Resistant organisms do not appear to develop readily.88 The substance is7 77 879808 18a81Edlinger and Faguet, Ann. Xnst. Pasteur, 1950,79, 436.Brown, Nature, 1950, 100, 527.Smith and Worrel, Fed. Proc., 1950, 9, 230; Archiv. Biochem., 1950, 28, 1, 232.Long, Bliss, Schoenbach, Chandler, and Bryer, Lancet, 1950,258, 1139.LBpine, Barski, and Maurin, Proc. SOC. Exp. Biol., 1950,73, 252.Glazko, Dill, and Rebstock, J . Biol. Chem., 1950,183, 679.Broschard, Dornbush, Gordon, Hutchings, Kohler, Krupka, Kushner, Lefemine,and Pidacks, Science, 1949, 109, 199.84 Saltzman, J .Lab. Clin. Med., 1950,35, 123.8 5 Schmerson, Proc. SOC. Exp. Biol., 1950, 74, 106; Alture-Werber and Loewe, J .86 Price, Randall, and Welch, Ann. N . Y . Acad. Sci., 1948, 51, 211; Whitlock andLab. Clin. Med., 1950, 35, 660.Tashman, J . Bact., 1950,59,314. a 7 Spicer, J . Lab. Clin. Med., 1950,36, 183.Gezon and Fasan, Proc. SOC. Exp. Biol., 1950,73, 10294 BIOCHEMISTRY.also active against Rickettsia and certain viruses,89 and against E . hiatoty-timW Like chloromycetin, aureomycin is absorbed from the gastro-intestinaltract and has a relatively low toxi~ity.9~192 Its LD,, in mice, when givensubcutaneously, is 30004000 mg. /kg. In high concentrations aureomycindisturbs mitosis in tissue cult~res.~3Terramycin. An amphoteric antibiotic named terramycin has beenisolated in crystalline form from the culture fluid of Streptomyces rimosus.Its probable molecular formula is C22H24-,609N2,2H20.It shows absorptionmaxima at 247,275, and 353 mp., and contains three ionisable groups, forminga hydrochloride and a disodium salt. It is relatively stable in aqueoussolution at pH 1-9 when stored at 5O.94 Terramycin differs from aureo-mycin in containing no chlorine and in being much more stable in aqueoussolution. Terramycin is active in vitro against a variety of Gram-positiveand Gram-negative bacteria and against Rickettsia, and is bactericidal insufficient con~enfration.~~ It has a relatively low toxicity, the LD,, of thehydrochloride being about 800 mg,,’kg.when given subcutaneously in mice.The substance is absorbed from the gastro-intestinal tract. It shows markedchemotherapeutic activity in mice infected with sensitive bacteria.Os# 97This antibiotic was isolated in crystalline form fromcultures of an unidentified species of Streptomyces. It appears to have themolecular formula C28H4,0gN2 and to be a nitrogenous phenoLg8 It isfungicidal,99 insecticidal, and acaricidal.100 A dose of‘ 30 mg.jkg. given bystomach tube to rats proved fatal.lo1A red crystalline antibiotic named actinomycin, whichappeared to have the approximate formula C41H560,1N8, was isolated fromActinomyces antibioticus.lo2 An identical or very similar substance from aspecies of Streptomyces, which has the approximate formula C41H,8011N8, hasAntimycin A .Actinomycin.89 Wong and Cox, Ann.N . Y . Acad. Sci., 1948,51, 290.Hewitt, Wallace, and White, Science, 1950, 112, 144; Watt and Van de Grift,J. Lab. Clin. Med., 1950,36, 741.91 Harned, Cunningham, Clark, Cosgrove, Hine, McCauley, Stokey, Vessey, Yuda,and SubbaRow, Ann. N.Y. Acad. Sci., 1948,61, 182.Q2 Schoenbach, Bryer, and Long, ibid., p. 267.93 Keilova-Rodova, Experientia, 1950, 6, 428.94 Finlay, Hobby, P’an, Regna, Routien, Seeley, Shull, Sobin, Solomons, Vinson, andKane, Science, 1950,111,85 ; Regna and Solomons, Ann. N . Y . A d . Sci., 1950,53,229.9 5 Hobby, Lenert, Pikula, Kiseluk, and Hudders, ibid., p. 266.*6 Herrell, Heilman, Wellman, and Bartholomew, Proc. Mayo Clin., 1950, 25, 183.9 7 Werner, Knight, and McDermott, Proc.SOC. Exp. Biol., 1950, 74, 261; P’an,Reilly, Halley, Richard, Pekich, and Pollets, J . P b r m . Exp. Ther., 1950, 99, 234;Hobby, Dougherty, Lenert, Hudders, and Kiseluk, Proc. SOC. Ezp. Biol., 1950, 73,503; Hobby, Reed, Rinne, Powers, and D’Ambrosia, ibid., p. 511; P’an, Scaduto,and Cullen, Ann. N.Y. Acad. Sci., 1950, 53, 238; Schoenbach, Bryer, and Long,ibid., p. 245 ; Welch, ibid., p. 253.O 8 Dunshee, Leben, Keitt, and Strong, J . Amer. Chem. Soc., 1949, 71, 2436.99 Leben and Keitt, Phytopath., 1948, 38, 899.loo Kid0 and Spyhalski, Science, 1950, 112, 172.101 Ahmad, Schneider, and Strong, Arch. Biochem., 1950, 28, 281.lop Waksman and Tishler, J . Bid. Chem., 1942,142, 619ABRAHAM AND NEWTON : ANTIBIOTICS.296been called actinomycin B or (' antibiotic X-45." lo39 lo4 Actinomycin Byields threonine, L-proline, D-valine, N-methylvaline, and sarcosine onhydrolysis, and appears to be a peptide associated with a quinone system.Actinomycin contains the same amino-acids as actinomycin B.An antibiotic from an actinomycete, which hits the probable formulaC,oH,7O11N7, has been called actinomycin C.lo6 It differs from actinomycinand actinomycin B in containing D-isoleucine or D-alloisoleucine in place ofD-Vahe.l'* 106This is a red crystalline antibiotic, isolated from a strainof Actinomyces, which has the formula C,,H,,O,, and is thought fo contain ahydroxy-quinone system. It prevents the growth of Staph. aurem at adilution of 1 in 1OS.1O6Prwtimmycin.The crude basic antibiotic first described in 1942 lo7has been separated into three active substances, proactinomycin A, B andC, which appear to have the molecular formulae C,,H,,O,N, C,,H,,O,N, andC,,H,lO,N respectively.lo8 It was thought unlikely that any of the threesubstances would be useful clini~alIy.1~~Other antibiotics recently reported to be formed byac tinom y cetes are fungicidin, lo streptocin ,I1 viom y cin, l2 m ycom ycin ,113and an antibiotic resembling xanthomycin.114Antibiotics from Bacteria-Many of the antibiotics obtained frombacteria are polypeptides. Recent work has shown that they often occur aafamilies of closely related peptide~,2~-~~ although in the case of the poly-myxins each pure strain of B. polymym is said to produce a single polypeptideantibiotic.l16 None of these substances has found an established place inmedicine ; the bacitracins, polymyxins, and licheniformins have chemo-therapeutic properties, but their renal toxicity has prevented their generaluse in man.Nevertheless, the detailed structure of the polypeptide anti-biotics is of great interest because of its bearing on the structure of proteins,and the substances continue to be the subject of extensive chemicalinvestigations.llsCrude bacitracin, obtained from a strain of B.Zicheniformis, was fractionated by counter-current distribution, under acidActinorhodin.Miscellaneous.Bucitracin (AyJivin).lo3 Lehr and Berger, Arch. Biochem., 1949, 23, 503; Dalgliesh and Todd, Nature,1949, 184, 830; Abstracts of Communications a t 1st Intern.Congr. Biochem.,Cambridge, 1949, p. 246.lo4 Dalgliesh, Johnson, Todd, and Vining, J., 1950, 2946.lo6 Brockmann and Grubhofer, Nuturwiss., 1949, 12, 376.lo6 Brockmann, Pini, and Plotho, Ber., 1950,83, 161.lo' Gardner and Chain, Brit. J. Exp. Path., 1942, 23, 123.108 Marston, ibid., 1949, 30, 398.118 Hazen and Brown, Science, 1950,112,423.111 Kupferberg, Styles, Singher, and Waksman, J. Bact., 1950, 69, 523.112 Steenken and Wolinsky, A m r . J. Med., 1950,9,633.113 Jenkins, J. Lab. Clin. Med., 1950, 36, 841.114 Mold and Bartz, J. Amr. Chem. SOC., 1950,72, 1847.116 Brownlee, Symposia SOC. Exp. Biol., 1949, No. 111, p. 81.116 Synge, Quart. Reviews, 1949, 3, 246.100 Marston and Florey, ibid., p.407296 BIOCHEMISTRY.conditions, into a major and two minor components.2Q The bulk of theantibacterial activity was accounted for by the major component but analysisof the distribution curve indicated that some of the material in the majorband was not homogeneous. Further information about the nature ofbacitracin has come from studies of the polypeptide antibiotic ayfivin alsoproduced by a strain of B. li~heniformis.~~1 117 Counter-current distributionunder neutral conditions of a mixture of crude bacitracin and ayfivin indicatedthat the two antibiotics contained essentially the same components, and thename ayfivin has consequently been abandoned. The major componentappearing in the distribution under acid conditions could be resolved underneutral conditions into three biologically active components, which werenamed bacitracin A, B, and C 3 0 Bacitracin A was present in the largestamount.Bacitracin C was found to have about the same antibacterialactivity as A, while B was only a third as active. Preliminary investigationsof these substances indicate that they have different toxicities for mice.ll*Licheniformin. Crude licheniformin 119 has been fractionated bycounter-current distribution into three distinct polypeptides (A, B, and C)that appear to have cyclic s t r ~ c t u r e s . ~ ~ A and B contain aspartic acid, serine,glycine, lysine, arginine, proline, valine, and phenylalanine. C contains inaddition glutarnic acid. The three polypeptides have different toxicities forthe mouse, and their toxicity does not parallel their antibacterial activity.They all cause damage to the kidneys.Polymyxins A (aerosporin),B, C, D, and E are similar polypeptides, produced by strains of B.polymyxa,which are active against certain Gram-negative bacteria and dermato-mycetes.3~ lZo* lzl Circulin is formed by a strain of B. circulans.122 All thesesubstances appear to be cyclic polypeptides. On hydrolysis they yieldthreonine, ay-diaminobutyric acid, varying amounts of leucine, phenyl-alanine, and serine, and (+ )-6-methyloctanoic acid.123 The 6-methyl-octanoic acid, which has now been synthesised,l** is thought to be joined tothe rest of the molecule through an ester linkage in circulin and an amidelinkage in polymyxin A and D.An antibiotic named polypeptin has been obtained from astrain of B. circulans as a crystalline ~u1phate.l~~ Counter-current distri-bution indicates that the preparation is about 90% pure.126 Polypeptin isa strongly basic polypeptide of molecular weight about 2000.Paper chro-matograms of hydrolysed material are reported to indicate the presence of anumber of amino-acids, but only ay-diaminobutyric acid has been specificallyPolymyxins (Aerosporin) and Circulin {&I 9).Polypeptin.117 Sharp, Arriagada, Newton, and Abraham, Brit. J . Exp. Path., 1949,30,444.118 H. W. Florey, personal communication.1l0 Callow, Glover, D'Arcy Hart, and Hills, Brit. J . Exp. Path., 1947, 28, 418.120 Ann. N . Y . Acad. Sci., 1949, 51, 855-997.121 Serri, Compt. rend.Soc. Biol., 1949, 143, 362.122 Peterson and Reineke, J . Biol. Chem., 1949,181, 95.123 Wilkinson, Nature, 1949,164, 622.1z5 Howell, J . Biol. Chm., 1950,186, 863.126 L. C. Craig, personal communication to 8. F. Howell.12* Crombie and Harper, J . , 1950, 2685ABRAHAM AND NEWTON : ANTIBIOTICS. 297mentioned. The ultra-violet absorption spectrum of polypeptin sulphateindicates that the substance does not consist only of known amino-acidslinked in a peptide chain. Polypeptin is active against both Gram-positiveand Gram-negative bacteria and against fungi. It is also haemolytic andextremely toxic.Crystalline gramicidin has been resolvedinto at least four components by counter-current di~tributi0n.l~’ X-Rayanalysis of crystals of gramicidin A and B show that they may be both builtup of a repeating unit of molecular weight 3800, which is compatible with theresults of chemical analysis.128 An outstanding problem presented by thecomposition of the gramicidins is how molecules that have neither acidic norbasic properties can be made up only of monoamino-monocarboxylic acidsand ethanolamine.It has been reported that the free OH groups in grami-cidin do not come from the ethanolamine residues. A possible structure inwhich the ethanolamine is involved in an O-peptidyl linkage has beensuggested (XIII), but this structure stillleaves the question of the masked basicGramicidin S is quite distinct from the\CHR~ (xIII.) other gramicidins and contains only five/ different amino-acids.The smallest ob-served unit in crystals of this compound corresponds to a decapeptide. Theantibacterial properties of gramicidin S appear to depend on its cyclicstructure, for various derivatives of an open-chain pentapeptide containingamino-acids of the same configuration and in the same sequence as those inthe antibiotic showed little activity.130Marcescin has been obtained as a, highly active powder fromculture fluids of Serratia m a r ~ s c e n s . l ~ ~ The crude material was found to bea complex which could be separated on a buffered silica-gel column into anacidic and a basic fraction. The antibacterial activity was associated withthe latter, which was shown to be a strongly basic polypeptide of considerablestability. Marcescin is highly active against certain Gram-positive organisms,but it is hzemolytic and very toxic to mice.Nisin. Further studies on nisin, an antibiotic produced by a strain ofStrep. lactis, have indicated that the needles previously observed in purifiedmaterial 132 may not have been true crystals, and that the most active prepar-ations are a mixture of at least two polypeptides.133 The amino-acidsalanine, valine, leucine, and isoleucine were recognised in a hydrolysate ofpurified nisin.Anagar diffusion method for the assay of nisin has been described.l=Gramicidins and Gramicidin 8.R*HC\C/OH // groups unsolved.129HN/ \O*CH2*CH2*N \Marcescin.Cystine and aspartic acid were also probably present.12’ Gregory and Craig, J . Biol. Chem., 1948, 172, 839.Hodgkin, Cold Spring Harbor Symp., 1949, 14, 65.lZ0 Synge, ibid., p.191.130 Harris and Work, Biochem. J . , 1950,48, 582.131 Fuller and Horton, J . Cfen. Microbiol., 1950, 4, 417.132 Berridge, Lancet, 1947, 253, 7 . 133 Idem, Biochem. J . , 1949, 45, 486.Friedman and Beach, Proc. SOC. Gen. Microbiol., 1951,5, V298 BIOCHEMISTRY.lodinin. This antibiotic from Chrombacterium iodinum has finallybeen proved to be the di-N-oxide of 1 : 5-dihydroxyphena~ine.1~6MisceZZuneous. The following antibiotics from bacteria have not yetbeen well characterised. An antibiotic from Bacillus nutto,136 an antibioticfrom B. b r e z ~ i s , ~ ~ ~ various c~licines,~~* diplomycin,139 fungocina,lU Rhizoc-tonia, and Aspergillus factors,141 and ~ t r e p t o t a s i n .~ ~ ~Mode of Action and Acquired Resistance.-The mechanisms by which theantibiotics exert their antibacterial effects are of fundamental interest tobiochemistry ; they are also of practical importance, for a better understand-ing of them may help to place chemotherapy on a more rational basis.Some antibiotics, such as tyr0cidine,l4~ may rupture the surface mem-branes of the bacterial cell or denature cell proteins in an unspecific manner,but there can be no doubt that many others, including those important inmedicine, act in a more subt,le fashion. Possibly these substances interferewith the function of one specific enzyme. Since penicillin is vastly moreeffective against growing than against resting organisms, and the former arealso more susceptible to streptomycin and chl~romycetin,~~~ special import-ance may attach to enzymes concerned with the synthesis of new cell material.The changes in morphology that sometimes occur in the presence of anti-biotics 146 show that cell growth and cell division may be inhibited todifferent degrees.One possible approach to the,subject is to study the fate of the antibioticitself when it comes into contact with sensitive bacteria.This is a difficultprocedure because very small quantities of substance are involved, but itmay be facilitated by the use of antibiotics containing a radioactive atom.146With penicillin containing 35s it has been found that sensitive organisms bindmore of the drug than do resistant ones, and growing organisms more thanresting ones.Sensitive bacteria are able to concentrate penicillin from thesurrounding medium so long as it is present at a higher dilution than thata t which it exerts a maximum bactericidal effect. Since the radioactivity155 Clemo and Daglish, J., 1950, 1481.13b Ishidate, Shibata, and Hagiwara, J . Pharm. Soc. Japan, 1949, 69, 373.lS7 Anzai, Nagaki, Date, Tsukakoshi, Hatori, and Nakamura, Kitisato Arch.E':E":E':E':E':E':E':E. Med., 1948, 21, 23 ; Nagaki and Yajima, ibid., 1948, 20, 28.138 Chabbert, Ann. Inst. Pasteur, 1950, 79, 51 ; Fredericq, Compt. rend. SOC. Biol.,1950, 144, 297, 299, 435, 437, 439, 728, 730; Mondolfo, Bull. SOC. Ital. Biol. sper., 1948,24, 1086.130 Noster, Zentr. Bakt., 1949: 153, 32; Enell, Lingen, and Melin, Acta Paediatr.,Stockh., 1950,39, 251.140 Cer6os, Ministerio de Agr.y Ganed. (Argentina) Pub. Tec. No. 36.141 Michener and Snell, Arch. Biochem., 1949, 22, 208.142 SheTwood, Russell, Jay, and Bowman, J . Infect. Dis., 1949,84, 88.143 Fong and Krueger, J . Ben. Physiol., 1950,33,311.144 Edlinger, Ann. Inst. Pasteur, 1950, 78, 417.145 Fleming, Voureka, Kramer, and Hughes, J. Ben. Microbial., 1950, 4, 257; Leva-diti, Vaisman, and Henry-Eveno, Bull. Acad. Nat. Med., 1950,134, 369; Warbasse andJohnson, J . Bact., 1950, 60, 279.146 Cooper and Rowley, Nature, 1949,163,480; Cooper, Rowley, and Dawson, ibid.,1949,164, 842; Maass and Johnson, J . Bact., 1949,57,415ABRAHAM AND NEWTON : ANTIBIOTICS. 299is not ertsily removed from the cells it appears that penicillin enters intochemical combination with a cell component, which is present in very smallamounts in resting organisms but which increases during g r 0 ~ t h .l ~ ~An alternative approach is to look for enzyme systems in the cell whichare affected by the antibiotic. It is often difficult here to distinguish betweenthe primary action of the drug and one of the innumerable changes that musteventually follow it. The interesting discovery that certain Gram-positiveorganisms are no longer able to .concentrate amino-acids from their environ-ment after contact with penicillin during growth suggested the possibilitythat penicillin acted by preventing amino-acid a~simi1ation.l~~ It does notnow seem probable that this is the first effect of the drug, for other organismsthat are highly sensitive to penicillin do not need pre-formed amino-acidsfor growth.149 Nevertheless, it is not unlikely that the antibacterial effect ofpenicillin is closely related to a disturbance of protein synthesis.Staphy-lococci respiring in glucose and various amino-acids do not increase theirprotein in the normal manner when penicillin is present, but appear insteadto produce extracellular polypeptide.lW The effect of penicillin on a Gram-negative bacterium grown in L-leucine, glycine, L-leucine plus glycine, orL-leucylglycine was thought to indicate that it prevented the incorporationof glycine into a peptide.151 On the other hand, staphylococci growing inthe presence of .penicillin also undergo changes in their nucleotide-nucleicacid balance and it has been suggested that this may really be the primaryeffect of the drug.152 An enzyme system in Staph.aureua concernedwith the catabolism of uridylic and guanylic acids is inhibited bypenicillin .153Streptomycin has been found to affect certain metabolic activities ofresting sensitive bacteria. It inhibits the complete oxidation of stearic acidby resting cells of Bact. coli, apparently by stopping the condensation ofpyruvate with oxaloacetate and thus preventing the entry of pyruvate intothe tricarboxylic acid cycle of the terminal respiration system.154* 156 Theoxidation of higher fatty acids by Myw. tuberculosis Avian Type is inhibitedby streptomycin, but this is reported to take place by a different mechan-ism.165 It does notaffect the respiration or protein breakdown of resting or growing bacteria,but inhibits the action of esterases. Its action on the esterases of animalcells is much weaker than on those of sensitive bacteria and it has been sug-gested that the animal cell wall presents a barrier to the drug.166 Anti-Chloramphenicol also disturbs the metabolism of fats.ld7 Rowley, Cooper, Roberts, and Lester-Smith, Biochem.J., 1950,46, 157.14* Gale and Taylor, J . Gen. Microbiol., 1947, 1, 314.140 Hunter and Baker, Science, 1949,110,423 ; Grelet, Ann. Inst. Pasteur, 1949,77,263.I 5 O Hotchkiss, J . Expt. Med., 1950, 91, 351; Ann. N . Y . Acad. Sci., 1950, 53, 13.151 Simmonds and Fruton, Science, 1950,111, 329.15( Mitchell, Nature, 1949,164,259; Park and Johnson, J .Biol. Chem., 1949,179,585.153 Gros, Beljanski, and Machboeuf, Compt. rend., 1950, 231, 184.154 Umbreit, J . Biol. Chem., 1949, 177, 703; Ann. N . Y . A d . Sci., 1950, 53, 6 .155 Oginsky, Smith, and Solotorovsky, J . Bact., 1950, 69, 29.156 Smith, Worrel, and Swanson, ibid., 1949, 58, 803300 BIOCHEMISTRY.mycin A has proved to be the most powerful inhibitor of succinic dehydro-genase so far discovered, and unlike other inhibitors of this enzyme it isneither competitive nor involved in a reaction with SH groups.l0lSome antibiotics appear to inhibit a process by which the energy ofcellular oxidation is made available for synthesis, showing a resemblance inthis respect to 2 : 4-dinitrophenol.Gramicidin inhibits the uptake ofphosphate by staphylococci without depressing respiration,157 and alsoinhibits phosphorylation in the cyclophorase system. 158 Aureomycininhibits phosphorylation by mitochondria without affecting re~pirati0n.l~~Such effects are thought to be caused by a disturbance of the mechanism bywhich phosphate-bond generation is coupled to oxidative reactions.160The idea that an antibacterial substance may inhibit the utilisation of anessential metabolite of similar structure, which has played so important apart in our understanding of the mode of action of the sulphonamides, hashitherto found little application in studies of the antibacterial action ofantibiotics. It has been found, however, that phenylalanine and certainstructural analogues inhibit the action of chloramphenicol in a non-competitive manner,] 61 and that an antagonist to streptomycin anddihydrostreptomycin is produced during the growth of Pseudomnaspyocyanea.162Bacteria become more resistant to most antibiotics when grown in theirpresence, though the rapidity and extent of the change in sensitivity mayvary enormously.163 Resistance to penicillin, chloromycetin, aureomycin,and terramycin appears to develop gradually, whereas very high resistanceto streptomycin may suddenly be acquired in a single step.164 In additionto acquiring resistance, bacteria may become dependent on streptomycin 165and on chloramphenicol 166 for their growth.The interesting phenomenon of acquired resistance raises two relatedquestions.By what kind of mechanism does resistance develop? and inwhat way do resistant organisms differ from their sensitive ancestors ?It is now widely assumed that resistant organisms are formed by mutationindependently of the antibiotic, but that in its presence they replace themajority of sensitive organisms through the operation of natural selec-ti0n.1~~* 167 The direct evidence for this view comes largely from the applic-15' Hotchkiss, Adu. Enzymology, 1944, 4, 153.lK8 Cross, Taggart, Covo, and Green, J. Biol. Chem., 1949,177, 655.lS0 Loomis, Science, 1950, 111, 474.l 6 0 Loomis and Lipmann, J . Biol. Chem., 1948,173, 807.161 Woolley, Fed. Proc., 1950, 9, 249.163 Gezon, Fasan, and Collins, Proc. SOC. Exp. Biol., 1950, 74, 505; Garrod, Bull.Hyg., 1950, 25, 539; Miller and Bohnhoff, Ann.Rev. Microbiol., 1950, 4, 201 ; Coffey,Schwab, and Ehrlich, J . Infect. Dis., 1950, 87, 142.lti2 Lightbrown, Nature, 1950,166, 356.164 Bryson and Demerec, Ann. N . Y . Acad. Sci., 1950, 53, 283.165 Miller and Bohnhoff, J . Bact., 1947, 54, 467; Foley and Shwachman, J . Gen.Microbiol., 1950, 4, 141.Goche and Finland, Proc. SOC. Exp. Biol., 1950, 74, 824.167 Wyss, Ann. N . Y . Acad. Sci., 1950,53, 183 ; Cavalli and Maccacaro, Nature, 1950,166, 991ABRAHAM AND NEWTON : ANTIBIOTICS. 301ation of an ingenious statistical method 168 to the analysis of the numbers oforganisms that have become resistant to penicillin and s t r e p t ~ m y c i n . ~ ~ ~The interpretation of the results obtained with penicillin, however, has beendisputed,170 and some of the facts about the development of resistance tostreptomycin are thought to remain unexplained by the theory of spontaneousmutati0n.17~The alternative theory that resistance is induced by the drug has beendeveloped in an interesting manner on physico-chemical lines,172 but hasbeen criticised as Lamarkian.173 Although the force of this criticism inrelation to bacteria is far from evident, it would not be surprising if gene-likestructures were involved in some of the enzymic changes that increase theresistance of the bacterial cell to for the presence of specific genes iscertainly required for many biochemical reactions in Neurospora and incertain yea~ts.1'~ However, the acquisition of resistance may sometimesdepend on changes in the amount rather than in the nature of the enzymesystems in the cell, and whether these changes are unconnected with thepresence of the drug itself seems to be still an open q ~ e s t i 0 n .l ~ ~ Boththeories may well play a part in explaining the varied phenomena of acquiredresistance.In some cases bacteria with an acquired resistance to an antibiotic areable to dispense with a biochemical process that is inhibited by the antibioticin the original sensitive strain. As the resistance of Stuph. aurew to penicillinincreased the cells lost their ability to concentrate pre-formed amino-acidsfrom the medium and acquired a mechanism for synthesising the amino-acidsfrom ammonia and g1u~ose.l~~ When Bact.coli acquired resistance tostreptomycin it was no longer able to bring about the rapid oxidation ofstearate that had previously been inhibited by the It is thusreasonable to suppose that the development of alternative metabolic path-ways may be responsible for acquired resistance to these substances.Antibiotics in Medicine.-It is no exaggeration to say that the last fifteenyears have seen a revolution in the treatment of infectious disease. Thechange which began with the introduction of the sulphonamides has becomefar-reaching through the isolation of a variety of antibiotics having systemicchemotherapeutic properties. To-day the majority of bacterial infectionscan usually be treated with some success, and chemotherapy has also beenextended to rickettsia1 and some viral diseases.168 Luria and Delbruck, Genetics, 1943, 28, 491 ; Lea and Coulson, J .Genet., 1948,169 Demerec, J . Bact., 1948,56, 63; J . Clin. Invest., 1949,28, 891.l i 0 Eriksen, Acta Path. Microbiol. Scand., 1949, 26, 269.1 7 2 Hinshelwood, " The Chemical Kinetics of the Bacterial Cell," 1946, Clarendon1 7 4 Clark, Wyss, and Stone, Nature, 1950,166, 340.175 Beadle, Ann. Rev. Physiol., 1948, 10, 17 ; Tatum and Perkins, Ann. Rev. Micro-49, 264.Linz, Ann. Inst. Pasteur, 1950, 78, 105.Press, Oxford; Endeavour, 1949, 8, 151. Lurk, Bact. Reviews, 1947, 11, 1.biol., 1950, 4, 129. 176 Hinshelwood, Nature, 1950,166, 1089.Gale and Rodwell, J . Qen. Microbiol.,, 1949, 3, 127302 BIOCHEMISTRY.The clinical use of the more valuable antibiotics has recently been thcsubject of a number of revie~s.~O1 8op 96* 112* 178 Penicillin remains the drug ofchoice for infections caused by sensitive organisms such &g streptococci,sfaphylococci, pneumococci, neisseria, B.anthracis, and clostridia, and also byspirochaetes. Its very low toxicity makes it almost a perfect chemothera-peutic agent, although reactions due to sensitisation of patients are said tobe increasing.The use of streptomycin has tended to become more restricted, for thissubstance can cause permanent deafness and vestibular damage, and bacteriadevelop resistance to it with remarkable facility. Dihydrostreptomycin hasbeen claimed to be less toxic than streptomycin to the vestibular apparatus,but this is not universally accepted.Streptomycin is highly effective,however, in the treatment of plague and tularemia, and appears to bevaluable in combating the infection that is a significant factor in death fromradiation injury.179 It is still the best drug available for dealing withtuberculosis, and can save the lives of a significant proportion of patients withmiliary or meningeal tuberculosis, diseases that are almost invariably fatalwhen untreated. In the treatment of other forms of tuberculosis the drughas definite limitations. With pulmonary tuberculosis it appears best touse streptomycin together with p-aminosalicylic acid (PAS) .180 Whetherneomycin will prove superior to streptomycin remains to be seen, but since itappears to produce renal damage caution will be necessary in its clinical use.Chloramphenicol and aureomycin are valuable in the treatment of anumber of bacterial diseases that do not respond to penicillin.For example,chloramphenicol is highly effective against typhoid fever, and aureomycinagainst infections with penicillin-resistant staphylococci, while both sub-stances give good results with undulant fever. These antibiotics have alsobeen responsible for the extension of chemotherapy to rickettsia1 diseases,such as typhus, and to certain viral infections, such as psittacosis, lympho-granuloma venereum, and primary atypical pneumonia. No dangeroustoxic reactions appear to follow the use of the drugs, but aureomycin oftenproduces nausea and diarrhoea, and chloramphenicol may cause dry tongueand muscular weakness, Terramycin, which received extensive humantrials in 195O,l8l shows a close resemblance to aureomycin in its therapeuticand pharmacological properties.The polypeptide antibiotics bacitracin and polymyxin exert a chemo-therapeutic action in animals infected with certain Gram-positive and Gram-negative bacteria respectively, and both have been tried on a limited scale178 Garrod, Brit.Med. J., 1950,11, 722; Knight, N.Y. State J . Med., 1950,50, 2173;Herrell, Amr. J . Med. Sci., 1950, 219, 670; Germer, Deut. med. Woch., 1950, 75, 1132;Smadel, Amer. J. Trop. Med., 1950, 30, 357.170 Miller, Hammond, and Tompkins, Science, 1950,111, 719; Hammond and Miller,Ann. N . Y . Acad.Xci., 1950, 58,303.180 Brit. Med. J . , 1949,11, 1615; 1950,11, 1073.181 Yeager, Mansberger, Thomas, and Barnes, Ann. N . Y . Acad. Sci., 1950, 53, 319;Knight, ibid., p. 332 ; Smadel, Jackson, and Ley, ibid., p. 375 ; Kneeland and Melcher,ibid., p. 437; Caldwell, Spies, Wolfe, Lepper, and Dowling, J . Lab. CEin. Med., 1950,36, 747 ; Linsell and Fletcher, Brit. Med. J . , 1950,11, 1190VAN REYNINGEN : BACTERIAL TOXINS. 303in man, but the fact that they can cause serious damage to the kidneytubules has prevented their general use. The use of bacitracin and penicillintogether in caam where the two drugs exert a synergic action, however, isreported to be very effective,l82 and polymyxin B and E have proved valuablein the treatment of infection with Ps.p y o c y a n e ~ . ~ ~ ~Although many of the properties required by a systemic chemotherapeuticagent are well understood, the value of a new antibiotic t o medicine cannotyet be predicted with confidence on the basis of its antimicrobial activityin vitro and its pharmacological behaviour. The effective concentration ofpenicillin in vivo against streptococci and pneumococci is reported to be closeto that which would be expected from its activity in v i t r ~ , ' ~ ~ but no simplerelation is said to be found in the treatment of infection with B. n ~ v y i . ' ~ ~Aureomycin is stated to be five to ten times more effective against Stuph.u w e w in wivo than in vitro,l86 while helvolic acid, which can readily beintroduced into the blood in bacteriostatic concentrations, is unable to controlstaphylococcal infections in a satisfactory manner.187 On the other hand,although aureomycin compared favourably with streptomycin in its activityagainst a strain of Myco.tubercuEosis in oitro, it was quite ineffective againstinfection induced with this organism in mice.ls8 The reason why ohlor-amphenicol is much more effective than aureomycin against typhoid feveris also not apparent from the activity of these antibiotics in vitro.Whether bactericidal activity is essential in a powerful chemotherapeuticagent, or whether a bacteriostatic action may be sufficient, is still uncertain.When tested in vitro the antibiotics that are valuable in medicine usuallykill the majority of a population of sensitive bacteria but leave a smallnumber of survivors.87 How far the natural defence mechanisms of the bodycan contribute to the elimination of infecting organisms, and how far theirefficiency is affected by the various antibiotics, appear to be questions worthyof future study.E. P.A.G. G. F. N.1821831841851861 8 71883. BACTERIAL TOXINS.Two of the more important dates in the history of bacteriology are 1858and 1890, when Roux and Yersin discovered that sterile culture filtrates ofthe diphtheria bacillus contained a toxic substance, and von Behring showedthat the serum of animals injected with sub-lethal doses of this toxincontained a specific antitoxin. Since then many other toxic antigenicsubstances have been found in the culture filtrates of Gram-positive bacteriaJohnson and Meleney, Ann.AN. Y . Acad. Sci., 1950, 53, 42.Pulmki, Baker, Rosenberg, and Connell, J . Clin. Invest., 1949, 28, 1028.Eagle, Fleischrnan, and Musselman, J . Bact., 1950, 59, 625.Kley and Ercoli, Experientia, 1950,6, 153.Klein, Schorr, Tashman, and Hunt, J . Bact., 1950,60, 159.Florey and Jennings, 1946, unpublished.Stoinback, Baker, and Ducn, Proc. SOC. Exp. Biol., 1950, 74, 596304 BIOCHEMISTRY.and in the cells of Gram-negative bacteria, and also in snake venoms andplant seeds. The coagulases, fibrinolysins, hyaluronidases and proteases(and sometimes the haemolysins) listed in Table I are not always classifiedTABLE I.Toxins produced by principal toxin-producing pathogenic Gram-positivebacteria.Diseases associated with these organisms are given in squarebrackets.CZostridium botulinum : Four neurotoxins. [Botulism in man and animals.]CZ. oedematiens : (1) a-toxin, lethal, histotoxic ; (2) /?:toxin (lecithinase), lethal,haemolytic ; (3) y-toxin (lecithinase), lethal, haemolytic ; (4) b-toxin, haemolytic ;(5) c-toxin (lipase), lethal, haemolytic ; (6) I-toxin, haemolytic. [Gas gangrene inman; black disease and bradsot in sheep ; bacillary osteomyelitis in buffaloes.]CZ. septicum : A lethal haemolytic toxin. [Gas gangrene in man; blackleg and braxy insheep.]Cl. tetani : (1) Tetanospasmin, neurotoxic ; (2) tetanolysin, haemolytic. [Tetanus inman and animals.]C1. wekhii : (1) a-toxin (lecithinase), lethal, histotoxic, haemolytic ; (2) p-toxin, lethal;(3) y-toxin, lethal; (4) &toxin, lethal; (5) €-toxin, lethal, histotoxic ; (6) 7-toxin,lethal ( 9 ) ; (7) c-toxin, lethal, histotoxic; (8) &toxin, lethal, haemolytic ; (9) K-toxin(collagenase), lethal, proteolytic ; (10) h-toxin, proteolytic ; (1 1) p-toxin (hyaluroni-dam), spreading factor.[G? gangrene and enteritis necroticans in man; lambdysentery, struck and infectious enterotoxaemia in sheep.]Corynebacterium diphtheriae : A lethal, histotoxic toxin.Staphylococcus aureus : (1) a-toxin, lethal, histotoxic, haemolytic ; (2) p-toxin, lethal,haemolytic ; (3) y-toxin, lethal, hsmolytic ; (4) b-toxin, hsmolytic ; (5) hyaluronidase,spreading factor ; (6) coagulase ; (7) fibrinolysin. [Pyogenic infections in man andanimals .]Streptococcus pyogenes : (1) Dick toxin, lethal, erythrogenic ; (2) Streptolysin-0, lethal,hsmolytic ; (3) streptolysin-S, lethal, haemolytic ; (4) hyaluronidase, spreadingfactor ; (5) streptokinase, fibrinolytic. [Scarlet fever ; tonsillitis, pyogenicinfections, etc., in man.]as toxins, but their exclusion from this category is unwarranted.Theirtoxicity is either unknown or it is intermediate in the range of toxicity ofthe conventional toxins, which stretches from about 0.2 Minimal LethalDoses (per kg., mouse) per mg. (Shigelh shigae endotoxin) to 1.2 x log MLD(per kg., guinea pig) per mg. (Clostridium tetani exotoxin). Nor should theybe excluded merely because their substrates can, in some cases, be definedin more definite anatomical or chemical terms than those of the classicaltoxins.Both groups of substances are biologically active and antigenic,and are not only toxic to the host but also (with the possible exception ofthe neurotoxins) helpful to the parasite in its invasion.Since the beginning of the last war much progress has been made in thestudy of the toxins, as is superficially evident from the Decennial Indices ofChemical L4bstracts ; the Index for 1937-1946 contains 11 columns of entriesunder ‘‘ Toxins ” compared with 3 columns for the previous decade. Thisprogress has recently been reviewed,l* 2* but it continues to receive scantattention in recent textbooks of pathology, bacteriology, and biochemistry.In this review only a few aspects of this subject can be dealt with, and anecessarily over-simplified account of the toxins of Gram-negative bacteria[Diphtheria in man.]1 Pappenheimer, Adu.Protein Chemistry, 1948, 4, 123.9 Pillemer and Robbins, Ann. Review Microbiol., 1949, 3, 265.3 van Heyningen, “ Bacterial Toxins,” Oxford, Blackwell Scient. Publ. Ltd., 1950VAN HEYNINGEN : BACTERIAL TOXINS. 305is included mainly in order that they can be compared with those of theGram-positive bacteria.Lecithinases.-The or-toxin of CZ. welchii, which plays a major role inthe toxaemia of gas gangrene, is a lecithinase.4 In particular it is a lecithin-phosphatase that catalyses the hydrolysis of the bond between phosphoryl-choline and glycerol stearate oleate in lecithin, and therefore it has beencalled lecithinase C.(Lecithinase A splits off the unsaturated fatty acid toleave lysolecithin, and lecithinase B splits off both fatty acids.s The namelecithinase C was once proposed for a hypothetical enzyme that split offcholine,6 but priority should be given to the enzyme whose existence isestablished.) The Cl. welchii lecithinase is.quite specific ; it does not attackkephalin, cerebroside, lysolecithin, a-glycerophosphorylcholine, or p-glycero-phosphate, but it attacks sphingomyelin very slowly.4* '9 It is inactive inthe absence of calcium or magnesium ions and is therefore inhibited byphosphate or citrate buffers. The reaction between toxin and antitoxinalone is unaffected by these ions, but in the presence of substrate it is affectedbecause the toxin-substrate reaction is favoured and consequently theequilibrium of the reaction, toxin + antitoxin toxin-antitoxin isshifted to the 1eft.O Lecithinase C can be quantitatively determined bymeasuring the amount of acid-soluble phosphorus liberated by it fromle~ithin,~ or manometrically by measuring the volume of carbon dioxideliberated by the action of phosphorylcholine on sodium hydrogen carbonate,8or less accurately but with greater convenience by measuring the turbidityit produces in a solution of crude egg-yolk 1ipoprotein.lOLecithinases are also produced by other anaerobic clostridia, wiz., Cl.oedemtiens (p- and y-toxins),ll CZ.haemolyticum (-q-toxin),ll* l 2 q l3 Cl.biferrnentuns.l4 They are probably also produced by Cl. sordellii, chauvoei,sporogenes, centrosporogenes, and tertium.15 The lecithinases of Cl.welchiiand CZ. oedemtiens are antigenically distinct, but Cl. oedemtiens p-toxin andCl. huemolyticum ?-toxin are apparently antigenically identical.12 Thereis some antigenic relationship between the lecithinases of CZ. welchii andCl. bifermentans.14The aerobic sporing bacilli, Bacillus anthracis, B. mycoides, and particularly23. cereus, also produce a lecithinase C, which will hydrolyse kephalin aswell as lecithin and sphingomyelin. Unlike the lecithinases of the anaerobes,its action on free lecithin (but not on lipoprotein lecithin, or on kephalin) isMacfarlane and Knight, Biochem. J . , 1941, 35, 884.Belfanti, Contardi, and Ercoli, Ergebn. Enzymforsch., 1936, 5, 213.Contardi and Ercoli, Biochem.Z . , 1933, 261, 275.Macfarlane, Biochem. J . , 1942, 36, 11 1 ; 1948, 42, 587.Zamecnik, Brewster, and Lipmann, J. Exp. Med., 1947, 85, 381.Oakley and Warrack, J . Path. Bact., 1941, 53, 355; Zamecnik and Lipmann, J.Exp. Med., 1947, 85, 395. lo van Heyningen, Biochem. J . , 1941, 35, 1246.l1 Oekley, Warrack, and Clarke, J . Gen. MicrobioE., 1947, 1, 91.l2 Macfarlane, Biochem. J., 1950, 47, 267.l3 Jasmin, Amer. J . Vet. Res., 1947, 8, 289.l4 E. M. Miles and A. A. Miles, J . Qen. Microbiol., 1947, 1, 385; 1950, 4, 22.l5 Crook, Brit. J . Exp. Path., 1942, 23, 37306 BIOCHEMISTRY.inhibited by normal serum.l6, l7 The hemolytic bacterial lecithinases havea different action from the lecithinases of certain snake venoms.The latterare lecithinases A whose action results in the formation of the hemolyticsubstance, lysolecithin. Neither of the products of hydrolysis by theformer is itself hzemolytic.CZ. wekhii lecithinase has been partially purified, but a quantitativestudy of the toxin-antitoxin flocculation showed that the product could notbe more than 50% pure. Half of the nitrogen of the purified product wasprecipitated by antitoxin, but since it was shown that toxin-antitoxinfloccules could carry down non-specific impurities it could not be assumedthat all the precipitated material was pure toxin-antitoxin.l* This workshowed that quantitative studies of the toxin-antitoxin flocculation re-action l9 can have their pitfalls. The diffusion constant of the toxin, 020, is7-41 x The partly purified toxin had MLD 200 (per kg.,mouse) per mg.The bacterial lecithinases are lethal, hemolytic, and histotoxic, andalthough the fundamental biochemical activity underlying these biologicalactivities has been defined, a complete explanation of their behaviour inbiological systems is still missing.Lecithinases from different species oforganisms can differ in their behaviour in the cells of one animal, and cellsfrom different animals can react differently to an enzyme from one source.The lecithinases of CZ. welchii and CZ. oedematiens have the same action onlecithin in vitro but they differ in their action on the red blood cell. CZ.welchii a-toxin haemolyses sheep red cells readily and horse red cells slowly ;CZ.oedematiens y-toxin hzemolyses sheep cells slowly and horse cells readily ;CZ. oedematiens &toxin haemolyses both species of cells readily.ll Table I1cm.2/sec.18The other bacterial lecithinases have not been purified.TABLE 11.Reactions of different lecithinases with red blood cells from different species.(Adapted from Macfarlane, Biochem. J . , 1960, 47, 270.)Reaction rates.7 A \ Hydrolysis of Hydrolysis ofLecithinme (amounts phospholipids in phospholipids ex-intact cells. tracted from cells.lecithin in vitro). Sheep. Horse. Sheep. Horse. Sheep. Horse.CLwelchiia ............... +++ + +++ + +++ +++Cl. oedematiens y ......... + +++ + +++ +++ +++CL oedematiens ......... +++ +++ +++ +++equipotent in action on H~molysis.shows that the species differences in hemolysis of red cells occur only inthe intact cells; they are not apparent when the lecithinases act on thelecithin in the phospholipids isolated from the cells.The species differencesl6 McGaughey and Chu, J . Qen. Mimobiol., 1938, 2, 234.Chu, ibid., 1949, 3, 255.van Heyningen and Bidwell, Biochem. J., 1948, 42, 130.19 Pappenheimer and Robinson, J . Immunol., 1937, 32, 291VAN HEYNINQEN : BACTERIAL TOXINS. 307therdfore must be concerned with the nature of the intact cells and theaccess that the enzymes have to their substrates in them. Such access couldbe controlled by properties of the enzyme, such as molecular weight, shape,and charge, which would not necessarily affect its action on isolated lecithin.The bacterial lecithinases can also differ in their toxicity to animals.It has been observed that amounts of lecithinase from three different strainsof Cl.bifermentuns that were equipotent in their enzyme activity in vitrowere respectively 9, 60, and 70 times less toxic to mice, and less haemolyticto red cells, than the corresponding amount of Cl. welchii lecithinase. Inthis case there were differences not only between different species oforganisms, but between strains of the same species.14There are differences in species susceptibility to other toxins besides thelecithinases. Thus the guinea-pig is 1000 times more susceptible to diphtheriatoxin than the mouse 1 ; some workers 20 have found that the guinea-pig isG000--8000 times more susceptible to the toxin of Cl.botulinum type B thanthe mouse, others 21 have found only a 3-fold difference ; the ratio of guinea-pig toxicify/rabbit toxicity has been found to vary from 3 to 354 withdifferent samples of toxin from Cl. tetuni.22 The mitis and gravis types ofCorynebacterium diphtheriae cause diseases that differ considerably in severityalthough no differences have yet been observed in their toxins. It isconceivable that while these toxins are equally toxic to guinea-pigs theymay differ in their toxicity to man.Differences in toxicity of toxins having the same biochemical actioncould also be caused by the presence, in the body, of inhibitors, as is the casewith the B. cereus lecithinase. This lecithinase is 4-8 times less toxic tomice than the CZ.wekhii lecithinase because it is inhibited by a constituentof normal serum. In the absence of serum it is just as hEmolytic as theCZ. welchii 1ecithinase.l' The same argument does not apply to the CZ.bifermentam lecithinase because this enzyme is not inhibited by normalserum and it is also less hEmolytic than the CZ. welchii enyme. Enzymeand toxin activators in the body should also be considered, although thesehave not yet been identified. It has been shown that constituents of brothand serum may " potentiate " botulinus and tetanus toxins, and that thispotentiation is not merely a protection of the toxins against denaturation indilute solution .23The hemolysis that is caused by lecithinase acting on the red blood cellfollows the breakdown of cell ph~spholipid,~~ but it is not clear how thebreakdown of lecithin or phospholipid in other tissues of the body results inharmful effects. One means is suggested by the recent discovery that CZ.welchii lecithinase inactivates the magnesium-activated adenosine tri-phosphatase (Mg-ATPase) of muscle.This enzyme, which is distinct from*O Stevenson, Helson, and Reed, Canad. J . Res., 1947, 85, E , 14.21 Lamanna and Glassmann, J. Bacb., 1947,5;4, 576.22 Llewellyn Smith, Bull. Health Organ. League of Nations, 1942143, 10, 104.2s (a) Traub, Hollander, and Friedemann, J . Bact., 1946, 62, 169; (b) Wentzel,Macfarlane, Biochern. J., 1860, 47, 270. Sterne, and Polson, Nature, 1950,166,739308 BIOCHEMISTRY.the myosin-ATPase, has a phospholipid prosthetic group which is essehtialfor its activity and is destroyed by the lecithinase.2sDiphtheria Toxin.-The properties of this toxin, which is the first to havebeen obtained pure,26 are given in Table 111.Recent work on its mode ofTABLE 111.Properties of puri$ied toxins.Diphtheria toxin. Botulinus A toxin. Tetanus toxin.References U 1, 35, b 1, 36, c.Biological propsr ties Lsthal, histo toxic. L9 thal, neurotoxic, Lethal, neurotoxic.haemagglutinat-ing, precipitatesnormal serum.MLD/mg.(per kg. mouse) 3.5 620,000 200,000(per kg. guinea-pig) 3500 1,200,000 1,200,000x 107 (200)Isoelectric point 4.1 5.5 5.1Sedimentation const. 4-6 17.3 4.5Molecular weight 72,000 900.000--1,130,000 67,000Elementary analysisDiffusion const.6.0 cm.2/sec. 2.14 cm.2/sec. -Frictional coeff., f/fo 1-22 1.76 -C, 51-47; H, 6.75; C, 53-73; H, 7.02; N, 15.7; S, 1.04;N, 16.0; S, 0.75; N, 16.29; S, P, 0.065P, <0.03(%)0.437 ; P, 0.052-0.059a, Peterman and Pappenheimer, J. Phys. Chem., 1941, 45, 1 ; Pappenheimer,Lundgren, and Williams, J . Exp. Med., 1940, 71, 247; Pappenheimer, J. Bact., 1942,43, 273.b, Buehler, Borner, Schantz, and Lamanna, J. Bad., 1946, 51, 571; Putnam,Lamanna, and Sharp, J. Biol. Chem., 1946,165, 735.c , Dunn, Camien, and Pillemer, Arch. Biochem., 1949, 22, 374.TABLE IIIA.Properties of purijed toxins.Diphtheriatoxin. Botulinus A toxin.PerAmino-acid composition ,--A-, (-&-, mol.Alanine Arginine - ; 3.8 3.92; 4-62 394; 239Aspartic acid Cysteine -; - 20.26; 0.27 1370; 20Cystine Glutamic -; - 0.53; 15-57 40; 953G1 ycine Histidine - ; 2.4 1.38; 1.03 166; 60Leucine isoLeucine -; - 10.30; 11.94 708; 820Lysine Methionine 5.3; - 7.74; 1.06 477; 64Phenylalanine Proline _.- 1.17; 2.60 64; 203Serine Threonine - a - 4.36; 8.49 374; 642Tryptophan Tyrosine 1.4; 9.5 1.86; 13.50 82; 672Valine - 5.29 406%. %.acidTotanus toxin.Per(-A----, mol. %*- . , 3.36 -; 1315.3 ; - 76; -- ; 10.3 -. , 473-34; 1-15 30; 58.23; 9.36 43; 4810.0 ; 1.78 46; 84.91; - 20; -- ; 5.13 -; 290.91; - 3; -5.39 31production by the organism might provide a clue to its mode of a c t i ~ n . ~ ’ ? ~ ~The production of toxin is always accompanied by the production of extra-2: Kielley and Meyerhof, J.Biol. Chem., 1950, 183, 391.26 Eaton, J. Bact., 1936, 31, 367; Pappenheimer, J. Biol. Chem., 1937, 120, 543.2 7 Idem, ibid., 1947, 167, 251.2 8 Pappenheimer and Hendee, ibid., 1947, 171, 701 ; 1949, 180, 597VAN HEYNINGEN : BACTERIAL TOXINS. 309cellular porphyrin, and the production of both of these is inhibited when theiron content of the culture medium is above an optimum level of about100 pg./l. For every 4 atoms of iron in excess of this level 4 fewer moleculesof porphyrin and 1 fewer of toxin are produced. The excess of iron is takenup by the organism which at the same time acquires a greater concentrationof an intracellular haem protein which is probably identical with cytochromea respiratory pigment which is concerned in the oxidation of succinicacid.When the toxin production has fallen to half the maximum valueobtained in low-iron media the rate of oxidation of succiriic acid has increasedto half the maximum obtained with organisms grown in high-iron media.Since the proportions of iron : porphyrin : protein in cytochrome b are also4 : 4 : 1 it was suggested that the toxin might be the protein moiety ofdiphtherial cytochrome b. In the absence of iron the organism continues tosynthesise the porphyrin and the protein component of cytochrome b, butsince they cannot be built into cytochrome b without iron they are dischargedas waste products. Diphtheria toxin would then be very similar to, but notidentical with, the protein moiety of mammalian cytochrome b, and wouldexert its toxic effect by competitively inhibiting the synthesis of cytochromeb in the tissues of the host.But the iron content of the growth mediumdetermines, not only whether the porphyrin shall be intra- or extra-cellularand bound to protein or free, but what its constitution shall be. Spectro-scopic observations had led some workers 30 t? conclude that the extracellularporphyrin was coproporphyrin (*CH,*CH,*CO,H substituents in positions 2and 4), and others 27 that it was haematoporphyrin [*CH(OH)*CH, substi-tuents]. Recent more detailed examination shows that, it is very likely to bec~proporphyrin.~l The biological conversion of the *CH:CH2 substituentsof the protoporphyrin of the intracellular haem protein into the *CH( OH)*CH,substituents of haematoporphyrin would involve only a hydration, but theconversion of vinyl groups into propionic acid groups is considered to be lesslikely.32 Other experiments also suggest that the intracellular haem proteinand the extracellular coproporphyrin are not directly intraconvertible. Theessential results of this work 33 are summarised in Table IV.The diphtheriabacillus was grown in low- and high-iron media containing glycine labelledwith 15N, and it can be seen that the amount of isotope incorporated into theintracellular haem was more than twice as great as that incorporated intothe coproporphyrin in the toxic filtrate. These results further suggest thatthe coproporphyrin was synthesised later than the ha,em protein, after theisotope content of the glycine had been diminished by randomisation.Thiswork does not disprove the interesting hypothesis that diphtheria toxin isthe protein moiety of diphtherial cytochrome b, but it does suggest that thehypothesis needs elaboration.m Rawlinson and Hale, Biochem. J . , 1949, 45, 247.30 Wadsworth, Crowe, and Smith, Brit. J . Ezp. Path., 1935, 16, 201.31 Gray and Holt, J . Biol. Chem., 1947, 169, 235; Biochem. J . , 1948, 43, 191.aa Watson, Pass, and Schwartz, J . Biol. Chem., 1941, 139, 583.33 Hale, Rawlinson, Gray, Holt, Rimington, and Smith, Brit. J . Exp. Path., 1950,31, 96310 BIOCREWSTRY.The small amount of uroporphyrin that appears in culture filtrates(Table IV) belongs to the isomeric series of aetioporphyrin I , whereas thecoproporphyrin and the intracellular iron-protoporphyrin belong to the 111TABLE IV.Incorporation of 16N into intracellukr h e m and extracellular porphyrins of C .diphtheriae grown in mcdia containing glycine enriched with 15N.(Adapted from Hale et u Z .~ * )16N inCopro- 16N in intra- Uro-Toxin in porphyrin copro- cellular porphyrin 15N in uro-Fe in culture in culture porphyrin haem in culture porphyrinmedium filtrate filtrate (atoms yo (atoms % filtrate (atoms %(pg. I d . ) . (unitslml.) . (pg. /ml.). excess). excess). ( p g . /ml.). excess).0-147 55 1.90 0.51 - 0.04 0.460.753 5 0.16 0.28 1.26 0.02 0.34series. Moreover, Table IV shows that the ratio, units of toxin : pg. ofuroporphyrin, decreases 5.5-fold as the iron content of the medium increases&fold, while the ratio of toxin to coproporphyrin remains unchanged.Thissuggests that the uroporphyrin is not directly concerned with toxinproduction.Neurotoxins.-Toxins that act specifically on the nervous system areproduced by the Gram-positive anaerobes, Cl. botuliwm and GI. tetuni, andby rough and smooth variants of the Gram-negative aerobe, Shigella shigae.The neurotoxins of the anaerobes are 1.5 X lo4 to 3 x lo8 times as toxic asaconitine and are the most toxic substances known. They appear to bequite simple proteins, but we have no idea of their mode of action. Pharma-cological studies 34 suggest that they act on the peripheral motor nerves bypreventing the output of acetylcholine from the nerve endings.Tetanustoxin is thought to act on the central nervous system as well. Their actionis extremely slow, but they appear to be fixed quite rapidly on theirsubstrates. It is conceivable that they may be kinases, i.e., substances thatcatalyse the production of biologically active catalysts from inactin pre-cursors. The pharmacology of Sh. shigae neurotoxin has not yet beenstudied.The four types of CI. botulinum, A, B, C, and D, eachproduce it distinct toxin. The type-A toxin has been purified andcrystallised,36 and its chemical and physical properties are shown in Table 111.It can be calculated that about 20 million molecules of toxin (about 10molecules per nerve cell) will kill a mouse. Unlike all other bacterial toxins,which are effective only when administered parenterally, botulinus type-Atoxin is effective by mouth.Presumably this is because its toxicity is notdestroyed by the proteolytic enzymes of the gut, but the possibility still34 Ambache, J. Physiol., 1949, 108, 127; Ambache, Morgan, and Wright, ibid.,1948, 107, 45; Bvit. J . Exp. Path., 1948, 29, 408; Burgen, Dickens, and Zatman, J.Physiol., 1949, 109, 10.35 Lamanna, Eklund, and McElroy, J. Bact., 1946, 62, 1; Abrams, Kegeles. andHottle, J. Biol. Chem., 1946, 164, 63.Botulinus ToxinsVAN HEYNINGEN : BACTERIAL TOXINS. 31 1remains that the toxin is broken down into smaller toxic fragments. Suchfragments would pass through the wall of the gut more radily than amolecule with a molecular weight of a million.The type-B toxin, which is as toxic as the type-A toxin, has been obtainedin a purified but amorphous condition.21 Its diffusion constant (20") of7.22 x lo-' cm.2/sec.suggests a molecular weight of 60,000, and thereforethat it is less toxic per molecule. It is relatively insoluble in water abovepH 5.0-5.5, and its solubility is not increased by the addition of salt.The type-D toxin has recently been partly p~rified,~3(b) but very fewdetails are yet available. It appears to have the astonishing toxicity of1.3 x 1O1O MLD (per kg., mouse) per mg. This would mean that it isabout 20,000 times as toxic as the type-A toxin, or that 10 mg. would beabout the MLD for the entire human population of the world.The chemical and physical properties of tetanospasmin,the lethal toxin of CZ.tetani, are summarised in Table 111. This toxin waspurified and crystallised36 at the same time as botulinus type-A toxin andis equally toxic. Like diphtheria toxin, its production is inhibited by quitelow concentrations of iron, but some strains of CZ. tetani are apparently notaffected by iron.37 It is unlikely that the toxin is concerned with cyto-chrome b because the organism does not contain cytochromes. Whenkept, the purified toxin becomes less soluble and less toxic without losing itsantigenicity. At the same time its sedimentation constant changes from4.5 to 7 and it has been suggested that an atoxic dimer is formed. Thischange is catalysed by low concentrations of f~rmaldehyde.~~This toxin is unlikely to be as toxic as those of theanaerobes, the best preparation to date having only MLD 20 (per kg.,mouse) per mg.39 It is an intracellular toxin and appears in culture filtratesonly after the organism has autolysed.It is produced by both rough andsmooth variants of Xh. shigae and it has been suggested that it is the toxiccomponent of the toxic somatic antigen (see below).40 The production ofthe toxin is also inhibited by iron.39Ha,emolysins.-Oxygen-ZabiZe Haemolysins. The 0-toxin of Cl. welchii,the tetanolysin of CZ. tetani, the pneumolysin of the pneumococcus, and thestreptolysin 0 of the hzmolytic streptococcus are haemolysins that haveseveral properties in common in spite of their diverse sources. They arereversibly activated by reducing agents, irreversibly inhibited by cholesterol,and serologically related, i.e., any one of them is neutralised by the anti-toxin to any of the others.41 It has been shown that reducing agents arenecessary for the production of streptolysin 0 as well as its activity,42 and itis interesting that ascorbic acid is as effective as, e.g., cysteine in theTetanus Toxin.Shiga Neurotoxin.36 Pillemer, Wittler, Burrell, and Grossberg, J .Exp. Med., 1948, 88, 205.37 Mueller and Miller, J . Immunol., 1943, 47, 15; 1945, 50, 377.as Pillemer and Moore, J . Biol. Chem., 1948, 173, 427.39 Dubos and Geiger, J . Exp. Med., 1946, 84, 143.40 Boroff, J. Bact., 1949, 57, 617 ; Boroff and Macri, ibid., 1949, 58, 387.41 Todd, J . Path. Bact., 1934, 39, 299; Brit. J . Exp. Path., 1941, 22, 172.42 Slade and Knox, J .Bact., 1950, 60, 301312 BIOCHEMISTRY.production of the haemolysin, but completely ineffective for its activation.68None of these toxins has been obtained in a pure state, but streptolysin0 43 and pneumolysin44 have been partly purified. They appear to beproteins. These haemolysins are interesting not only because of theircommon immunological properties but also because their biological activityclosely resembles that of the chemically very dissimilar saponins : (1) Bothtypes of substance are haemolytic, cardiotoxic, and lethal. (2) The hzemolyticactivity of both is inhibited by cholesterol and by a benzene-soluble, heat-stable substance in hzemolysed red blood cells.44+ 45 (3) A single dose ofstreptolysin 0 has no effect on the isolated frog's heart, except that it releasesan inhibitory substance which is heat-stable, non-dialysable, and chloroform-soluble.If the inhibitory substance is washed away the heart is sent intosystolic contracture by a second dose of streptolysin 0. Pneumolysin and0-toxin have the same effect.46 (4) Mice injected with the bacterialhaemolysins, or with saponin, rapidly develop a temporary (non-immune)resistance specifically to lethal doses of both types of s~bstance.~' (5) Micecan be protected against streptolysin 0 by previous injection of cholester01.~~A saponin-like prosthetic group on the oxygen-labile haemolysins wouldnot only explain their biological relationship to the saponiris, but it couldact as a hapten and account for their close serological relationships.Thescanty available evidence does not rule out such a possibility. It must beborne in mind, however, that the saponins are active by virtue of theirsurface-active effects, and that cholesterol inhibits the hamolytic action ofother surface-active agents, such as sodium oleate.Oxygen-stable Haemolysins. There are many other oxygen-stablebacterial haemolysins besides the lecithinases which have been discussed.The haemolysis of red cells by the GI. septicum haemolysin is preceded by aninduction period during which the main reaction takes place, and the cellsswell up and become translucent without lysis. The lysis that then followsis independent of the haemolysin since it still takes place if antitoxin isadded a t the end of the induction period.On the other hand, the spontaneouslysis of toxin-treated cells is inhibited by sucrose, which does not inhibit theprimary reaction between the toxin and the cells.49 This phenomenon isreminiscent of the " hot-cold " haemolysis by CZ. welchii lecithinase. If asuspension of red cells is incubated a t 37" with a low concentration of thishaemolysin no apparent hzmolysis takes place ; but if the suspension is thencooled rapid hEmolysis ensues, and this hamolysis is not inhibited if anti-toxin is added before cooling.50 Hamolysis by streptolysin S (see below) isalso preceded by an induction period.43 Herbert and Todd, Biochem. J . , 1941, 35, 1124.44 Cohen, Halbert, and Perkins, J . Bact., 1942, 43, 607.45 Smythe and Harris, J .Immunol., 1940, 38, 283.46 Bernheimer and Cantoni, J . Exp. Med., 1945, 81, 295; Cantoni and Bernheimer,4 7 Bernheimer and Cantoni, ibid., 1947, 86, 193.48 Hewitt and Todd, J . Path. Bact., 1939, 49, 45.49 Rernheimer, J . Exp. Med., 1944, 80. 309,321, 333; J. Gen. Physiol., 1947,30, 337.50 van Heyningen, Biochem. J., 1941, 85, 1257.ibid., p. 307VAN HEYNINOEN : BACTERIAL TOXINS. 313Some strains of streptococci produce, in addition to streptolysin 0, ahaemolysin called streptolysin S because it was first found when the organismswere grown in serum-broth, or when organisms grown on plain broth wereextracted with serum. It has since been shown that serum can be replacedas an extractant by a crude solution of egg-yolk le~ithovitellin.~~ As atoxigenic factor in growth media serum can be replaced by ribonucleic acid,and even more effectively by an active factor (AF) obtained by digestingribonucleic acid with ribonuclease.Small amounts of maltose (~/20,000) orglucosamine (~/10,000) are also necessary. 52 Streptolysin S is also producedwhen washed " resting '' adult streptococcal cells are suspended in a milieucontaining AF, glucosamine, sodium, potassium, magnesium, and phosphateions, and a reducing agent. The process by which the haemolysin is thusproduced is probably enzymic. By the use of this method a highly activepreparation of streptolysin S was obtained which appeared to contain AFand a hexosamine. The AF in this product did not appear to be essentialfor hzemolytic activity because 40% of the total phosphorus could beconverted into inorganic phosphorus (by treatment with a phosphatase)without loss of activity. A mutant strain that did not produce streptolysinunder these conditions gave rise to an inactive substance with roughly thesame constitution.53The staphylococci produce at least three, and possibly four, oxygen-stable haemolysins. Little is known of their nature or their mode of action.They are differentiated immunologically and by their specificity for the redcells of different species. The f3-toxin is a '' hot-cold " haemolysin, like theCZ. wekhii lecithinase, but it is not known whether the lysis that takes placeon cooling is independent of the haemolysin. Lysis (at 37") of " p-conditioned " cells also takes place if they come in contact with a number ofdifferent agents, vix.: (i) Glycerol.54 (ii) Bacterial l i p a ~ e . ~ ~ (Possibly thelipase acts in this case by producing glycerol. Lipases cause hzemolysis ofnormal cells when sufficient fat is present by producing hemolytic soaps, butin this case no fat was present and normal cells were not hzcmolysed.)(iii) Certain broth constituents.a (iv) Bacterial p r ~ t e a s e . ~ ~ (Possibly theprotease produces substances similar to the broth constituents.) (v) A heat-stable substance produced by Strep. agala~tiae.~~The toxins of a largenumber of Gram-negative bacteria, e.g., the shigellae, salmonellae, neisseriae,brucella, produce roughly the same symptoms, and they appear to beroughly similar complexes.They are identical with the dominant 0 somaticantigens that are located on the surfaces of smooth variants of these bacteria.These antigens consist of polymolecular complexes of phospholipid, poly-Somtic Antigens of Gram-negative Bacteria.5751 Herbert and Todd, Brit. J. Exp. Path., 1944, 25, 242.52 Bernheimer and Rodbart, J. Exp. Med., 1948, 88, 481.53 Bernheimer, ibid., 1949, 90, 373.54 Llewellyn Smith, and Price, J. Path. Bact., 1938, 47, 361.5 5 Christie and Graydon, Austral. J. Exp. Biol. Med. Sci., 1941, 19, 9.56 Munch-Peterson and Christie, J. Path. Bact., 1947, 59, 377.5' For references see ref. 3314 BIOUHEMISTRY.saccharide, and conjugated protein. In their native state it is likely thatthey contain additional loosely- bound lipid and phospholipid, and thatthese complexes are aggregated to form particles of high molecular weight,solutions of which show anisotropy of flow.The polysaccharide-conjugated protein component of the complex issplit into a degraded polysaccharide and an acidic conjugated protein onacid hydrolysis; and into a viscous undegraded polysaccharide and asimple amphoteric protein on alkaline hydrolysis.The toxicity of thecomplex appears to be associated with the undegraded polysaccharide orwith the conjugated protein, according to the method of hydrolysis. Theundegraded polysaccharide is a hapten which combines readily with theconjugated protein to form an antigen which is immunologically indistin-guishable from the original 0 antigen.The conjugated protein will alsocombine with undegraded polysaccharide from heterologous species to formantigens whose specificity is determined by the polysaccharide. Theamphoteric protein will also combine with undegraded polysaccharide butthe resultant complex is not antigenic.The work that has been done on the toxic complexes of Gram-negativebacteria has been mainly concerned with their immunology and very littleis known about the toxic component.W. E. VAN H.4. GENERAL NUTRITION.Poule.--Proteirt and Amino-acids. The chick's tryptophan requirementwas met when the diet contained 0.18% of' L-trypt0phan.l The chick alsoutilised 17--40y0 of the D-tryptophan of an experimental diet.2 The grossand microscopic pathology of tryptophan deficiency was described.g TheL-threonine requirement was 0-45y0 of the diet, D-threonine not beingutilised.4 Raising the protein content of the diet augmented the chick'srequirement for lysine 6 and methionine.6 The chick required 0.6--043% ofDL-phenylalanine in the diet together with a similar amount of L-tyrosine orD- or L-phenylalanine to meet the tyrosine requirement.' The requirementof turkey poults was between 0.75 and 1025% of the diet for glycine,8 about0.5% for methionine, and 0.3% for cy~tine.~ Although methionine couldreplace cystine in the diets of poults, cystine could not adequately replacemethionine.. There was normal feather pigmentation in young poults ondiets containing 1.3% of lysine.1° The leucine requirement of the laying1 Wilkening, Schweigert, Pearson, and Sherwood, J .Nutrit., 1947, 34, 701.* Wilkening and Schweigert, J . BioE. Chem., 1947, 171, 209.3 Brown, Wilkening, and Schweigert, Proc. Soc. Exp. Biol., 1948, 68, 672.4 Grau, J . Nutrit., 1949, 37, 105.6 Almquist, Proc. SOC. Exp. Biol., 1949, 72, 179. ' Grau, J . Biol. Chem., 1947,170, 661.8 Kratzer and Williams, J . Nutrit., 1948, 85, 315,9 Kratzer, Williams, and Marshall, ibid., 1949, 37, 377.Idem, ibid., 1948, 36, 99.10 German, Schweigert, Sherwood, and James, Poultry Sci., 1949, 28, 166DUCKWORTH : GENERAL NUTRITION. 316hen appeared to be between 1-35 and 2.0% of the diet.ll The argininecontent of the diet should be 6% of its protein content.I2The leucine, holeucine , p hen y lalanine, valine , met hionhe, tryptophan ,histidine, arginine, threonine, and lysine contents of light and dark meat,and livers and gizzards of poultry, were reported.13 The arginine, histidine,lysine, and threonine contents of some common feeding stuffs weredetermined.14From a third to a half of native and added lysine was destroyed by auto-claving soya-bean oil meal.Less destruction took place with dry heat.There was only a slight loss of native or added lysine when isolated soya-bean protein was heated but all the lysine was destroyed on heating thiswith sucrose.16 Autoclaving lysine with glucose (" cerelose ") made itunavailable to chicks.16 However, there was no destruction of methionineautoclaved at 120" for 2 hours in 8% aqueous g1uc0se.l~ Refluxing purifiedsoya- bean globulin with aqueous glucose caused partial destruction of lysine,arginine, tryptophan, and histidine.ls Excessive heating (130"/60 minutes)impaired, and moderate heating (100"/30 minutes) improved, the nutritivevalue of soya-bean protein.The former, but not the latter, product wasimproved by adding methionine, cystine and 1~sine.l~ Products of auto-claving for 45 minutes at 4 lb./sq. in. were almost equal: in nutritive value tothose treated for 4 minutes at 15 lb.2O Drastic heating destroyed part ofthe lysine of soya bean and reduced the digestibility of the rest. Theabsorption and utilisation of methionine were also reduced.21 Processingthe meal did not affect the lysine, cystine, or methionine content of eggslaid by poultry fed on the diet.22 Wheat protein was a better source ofarginine, leucine, methionine, and tryptophan than the analysis indicatedbut was markedly deficient in l y ~ i n e .~ ~ The availability of tryptophan inraw soya-bean oil meal was 20%, in fish meal 40% and in casein loo%.%The inferiority of raw to heated soya-bean protein arose in part from thepresence of thermolabile enzyme inhibitors, of which there were at leastthree.26 The inhibitors, which were easily extracted, interfered with theaction of trypsin and erepsin, but not of pepsin or papain, in ~ i t r o . ~ ~ Auto-l1 Cravens, Poultry Sci., 1948, 27, 562.12 Almquist and Merritt, Proc. SOC. Exp. Biol., 1950, 73, 136.l3 Millares and Fellers, J . Amer.Dietetic ASSOC., 1948, 24, 1057.l4 Schweigert, Poultry Sci., 1948, 27, 223.l5 Evans and Butts, J . Biol. Chem., 1948, 175, 15.l6 Stevens and McGinnis, ibid., 1947, 171, 431.l7 Graham, HSU, and McGinnis, Science, 1949, 110, 217.1* Patton, Hill, and Foreman, ibid., 1948, 108, 659.l8 McGinnis and Evans, J . Nutrit., 1947, 34, 725.2 1 Evans and McGinnis, J . Nutrit., 1948, 35, 477.22 Evans, Davidson, and Butts, Poultry Sci., 1950, 29, 104.23 Jeppesen and Grau, ibid., 1948, 27, 588.24 Schweigert, Arch. Biochem., 1948, 19, 265.2Q Borchers, Ham, Sandstedt, Ackerson, Thayer, and Mussehl, Univ. NebTaekuClaudinin, Cravens, Elvehjem, and Halpin, Poultry Sci., 1948, 27, 370.25 Bowman, ibid., 1948, 16, 109.Agric. Exp. Stat. Res. Bull., No. 152, December 1947316 BIOCHEMISTRY.claving at 15-20 Ib.pressure for 20 to 30 minutes destroyed the factors.27~ 28p 29There was no evidence of preferential interference with the liberation ofspecific amino-acids during hydrolysis with ~ a n c r e a t i n . ~ ~ ~ 33 After pre-liminary digestion of raw and heated soya-bean oil med with pepsin therates of in-vitro digestion with pancreatin were the A highlypurified preparation of an antitryptic factor failed to impair the nutritivevalue when added to a normal soya-bean chick ration.31 The amino-acidspresent in the inhibitor have been identified.32If the free gossypol was not above 0.12% of the meal the inciusicn of10% of hydraulic-press cotton-seed meal in the ration did not depress thehatchability of eggs.34 The correlation between the nutritive value ofcotton-seed meals and their content of gossypol and gossypurpurin waspoor ; mechanical removal of pigment glands, hydraulic pressure, andextraction with diethyl ether (but not hexane) removed the toxicDiscoloration of eggs was prevented by isopropanol extraction of cotton-seed meal, but not by hydraulic-pressure treatment .36 Wafer-soaking oflinseed meal destroyed its toxicity for chicks 379 38 and turkeyMeals made from flax seed treated with gaseous ammonia before storagewere non-toxic to chicks.40 There was no trypsin inhibitor in linseed meal.41A new chemical method has been described for determining the digesti-bility of protein by poultry.Good reproducibility was found when resultson normal droppings were compared with those obtained by analyses onpure urine and faeces secured by the artificial-anus technique.42 TheDiakow and the Stotz methods did not yield comparable results.P3 Therewas a good correlation between the evaluations of feed protein by chemicaland growth methods.44The blastoderm of the chick embryo utilisedD-glucose, D-mannose, D-frUCfoSe, ~-galactose, and D-maltose, but not D-arabinose, D-xylose, D-ribose, L-sorbose, sucrose, lactose, trehalose, cellobiose,melifiose, raffinose, melezitose, or glycogen.The minimum concentrationsof the five sugars necessary for embryonic development were respectively :27 Borchers, Ackerson, and Mussehl, Poultry Sci., 1948, 27, 601.28 Desikachar, De, and Subrahmanyan, Ann.Biochem. Ezp. Med., 1948, 8 , 93.29 Ackerson, Borchers, and Mussehl, Univ. Nebraska Agric. E:E:E:E:E:E:E:E:E:E:E:, Stat. Res. Bull.,30 Leiner and Fevold, Arch. Biochem., 1949, 21, 395.31 Borchers, Ackerson, and Mussehl (with A. Moehl), ibid., 1948, 19, 317.32 Work, Biochem. J . , 1948, 42, xlix.33 Ingram, Riesen, Cravens, and Elvehjem, Poultry Sci., 1949, 28, 898.34 Heywang, Denton, and Bird, ibid., p. 610.3 5 Boatner, Altschul, Irving, Pollard, and Schaefer, ibid., 1948, 27, 315.36 Kuiken, Lyman, and Hale, ibid., p. 742.37 Mani, Nikolaiczuk, and Maw, Sci. Agric., 1949, 29, 86.38 MacGregor and McGinnis, Poultry Sci., 1948, 27, 141.a9 Kratzer, ibid., 1949, 28, 618.40 Altschul, Karon, and Schaefer, ibid., 1948, 27, 408.4 1 Mani, Nikolaiczuk, and Maw, Sci.Agric., 1949, 29, 91.42 Ekman, Emanuelson, and Fransson, Kgl. Lantbrukshtigsk. Ann., 1949, 16, 749.48 Hsie, Meld. Norges Landbruksh0gsk., 1948, 28, 399.44 March, Stupich, and Biely, Poultry Sci., 1949, 28, 718.Carbohydrate and Fat.No. 156, June 1948DUCKWORTH : GENERAL NUTRITION. 31720, 20, 50, 200, and 400 mg./100 ml.45 High levels of lactose in the dietrapidly stopped egg production but did not affect the hatchability of theeggs laid. Simultaneous high levels of fat in the diet prevented the effect.46Growth was improved by small additions of cellulose to inferior, but not tosuperior, purified diets, perhaps by permitting increased intestinal synthesisof essential ~ubstances.~' Small additions of sawdust also improved growthon synthetic rations.48 The adverse effect of added fibre in rations wasmore serious in cases where the ration was low in energy.49Phenol, resorcinol, phloroglucinol, and a-naphthol, but not benzoic acid,p-aminobenzoic acid, or chloral hydrate, increased urinary glucuronic acidexcretion.Over 75 yo of injected phenol, resorcinol, quinol, phloroglucinol,and catechol was rapidly excreted as glucuronides and ethereal s ~ l p h a t e s . ~ ~The incidence of spontaneous arteriosclerosis in chickens was reduced byremoving most of the fat and cholesterol from a normal diet.62 It wasintensified in proportion to the amount of cholesterol added to the diet.53Cholesterol-induced arteriosclerosis declined in severity when birds weretransferred to normal or cholesterol-free diet.54 Dosage with desiccatedthyroid reduced the incidence of arteriosclerosis in cholesterol-fed chicks ;dosage with potassium iodide yielded contradictory results.55 Feeding drycholesterol intensified coronary arteriosclerosis but did not increase theincidence of the c ~ n d i t i o n . ~ ~Experiments in which radio-calcium was fed showed that60-75% of the calcium of the egg came from the daily intake, and the restfrom skeletal stores.57 Variations in the uptake of radio-calcium 58 andradio-phosphorus 59 in shell, yolk, and white immediately after dosagedepended on the stage of maturity of the egg component at the time.Calcium deficiency increased the incidence and intensity of black pigment-ation in the down feathers of chicks with brown plumage, and in spite ofhigh vitamin D intake.60 Only 5% of the dietary radio-phosphorus [givenas Ca,(PO,),] appeared in the egg,61 presumably because of the endogenoussupply of phosphorus from resorbing bone.The greater utilisation ofinjected Na,PO, than of glycerophosphate-both labelled with radio-phosphorus-indicated that organic phosphate esters are not used directlyMinerals.4 5 Spratt, J . Exp. Zool., 1949, 110, 273.46 Couch, Barki, Sunde, Cravens, and Elvehjem, J . Nutrit., 1949, 38, 105.4 7 Lepp, Harper, and Elvehjem, Poultry Sci., 1949, 28, 372.4 8 Davis and Briggs, ibid., 1948, 27, 117.48 Robertson, Miller, and Heuser, ibid., p. 736.50 Sperber, Kgl. Lantbrukshogsk. Ann., 1948, 15, 108.5 1 Idem, ibid., 1949, 16, 446.5 2 Horlick, Katz, and Stamler, Amer.Heart J . , 1949, 37, 689.53 Idem, ibid., 1949, 38, 336.5 5 Dauber, Horlick, and Katz, Amer. Heart J., 1949, 38, 25.5 6 Paterson, Slinger, and Gartley, Arch. Pathol., 1948, 45, 306.ci7 Driggers and Comar, Poultry Sci., 1949, 28, 420.5 8 Spinks, Berlie, and O'Neil, Science, 1949, 110, 332.59 Spinks, O'Neil, Jowsey, Lee, and Reade, Canad. J . Res., 1948, 26, D, 163.6o McGinnis and Carver, PouEtry Sci., 1948, 27, 115.61 O'Neil, Jowsey, Lee, Reade, and Spinks, Science, 1948, 107, 295.54 Idem, J . Lab. Clin. Med., 1949, 54, 1427318 BIOCHEMISTRY.in phospholipin synthesis but that they are first hydrolysed and the thenfree phosphate is incorporated into the phospholipin molecule.62 Radio-phosphorus was uniformly distributed in relation to total phosphorus in chickembryos from the eggs of a hen fed with radio-phosphor~s.~~ The changesin ribonucleic and deoxyribonucleic acid phosphorus in the whole embryo 64and in heart and liver 65 during incubation have been determined.Orthophosphates, P-Ca,(PO,),, and mono- dLY, and tri-calcium phosphateswere excellent .sources of phosphorus snd slightly superior to bone meal.Defluorinated rock phosphate and superphosphate were slightly inferior tobone meal.Meta- and pyro-phosphates were of no value, except calciumdihydrogen pyrophosphate.66 Raw rock phosphate (3.3% of fluorine) did notinterfere with growth, maturity, or egg produotion of pullets, or fertility orhatchability of eggs. Although there was high storage of fluorine in thebone there was no increase in the fluorine content of the edible portion of theeggs and second generation chicks grew normally.67 Phytate-phosphoruswas poorly utilised and increased the requirement for vitamin D3.6*,6QCalcium and phosphorus requirements of young White Leghorn cockerelswere 1.07 and 0.65% in the ration respe~tively.~~ The requirements ofBroad-breasted Bronze turkeys appeared to be 1.2% of the ratio for calciumand about l.Oyo for phosph~rus.~~ Young chicks required from 0.38 to0.47% of phosphorus in the ration.68 If the ration was high in phytate-phosphorus it should contain o.4y0 of phosphorus in inorganic form.69Removal of the centre toes of the feet for analysis in vitamin D assayswas suggested. The operation had no effect on growth or calcification ofthe rest of the skeleton.72 Seasonal differences in the ash content of tibiaeof chicks (a source of error in vitamin D assays) were thought to result fromhigher D reserves in the summer- and autumn-hatched chick than in winter-hatched chick~,7~ Crooked keels in birds were not associated with inferiorbody or bone growth.74The clinical and neuropathological effects of magnesium deficiency weredescribed and resembled those in the rat.75 The pathology of potassiumdeficiency has also been described and the requirement found to be 0.24%in the ration ; lower potassium levels were satisfactory if phosphorus supplieswere not minimal.76 The upper safe limit of sodium chloride in the diet was62 Spinks, Lee, and O’Neil, Canad.J . Res., 1949, 27, B , 629.83 O’Neil, Jowsey, Lee, and Spinks, ibid., D, 223.1 3 ~ Davidson and Leslie, Biochem. J . , 1948, 43, xxviii.6 5 Novikoff and Potter, J . Biol. Chem., 1948, 173, 233.8 6 Gillis, Norris, and Heuser, J . Nutrit., 1948, 35, 195.67 Gerry, Carrick, Roberts, and Hauge, Poultry Sci., 1949, 28, 19.6~ Singsen, Storrs Agric. Exp. Stat. Bull., No. 260, 1948.60 Gillis, Norris, and Heuser, Poultry Sci., 1949, 28, 283.70 Mussehland Ackerson, Univ. Nebraska Agric. Exp.Stat. BulL.,No. 386, October 1947.7 1 Motzok and Slinger, Poultry Sci., 1948, 27, 486.72 Stadelman, Boucher, and Callenbach, ibid., 1949, 28, 161.7 3 Hill and Motzok, ibid., 1948, 27, 515.74 Buckner, Insko, Henry, and Wachs, ibid., 1949, 28, 644.75 Bird, J .Nutrit., 1949, 39, 13. 76 Gillis, ibid., 1948, 86, 351DUCKWORTH GENERAL NUTRITION. 3193y0.77 As in the case of fluorine, the resistance of the fowl to bromine ishigh, an effect being noted only on the gonads and without modification ofsecondary sex characteristi~s.7~ Repeated radio-iodine injections causeddestruction of the thyroid inCholine and ethanolaminesupplements to practical rations already containing 0.14 and o*17y0 ofcholine did not improve egg production or egg quality.80 There was no lossof choline in high-protein poultry feeds stored a t 70" F. for up to 6 months.s1Choline and methionine requirements for growing chicks were divided intotwo parts : essential and replaceable. The essential requirement of cholinewas O.lOyo of the diet, and of methionine 0450%.These were consideredto be the basic amounts required for tissue construction. The replaceablerequirements needed for methylation processes were 0.25 yo of methionineand 0.45% of choline chloride. Excess of methionine depressed growthunless glycocyamine was given as a methyl acceptor. Excess of choline upto 1.6% did not depress growthes2 Studies of the replacement of choline bybetaine in incipient perosis were reported.83 Choline and methionine wereto some extent inter~hangeable.~~ Betaine, added to a simplified diet, hada greater supplementary effect than either choline or methi~nine.~~ Ethanol-amine could replace partly the choline of chick rations.86 The fat of turkeycarcases held in cold storage was less liable to rancidity when their rationshad been supplemented with choline or ethan~larnine.~~.88Unidentified chick-growth factors have been found in condensed fishsolubles (made by evaporating the " stickwater " produced during oilextraction of fatty fi~h),~99 91 9 9 2 p 93* 949 100 fish meal,Q59 96 liver prepar-a t i o n ~ , ~ ~ ~ 94 cow dung,@* 99 distiller's by-product^,^^ meat meals,96 casein,Q7Non-vitamin and Unidentiified Dietary Factors.7 7 Barlow, Slinger, and Zimmer, Pou2try Sci., 1948, 27, 542.70 Winchester, Comar, and Davis, Science, 1949 110, 302.*O Gish, Kummerow, and Payne, Poultry Sci., 1949, 93, 305.Cooley and Christiansen, ibid., 1948, 27, 822.82 McKittrick, Arch. Biochem., 1947, 15, 133.84 Gerry, Carrick, and Hauge, Poultry Sci., 1948, 27, 161.8 5 Mishler, Carrick, and Hauge, ibid., 1949, 28, 24.8 6 Kummerow, Weaver, and Honstead, ibid., p.475.8 7 Hite, Kloxin, Kummerow, Vail, and Avery, ibid., p. 244.8 8 Hite, Kloxin, and Kummerow, ibid., p. 249.Borgatti, Arch. Sci. biol., Napoli, 1947, 32, 67.*3 Idem, ibid., 1948, 18, 437.Robblee, Nichol, Cravens, Elvehjem, and Halpin, ibid., 1948, 27, 442.Nichol, Robblee, Cravens, and Elvehjem, J . Biol. Chem., 1949, 177, 631.91 Hnie, Tidsskr. norske Landbmk, 1949, 56, 23.g2 Mishler, Carrick, and Hauge, Poultry Sci., 1948, 27, 263.O3 Robblee, Nichol, Cravens, Elvehjem, and Halpin, J . Biol. Chem., 1948, 173, 117.g4 Nichol, Robblee, Cravens, and Elvehjem, Poultry Sci., 1948, 27, 438.O5 Combs, Heuser, and Norris, ibid., p.238.s6 Wiese, Petersen, and Lampman, ibid., p. 466.O 7 Csonka and Olsen, J . Nutrit., 1949, 39, 485.Schlamb and Winter, Poultry Sci., 1948, 27, 492, 498.Bird, Rubin, and Groschke, J . Biol. Chem., 1948, 174, 611.loo Hill, Poultry Sci., 1948, 27, 536320 BIOCHEMISTRY.and whey.loO Certain of these factors are undoubtedly to be identified withvitamin B12. In some cases confusion has arisen because of failure to depletethe hen's reserves before collecting the eggs for hatching. Purified dietsfor turkey poults were satisfactory for short periods in early life,lol but notfor 2 generations.lo2 Condensed fish solubles provided an essential factorfor poults.lo3 Dietary factors essential for the chick were supplied by oldbuilt-up floor litter.lM The growth-promoting action of cellulose could bereproduced by adding to the diet 10% of sawdust + either O.lyo of lzevulicacid or 0.1 to 0.3% of furfuraldehyde, but not by D-xylose, furamide, or~-cellobiose.~~~ Soil (as a source of B.subtilis factor),lo6 added a t the rateof 1% to a diet deficient in animal-protein factor(s), stimulated growth andreduced mortality.Unidentified hatchability factors have been found in fish meals, l 0 7 ~ lo8cow dung, log and built-up floor litter.l1° Methods of concentrating thefactor(s) in fish meal were reported lo8 and an assessment made of theamounts of fish meal necessary to provide enough of the factor(s) to maintainhatchability. lo7Growth of chickens and turkeys was accelerated by 4-hydroxy-3-nitro-phenyl-, p-hydroxyphenyl-, and phenyl-arsonic acid.lll* lS6* lS7 Aureo-mycin, aureomycin mash (with its antibiotic activity destroyed), strepto-mycin, and succinoylsulphathiazole also stimulated growth.111Thyroprotein supplements in the diet hadlittle effect on growth or feed efficiency of chicks although feathering ratewas improved.1l2 Continuous feeding with thyroprotein delayed birds inreaching 50% egg production.l13 Some workers found no effect on eggproduction,ll** 115, n4* lZ9 others a beneficial effect.l16* ll7~ 118* lZ6 Reportedadvantages of dosage were : delayed moulting and improved health,l16improved shell quality,l14* 124 and higher production in oldSimultaneous dosage with thyroprotein, an estrogen [di(methoxyphenyl)-hexene] and an androgen (in dried cow dung lS5) gave no added advantage.llsFeeding dried pig thyroid checked growth and induced precocious maturity.l*OHormone Administration.101 Scott, Heuser, and Norris, Poultry Sci., 1948, 770.102 Jukes, Stokstad, and Gilbert, ibid., p.434.103 German, Schweigert, Pearson, and Sherwood, ibid., p. 113.104 Kennard and Chamberlin, ibid., p. 240.105 Davis and Briggs, Fed. Proc., 1948, 7, 284.lo8 Stephenson, McGinnis, Graham, and Carver, Poultry Sci., 1948, 27, 827.107 Lindstrom, Petersen, Wiese, and Moore, ibid., 1949, 28, 552.108 Pensack, Bethke, and Kennard, ibid., p. 398.109 Groschke, Rubin, and Bird, ibid., 1948, 27, 302.110 Kennard, Bethke, and Chamberlin, ibid., p. 477.111 Stohtad and Jukes, PTOC.SOC. E:E:E:E:E:E:E:E:E:E:E:. Biol., 1950, 73, 523.112 Wheeler, Hoffmann, and Graham, Poultry Sci., 1948, 27, 103.113 Wheeler and Hoffmann, ibid., p. 509.114 Hoffmann and Wheeler, ibid., p. 609.115 Hutt and Gowe, ibid., p. 286.118 Moore and Rees, Vet. J., 1948, 104, 156.117 Turner and Kempster, Poultry Sci., 1948, 27, 453, 11* Turner, ibid., p. 613DUCKWORTH : GENERAL NUTRITION. 321Chicks from eggs of hens fed with thyroprotein 119* 121* 122* lZ3* 126 orthiouracil l22,1*3 had enlarged thyroids. The length of incubation time ofthese eggs was increased,l231125 in proportion to the dosage of the hen.f1sThiouracil also increased incubation time.lg3 The degree of enlargementof the chick's thyroid varied with the iodine content of the thyroproteingiven to the mother, and not with its thyroxin content.12s Incubation timereturned to normal soon after thyroprotein was left out of the diet.122Injection of incubating eggs of thyroprotein-fed hens with 6 pg. of (&)-thyroxine reduced the chick thyroids to normal size.122 Injection of normaleggs with 1 pg.of thyroxine impaired thyroid demlopment.l27 High levelsof potassium iodide injection of eggs increased thyroid size and extendedincubation time; 128 low levels had no effect.122 Thyroprotein feeding didnot affect fertility or hatchability of eggs.lz9> 132 Semen volume decreasedin thyroprotein-fed cocks,130 and semen quality might 131 or might not I3O beimpaired. It reduced thyroid size in hens, possibly by inhibiting theproduction of pituitary thyrotrophic h0rm0ne.l~~Fast- and slow-growing strains of birds in each of two breeds had theirown optimum levels of thyroid activity for growth, feathering, and feedutilisation.The growth response to thyroprotein was negative in a fast-growing strain and positive in a slow-growing strain of New Hampshirechicks.13* Thyroid activity was higher in birds with high than in those withlow egg production,135 and there was an indication of seasonal variation. 136It is possible that some of the conflict between results in studies of thyro-active materials may have arisen through failure to take such factors intoaccount. Thyroxine secretion was much higher in ducklings thanMoulting could be forced in turkeys by thyr0pr0tein.l~~ Thyroid size washighly variable in turkeys.139Thiouracil alone in the diet reduced growth in cockerels 140 and turkeys,141but when combined with thyroprotein yielded normal growth and superiormarket grade.142 Thiouracil plus diethylstilbcestrol gave the best weight119 Wheeler and Hoffmann, Endocrinology, 1948, 43, 430.lZo Spisni and Sala, Zootec.Vet., 1949, 4, 730.lZ1 Wheeler and Hoffmann, Endocrinology, 1948, 42, 326.lg2 McCartney and Shaffner, ibid., 1949, 45, 396.lZ3 McCartney and Shaffner, Poultry Sci., 1949, 28, 223.lZ5 Wheeler and Hoffmann, Proc. SOC. Exp. Biol., 1949, 72, 250.lZ6 Turner and Kempster, P a l t r y Sci., 1949, 28, 826.lZ7 Booker and Sturkie (with Booker), ibid., p. 147.lZ8 Wheeler and Hoffmann, Endocrinology, 1949, 45, 208.lZ9 McCartney and Shaffner, Poultry Sci., 1950, 29, 67.130 Huston and Wheeler, ibid., 1949, 28, 262.131 Shaffner, ibid., 1948, 27, 527.132 Wheeler and Hoffmann, ibid., p.685.13' Glazener, Shaffner, and Jull, ibid., 1949, 28, 834.135 Booker and Sturkie, ibid., p. 757.13' Hoffmann, ibid., 1950, 29, 109.138 Blakely, Anderson, and MacGregor, ibid., 1949, 28, 757.140 Moreng and Shaffner, ibid., p. 504.142 Henderson, Carver, and Stephenson, ibid., 1948, 27, 667.lZ4 Godfrey, ibid.,p. 867.133 Turner, ibid., p. 155.136 Turner, ibid., 1948, 27, 146.138 Kosin and Wakely,ibid., 1948,27,670.141 Blakely and Anderson, ibid., p. 185.REP.-VOL. XLVII. 322 BIOUHEMISTRY.gains, the highest feed efficiency and the best market grade.f42 Goitrogenswere rapidly absorbed and excreted in birds and mammals so that toxiceffects on consumers would be ~nlike1y.l~~ Thiouracil greatly reducedgrowth in ducklings and caused great enlargement of the thyroid.137Addition of 0.3% of thiouracil to the diet had no effect on fertility of eggsbut reduced production and hatchability ; 0.1 yo additions had no detrimentaleffect .1z9Clhtrogens fed in diets containing 18% of protein did not improve growthbut when added to 14% diets improved skin appearance.When diethyl-stilbestrol, but not dienoestrol diacetate, was given, the liver and abdominalfat contained enough estrogen to cause =estrus phenomena in menopausalwomen eating these tissues.144s148 Implantation of 30 mg. of diethyl-stilboestrol improved the growth of cockerels lP5 but stilboestrol dipropionatefailed.149 Injection of diethylstilbestrol raised plasma lecithin and cephalinmarkedly 146 and increased the incidence of atheroscler~sis.~~~ Treatmentof immature pullets with mtradiol dipropionate produced the changes in theoviduct and blood, similar to those in the normal laying bird, but no increasedcalcium and phosphorus retention.Testosterone had no such effect.Simultaneous dosage with estrogen and androgen produced oviduct andblood changes and increased calcium and phosphorus retentions. 150Di(methoxypheny1)hexene (and chlorotriphenylethylene) increased fatnessof turkeys.l5l* 152 High dosage levels impaired egg production and induced'( leg-weakness," but a t 0.0125% in the diet egg production was irnpr0~ed.l~~Di( methoxypheny1)hexene reduced iodine turnover in the thyroid.154Feed efficiency (bodyweight gain/feed consumption)was higher in fast-growing than in slow-growing strains within a breed, incrossbred than in purebred birds, in outbred than in inbred birds, in NewHampshires than in Barred Plymouth 159* 160 Environmentaltemperature ranges for maximal feed efficiency in chicks have been deter-mined.Maximal feed efficiency characterised maximal growth rates.Variations in humidity from 35 to 75o/b were without effect on growth orefficiency.' Provided that supplies of individual amino-acids wereFt?ed Eficiency.143 Pipes and Turner, Univ. Missouri Agric. Exp. Stat. Res. Bull., No. 422, July 1948.u4 Bird, Off.Rep. Eighth World's Poultry Congr., 1948, p. 131.145 Bos, Tijdschr. Diergeneesk., 1950, 75, 150.1413 Ranney, Entenman, and Cheikoff, J . Biol. Chem., 1949, 180, 307.147 Horlick and Katz, J . Lab. Clin. Med., 1948, 33, 733.14* Gowe, Poultry Sci., 1949, 28, 666.14B Kelly and Roberts, Vet. Rec., 1950, 62, 44.150 Common, Rutledge, and Hale, J. Agric. Sci., 1948, 38, 64.151 Thayer and Davis, Poultry Sci., 1948, 27, 176.lS2 Davis and Thayer, ibid., p. 79.154 Epstein and Wolterink, ibid., 1949, 28, 763.156 Morehouse, ibid., 1949, 28, 375.15' Bird, Groschke, and Rubin, J . Nutrit., 1949, 37, 215.15* McCartney and Jull, Poultry Sci., 1948, 27, 17.l60 Headley, Univ. Nevada Agric. Exp. Stat. Bull., No. 180, July 1948.l a 1 Barott and Pringle, J .Nutrit., 1949, 37, 153.15s Turner, ibid., p. 593.1 5 5 Turner, ibid., 1948, 27, 789.159 Hess and Jull, ibid., p. 24DUCKWORTH : QENERAL .NUTRITION. 323adequate, little advantage in feed eficiency was gained by raising theprotein level in chick rations from 20 to 28%.182 Feed dilution with cellulosebeyond a 10% level decreased feed efficiency.la Poults retained 42.6%,23.4%, and 3103% respectively of the nitrogen, calcium, and phosphorusconsumed during the first eight weeks of life?* Higher feed efficiencieswere found in male than female turkeys.lm By use of the general equationY = C + blxl + bzx2 + b3x3 (Y = total feed consumed; x1 = eggs laid,in g. ; x2 = average body weight ; x3 = change in bodyweight during laying)it was found that 71% of feed consumed was for maintenance, 27% for eggproduction, and 2 yo (non-significant) for weight increase, in pullets with 72 yoegg production.Lower-producing birds used the feed fraction for eggproduction as efficiently as did high-producing birds.lsSblwine.-Protein and Amino-mi&. Addition of 0.4% of DL-tryptophanto a purified diet met the requirement of pigs weighing 50-100 lb.168~1s7The lysine requirement of weanlings was met by 0.6% of L-lysine,ls8 or amaximum of 2 yo of ~ ~ - 1 y s i n e . ~ ~ ~ Hexahomoserine (2-amino-6-mercapto-hexanoic acid) inhibited growth and reduced the red blood-cell count.170The animal-protein needs of bacon pigs were met by giving 7% of fish mealin the diet up to 90-100 lb. liveweight.blinerals. When a ration containing @15y0 of calcium and 1.69% ofphosphorus was fed, the blood-calcium and alkali reserve were lowered, andthe blood- and urine-phosphorus were raised.172 The rachitogenic substancein yeast was heat stable and did not reduce intestinal breakdown of phyticacid.173 Protein deficiency caused a mild normocytic, normochromic anzmiaand a marked reduction in iron-binding capacity of the serum. Irondeficiency caused a severe microcytic, hypochromic anzmia and a reductionin b10od-iron.l~~ In piglets housed in cold conditions and given ironsupplements the anamia was a consequence of liver damage.17s* 176Growth factors (for baby pigs)in liver extract,177 (for weanling pigs) in meat scraps, whey products, andcondensed fish s~lubles,~~* and (for growing and fattening pigs) in condensedThereafter none was needed.171Unidenti$ed and Non-vitamin Factors.Singsen, Poultry Sci., 1949, 28, 713.163 Davis and Briggs, ibid., 1948, 27, 658.164 Ackerson and Mussehl, Univ. Nebraska Agric. Exp. Stat. Res, Bull., No. 151,la5 Joshi, Shaffner, and Jull, Poultry Sci., 1949, 28, 301.lea Beeson, Mertz, and Shelton, J. Animal Sci., 1949, 8, 532.le7 Idem, Science, 1948,107, 599.la8 Loosli, Williams, and Maynard, Fed. PTOC., 1949, 8, 379.16g Metz, Shelton, and Beeson, J. Animal Sci., 1949, 8, 624.1x1 Mertz, Beeson, Waltz, and Gaudry, Proc. SOC. Exp. Biol., 1948, 69, 609.171 Woodman and Evans, J. Agric. Sci., 1948, 38, 354.17a Liegeois and Derivaux, Compt. rend. SOC. Biol., 1949, 143, 126.July 1947.Braude, Henry, and Kon, Brit.J . Nutrit., 1948, 2, 66.Csrtwright and Wintrobe, J . Biol. Chern., 1948, 176, 571.175 Howie, Biggar, Thomson, and Cook, J . Agric. Sci., 1949, 39, 110.176 Naftalin and Howie, J. Path. Bact., 1949, 61, 319.178 Dyer, Krider, and Carroll, J . Animal Sci., 1949, 8, 541.Neumann, Krider, and Johnson, Proc. SOC. Ezp. Biol., 1948, 69, 513324 BIOCHEMISTRY.fish solubles and liquid Baby pigs required aboutO*lyo of choline in the ration.lsO Choline was riot required in baby-pig“ synthetic milk” diets if the methionine content was 1.6% of the drymatter. At half this methionine content 0.1% of choline was needed.lsl“ Synthetic milk ” plus serum or plasma as a colostrum substitute failed tosupport life in new born pigs.lS2 Given “ synthetic milk ” on the third dayof life they grew normally.la3 Aureomycin 18** us and streptomycin lS6stimulated growth in pigs.Hormone Administrution. Thyroprotein increased the growth rate ofpigs.la7* lS8* I n restricted feeding, growth and feed efficiency werereduced thereby.lS7 Improved feed efficiency and growth was associatedwith a higher percentage of fat and lower percentages of moisture, protein,and minerals in the carcase.188 Reduced growth rate and poorer feedefficiency were reported.lWFeeding anti-thyroid drugs gave conflicting results. Some foundthiouracil lgl* lg2 or methylthiouracil 191 improved growth and/or feedefficiency. Others found detrimental effects for these drugs andthiourea.lsgl lg3, lg4 Beyond a slight reduction in skeletal growth and carcase-dressing value there was no effect on carcase quality.lg6 Fresh liver, butnot lean shoulder meat, of methylthiouracil-fed pigs was goitrogenic torats.lgl Combined thyroprotein and stilboestrol feeding improved growthand feed efficiency in fattening pigs.lg6Protein requirements of cattle (andsheep) were factorised in a statistical study of recorded data and the concept“ available protein ” introduced.The available protein is the metabolisableprotein of the feed taxed for the loss of body-nitrogen which its ingestionentails and thus available for covering endogenous losses and the demandsof other anabolic processes.lg7 New estimates of the protein requirements ofgrowing heifers were given lg8* lg9 with discrimination between requirementshave been found.Cattle.-Protein (inchding Urea).170 Geurin, Hoefer, and Beeson, J.Animal Sci., 1950, 9, 94.180 Neumann, Krider, James, and Johnson, .I. Nutrit., 1949, 38, 195.181 Nesheim, Krider, and Johnson, J . Animal Sci., 1949, 8, 627.Bustad, Ham, and Cunha, Arch. Biochem., 1948, 17, 240.183 Lehrer, Moore, Wiese, and Pahnish, J . Animal Sci., 1949, 8, 107.Carpenter, Arch. Biochem., 1950, 27, 469.185 Jukes, Stokstad, Taylor, Cunha, Edwards, and Meadows, ibid., p. 324.Luecke, McMillan, and Thorp, ibid., p. 326.187 Nordfeldt and Hydh, Kgl. Lantbrukshogsk. Ann., 1948, 15, 173.lE8 Perry, Beeson, and Andrews, J . Animal Sci., 1950, 9, 48.Beeson, Andrew$, Perry, and Witz, ibid., 1949, 8, 508.lS0 Vander Noot, Reece, and Skelley, ibid., 1948,7, 84.192 Terrill, Krider, Carroll, and Hamilton, ibid., 1949, 8, 501.lS3 Braude and Cotchin, Brit.J . Nutrit., 1940, 3, 171.lg4 Willman, Asdell, and Loosli, J . Animal Sci., 1949, 8, 191.lS5 Terrill, Hamilton, Krider, and Carroll, ibid., 1950, 9, 58.lS8 Braude, Nature, 1948, 161, 856.lS7 Blaxter and Mitchell, J . Animal Sci., 1948, 7, 351.lQ8 Steensberg, Brit. J . Nutrit., 1947, 1, 139.lg9 Jensen, Steensberg, and Wnther, Forsogslab. Ksbenhauit Beretn. No. 237, 1949.lgl Idem, ibid., 1950,9, 54DUCKWORTH GENERAL NUTRITION. 325for high- and low- quality rations. lg7 The digestible crude protein require-ment per kg. of milk containing 4% of butterfat was 55 to 60 g.200 Dis-crepancies between protein digestibilities of rations computed from tablesand found in experiment may be serious.201 That roughage intakes ofgrazing cattle can be estimated from the nitrogen content of faxes 202 wasdenied .203When suchurea was fed in rations to cattle and sheep, 15N was present in excess ofnormal in various body proteins, indicating the use of urea-nitrogen inprotein synthesis.205* 206 Adding urea to the rations of beef steers improvednitrogen retention and did not affect feed digestibility. There was noammonia in expired or regurgitated air.207 Urea-feeding raised the ammoniaand urea content of the bl00d.~~~1 208There was little or no difference betweencattle and sheep in their capacity to digest common feeds.209* 210* 211 Raisingthe plane of nutrition depressed the digestibility of carbohydrates in linseedoil meal 212 and barley.213 Only excessive barley feeding reduced proteindigestibility.2'2 The protein required for efficient roughage digestion waslow but was increased by additions of The digestibility of mixedrations (protein concentrate, carbohydrate concentrate, and roughage) wasunaffected by altering the nutritive ratio.215The depressing effect of lignin on roughage digestibility difTered markedlybetween species of herbage.216 Digestion in the rumen was studied, usinglignin as an (almost) inert marker.Roughage carbohydrates (except lignin)and protein were rapidly digested during the first 6 hours in the rumen, andcellulose was attacked mainly in the second 6 hours.There was littledigestion after 12 hours and only slight additional digestion in the c i e c ~ m . ~ ~ The use of markers (Cr203 218 and lignin, silica and iron 219) in digestibilitytrials was studied. A statistical analysis was made, of European data, toThe synthesis of urea containing 15N has been described.204Carbohydrate (and Energy).)O0 Ulvesli, Norges Landbruksh0gsk. Foringsforsnk. Beretn., No. 65, 1949.aol Poijiirvi, Maataloust. Aikakausk, 1947, 19, 108.202 Gallup and Briggs, J . Animal Sci., 1948, 7 , 110.403 Forbes, ibid., 1949, 8, 19.204 Leitch and Davidson, Sci. Agric., 1949, 29, 173.*05 Watson, Davidson, and Kennedy, ibid., p. 185.206 Watson, Kennedy, Davidson, Robinson, and Muir, ibid., p. 189.207 Dinning, Briggs, and Gallup, J .Animal Sci., 1949, 8, 24.208 Dinning, Briggs, Gallup, Orr, and Butler, Amer. J . Physiol., 1948, 153, 41.209 Watson, Davidson, Kennedy, Robinson, and Muir, Sci. Agric., 1948, 28, 357.210 Axelsson, Kgl. Lantbrukshogsk. Ann., 1949, 16, 84.211 Idem, Svenslc Jordbruksforsk. Arsbok, 1947.212 Watson, Kennedy, Davidson, Robinson, and Muir, Sci. Agric., 1949, 29, 263.Watson, Davidson, Kennedy, Robinson, and Muir, ibid., p. 400.Burroughs, Gerlaugh, Edgington, and Bethke, J . Animal Sci., 1949, 8, 9.215 Watson, Kennedy, Davidson, Robinson, and Muir, Sci. Agric., 1947, 27, 600.216 Phillips and Loughlin, J . Agric. Res., 1949, 78, 389.a17 Hale, Duncan, and Huffman, J . Nutrit., 1947, 34, 733.a18 Jarl, Kgl. LantbruksMgsk. Ann., 1949, 16, 785.Druce and Willcox, Empire J , Exp.Agric., 1949,17, 188326 BIOCHEMISTRY.relate methane production to dry matter, digestible carbohydrate, anddigestible crude fibre of the ration.220Minerals. Adding calcium carbonate to a calcium-low ration had littleor no effect on the digestibility of the ration.221 Oxalic acid, in paddystraw, hindered the utilisation of dietary calcium and provoked severealkalosis, probably through conversion of oxalic acid into carbonate andhydrogen carbonate in the rumen.222 The giving of calcium supplements,with or without trace elements, to preparturient cows had no effect on thecalcium and phosphorus content of the blood of the cows and their calvesat the time of parturition.223 Supplementing the ration with calcium hadno effect on the total output of lipids in faxes, but the ratio of neutral fatsand sterols to soaps and free fatty acids was altered.224Maximum output of radio-phosphorus in milk was reached 5 hours afterinjection, the level of dosage affecting neither the time required to reachmaximum output nor the magnitude of output.About 20% of the injecteddose was recovered in the milk during 7 days after dosage. Casein labelledwith radio-phosphorus (at about 2 pc. per g.) could be collected during thefirst 3 days after dosage,2z6 Giving 35 g. of phosphorus daily as NaH2P0,,Na,HPO,, or CaHPO, to cows in milk had no effect on faeces consistency orurine pH. Urinary acidity in silage-fed cows was reduced by Na2HP0, andCaCO, supplements.226 Fused Ca3(PO,J2 should not contain over 0.24% offluorine and should be used with care ; over 0.2% of fluorine in the concentrateration was dangerous to dairy cows.227Long-term feeding of growing calves indicated that about 50 mg.ofcobalt daily per 100 Ib. liveweight approached toxic levels, although animalswere variable in their response to dosage Studies with radio-cobalt indicated that adults did not store cobalt and that the main functionof cobalt was in the rumen. However there was liver storage of cobalt inthe fcetus.229 Prepartum dosage with cobalt greatly increased the cobaltcontent of colostrum. Cobalt feeding of calves raised the cobalt content ofall body parts, but particularly of the liver and kidney.230 Oral dosagegave small, and intravenous dosage large, retentions of radio-copper, mostbeing held by the liver.229*231 In lactating cows retentions of manganesewere surprisingly high (154 mg.daily), and independent of intake.2s2Sao Axelsson, Kgl. LantbrukshBgsk. Ann., 1949, 18, 404.221 Mukherjee and Chatterjee, Indian J . Vet. Sci., 1947, 17, 85.222 Talapatra, Ray, and Sen, J . Agric. Sci., 1948, 38, 163.22s Reid, Ward, and Salsbury, J . Nutrit., 1948, 38, 75.224 Ward and Reid, ibid., 1948, 35, 249.225 Kleiber, Smith, and Ralston, PPOC. SOC. Exp. Biol., 1948, 89, 354.22u Axelsson and Kivimae, Kgl. Lantbrukshdgsk. Ann., 1949,18, 101.227 Mather, Pratt, and Holdaway, J . Dairy Sci., 1949, 82, 228.228 Keener, Percival, Morrow, and Ellis, ibid., p. 527.Comar, Proc. Auburn Conf.on the Use of Radioactive Isotopes in AgriculturalResearch, December 18-20, 1947, p. 137.230 Ward, B e ~ e , Webster, Duncan, and Huffman, J . AnimaZ Sci., 1949, 8, 632.'** Reid and Ward, J . Nutrit., 1948, 85, 691.Comar, Davis, and Singer, J . Biol. Chem., 1948, 174, 906DUCKWORTH : GENERAL NUTRITION. 327Hormone Administration. Thyroprotein dosage improved milk andbutterfat production in cows 233,2u* 235,238, 237* 238 except at low dosagelevels.239 Feeding thyroprotein in successive years had less favourableeffects in later lactations.2a A comparison of the effects of oral dosage with(--)-thyroxine and iodocasein indicated either that (-)-thyroxine was moreefficiently used than iodocasein or that estimates of the thyroidal activity ofiodocasein are errone0us.m The energetic efficiency of producing the extramilk was much lower than that of normal milk p r o d u ~ t i o n .~ ~ ~ * ~ ~ l Severeweight losses by cows dosed with thyroprotein in hot weather could not beprevented by giving extra food.242Prolonged thyroprotein dosage of young stock increased body size, withjoint stiffness and osteoporosis.243 Intravenous thyroxine injection inhibitedfattening in non-pregnant non-lactating cows and increased the hardness ofbone.244 Thyroxine injection had an adverse effect on nitrogen and calciumbut not on phosphorus balances of lactating cows.245 The preparation ofiodinated casein containing radio-iodine was described.246 Additions ofthiouracil, nux vomica, and arsenious oxide improved feed efficiency infattening beefSheep.--Protein (incEwling Urea).No advantage was gained by feedingover 10.3% of protein in lamb rations.248 On feeds containing 9.5, 10.4, andl l . O ~ o of protein the daily weight gains of lambs were 0-27,0*30, and 0-30 lb.respectively with daily nitragen retentions of 2.7, 3-4, and 3.6 g.249 Noimprovement was produced in reproductive performance, birth weight oflambs, or subsequent lamb growth 250 by increasing the daily protein intakeof pregnant ewes from 0.15 to 0-31 lb. The protein of rumen bacteria wasabout one-third less digestible than casein but equal to it in biological value(rat a ~ s a y ) . ~ ~ l It was rich in methionine and ~ystine.~62 40% of dietaryzein was converted into microbial protein in the r ~ m e n .~ ~ ~233 Allen, DOW, Logan, and MacKenzie, Sci. Agric., 1948, 28, 340.234 Thomas and Moore, J . Dairy Sci., 1948, 31, 661.*s5 Thomas, Moore, and Sykes, ibid., 1949, 52, 278.236 Opichal, Chumchal, and Kopeckf, Sborn. Ces. Aka4 ZemBd., 1949, 21. 280.2s7 Moustgaard and Thorbek, Forssgslab. Ksbenhuvn Beretn., 1949, No. 240, 1-45.238 Lanik and Isajev, Sborn. Zes. Akad. Zeindd., 1949, 22, 65.239 Swanson and Knodt, J . Dairy Sci., 1949, 32, 257.240 Bailey, Bartlett, and Folley, Nature, 1949, 163, 800.241 Thorbek, Hansen, and Moustgaard, J . Animal Sci., 1948, 7 , 291.242 Gardner and Millen, J . Dairy Sci., 1948, 31, 660.24s Dyrendahl, Thesis, Univ. Stockholm, 1949, p. 116.244 McQuillan, Trikojus, Campbell, and Turner, Brit. J .Exp. Puthol., 1948, 29, 93.245 Owen, Biochem. J . , 1948, 43, 235, 243.246 Courrier, Roche, Deltour, Marois, Michel, and Morel, Bull. SOC. Chim. biol., 1949,a47 Kline, Ensminger, Cunha, Heinemann, and Ham, J . Animal Sci., 1949, 8, 411.248 Klosterman, Thesis, Cornell Univ., 1946.250 Jordan, Klosterman, and Wilson, J . Animal Sci., 1949, 8, 623.261 Reed, Moir, and Underwood, Austral. J . Sci. Res., 1949, 2, B, 304.f5a Johanson, Moir, and Underwood, Nature, 1949, 163, 101.2s3 McDonald, J . Physiol., 1948, 107, 21P.31, 1029.240 Briggs, Thesis, Cornell Univ., 1946328 BIOCHEMISTRY.Urea could replace part of the nitrogen in the rations of lambs weighing50 Ib. or more. I n rations containing 12% of crude “protein ” (totalnitrogen x 6.25) urea could provide half the nitrogen, provided that aquarter of the nitrogen was present as pre-formed protein.254* 255 Rumencontents of sheep given urea as the sole source of nitrogen contained largequantities of 10 “ essential ” amino-acids, in amounts similar to thosefound when casein was given.266 Dietary supplements of thiodiglycollicacid improved nitrogen balances.257 Loss of dietary nitrogen throughabsorption of ammonia from rumen contents was partly compensated for bysalivary urea reaching the r ~ m e n .~ ~ ~ Nitrogen fixation by rumen organismswas demonstrated.259 The rate of reduction of nitrate to nitrite in rumencontents depended on the nature of the diet.260 Preparation of purifieddiets for studies of amino-acid needs of ruminants was described.261Carbohydrate and Fat (including Energy).Formulae for predicting thedigestibility of herbage organic matter from faecal nitrogen,262 and thenitrogen content of herbage grazed from fmal nitrogen,263 were given. Arelation was reported 264 between starch equivalent (S.E.) and crude fibre(2) of herbage, vix., S.E. = 73.56 - 0*62x, but it seems to be tautological :the S.E. is essentially the digestible organic matter less a crude fibrecorrection value proportional to the crude fibre content. Formuls forestimating the digestible crude protein and starch equivalent of grasssilages z65 and dried grass 266 were given.Herbage lignins lost aldehyde groups during digestion, but methoxyland nitrogen contents were unchanged. The nitrogen was considered to bepresent in lignin as part of a heterocyclic structure, and not as protein orprimary or secondary amine.267 This view was rejected, specific amino-acids being identified in lignin.268 Lignins of young clover were morereadily digested and metabolised than those of mature clover, as judgedfrom the proportions of hippuric and benzoic acids voided in urine.269About half of the pentosans of hay were digested in the stomach (mainly therumen), and the remainder in the intestines.270 The digestibilities of pecticacids and pectins were reported.271 Addition of green lucerne or concentrates254 Hamilton, Robinson, and Johnson, J .Animal Sci., 1948, 7, 26.255 Briggs, Gallup, Heller, and Darlow, ibid., p. 35.256 Thomas, Loosli, Ferris, Williams, and Maynard, Fed.PTOC., 1949, 8, 398.2 5 7 Ferrando and Thomas, Bull. SOC. Chim. biol., 1948, 30, 228.2 5 8 McDonald, Biochem. J . , 1948, 42,584.260 Sapiro, Hoflund, Clark, and Quin, Onderstepoort J. Vet. Sci., 1949, 22, 357.Thomas, Loosli, Williams, and Maynard, J . Animal Sci., 1948, 7, 534.262 Lancaster, Nature, 1949, 163, 330.264 Hallsworth, J . Agric. Sci., 1949, 39, 254.285 Dijkstra, Versl. Landbouwk. Onderzoek., 1949, No. 55.10, pp. 15.266 Idem, ibid., 1949, No. 54.11, pp. 48.2 6 7 Bondi and Meyer, Biochem. J . , 1948, 43, 248.268 De Man and De Heus, Rec. Trau. chim., 1950, 69, 271.269 Pazur and Delong, Sci. Agric., 1948, 28, 39.270 Marshall, Brit. J . Nutrit., 1949, 3, 1.3 7 1 Leroy and Michaux, Compt. rend., 1949, 220, 1034.Tbth, Experientia, 1948, 4, 395.263 Raymond, ibid., 1948, 161, 937DUCKWORTH : GENERAL NUTRITION.329improved cellulose digestibility in veldt hay.272 The effect of the nature ofthe ration on the types and numbers of rumen organisms was reported.273The volatile acids of' rumen liquor were acetic acid about 75%, propionicacid 13-15y0, and butyric acid 7-10y0.274 At the height of fermentationafter feeding, acetic acid formed 86-95% of the total volatile acids inblood, but propionic, butyric, and at least one higher acid were also present.276Absorption of acetate ion was prevented by making the rumen contentsalkaline.276Onrefeeding a8fter starvation there was a transitory production of hydrogen,preventable by introducing normal rumen liquor into the rumen of thestarved sheep.Methane production was most rapid during the 4 hoursafter feeding. I n starvation, methane production ceased after 3-5Methane production (ing.) = 2 . 4 1 ~ + 9.80, where x is the hundreds of g. of carbohydrates digested.278Replacing carbohydrate by fat in isocaloric rations did not affect theutilisation of protein and energy, but methane production and dry matterdigestibility were slightly depressed.278The fasting metabolism per kg. W0'73 (W = bodyweight) of sheep was74.5 kcals. and the basal metabolism 59 kcals.,279 or 860 kcals./sq.m./24 hr.280The effects of the plane of nutrition on pregnant ewes, on the growth of theirfoetuses and lambs, and on milk production were reported.281* 282p 283* 284Changes in organ and tissue weights of ewes transferred from super-maintenance to submaintenance rations, and vice versa, were determined.285The influence of plane of nutrition on wool growth and quality was studied.286The daily calcium and phosphorus requirements of adultwethers were suggested to be 6.0 and 1.0-1.5 g.Ureafeeding did not alter the calcium and phosphorus requirements of lambs.288The copper requirement of sheep for the production of wool with satisfactorytextile properties was 5-10 mg. daily. Higher dosages may be bene-ficial.289*290 Attempts to deplete sheep of copper by injections of' BALgave inconsistent results.291 Cobalt-deficient lambs were benefited byNo hydrogen was evolved in the rumen when chopped hay was fed.or a t least reached a very lowMinerals..2722 7321427527727821928088128228328628 728828929029 1Louw, Bodenstein, and Quin, OndeTStepOOTt J .Vet. S C ~ . , 1948, 23, 239.Gall, J . Animal Sci., 1949, 8, 619.Schambye and Phillipson, Nature, 1949, 164, 1094.Reid, ibid., 1950, 165, 448.Pilgrim, Austral. J . Sci. Res., 1948, 1, B , 130.Swift, Bratzler, James, Tillman, and Meek, J . Animal Sci., 1948, 7 , 475.Marston, Austral. J . Sci. Res., 1948, 1, B , 93.Blaxter, J . Agric. Sci., 1948, 38, 207.A. M. Thomson and W. Thomson, Brit. J . Nutrit., 1949, 2, 290.Wallace, J . Agric. Sci., 1948, 38, 93.Idem, ibid., p. 243. 284 Idem, ibid., p. 367.Marston, Austral. J. Sci. Res., 1948, 1, B , 362.Axelsson and Eriksson, KgE.Lantbwksh&gek. Ann., 1949, 16, 71 1.Gallup and Briggs, J . Animal Sci., 1949, 8, 619.Palmer, J . Agric. Sci., 1949, 89, 265.Marston and Lee, Austral. J . Sci. Res., 1948, 1, B , 376.Stewart and Robertson, Biochem. J., 1948, a, xxii.276 Gray, J . Exp. Bid., 1948, 25, 135.Robinson, ibid., p. 345330 BIOCHEMISTRY.injection, but not by oral dosage, with liver extract. Injections of folicacid and vitamin B,, were without effect.292Studies using radioactive thyroprotein showedthat maximum absorption was reached 10 hours after oral dosage, Intra-venously administered thyroprotein disappeared from the blood in 7 ho~rs.29~Thyroprotein improved wool production,29P but not growth or feed efficiencyin fattening lambs.296 It raised the basal metabolism of ewes, the relationbetween dosage (in mg.per kg. W0’73) being linear with an increase of0.128% per mg.280 Overdosages reduced the digestibility of the ration,increased nitrogen losses in proportion to dosage and provoked large lossesof skeletal calcium and phosphorus.296Thiouracil, methylthiouracil, and propylthiouracil had either no effect,296or a detrimental effect,297 on growth and feed efficiency of fattening lambs.In aged ewes fattening was improved by methylthio~racil.~~7 Growth andfeed efficiency of wether lambs were improved by stilbcestrol and testosteroneimplants.298The nutritive value of wheat gluten 300and oat protein 301 was improved by lysine supplements. The biologicalvalue for infants of the proteins of breast milk was higher than those ofproteins of cow’s milk, mainly because of higher digestibility.Hydrolysedliver proteins were intermediate.302 Infants retained 50-90 yo of intraven-ously injected serum protein.303 Increasing calories (as glucose) above normalimproved nitrogen retention in parenteral alimentati~n.~M The averagedaily protein requirement of young women eating a high cereal diet was31.7 6 1.6 g., or 25 g./lOOO kcals. of basal heat, the average protein index(biological value x true digestibility) of the diet being 61.4 24.305Nitrogen retentions of young women were higher when part of the animalprotein intake (meat and milk products) was taken a t each meal than whenrestricted to two of the day’s meals.306Heavy work (mining) did not raise the nitrogen requirement aboveresting levels of 7.0-8.0 g.but productivity declined.307 Casein was aseffective as meat in restoring capacity to work in subjects who had beenHormone Administration.Hexcestrol impaired appetite in ewes.299Man.-Protein and Amino-acids.202 Becker and Smith, J . Animal Sci., 1949, 8, 615.193 Campbell, Andrews, and Christian, ibid., p. 638.294 Labarthe, Bertone, and Washburn, ibid., p. 624.296 Blaxter, J . Agric. Sci., 1948, 38, 1.297 Vander Noot, Reece, and Skelley, J . Animal Sci., 1950, 9, 3.298 Andrews, Beeson, and Harper, ibid., 1949, 8, 578.Z9* Austin, Whitten, Franklin, and Reid, Austral. J . Exp. Biol. Med. Sci., 1947,25,343.300 Hoffman and McNeil, J . Nutrit., 1949, 38, 331.302 Rossier and Beauvillain, Compt.rend. SOC. Biol., 1949, 143, 976.3O3 Pliickthun, 2. Kinderheilk., 1949, 66, 496.304 Ellison, McCleery, Zollinger, and Case, Surgery, 1949, 26, 374.305 Bricker, Shively, Smith, Mitchell, and Hamilton, J . Nutrit., 1949, 87, 163.306 Leverton and Gram, ibid., 1949, 89, 67.307 Kraut and Lehmann, Biochem. Z., 1949,319, 228.Barrick, Beeson, Andrews, and Harper, ibid., p. 243.Kuether and Myem, ibid., 1948, 35, 651DUCKWORTH : UENERAL NUTRITION. 33 1on low-protein diets, but meat extracts were ineffective.308 Reducingdietary protein from 90-95 g. (46 g. of animal protein) to 76 g. (19 g. ofanimal protein) daily did not affect output in heavy workers, but reducingintakes from 80-90 g. ( 3 0 4 0 g. of animal protein) to 70 g. (10 g.of animalprotein) did.309Wholemeal bread, water-extracted bran, and the aqueous extract ofbran raised fecal output of nitrogen above normal, but filter paper didThe increase was bacterial nitr~gen.~ll A metabolic fecal protein (namedfecanin) was isolated from infant stools and found to be rich in essentialamino-acids. Its amino-acid content was unaffected by the nature of thedietary protein .Tentative minimum daily requirements of adults for L-tryptophan(0.25 g.), L-phenylalanine (1.10 g.), L-lysine (0.80 g.), L-threonine (0-50 g.),L-valine (0.80 g.), L-methionine (1.10 g.), L-leucine (1.10 g,), and L-isoleucine(0.70 g.) were determined. Histidine was not essential, D- and L-methioninewere equally effective, and D-phenylalanine was partly utilised.Dietscontaining casein, hydrolysed casein, and pure amino-acids were found toraise the caloric requirement for nitrogen equilibrium in these experiments.313About 200 mg. of L-tryptophan daily met adult requirement and acetyl-L-tryptophan had about the same nutritive value. D-Tryptophan and acetyl-D-tryptophan were ineffective.314 Comparable results were found forinfants.316 The sulphur-amino-acid needs of infants were met by 85 mg.of L-methionine plus 15 mg. of L-cystine, or 65 mg. of L-methionine plus50 mg. of L-cystine per kg. bodyweight daily.31s The infant's daily need forL-isoleucine was 90 mg./kge317After rapid intravenous injection of 50 g. of amino-acids (withoutglucose) 9.5% of the a-amino-nitrogen appeared in the urine, with from 1 to16% of individual amino-acids, and a total of 4.3% of the 10 " essential "a m i n o - a c i d ~ .~ ~ ~ The distribution of amino-acids in the urine did not reflectthe pattern in the blood or injected solution.319 When DL-methionhe wasinjected intravenously only the D-isomer appeared in the urine.322 Amino-acid-nitrogen wastage was greater with injected enzymic hydrolysates ofcasein than with acid hydrolysates or amino-acid mi~tures.~~OMales eating diets providing 1 g. of protein/kg. of bodyweight dailyexcreted leucine 25.8, isoleucine 17.5, valine 2043, threonine 60, arginineKraut, Lehmann, and Szakall, Biochem. Z., 1949, 319, 247.Lehmann and Michaelis, ibid., 1949, 520, 99.slo Fournier, Bull. SOC. Chim. biol., 1949, 31, 407.81a Albanese, Davia, Lein, and Smetak, J .Biol. Chem., 1948, 176, 1189.s13 Rom, Fed. Proc., 1949, 8, 546.314 Baldwin and Berg, J . Nutrit., 1949, 89, 203.s16 Albanese, Davis, and Lein, J. Biol. Chem., 1948, 172, 39.316 Albanese, Holt, Davis, Snyderman, Lein, and Smetak, J . Nutrit., 1949, 37, 611,817 Idem, ibid., 1948, 35, 177.s18 Eckhardt and Davideon, J. CEin. Inve~t., 1948, 27, 727.slQ Harper, Proc. SOC. Exp. Biol., 1949, 72, 184.s*O Smyth, Levey, and Lasichak, J . Clin. Inveet.. 19.48, 27, 412.sll Idem, ibid., p. 411332 BIOCHEMISTRY.25.8, histidine 263.3, lysine 100, and methionine 8.3 mg. daily in the urine, thetotal being 2.46% of that ingested.321 Only methionine levels in the bloodfluctuated in relation to the amino-acid pattern of the diet.Adding DL-methionine to the diet caused a rise in free and combined non-precipitablemethionine and lysine in plasma. Urinary outputs were low (about 265%of intake), with only histidine and threonine fluctuating slightly in relationto intake.323 Urinary excretion of amino-acids was largely independent ofprotein intake (0-150 g. daily).324 D- and L-Methionine were absorbed a tthe same rate, but more rapidly from solutions than from tablets.325 Faecaloutput of amino-acids bore little relation to dietary intake.326The digestibilities of rape-seedand cotton-seed oils were 99.0 and 96.5% respectively, 51-6 g. being eatendaily. Butter, olive oil, lard,and margarine failed to raise urinary excretion of oxalicThe total specific dynamic action (S.D.A.) of a 993-kcal.high-protein testmeal during 16 hours post prandium was 17.0y0 of its energy content. Thecorresponding value for a high-carbohydrate meal was 9.6%. Suchincrements were of little practical importance to workers in hot or coldenvironments.329 Work reduced and shortened the S.D.A. of food,particularly if done before aEnergy expenditures (including basal metabolism) of boys a t quiet playwere 3.1 (age 7-8 years), 2.6 (9-11) and 2.1 (12-14) kcals./kg. of body-weight/hr. and when cycling were 6.6, 5-1, and 4.5 kcals. respectively.331Energy expenditures of boys (9-1 1) sitting listening, sitting singing, standingsinging, standing drawing, and dressing and undressing were 2-07, 2-23,2-35, 3.19, and 4-29 kcals./kg./hr.respectively. Corresponding values forgirls (9-11) were 1.80, 2.06, 2.13, 2.62, and 4 ~ 0 4 . ~ ~ ~ ~ ~ ~ ~ The average basalmetabolic rates (B.M.R.) of the children ranged from 1.44 to 1.50k ~ a l s . / k g . / h r . ~ ~ ~ The B.M.R. of girls and young women living in subtropicalconditions and a t an altitude of 2400 ft. declined from 36.4 (14 years) to31.2 (18 years) kcals./sq.m./hr. with little change from 18 to 23 years ~ f a g e . ~ ~ Basal heat production of healthy lean Brazilian adult males was not pro-portional to the body surface. The relation between basal heat productionand body weight ( W ) was found to be : Kcals. per hr. = 2.038 W083 (coldCarbohydrate and Fat (including Energy).There was no effect on nitrogen balance.327Sz1 Sheffner, Kirsner, and Palmer, J.Biol. Chem., 1948, 175, 107.322 Kinsell, Harper, Barton, Hutchin, and Hess, J. Clin. Invest., 1948, 27, 677.323 Kirsner, Sheffner, and Palmer, ibid., 1949, 28, 716.3z4 Eckhardt and Davidson, J. Biol. Chem., 1949, 177, 687.326 Harper and Uyeyama, Proc. SOC. Exp. Biol., 1948, 68, 296.326 Sheffner, Kirsner, and Palmer, J. Biol. Chem., 1948, 176, 89.327 Deuel, Johnson, Calbert, Gardner, and Thomas, J. Nutrit., 1949, 38, 369.328 Kabelitz, Klin. Woch., 1943, 22, 439.3eo Glickman, Mitchell, Lambert, and Keeton, J. Nutrit., 1948, 36, 41.s30 Wachholder, P$iigers Arch., 1949, 251, 485.331 Taylor, Lamb, Robertson, and MacLeod, J. Nutrit., 1948, 35, 51 1.332 Taylor, Pye, Caldwell, and Sostman, ibid., 1949, 88, 1.33s Idem, ibid., 1948, 36, 123.334 Thompson, Cox, and Ridgway, ibid., p.50DUCKWORTH 1 GENERAL NUTRITION. 333climate value is WoSs). For better proportioned subjects the relation, kcals.per hr. = 2.334 wasEstimates of energy needs for h o u s e w ~ r k , ~ ~ ~ ~ 3379 339 sedentary occu-p a t i o n ~ ; ~ ~ ~ light,337 medium,337 and heavy 337* 338 work have been given.Resting-fasting metabolism (Erhaltungsumsatz) was increased or decreasedby raising or lowering food intakes. Errors may be introduced in estimatesof energy needs for work if the resting-fasting rate is assumed to beconstant .340Comparison of bone meal and dried skimmed milk as sourcesof calcium for adults yielded inconclusive results.341 High utilisation ofwhey-calcium was found.342 Organic and inorganic, soluble and insoluble,calcium salts were all absorbed equally well, as judged by blood-calciumlevels. Magnesium favoured calcium a b s o r p t i ~ n .~ ~ Negative calciumbalances were found when crude or refined coconut oil was fed to adults.344Wholemeal bread and oatmeal provoked strongly negative calcium balancesin subjects living almost exclusively on these cereal pr0ducts.~~~9 346 Evidencewas presented to indicate that subjects became adapted to high phytate-phosphorus diets in time, and that calcium balances improved.347 Anoptimum pH of 5.2 was found for faecal p h y t a ~ e . ~ ~ ~ 80% of the calcium inrectal suppositories was absorbed and at a rate comparable with that ofintramuscularly injected calcium. The tone of the uterus was the criterion.34sAdaptation in the adult allowed calcium equilibrium to be maintained onintakes of about 0-5 g. daily.360 Older women (52-74 years) required 1-07 g.of calcium daily for equilibrium.351Gastric reductions of Fe+++ to Fe++ of sO-90~o were found in stomachcontents after eating bread, meats, and fruit.Milk and eggs gave irregularresults.352 More Fe+++ than Fe++ was dialysable from gastric juice betweenneutrality and pH 3.5. Mucin was less important than other gastric juicecomponents in forming stable iron complexes.353 Iron absorption wasMinerah.335 Galvao, J. Appl. Physiol., 1948, 1, 385.336 Droese, Kofranyi, Kraut, and Wildemann, Arbeitsphysiologie, 1949, 14, 63.337 Lshmann, Muller, and Spitzer, ibid., p.16G.338 Kraut, Bauer, Droese, Spitzer, and Wildemann, ibid., p. 147.330 De Langen, Nederland. Tijdschr. Geneesk., 1949, 93, 3247.340 Wachholder, PJliigers Arch., 1948, 250, 534.341 Drake, Jackson, Tisdall, Johnstone, and Hurst, J. Nutrit., 1949, 37, 369.34a De and Som, Indian J. Vet. Sci., 1948, 18, 241.343 Lafontaine, Compt. rend. Xoc. Biol., 1948, 142, 1089.344 De and Karkun, Indian J. Dairy Sci., 1949, 2, 114.345 McCance and Walsham, Brit. J. Nutrit., 1948, 2, 26.346 McCance and Glaser, ibid., p. 221.347 Walker, Fox, and Irving, Biochem. J., 1948, 42, 452.348 Courtois and PBrez, Bull. SOC. Chim. biol., 1949, 31, 1373.34B Sauter, Schweiz. Med. Woch., 1948, 78, 404.350 Kraut and Wecker, Biochem. Z . , 1948, 318, 495.3.51 Roberts, Kerr, and Ohlson, J .Amer. Dietectic ASSOC., 1948, 24, 292.352 Bergeim and Kirch, J. Biol. Chem., 1949, 177, 591.353 Lederer, Aata pntro-enterol. belg., 1949, 12. 233334 BIOUHEMISTBY.improved by adding beef to the diet of women.sM Urinary output of ironwas reduced by dosage with sodium hydrogen carbonate and increased byammonium chloride.356 Iron needs of girls (13-14 years) were 12-13 mg.daily.356 The needs of women (18 years) were between 7 and 10.4 mg.daily, the higher level being more satisfactory for repairing menstruallosses. Beef promoted iron retention.367* 358 Positive iron and copperbalances were found in women (17-22 years) on iron intakes 64-13.6 mg.,and copper intakes of 6.5-13.0 mg. daily. The amounts of iron and copperretained were similar.36Q The so-called ‘‘ physiological ” anEmia ofpregnancy was caused at least in part by inadequate iron reserves.36o Theiron reserve of the adult male was estimated at 600 mg.361 Iron losses insweat were low, probably not exceeding 0.02-0*06 mg.daily.362Addition of sodium citrate to a diet low in sodium chloride increasedsodium but not chloride output. Urinary potassium output was increasedby sodium citrate but not by sodium When lithium chloridewas used in food as a taste substitute for sodium chloride toxic symptomsdid not appear until serum-lithium levels rose to 1.0 m. e q ~ i v . ~ 6 ~ Radio-sodium studies indicated that the sodium transfer across the placenta duringthe twelfth week of pregnancy was 160 times, and during the fortieth week1100 times the foetal retenti~n.~~b After feeding of potassium citrate tochildren, renal clearances of potassium tended to vary inversely with theplasma-potassium level.In the adult given potassium dihydrogen phosphateor potassium citrate the plasma-potassium levels fell and clearances rose.Clearance with little change in plasma-potassium followed ingestion ofpotassium ~hloride.~6~Radio-iodine was not collected by the foetal thyroid before the fourteenthweek of pregnan~y.~67 The anti-thyroid activity of a large range of foodswas tested, by using radio-iodine. The inhibitory effect was greater invegetable than in animal products, swede, turnips, beets, cabbage, lettuce,and spinach being most potent.368J. D.854 Johnston, Frenchman, and Boroughe, J.Nutrit., 1948, 55, 453.5s5 Barer and Fowler, J. Lab. Clin. Med., 1949, 54, 932.366 Schlaphoff and Johnston, J. Nutrit., 1949, 59, 67.357 Johnston, Frenchman, and Boroughs, ibid., 1949, 38, 479.s58 Frenchman and Johnston, J. Amer. Dietetic ABSOC., 1949, 25, 217.350 Holt and Scoular, J. Nutrit., 1948, 35, 717.a60 Wills, Hill, Bingham, Miall, and Wrigley, Brit. J. Nutrit., 1947, 1, 126.361 Hynes, J. Clin. Pathol., 1949, 2, 99.a62 Johnston and Hagan, Fed. Proc., 1949, 8, 387.364 Talbott, Arch. Int. Med., 1950, 85, 1.365 Flexner, Cowie, Hellman, Wilde, and Vosburgh, Amer. J. Obstet. Gynecot., 1948,366 Wilson, Arch. Dis. Childhood, 1948, 25, 176.367 Chapman, Corner, Robinson, and Evans, J. Clin. Endocrinol., 1948, 8, 717..568 Greer and Astwood, Endocrinology, 1948, 45, 105Leaf, Couter, and Newburgh, J.Clin. Invest., 1949, 28, 1082.55, 469HERBERT 3356. OXIDISING) ENZYMES.The number of papers on oxidising enzymes published during the last threeyears has been so large that it has not been possible to refer to all of themwithin the space provided ; where a choice has had to be made, papers on theless well-defined enzymes have been omitted. The report deals primarilywith the properties of enzymes, and not with their physiological functions orchanges in various pathological or nutritional conditions. Of the referencescited, 63% are from American laboratories, 12% from British or Common-wealth laboratories, and 12% from Sweden. Clearly the study of thisimportant field is being neglected in this country.Books and Review Articles.-Second editions have appeared of Sumnerand Somers’s useful book,l and the collaborative work on respiratoryenzymes edited by H.A. Lardy; the latter has been almost completelyrewritten. Lemberg and Legge’s book on tetrapyrrole compounds (3182references) contains detailed accounts of a11 the iron-porphyrin enzymes andwill be invaluable to workers in this field; three excellent review articles onhaemoproteins have also appeared. 4 Dixon’s book contains (besides muchother matter of interest) an account of the author’s theoretical treatment ofphosphorylation reactions according to a “ scale of phosphorylationintensitx ” or rP scale, analogous to the rH scale or reducing intensity.AnEnglish translation of Warburg’s “ Schwermetallen ” has now appeared.The new edition of W. A. Waters’s book on free radicals has a long chapteron biochemical and enzymic mechanisms ; three review articles on mechan-isms and kinetics of enzymic reactions have also appeared.8 Review articleshave appeared on lipo~idase,~ the enzymes of snake venom (includingL-amino-acid oxidase) , l o and the citric acid cycle.llGeneral Trends.-The bulk of the work done during the review periodhas proceeded along well-tried lines, but some new trends are emerging, ofwhich the following appear to be significant : (a) The detailed physico-chemical study of pure enzymes by the methods of the protein chemist hasbeen elaborated. ( b ) A new approach to enzyme kinetics has been initiatedby the work of Britton Chance, described in the section below on haemo-proteins.This treatment, combined with the physico-chemical studies,seems most likely to provide an answer to the fundamental problem of allenzyme studies, namely, the problem of how enzymes work. (c) Increasingattention is being paid to insoluble enzyme complexes (“ succinoxidase,”l “ Chemistry and Methods of Enzymes,” Academic Press Inc., 1947.“ Respiratory Enzymes,” Burgess Publ. Co., 1949.“ Haematin Compounds and Bile Pigments,” 1949.Theorell, Adv. Enzymol., 1947, 7 , 265; Granick, ibid., p. 305; Wyman, Adv.Warburg, “ Heavy Metal Prosthetic Groups and Enzyme Action,” Clarendon Press,Protein Chem., 1948, 4, 410.Oxford, 1949.“Multi-enzyme Systems,” Camb.Univ. Press, 1948.’ “ Chemistry of Free Radicals,” Clarendon Press, Oxford, 1948.Michaelis, Adv. Enzymol., 1949, 9, 1 ; Stearn, ibid., p. 25; LuValle and Goddard,* Bergstrom and Holman, Adv. Enzymol., 1945,8,426. Quart. Ref. Biol., 1948,23, 197.lo Zeller, ibid., p. 459. l1 Martins and Lynen, ibid., 1950, 10, 167336 BIOCHEMISTRY." cyclophorase," etc.), and their relationships to mitochondria and otherformed elements of the cell.Methods.-Increasing use is being made of spectrophotometric techniquefor following the course of enzyme-catalysed reactions ; there are few enzymeswhich cannot be studied by this method. A disadvantage of the techniqueis the difficulty of adequate temperature control in most commercialspectrometers.Essenti-ally this is a micro-modification of the original instrument of Hartridge andRoughton, reduced t o a scale which permits complete measurements to bemade with 0.2 ml.of solution. Monochromatic illumination with highlystabilised light sources, photocells, and high-gain amplifiers, permits theautomatic recording of minute spectral changes (log I,/I - lo4). Reactionswith half-times of a few milliseconds can be followed by the " rapid-flow "technique, and slower reactions by a " stopped-flow " method.Some advances in methods for isolation and purification of' enzymes havebeen recorded, though this subject remains more a skilled form of cookerythan a science. Low-temperature fractionation with organic solvents isbeing increasingly used; the classical researches of the Harvard school inthe application of this technique to plasma-protein fractionation are reviewedby Ed~a1l.l~ So far, ethanol and methanol have been used almost exclusivelyfor this purpose, and the experiments of Dixon and Askonas l5 which givecomparative data for a number of solvents are therefore particularly welcome.Svensson l6 has reviewed preparative-scale electrophoresis methods.Animproved version l7 has been described of Kirkwood's apparatus whichfractionates protein mixtures by a combination of electrophoretic transportand vertical convective transport; components move from an upper to alower reservoir at rates depending on their electrophoretic mobilities. I nan interesting apparatus described by Svensson and Brattsten,l* the mixtureflows downwards through the space between two vertical glass plates, packedwith " Celite " to avoid convection, while a horizontal electric field is main-tained between the ends of the cell. Both these methods are in the develop-mental stage, but the latter in particular, on account of its simplicity, affordspromise for the future.Two-phase partition methods, so successful in otherfields, have not hitherto been applied t o protein separation owing to thedifficulty of finding non-miscible solvent pairs which will both dissolve pro-teins. Herbert and Pinsent l9 used the two phases which are formed in certainconcentration regions with three-component systems of the type water +salt + water-miscible solvent, in this case water-ammonium sulphate-ethanol ; the method was successfully applied in the isolation of crystallinebacterial catalase.More work needs to be done before it can be said whetherI n a different category is Chance's rapid-flow instrument, 12- l3l2 Chance, Acta Chem. Scand., 1947, 1, 236.13 Idem, Biochem. J . , 1950, 46, 387.1 5 1st Intern. Congr. Biochem., Cambridge, Aug. 1949 ; Askonas, Biochem. J . , 1951,1 7 Cann, Brown, and Kirkwood, J . Amer. Chem. SOC., 1949,71, 1609.18 Arkiv Kemi, 1949,1,401.l4 Adv. Protein Chem., 1947, 3, 384.l6 Adv. Protein Chem., 1948, 4, 251.lS Biochem. J., 1948, 43, 193.in the pressHERBERT : OXIDISING ENZYMES. 337this will prove a generally applicable method on the preparative scale, butMartin and Porter 2o have shown that it may be applied to the partitionchromatography of proteins on a analytical scale, using columns packedwith (‘ Hyflo.” By this technique, using water-ammonium sulphate-( ( Cellosolve ” mixtures, they have shown that crystalline and electro-phoretically homogeneous ribonuclease contains two enzymically activecomponents.Adsorption chromatography of proteins has been little prac-tised, since the best protein absorbents are gels whose physical propertiesare unsuitable for columns. Tiselius, however, has used strong salt solutionsto promote adsorption of proteins on paper.21 Mitchell, Gordon, andHaskins 22 separated the adenosine deaminase, amylase, and phosphatase ofa takadiastase preparation on a “ chromatopile ” (a column made from astack of filter papers), using an ammonium sulphate solution of graduallydecreasing concentration.Ion-exchange chromatography on a colourlesscarboxylic acid resin of a new type has been used to purify cytochr~me-c.~~The separation and purification of enzymes associated with insolubleparticles has always been a baffling problem and has succeeded in only a fewcases. A recent note by Morton 24 describes the use of n-butanol to obtaintrue solution into distilled water, or buffer solutions, of a wide range ofenzymes from insoluble particles, apparently by removal of lipid and othermaterials. If this proves a general method for dealing with insoluble enzymesit will be invaluable to workers in this difficult field.Coemymes.-[Abbreviations : Diphosphopyridine nucleotide (coenzyme1) is referred to in general as DPN; where the reference is specifically to itsoxidised or reduced form, DPN+ or DPNH is used respectively.TPN,TPN+ , and TPNH are used similarly for triphosphopyridine nucleotide(coenzyme 2), and NMN, NMN+, and NMNH for nicotinamide mono-nucleotide. AMP, ADP, and ATP are used for adenosine mono-, di-, andtri-phosphates, FAD for flavine adenine dinucleotide, FMN for flavinemononucleotide, and DPT for diphosphothiamine.]New or improved methods have been described for the preparation ofDPN 2s and TPN.26 Results reported by Slater 27 indicate that LePage’swidely used assay method 28 for DPN may give spuriously high results withcrude DPN preparations ; these may contain substances which give an absorp-tion band a t 340 mp.on reduction with sodium dithionite but are enzymicallyinactive ; the Reporter’s unpublished observations confirm this. Reductionwith sodium borohydride has been advocated for assay but this2o Biochem. J., 1951, in press.21 Tiselius, Arkiv Kemi, Min., Geol., 1948, 26, B, No. 1 ; Chem. Eng. News, 1949,22 J . Biol. Chem., 1949, 180, 1071.23 Paleus and Neilands, Actu Chem. Scund., 1950,4, 1024.a4 Nature, 1950,166, 1093.25 Hogeboom and Barry, J . Biol. Chem., 1948, 176, 935; Clark, Dounce, and Stotz,28 LePage and Mueller, ibid., 1949, 180, 975,z a .I. Biol. Chem.. 1947, 168, 623,27, 1041 ; Shepherd and Tiselius, Discuss. Paraday SOC., 1949, No. 6.ibid., 1949, 181, 459.Biochem. J . , 1950, 40, 484.29 Mathews.ibid., 1948, 176. 229338 BIOCHEMISTRY.produces similar absorbing but unreactive products. Enzymic reductionmethods would seem preferable, both for assay and for preparation of DPNHor TPNH ; convenient techniques using alcohol deh ydrogenase are describedby Bonnichsen30 and Ra~ker.~l Enzymic methods have also been used toobtain accurate values for the molar extinction coefficients of DPNH andTPNH.32Biochemists working with crude tissue extracts have often been botheredby the presence of enzymes catalysing the breakdown or interconversion ofDPN and TPN. There are several of these enzymes, and Kornberg and hiswssociates have characterised and partly purified four of them :(1) An enzyme 33 obtained in partly purified form from yeast and livercatalyses the reversible reaction :"y:'} + ATP 3 DzP' } + pyrophosphate (i)"H DPNHThe reaction proceeds readily in either direction (equilibrium constantK = 0.45). This enzyme appears to be specific for all four reactants.(2) A second also present in yeast, catalyses a similar reversiblereaction with flavine mononucleotide (FMN), which reacts with ATP to giveFAD :M A + FMN + ATP - FAD + pyrophosphate.. . (ii)(3) An enzyme 35 of the phosphokinase type, also found in yeast, catalysesthe synthesis of TPN+ from DPN+ by direct phosphorylation with ATP;DPNH reacts similarly."?:+}+ ATP y; ':+}+ ADP . . (iii)DPNH TPNHThis reaction throws important light on the structure of TPN, indicatingthat it is not a triphosphoric acid derivative, but a pyrophosphate like DPNwith the third phosphate group attached to some other part of the molecule(see below).(4) A " nucleotide pyrophosphatase ", obtained in highly purified formfrom potatoesY36 cleaves DPN+ or DPNH at the pyrophosphate linkage,forming AMP and the oxidised or reduced form of nicotinamide mononucleo-tide (NMN+ or NMNH)."z?'} + H,O --j "EN') + A M P . . . (iv)DPNH NMNHTPN, FAD, DPT, ATP, and probably ADP are also attacked, though atThe same enzymeJo Acta Chem. Scand., 1950, 4, 714.se Horecker and Rornberg, ibid., 1948,175, 385.as Kornberg, ibid., 1950,182, 779.35 Kornberg, ibid., p, 805,slower rates, all being split a t the pyrophosphate linkage.s1 J . Biol. Chem., 1950,182, 313.Schrecker and Kornberg, ibid., p.795.Bornberg and Pricer., ibid., p. 763HERBERT : OXIDISING ENZYMES. 339is believed to attack all these compounds; crude extracts also contain en-zymes specifically attacking ATP and DPT. The products formed by theaction of this enzyme on TPN are NMN, and an adenosine diphosphate whichis not identical with ordinary ADP and is not a pyrophosphate compound;i.e., its two phosphate groups are attached to adenosine a t different points.37One of these is attached to C(5) of the ribose, and is cleaved by a specificadenosine4 phosphatase (isolated from potatoes) ; the monophosphateresulting is identical with the adenylic acid a recently isolated by Carter 38and Cohn 39 from yeast nucleic acid, which is probably adenosine2phosphate.The adenosine diphosphate arising from TPN is thereforeadenosine4 : 5 diphosphate, and the structure of TPN is probably :CHHi:-j OHHV*OOj~-OHH9-1 HV*OHHCOHi OH$!*OH IHF 9- QH HV CH2-O-~-O-~-O-CH20 0This work is an outstanding example of the use of enzymes in the elucidationof chemical structure.Mehler et a1.40 have tested thecoenzyme specificity of a number of dehydrogenases. The ratioactivity with DPN was found to be 135-220 for lactic dehydrogenase activity with TPN(three sources), 25-34 for malic dehydrogenase (two sources), and about 1for glutamic dehydrogenase (liver). isoCitric acid and glyceraldehyde-3 phos-phate dehydrogenases were completely specific for TPN and DPN respectively.Lactic and malic dehydrogenases had previously been thought to be com-pletely specific ; possibly other “ specific ” dehydrogenases might prove tobe more or less unspecific if tested equally rigorously.DPN Dehydrogenases.-AZcohoZ dehydrogenase of yeast has been crystal-lised by Racker 31 by a simple method giving good yields, which should makeit a readily available enzyme.The effect of pH was studied on the equili-brium :The true equilibrium constant of the reaction is obviously K =[CH,*CHO][DPNH][H+]/[C,H,*OH][DPN+], and K has the constant value1.15 x 10-l1 over a wide pH range. The equilibrium constant is usvallyCoenzyme Specificity of Dehydrogenuses.C2H,*OH + DPN+ CH,*CHO + DPNH + H+ (v)s7 Kornberg and Pricer, J Biol. Chem., 1960, 186, 657.s8 J . Amer. Chem. SOC., 1050, 72, 1466.?e Ibid., p.1471. 40 J . Biol. Chem., 1948,174, 96340 BIOCHEMISTRY.(wrongly) written as K' = [CH,*CHO][DPNH]/[C,H5*OH][DPN+], and isobviously pH-dependent; in fact a rise in pH of 1 unit causes a 10 foldincrease in the value of K'. Similar relations were found for lactic dehydro-genase, and should apply generally. At a fairly alkaline pH, in the presenceof excess of alcohol, reaction (v) goes almost completely from left to right, andadded DPN+ is quantitatively reduced to DPNH which may be determinedspectrophotometrically ; this makes a convenient assay method for DPN.The enzyme may also be used for the preparation of DPNH from DPN'.Bonnichsen 41 has crystallised the alcohol dehydrogenase of horse liver ;the apparently pure enzyme has, curiously, a turnover number for DPN verymuch lower than that of the yeast enzyme.He has also described enzymicmethods for assay of DPN and preparation of DPNH.30Racker 42 has discovered and purified to a considerable extent the pre-viously unknown acetczldeh yde dehydrogenase, which occurs in liver andcatalyses the virtually irreversible reaction :CH,*CHO + H20 + DPN' -+ CH,*C02H + DPNH + H+ (vi)A mixture of this enzyme with alcohol dehydrogenase behaves as an" aldehyde mutase," catalysing the dismutation of acetaldehyde accordingto the equation :2CH3*CH0 + H20 --+ CH,*CH,*OH + CH,*CO,H (vii)which results from adding equations (v) and (vi). There would seem to benow no grounds for postulating the existence in liver of a distinct aldehydemutase enzyme.Crystalline D-glyceraldehyde 3-phosphate dehydrogenuse of rabbit skeletalmuscle was isolated by the Cori's and their associate^.^^ It is electrophoretic-ally homogeneous.Accurate values are given for its catalytic activity, andits amino-acid content has also been determined.u This enzyme has a num-ber of unusual properties. The crystalline enzyme as isolated contains onemole of firmly bound DPN per 50,000 g. of protein ;45 this is not removed bydialysis or repeated recrystallisation, though it can be removed by treatmentwith phosphatase or " Norit." The DPN-free protein retains its activityafter these treatments, but will no longer crystallise unless DPN is added;the crystals then formed contain the original amount of bound DPN. Thisbound DPN is reduced, with appearance of an absorption band at 340 mp.,when glyceraldehyde-3 phosphate + phosphate (or arsenate) is added.Toobserve this spectrophotometrically, enzyme concentrations of 2 - 4 mg. /ml.are needed, the reaction being then too fast for its kinetics to be followed;if glyceraldehyde is used as substrate, however, the reaction is slow enoughBonnichsen and Wassen, Arch. Biochern., 1948, 18, 361 ; Bonnichsen, Acta Chem.43 G . T. Cori, M. W. Slein, and C. F. Cori, ibid., 1948,173, 605.44 Velick and Ronzoni, ibid., p. 627.Scand., 1950, 4, 715. 42 J. Biol. Chem., 1949,177, 883.4 5 Taylor et d,* ibid., p. 619.* References with four or more co-authors are cited bv the name of the first authoronlyHERBERT : OXIDISING ENZYMES. 341to be followed.reaction then occurs : 46glyceraldehyde + enzyme-DPN+ -+ glyceric acid +one molecule each of substrate and DPN reacting for each molecule ofprotein.The kinetics show that neither DPN+ nor DPNH can dissociate toa detectable extent, which means a dissociation constant of a t most lo-'-probably much less. If DPN+ is added to the DPN-enzyme the added DPN+is also reduced and the overall rate increases ; when the DPN+ concentrationis large compared to that of the enzyme, the reaction is :glyceraldahyde + DPN+ + glyceric acid + DPNH + H+ (ix)The enzyme here acts catalytically, and the kinetics indicate that DPN+and DPNH form reversible compounds with the enzyme, with equal dissoci-ation constants of 4 x The simplest hypothesis covering theseapparently contradictory facts is that the enzyme has two binding sites forDPN, one with comparatively low affinity (dissociation oonstant 4 xand the other with very much higher affinity.That DPN bound at thelatter site does dissociate to a finite (though very small) extent is presumedbecause ( a ) bound DPN exchanges with added DPN labelled with 32P, and( b ) bound DPNH is oxidised on addition of lactic dehydrogenase andpyruvate. These interesting observations are referred to again under" cyclophorase " (see below). Another interesting property of this enzymeis described by Rapkine et ~ 1 . ~ ~ Crystalline preparations lose activity onstorage a t room temperature or 0" ; such partly inactivated " aged " speci-mens are re-activated to a considerable extent by being heated for shortperiods at 55-65".The percentage reactivation by heat increases with ageand extent of previous inactivation, though the original activity of thefreshly crystallised enzyme is never regained in full. Heat-activation isincreased by addition of inert protein, formed on ageing. To explain theseunique phenomena the hypothesis is advanced that heat-activation " is aresult of the interaction between mercapto groups of the inactive protein anddisulphide groups of the active protein."L-a-GZym-ophosphk dehydrogenase of rabbit skeletal muscle has beenisolated by fractional crystallisation of Myogen A preparation^.^^ Itcatalyses the reaction :a-glycerophosphate + DPN+ =+In the presence of arsenate the following stoicheiomtricenzyme-DPNH + H+ (viii)enzymedihydroxyacetone phosphate + DPNH + H+ (x)The equilibrium position lies far to the right (K' = 1.4 x lo4 at 20" andpH 7 ) ; in the presence of excess of DPNH, dihydroxyacetone phosphate iscompletely reduced, and it may be assayed by this system.O 6 C.F. Cori, S. F. Velick, and G. T. Cori, Biochim. Biophys. Acta, 1950, 4, 160. '' Rapkine, Shugar, and Siminovitch, Arch. Biochem., 1950, 86, 33,u Baranowski, J . Biol. Chem., 1949, 180, 636342 BIOOHEMISTRY.The substrate specificity of crystalline lactic dehydrogenase has beenstudied by following spectrophotometrically the oxidation of DPNH byvarious keto-a~ids.~~ a-Ketobutyric acid is reduced nearly as fast aspyruvic acid, but for higher a-keto-acids the rate falls off rapidly with chainlength.The straight-chain ay-diketo-acids (from C, to Cll) are also reduced,all at about the same rate, equal to about one-tenth of the rate for pyruvicacid; only the a-keto-group is reduced.The formic dehydrogenase of green peas has been partly purified.s0 Itcatalyses the reaction ;As expected from the calculated free energy change (AFo = -6310 cals.),reaction (xi) is virtually irreversible, and when it was carried out in the pres-ence of NaH14C0, only minute amounts of 14C were incorporated into theformate. It is therefore unlikely that reversal of reaction (xi) can be asignificant pathway of C0,-fixation. The enzyme should be useful for DPNassay, since it is completely specific and reduction of DPN+ goes to comple-tion.Similar enzymes are present in small amounts in liver and kidney.Glucose dehydrogenase of lamb liver has been partly purified 6 1 p s2 and itscarrier systems studied.61* s3 Papers on this topic, discussing whether ornot cytochrome-c, diaphorase, etc., are involved in the electron transportbetween glucose and oxygen, have appeared at intervals ever since thediscovery of the enzyme (cf. Harrison et The impression is given bysome authors that the problem is in some way specifically connected withglucose dehydrogenase, whereas it applies to all DPN-dehydrogenases ; the realproblem (discussed in a later section) is the mechanism of aerobic oxidationof DPNH, irrespective of the particular dehydrogenase system reducingDPN+.A heat-stable malic dehydrogenase has been obtained by Militzer et aLS6from a thermophilic organism of the Bacillus group; the dehydrogenase inmesophilic bacilli is not heat-stable.A soluble liver enzyme has been obtained by Sweat et al.66 which inthe presence of DPN oxidises the >CH*OH group of testosterone to >CO,giving androstenedione.Further purification of the enzyme is needed todetermine if it is a simple DPN-dehydrogenase; if so, it will be the firstdehydrogenase known to act on a sterol.TPN Dehydmgenases.-isoCitric acid dehgdrogenuse of pig heart has beenstudied extensively by Ochoa and his associates. The previously unknownsubstance oxalosuccinic acid was synthesised s7 (it was also independently4o Meister, J .Biol. Chem., 1950, 184, 117.50 Mathews and Vennesland, ibid., 1950, 186, 667.51 Eichel and Wainio, ibid., 1948,175, 155.s4 Brunelli and Wainio, ibid., 1949, 177, 75.65 Renvall, Acta Chem. S a n d . , 1950, 4, 738.54 Harrison and Hawthorne, Biochem. J . , 1939, 33, 1573.6 6 Arch. Biochem., 1949, 24, 75.5 6 Sweat, Samuels, and Lumry, J . Biol. Chem., 1950,186, 75.Ochoa, ibid., 1948,174, 115.H*CO,-+ DPN+ j CO,+ DPNH . . . (xiHEBBERT : OXIDISING ENZYMES. 343synthesised by Lynen et ~ 1 . ~ ~ ) and found 50 to be the primary oxidationproduct of isocitric dehydrogenase (eqn. xii). It is decomposed by a secondenzyme " oxalosuccinic decarboxylase " 8o .into a-ketoglutaric acid (see xiii).isocitrate + TPN+ + oxalosuccinate + TPNH + H+ (xii)Mn++oxalosuccinate + H,O -.....a-ketoglutarate + HC0,- (xiii)bBoth reactions are reversible, but only the second needs Mn++. While(xi;) proceeds readily in either direction, the equilibrium of (xiii) lies far to theright ; nevertheless it was possible to reverse both reactions, bringing abouta fixation of carbon dioxide in isocitric acid, by coupling the above systemwith glucose-6 phosphate dehydrogenase and its substrate ; this systemperforms the function of keeping all the TPN present in the reduced state.69This reaction may be of importance in C0,-fixation; the left-to-right reac-tions (xii) and (xiii) are certainly important steps in the citric acid cycle.While the enzymes catalysing (xii) and (xiii) have been given different names,it is by no means certain that they are in fact different enzymes.Theyalways occur together and attempts to separate them have so far beenunsuccessful ; on purification the ratio of their activities is not altered.s1Their instability, however, has so far prevented extensive purification.Ochoa suggests that this may be a case of a single enzyme with a doublefunction, as has been suggested for the " malic enzyme" (see below).Enzymes catalysing reactions (xii) and (xiii) have been found by Venneslandand her co-workers in pigeon liver 62 and in plants,Gs and C0,-fixation by thesystem has been demonstrated with 14C0,. Here again, it is uncertainwhether one or two enzymes are concerned.A 6-phosphogluconic acid dehydrogeme has been obtained in soluble formin rat liver extracts,M and separated by ammonium sulphate fractionationfrom glucose and glucose-6 phosphate dehydrogenases.Partial purificationof a similar enzyme from yeast has also been reported.s6The " malic enzyme " is the name given by Ochoa et aZ.401 66 to an enzymeor enzyme system of pigeon liver that catalyses the reversible overallreaction :M d malate" + TPN+ - pyruvate- + CO, + TPNH (xiv)The equilibrium of this reaction lies to the right, but it has been possible toreverse it, with a resulting net fixation of carbon dioxide; this reaction,rather than (xii) and (xiii), is thought by Ochoa et at?. to be a major pathwayof C0,-fixation. 66 Reaction (xiv) involves a simultaneous oxidation and6 859606 16863646666Lynen and Schere, Annalen, 1948,560, 163.Ochoa, J .Biol. Chem., 1948,174, 133.Ochos and Weisz-Tabori, ibid., p. 123.Grafflin and Ochoa, Biochirn. Biophys. Acta, 1950, 4, 205.Grisolia and Vennesland, J . Biol. Chem., 1947, 170, 461.Ceithaml and Vennesland, ibid., 1947,178, 133.Dickens and Glock, Natwe, 1950,166, 33.Horecker, Fed. PYOC., 1950, 9, 186.Ochoa, Mehler, and Kornberg, J . BioE. Chem., 1948,174,979344 BIOCHEMISTRY.decarboxylation of malate. All enzyme preparations which catalyse it willalso decarboxylate oxaloacetic acid (eqn. xv) :Mn++ oxaloacetate‘ + H+ --+ pyruvate- + C02 . . (xv)Malic dehydrogenase, though a DPN-enzyme, also reacts fairly rapidly withTPN according to eqn. (xvi) :and this reaction followed by (xv) would give the overall reaction (xiv).However, the (( malic enzyme ” seems not to be a simple mixture of malicdehydrogenase and oxaloacetic decarboxylase, but a single enzyme catalysingboth reactions (xiv) and (xv).The ratio of these two activities remainsconstant on extensive purification, and the enzyme in the absence of Mn++does not have the properties of a malic dehydrogenase.66 Moreover,artificial mixtures of pure malic dehydrogenase and purified oxaloaceticdecarboxylase of Micrococcus lysodeikticus 671 68 [which catalyses reaction(xvi) only] do not have all the properties of the (‘ malic enzyme,” 4°-669 68which seems, therefore, to be a “ double-barrelled ” enzyme of a new type.A similar ( ( malic enzyme,” but utilising DPN instead of TPN, has beenfound in Lactobacillus arabino~us.~~ Vennesland and her co-workers 70* 71* 72have described apparently similar systems in parsley root and other plants,though it is not so certain in these cases that a single enzyme is concerned.In addition to reactions (xiv) and (xv), the plant enzyme systems catalysereaction (~vi),~O which the “ malic enzyme ” of pigeon liver does not ; how-ever, the malic dehydrogenase and oxaloacetic decarboxylase activities arealways closely associated.72Flavoprotein Enzymes.-L-Bmino-acid Oxidase of Snake Venom (“ Ophio-amino-acid Oxidme ”).Zeller’s review 10 covers the literature on this enzymeto 1948. Since then the enzyme has been isolated in a pure state frommocassin venom by Singer and K e a r n e ~ .~ ~ Homogeneity is indicated byultra-centrifuge, electrophoresis, and solubility data. The prosthetic groupis FAD,74 and analysis indicates one mole of FAD per 62,000 g. of protein;the molecular weight from ultra-centrifuge data is also 62,000. The sameauthors have described an interesting type of inhibition of the enzyme bymultivalent anions such as phosphate and arsenate. 75Some new amino-acid oxidases frommoulds have recently been described. These have not been purified andtheir chemical nature remains unknown ; they are included in this section onflavoprotein enzymes on grounds of analogy only.This organism6’ Herbert, Biochem. J . , 1950, 47, i.6 8 Idem, SOC. Exp. Biol. Symp. Carbon Dioxide Fixation, 1950.&@ Korkes and Ochoa, J .B i d . Chem., 1948,176,463.70 Vennesland, Gollub, and Speck, ibid., 1949,178, 301.7 1 Vennesland, a i d . , p. 591.72 Conn, Vennesland, and Kraemer, Arch. Biochem., 1949,23, 179.73 Ibid., 1950, 29, 190.7 5 Kearney and Singer, ibid., 1949, 21, 242.malate- + TPN oxaloacetate + TPNH + H+ (xvi)Amino-acid Oxidases from Moulds.(a) D - and L-Amino-acid oxidases of Neurospora crassa.74 Singer and Kearney, ibid., 1950, 27, 348HERBERT : OXIDISING ENZYMES. 345was previously known to produce a D-amino-acid ~ x i d a s e . ~ ~ It has nowbeen found that some strains produce an L-amino-acid oxidase but not theD-enzyme, while other strains produce both enzymes.77 The L-enzyme, butnot the D-enzyme, is excreted in considerable amounts into the culturemedium.(b) D- and L-Amino-acid oxidases of Penicillium qnd Aspergillus s p c k s .L-Amino-acid oxidases were discovered by Knight 78 in a number of Peni-cillium and Aspergillus species. Their properties were all very similar, butdiffer somewhat (e.g., in specificity) from the Neurospora enzyme; they arenot excreted into the culture medium, remaining firmly attached to themycelium.More recentl~,7~ D-amino-acid oxidases were found in eightPenicilliurn strains and in Aspergillus niger. These also are not excretedinto the culture medium, but active cell-free extracts could be obtained bygrinding the mycelium with sand. Bender and Krebs 8o have studied therate of oxidation of 48 amino-acids, among them 27 not occurring naturally,by the D-amino-acid oxidases of sheep kidney and Neurospora crassa and theL-amino-acid oxidases of Neurospora crassa and cobra venom.Considerabledifferences in specificity and relative rates of oxidation were found.D-Aspartic acid oxidase differs from the above amino-acid oxidases, butresembles glycine oxidase, in being specific for a single amino-acid. It hasbeen obtained in soluble form in liver or kidney extracts,81 but is distinctfrom Krebs’s classical D-amino-acid oxidase. It has not been greatly purified,but was identified as a flavoprotein by resolution into the apoenzyme, whichis inactive unless supplemented with FAD ; FMN is ineffective.82(The Reportersuggests that the use of the name “notatin” for this enzyme should beabandoned in favour of “ glucose oxidase,” which besides being descriptiveand conforming to normal enzyme nomenclature, has clear historicalpriority.) Keilin and Hartree 83 have made a detailed study of the propertiesof this enzyme, purified according to the method of Coulthard et aLs4 Theprosthetic group was identified as FAD, and direct analysis (corrected forca.8% of impurity in the enzyme) gave one mole of FAD per 75,000 g.protein. The molecular weight by ultra-centrifuge measurements is 152,000 ;hence the enzyme molecule contains two molecules of FAD. The specificitytowards a large number of compounds was tested; a few sugars and somemethylated glucoses are oxidised at very low rates, but to all intents andpurposes the enzyme may be considered specific for glucose.This makes itan invaluable reagent for glucose analysis; Keilin and Hartree 85 have alsoshown how it may conveniently be used to follow manometrically the actionof other enzymes which liberate glucose from its derivatives (e.g., glucosidases,Glucose oxidase of Penicillium notatum (“ notatin ”).78 Horowitz, J . Biol. Chem., 1944,154, 141.7 7 Bender, Krebs, and Horowitz, Biochem. J . , 1949,45, xxi. ’* J . Bact., 1948, 55, 401.Emerson, Puziss, and Knight, Arch. Biochem., 1950, 25, 299.Biochem. J . , 1950, 46, 210.Biochem. J . , 1948,42, 221.81 Stilletal., J. Biol. Chem., 1949, 179, 831.84 Ibid., 1945, 39, 24.82 Still and Sperling, ibid., 1950, 182, 585.85 Ibid., 1948, 42, 230346 BIOCHEMISTRY.maltase, invertase, glucose-phosphatases). Bentley and Neuberger e6used water enriched with H,lSO to study the reaction mechanism of theenzyme.The oxygen atoms of the hydrogen peroxide produced werederived entirely from molecular oxygen, without any detectable admixtureof oxygen derived from water. Polarimetric studies also showed theinteresting fact that the primary oxidation product is not gluconic acid but8-gluconolactone, which is hydrolysed to gluconic acid by a non-enzymicmechanism. The overall action of the enzyme must therefore be formulatedas : --+ H,O,HO&H 11 1 o + o , =The enzyme oxidases p-glucose 1.3 times as fast as a-glucose; suggestive,though not conclusive, evidence was obtained that the enzyme catalysesthe mutarotation of glucose, and that or-glucose is converted enzymicallyinto the p-form before being oxidised.TPN-Cytochrome c rediictuse is an important enzyme isolated from pigliver by Hore~ker.~’ In tissue extracts it is associated with small particles(probably mitochondria, cf.ref. 177), and digestion with trypsin was necessaryto bring it into solution. The pure enzyme contains one mole of FAD per68,000 g. of protein, differing from the corresponding yeast enzyme whoseprosthetic group is FMN. When resolved into the apoenzyme, however,this will combine to form active enzymes with both FAD and FMN, andcuriously, the “ artificial ’’ FMN-enzyme is the more active. The corres-ponding yeast apoenzyme also forms an “ artificial ” enzyme with FAD,and this is less active than the natural FMN-enzyme.The enzyme is reducedspecifically by TPN; the reduced enzyme is oxidised >50 times faster byferricytochrome-c than by oxygen.Xunthine oxiduse has been isolated from milk by an improved methodwhich gives preparations three times more active than any previouslydescribed, but still containing a little lactoperoxidase.88 Ferricyto-chrome-c is reduced by the enzyme with either aldehydes or purines assubstrates ; reduction is slow anaerobically but is accelerated by oxygen.The mechanism of this effect is not clear. Kalckar et aLe9 reported the oxid-ation of xanthopterin to leucopterin by a partly purified milk enzyme; this“ pterin oxidase ” and also xanthine oxidase were strongly inhibited bypteroylglutamic acid. Lowry et aLgO showed that this was due to traces ofan inhibitor, 2-amino-6-formyl-4-hydroxypteridine, in the pteroylglutamicacid preparations ; this substance also inhibits xanthine oxidation byxanthine oxidase.Purified preparations of the latter oxidise 2-amino-4-hydroxypteridine to isoxanthopterin as well as oxidising xanthopterin ;“ pterin oxidase ” is probably identical with xanthine oxidase. This is8’1 J . Biol. Chem., 1950, 183, 593. 86 Biochem. J., 1949, 45, 584.8 8 Horecker and Heppel, ibid., 1949, 178, 683.sB Kalckar and Klenow, ibid., 1948, 172, 349.Bo Lowry, Bessey, and Crawford, ibicl., 1949,180, 389, 399HERBERT : OXIDISING ENZYMES. 347confirmed by Kalckar et aZ.,91 who find that 6-formylpteridine is also a stronginhibitor of both xanthine and xanthopterin oxidation. Krebs and Norris 92confirm the probable identity of xanthine and pterin oxidases.Xanthopterinis oxidised more slowly than xanthine, but has a much higher affinity forthe enzyme ; it thus acts as a competitive inhibitor of xanthine oxidation.Copper-protein Enzymes.-PhewICt8eS. The hitherto obscure relationbetween the enzymes oxidising monophenols (“ tyrosinase,” “ monopheno-lase,” “ cresolase ”) and those oxidising o-dihydric phenols (“ catecholase,”“ polyphenol oxidase ”) is considerably clarified by two papers from Nelson’slaboratory. Mushroom tyrosinase can be separated into fractions of highand relatively low catecholase-to-cresolase activity ratios.gs Several prepar-ations with different activity ratios were homogeneous on electrophoresisand ultra-centrifugation but had different electrophoretic mobilities andcopper contents.94 The preparations with relatively high catecholaseactivity had properties differing from those of the enzyme as it occurs inmushroom juice.It is suggested94 that natural mushroom tyrosinase is asingle protein complex possessing both catecholase and cresolase activitiesand containing 4 copper atoms; the cresolase activity is associated withparts of the molecule which are split off by the purification procedure usedfor obtaining “ high catecholase ” preparations, The simultaneous oxid-ation of o-dihydric phenols is assumed necessary for the ozfidation of mono-phenols. It remains to be seen whether this hypothesis applies to otherphenolases.It is significant that catecholases with no monophenolaseactivity have been obtained, but preparations with only monophenolaseactivity have never been reported. Eiger and Dawsong5 have isolatedsweet-potato phenolase, which only oxidises o-dihydric phenols ; potatoslices oxidise monophenols readily, but this activity is lost as soon as thecell is destroyed. Kendalg6 also thinks that mushroom tyrosinase is asingle enzyme with different centres oxidising mono- and di-hydric phenols,but believes the two activities to be independent ; i.e., o-dihydric phenols arenot necessary for monophenol oxidation,Mammalian tyrosinase of mouse melonoma possesses both activities, butit is associated with insoluble cytoplasmic particles and no purification hasyet been possible.97 Schacter 98 describes a catecholase in human serumwhich oxidises only dihydric phenols. Studies have appeared on the pig-ments produced by secondary reactions when oxidation of phenols occurs inthe presence of certain amino-acids and amines,98*99 on the oxidation ofnumerous substituted phenols by tyrosinase,100 and on the oxidative in-activation of poison-ivy allergens by lac case. 101 A micro-spectr opho t o -91 J .Biol. Chem., 1948,174, 771.93 Mallette et al., ibid., 1948,16, 283.95 Ibid., 1948, 21, 181.97 Lerner et al., J . Biol. Chem., 1949, 178, 185; DuBuy et al., J . Nut. Canc. Inst.,Jackson and Kendal, Biochem. J . , 1949,44, 477 ; James et al., ibid., 1948,43, 626.92 Arch. Biochem., 1949,24, 49.94 Mallette and Dawson, ibid., 1949, 23, 29.g6 Biochem.J . , 1949, 44, 442.s8 J . Biol. Chem., 1950,184, 697. 1949, 9, 325.loo Beevers and James, ibid., p. 636; Cushing, J . Amer. Chem. Soc., 1948,70, 1184.lol Sizer, Arch. Biochem., 1949,20, 103348 BIOCHEMISTRY.metric method for catecholase assay using dichlorophenol-indophenol ashydrogen-acceptor is reported.lo2Dodds Io3 has partly purified the ascorbic acidoxidase of cucumber juice, and has compared its catalytic activity towards anumber of compounds with that of inorganic copper. Of the compoundstested, Cu++ ion only catalysed the oxidation of enediols, all at roughlysimilar rates ; the oxidase was more specific, only oxidising enediols with anadjacent carbonyl group and ring system, and these were attacked at widelydiffering rates.‘( Caerulosin,” a blue protein with a haemocyanin-typeabsorption spectrum, has a molecular weight of 151,000 and contains 8 copperatoms per molecule.104 It has been isolated from human and pig serum andaccounts for most of the serum-copper, but so far has not been found topossess any enzymic activity ; it is not identical with haemocuprein.Iron-porphyrin Proteins.-Catalase. (a) New catalases. Crystallinecatalases have now been isolated from nine different sources. They are allproteins of molecular weight ca. 230,000 containing 4 haematin groups permolecule ; these may all be protohaematin, or a varying proportion, in someliver catalases, may be “ bile pigment haematin ’’ (verdohaematin).Bonnichsen 105 crystallised catalase from horse erythrocytes and horse liver ;the former contained no bile pigment haematin while the latter containedca. 0.2y0 (18% of the total haematin).The protein components of the twoenzymes, however, were immunologically identical and had the same amino-acid contents ; they were immunologically distinct from human- bloodcatalase. Bonnichsen also isolated crystalline catalases from human erythro-c y t e ~ , ~ ~ ~ human liver,lo6 guinea-pig liver,lo7 and horse kidney.lo6 None ofthese contained any bile pigment haematin, which is therefore not an invari-able component of liver catalases ; Bonnichsen lo’ believes it to be an artefactarising during isolation. Catalases containing bile pigment haematin have anincreased end-absorption in the red spectral region, and a decreased absorp-tion at 405 mp., compared with catalases containing only protohaematin.lo7Bonnichsen’s preparation from human erythrocytes Io5 appeared to containonly 0.83% of total haematin, corresponding to only 3 haematin groups permolecule, but the same enzyme crystallised by a different method by Herbertand Pinsent 108 had 4 haematin groups per molecule.Herbert and Pinsent l9isolated a crystalline catalase from a bacterium, Micrococcus Zysodeikticus,which has an extremely high catalase content ( 1--2yo of the total dry weight).A two-phase partition method (cf. ‘( Methods ” section) was successfullyused in the isolation of this enzyme, which contained 4 protohaematins permolecule and no bile pigment haematin; its sedimentation constant in theultra-centrifuge log was the same as that of human-erythrocyte catalase, andits molecular weight is 230,000.It is an unfortunate fact that mostOther Coppek-proteins.(b) Activities and assay methods.lo2 Smith and Stotz, J .Biol. Chem., 1949,179,856.lo( Holmberg and Laurell, Acta Chem. Scand., 1948, 2, 550.lo5 Arch. Biochem., 1947, 12, 83.lo6 Acta Chem. Scand., 1947, 1, 114.lo8 Biochem. J . , 1048, 43, 203.loS Arch. Biochem., 1948,18,51.Ibid., 1948, 2, 561.Cecil and Ogston, ibid.,p. 205HERBERT : OXIDISING ENZYMES. 349catalase assays carried out before 1947 have been more or less unsatisfactory.The best known method is that of von Euler and Josephson,llo and catalasoactivities measured by this method have been expressed as ( ( Katalase-fahigkeit ” (Kat.f.). It involves exceedingly dilute enzyme solutions andthere is considerable enzyme destruction during the assay period, shown by aprogressive fall in the observed values of the first-order velocity constant, k ,whose initial value has t o be determined by an uncertain extrapolation tozero time.Furthermore, Kat.f. is defined in a confusing manner which hasled many workers to make mistakes in its meaning (cf. ref. 3, p. 411).Bonnichsen, Chance, and Theorell ll1 have devised a satisfactory assaymethod which depends simply on using high catalase concentrations ; allthe hydrogen peroxide is then decomposed before any appreciable enzymedestruction occurs, and the value of k remains constant throughout the reac-tion.These authors used a rapid titration method over short (ca. 15 scc.)time intervals; Lord Rothschild 112 used the same principles in a rapidmanometric method, employing a Barcroft- type manometer specially modi-fied for readings over short time intervals. Chance 113 used the Beckmanspectrophotometer to follow catalase activity by measuring the decrease inabsorption due to hydrogen peroxide at 215 mp. ; by modifying its electroniccircuits and coupling it t o a pen-recorder the instrument could be madeautomatic, and reaction half-times of 5 seconds or less could be accuratelymeasured.l14 By use of the rapid titration method, the kinetics of catalatichydrogen peroxide decomposition were found ll1 to follow the equation-dS/dt = kES, where S, E , are substrate and enzyme concentrations inmoles/l., t is time in seconds, and k is a characteristic velocity constant ofthe enzyme.The authors advocate the use of k, expressed in the properunits (1. mole-l sec.-l) instead of Kat.f. for the designation of catalase activi-ties, and there is much to be said for this. As the above equation implies,the decomposition of hydrogen peroxide is always a first-order reaction. Theusual (‘ saturation ” of the enzyme with change to a zero-order reaction at-high substrate concentrations is not observed with catalase, which has no(‘ Michaelis constant ” in the ordinary sense; these kinetics appear to beunique, and are a consequence of the unusual mode of action of the enzyme(see below). Other unusual properties of the enzyme are the very lowtemperature coefficient (activation energy only ca.1700 cals.) and the constantactivity over the pH range 3-5-8 ; the higher temperature coefficients andapparent pH optimum found by earlier workers, like the apparent (‘ Michaeliseonstant,” areal1 artefacts caused by enzyme destruction under unsuitableassayconditions. Feinstein 115 has described a catalase assay method using per-borate as substrate ; the catalase apparently attacks, not the perborate itself,but the hydrogen peroxide arising from its spontaneous decomposition. TheReporter sees no advantages in this method, which introduces a new variableinto the system and does not avoid the important factorof enzyme destruction.110 Annalen, 1927, 452, 158.*12 J .Ecp. Biol., 1950, 26, 399.114 Chance and Herbert, Biochem. J., 1950,46,40!2.1 1 1 Acta Chem. Scand., 1947, 1, 685.113 J . Biol. Chem., 1949, 179, 1299.115 J . Biol. Chem., 1949,180,1197350 BIOCHEMISTRY.When assayed by reliable methods, all erythrocyte, liver, and kidneycatalases containing 4 protohaematin groups per molecule have aboutthe same activity (k = 3.5 x lo7 l.mole-lsec.-l; Kat.f. = 60,000).19~107Liver catalases containing verdohaematin have lower activities per molecule,but roughly the same activity per molecule of profohmmatin. Since allthese catalases are of mammalian origin their equal activities are perhapsnot unexpected ; bacterial catalase, however, has ca.50% greater activity(k = 5.3 x lo7; Kat.f. = 97,000).19 It would be interesting to have datafor plant, invertebrate, and fungal catalases.(c) Kinetics and .mode of action. A series of important papers byChance 12, 13* 113- Il4* ll6? 117* 118 has greatly increased our knowledge of thisenzyme. Combination of the spectrophotometric rapid-flow apparatus 12with a platinum micro-electrode l3 allowed changes in oxygen, hydrogenperoxide, free catalase, and catalase-substrate complexes to be automaticallyrecorded during millisecond periods. The main conclusions are as follows :Catalase combines with hydrogen peroxide to form a green primary com-pound,12 “ catalase-H,O, complex I,” in which H,O, replaces the OH groupnormally attached to the catalase iron atoms (equation xvii).ComplexI then reacts with a second molecule of hydrogen peroxide with theformation of oxygen and the regeneration of free catalase (“ catalatic ”reaction, equation xviii) :L.,_ Fe*OH + H,O, ----- Fe*OOH + H,O . . . (xvii)Fe-OOH + H,O, -$ Fe-OH + H,O + 0,. . (xviii)k:kThe overall rate of destruction of hydrogen peroxide is proportional tothe steady-state concentration of complex I. The fraction of the totalenzyme present as complex I depends only on the velocity constants of itsformation (k,) and breakdown (k4), and is independent of the hydrogenperoxide concentration; hence the unique kinetics of catalase and the lackof any Michaelis saturation effect (see previous section). For erythrocytecatalase the steady-state ratio Fe*ooH ~ is almost exactly 0.25, i.e., on the total Feaverage one of the four haematins in each molecule is bound to peroxide.ll6This is a purely statistical effect, however, for with bacterial catalase 1.6 ofthe 4 haematins are bound to peroxide in the steady state.l14 This isbecause reaction (xvii) is faster with the bacterial enzyme ; hence the steady-state concentration of complex I is higher, and the overall activity is greater.Results with the bacterial enzyme 114 support the view that all four haematinsreact independently, disproving an earlier suggestion 113 that combination of asingle haematin with hydrogen peroxide gave the molecule “ special proper-ties.” Bacterial and mammalian catalases display other interesting differ-ences which suggest that the haematins of the former are more deeply“ buried ” in the protein molecule.114116 Chance, J .Biol. Chem., 1949, 179, 1311.117 Idem, ibid., p. 1331,1341 ; 180,865,947; 1950,182,643. 118 Idem,ibid.,p. 649HERBERT : OXIDISINC ENZYMES. 35 1The Fe*OOH-complex I will also react with a number of compounds 11*including methanol, ethanol, propanol, formaldehyde, formate, and nitrite,llgwhich are oxidised (“ peroxidatic ” reaction) :Fe-OOH + CH,*CH,-OH -% Fe-OH + CH,*CHO + H,O (xix)The catalatic reaction (xviii) is 104-105 times as fast as the peroxidaticreaction (xix); hence oxidation of ethanol, etc., only occurs when theconcentration of free hydrogen peroxide is very small. This can be achievedby continuous generation of hydrogen peroxide in very low concentration byglucose oxidase-glucose-0, or similar systems,l14 when “ coupled oxidation ”of ethanol occurs, as discovered by Keilin.Chance’s work provides a quanti-tative explanation of coupled oxidations, and emphasises the fundamentalsimilarity of catalatic and peroxidatic action. No valency changes of theiron atoms are postulated in his theory of catalase action. In experimentswith methaemoglobin as a “ catalase model,” however, Keilin and Hartree 120showed that initial formation of a MetHb*OOH complex is followed by itsreduction to haemoglobin during oxygen evolution ; they suggest a similarreduction of catalase-Fe in the catalatic reaction (xviii).If complex I remains for long in contact with hydrogen peroxide it isslowly transformed to a red “ complex 11,” which is catalytically inactive.17Formation of complex I1 is responsible for the inactivation of catalaseunder the conditions of Kat.f.determinations l3 (see previous section).George,121 using a manometric technique, describes an initial burst ofoxygen-evolution (a-activity) soon falling to an almost steady state (p-activity). This also may be caused by formation of a secondary complex.The inactivation of catalase by ascorbic acid is almost certainly due toformation of complex IT, produced by the hydrogen peroxide arising fromautoxidation of ascorbic acid.Catalase combines similarly with methyl and ethyl hydrogen peroxidesto form green primary complexes formulated as Fe*OOR (R = CH, or C,H,),which slowly change to red secondary complexes.117 The primary complexesreact with hydrogen peroxide or alcohols in the same way as the primaryFeoOOH complex : 117Fe*OOR + H,O, --+ Fe*OH + R*OH .. . . (cf. xviii)Fe*OOR + CH,*CH,OH + Fe*OH + R*OH + CH,*CHO (cf. xix)Chance 113 has studied the combination of catalase with cyanide;inhibitions by azide and hydroxylamine were studied by Foulkes andLemberg ,123Peroxidaim.-The kinetics of horse-radish peroxidase have been studiedin a series of papers by Chance, 124-126 and are rather similar to those ofllg Heppel and Porterfield, J . Biol. Chem., 1949,178, 549.120 Nature, 1950, 166, 513.lZ2 Foulkes and Lemberg, Austral. J . Exp. Biol. Med. Sci., 1948,26,307.lZs Idem, Enzymologia, 1949, 13, 302.la4 Arch. Biochem., 1949, 21, 416; 22, 224; 24, 389, 410.lZ5 Chance, J .Amer. Ch,em. Soc., 1950,72, 1577.lZ1 Biochem. J . , 1949, 44, 197.la6 Idem, Fed. Proc., 1950, 9, 160352 BIOCHEMISTRY.catalase ; the sequence of reactions is as follows (Fe-OH represents the ironatom of peroxidase with its OH group, and A*H, an acceptor * such asascorbic acid or pyrogallol) :Fe-OH + H,O, 2% Fe*OOH (I) + H20 . (xx)Fe*OOH (I) -% Fe*OOH (11) . . . (xxi)Fe*OOH (11) + A*H2 -2% Fe*OH + H20 + A . (xxii)The primary complex I is green and spectrally similar to catalase complexI. It rapidly changes to a pale red complex TI, which reacts with the acceptor,oxidising it and regenerating free peroxidase. Similar complexes Fe*OOR (Iand 11) are formed with alkyl hydrogen peroxides, the latter reacting similarly,but more slowly, with acceptors as in (xxii). Peroxidase complex 11 thusdoes not correspond to catalase complex 11 (which is inactive) and theirspectra are different.On long contact with hydrogen peroxide in absence ofan acceptor, I1 slowly changes to an inactive complex 111, which is brightred and has a similar spectrum to the inactive catalase complex 11. Onedetail which remains unexplained is that the change I --+ I1 (xxi), thougha first-order reaction, is accelerated by acceptors. Lactoperoxidase behavesin an essentially similar manner,125 which is interesting since it has a differentprosthetic group (an unidentified green haematin which Lemberg thinks maybe monoazahaematin). Another interesting finding 126 is that both theseperoxidases and also myeloperoxidase will oxidise ferro-cytochrome-c a tabout one-tenth of the rate of its oxidation by the yeast enzyme cytochrome-cperoxidase ; re-investigation of the specificity of the latter enzyme seemscalled for,Theorell et ~ 1 .1 2 7 have extended previous work on " artificial " peroxidasesfrom horse-radish peroxidase, made by splitting off its protohaematingroups and coupling the apo-enzyme with other iron-porphyrins. Several ofthe products are enzymically active, including the compound with mono-azahaematin. The prosthetic group of myeloperoxidase is a green haematin,different from that of lactoperoxidase. According to Lemberg and Purdom 12*i t closely resembles the as yet unidentified prosthetic group of choleglobin ;it is definitely not verdohaematin, and use of the name " verdoperoxidase ''for this enzyme should be abandoned.Smith et ~ 1 . l ~ ~ have developed acolorimetric assay method for peroxidase using dichlorphenol-indophenol.Ettori l30 has converted the pyrogallol method into a manometric procedureby measuring carbon dioxide output instead of purpurogallin formation.Cytochrome-c.-Cytochrome-c preparations, obtained from beef, pig, andchicken hearts by the trichloroacetic acid method, contained 0-37-38% of12' Theorell and Maebly, Acta Chem. Scund., 1950, 4, 422.lZ8 Abstr. 1st Intern. Congr. Biochem., 1949, p. 348.lag Smith, Robinson, and Stotz, J . Biol. Chem., 1949,179, 881.130 Biochem. J . , 1949, 44, 35.* In Chance's nomenclature which is used here, the " substrate " is the molecule(HOOK or ROOH) which combines with the enzyme, and the " acceptor " is themolecule (AH,) which reacts with the enzyme-substrate compoundHERBERT : OXIDISING ENZYMES.353iron and showed on electrophoresis a second component which was colourless,iron-free, and enzymically inactive.13f Since it migrates independentlyof cytochrome-c over the pH range 3.9-1 1.9, it is presumably an impurity andnot a dissociated part of the cytochrome-c molecule. The iron contents ofthe pure cytochrome-c components varied from 0.42 to 0.45 yo for the differentspecies ; these variations are considered significant .132 Horse cytochrome-chad an initial iron content of 0.45% and was homogeneous electrophoretically.The pure cytochromes of the different species had slightly different mobilitiesand could be separated by electrophoresis.Paleus and Neilands 23 have alsoseparated a colourless impurity from cytochrome-c on an ion-exchangecolumn. Paul 133 has made observations on the stability of cytochrome-con drying and at extreme pH values, and describes a succinoxidase prepar-ation of low cytochrome-c content, suitable for the biological assay ofcytochrome-c.Haematin-c is attached to its protein by thio-ether linkages with cysteine,and is not split off by treatment with hydrochloric acid and acetone; itmay be split, however, by treating cytochrome-c with silver ~u1phate.l~~This introduces a hydroxyl group into the side-chain, giving a haemato-haematin-c which may be converted into a haematoporphyrin-c ; the exactstructure of these has not yet been determined.Tsou 135 has obtained amodified cytochrome-c by peptic digestion, which does not remove theprosthetic group.Ferricytochrome-c combines with azide in a 1 : 1 molecular ratio to forma spectroscopically distinct complex; the effect of pH on the dissociationconstant of the complex shows that combination takes place with azideanion.136 Unlike the cyanide-ferricytochrome-c complex, which is formedvery slowly but has a low dissociation constant, the azide complex is formedrapidly, but the dissociation constant is so high ( 0 . 1 5 ~ . ) that negligibleamounts of it are formed with the low azide concentrations used for mostinhibition studies.Two new studies are reported on the oxidation-reduction potential ofcytochrome-c and its variation with pH; these are not in complete agree-ment with each other, or with accepted views on the dissociating groups ofcytochr~me-c.~~** 139 Reference has already been made to the reduction ofcytochrome-c by xanthine oxidase 88 and its oxidation by peroxidases; 126it is not known whether these reactions are of physiological significance.New C*chromes.-A new pigment “cytochrome-e” with a band at550 mp.(between the b and the c bands) has been observed by Keilin andHartree 139 in heart-muscle succinoxidase preparations ; owing to its lowconcentration it only becomes visible on cooling to liquid-air temperatures.131 Tirt and Reiss, J.Biol. Chem., 1950, 182, 385.13* Idern, ibid., p. 397.134 Paul, ibid., 1949, 3, 1178.13* Horecker and Stannard, J. BioE. Chem., 1948,172, 589.13’ Paul, Arch. Biochem., 1947,12, 441.13* Rodkey and Ball, J. Biol. Chem., 1950, 182, 17.139 Keilin and Hartree, Nature, 1949, 164, 254.133 Acta Chem. Scand., 1948, 2, 531, 557.135 Nature, 1949, 164, 1134.REP .-VOL . XLVII . 354 BIOCHEMISTRY.It is widely distributed in animal tissues, yeasts, and bacteria. The " cyto-chrome-b, " band which seemingly replaces bands b and c in certain bacteria isresolved a t liquid-air temperatures into b, c, and e bands, with the lastgreatly predominating. This cytochrome has not yet been obtained insolution, and its function is still unknown. Hill 140 and Davenport 141 haveobtained in soluble form, and purified to a considerable extent, a new pig-ment " cytochrome-f " from green plants.Its oxidation-reduction potentialis considerably higher than that of other cytochromes, so much so that it israther difficult to obtain it in the oxidised form. Its possible connection withphotosynthetic oxygen-production is an intriguing possibility.Electron Transport between Substrates and Oxygen.-The scheme belowattempts to summarise present views on this subject, the arrows indicatingpaths of electron transport ; the detailed pathways are discussed in turn.succinate substrate substrateL..--._ ----........--.-.--- TPN-succinic DPN TPN dehydrogenase; $ E;irogenase + dehydrogenase4 I 1 _ - - - - - - _ .J... ____.-_-.._._.__._-...___...__-.. _-----------. J.TPN-cytochrome-creductase1 cytochrome-b- - - - - - - - _.____.__.--..--- --._--_- .______---- __----..--._.-, factorI + Ic ;':---------- .1: cytochrome-cmethylene-blue: cytochrome-a ;14,i cytochrome-a, i0 2In the above scheme, thecomponents within the dotted line are all present in bound form (attachedto insoluble submicroscopic particles) in standard heart-muscle succinoxidasepreparations. So far only cytochrome-c, diaphorase, and TPN-cytochrome-creductase have been separated from the particles in soluble form and purified.Numerous claims to have brought cytochrome oxidase into true solution,and separated it into different factors are refuted in two important papersby Keilin and Hartree.142* 143 Most of such " purified " preparations have alower Qo, than the starting-material, and reported activation by variousComponents of the Xuccinoxidase Sydem.Hill, SOC.Exp. Biol. Symp. Carbon Dioxide Fixation, 1950.Ibid., 1949, 44, 205.1 4 1 Davenport, Nature, in the press.142 Biochern. J., 1947, 41, 500, 502HERBERT : OXIDISING ENZYMES. 355factors could be reproduced by adding indifferent proteins, or by precipitat-ing gelatinous calcium or aluminium phosphates in the preparation. (Re-ported “activation” by these ions only occurs in a phosphate buffer.)Preparations partly inactivated by freezing or drying could be reactivatedby the same means, a n t addition of serum proteins protects the preparationsagainst inactivation on drying.l@ It is considered that the catalysts in thecolloidal particles, as in the intact cells, are more or less rigidly held togetherin a framework or mosaic which ensures their mutual accessibility.Forexample, endogenous cytochrome-c in the preparations is reduced muchfaster by succinate than is added cytochrome-~,~~~ but it is reduced moreslowly by chemical reducing agents such as ascorbic acid; 14p i t would seemto be “ built in ” to the colloidal framework. Disruption of the mosaiclowers the overall activity without necessarily destroying or removing anyindividual catalyst ; flocculent precipitates of foreign materials can reactivateby providing new surfaces for re-orientation. Some such “ mosaic ”concept is now fairly generally accepted, and has been further extendedby Green to his “ cyclophorase ” preparations (see below).Kidneysuccinoxidase is essentially similar to the heart-muscle preparation ; it wasformerly thought to have a different cytochrome system (“ cytochrome-b, ”in place of b and c) but this was not ~0nfirmed.l~~Electron Transport from Succinate to Cytochrome-c. It is well known thatcytochrome-c is 8 link between the succinic dehydrogenase and cytochromeoxidase systems. Important contributions by Slater 145* 1469 14’ indicatethat : (i) cytochrome-b is an intermediary carrier between succinate andcytochrome-c, and between succinate and dyes such as methylene blue; (ii)a new respiratory catalyst, inactivated by “ BAL ” (2 : S-dimercaptopro-panol) is an intermediary carrier between cytochrome-b and cytochrome-c,but not between cytochrome-b and methylene blue; this is the “ BAL-labile factor ” in the scheme on p. 354.An unknown haematin compoundaccounts for m. 20% of the total haematin of heart-muscle preparations,145and this is destroyed by BAL treatment.14’ It is tempting to identify thiscompound both with Slater’s factor and with Keilin and Hartree’s newly-discovered cyto~hrome-e,~~~ but there is no real evidence for this so far.Studies on diphtherial and ox-heart succinoxidase preparations ledPappenheimer and Hendee 14* to suggest that succinic dehydrogenase andcytochrome-b are identical. There appears to be no evidence contradictingthis, and Slater 146 found a correlation between the dehydrogenase and cyto-chrome-b contents of different heart and kidney preparations ; actual proofof identity is, however, lacking.* Wainio et ~1.14~ claim the preparation ofcytochrome-b in “ soluble ” form and separated from other components ofu4 Borei, Biochem.J., 1950, 47, 227. 145 Ibid., 1949, 45, 1.14’ Ibid., p. 14. Ibid., p. 8.Pappenheimer and Hendee, J . Biol. Chm., 1949,180,597.Eichel, Wainio, and Person, ibid., 1950, 183, 89.* [Added in proof.] Tsou (Biochem. J., 1951, in the press) has found that potassiumcyanide slowly inactivates succinic dehydrogenase without apparently affecting cyto-chrome-b, and concludes that they are not identical356 BIOCHEMISTRY.the cytochrome system by fractionation with deoxycholate, but they give nodata on its succinic dehydrogenase activity.(The criterion of “ solubility ”adopted is non-sedimentation in 1 hour a t 25,000 g., which does not seemadequate to the Reporter to prove true solubility, in view of the well-knownpeptizing action of bile salts.)Warburg (ref. 6, p.70) describes improved measurements showing that the cytochrome-c turn-over in living yeast cells is fast enough to account for ca, 95% of the totaloxygen uptake. In other words, effectively all electron transport betweensubstrates and oxygen passes over cytochrome-c. The electron-transferroute for TPN systems has been made fairly clear by the isolation of TPN-cytochrome-c reductase (see scheme on p. 354). The route for DPN systemsis not so well understood.Straub’s diaphorase oxidises DPNH but does notreduce cytochrome-c. Either (i) it plays no part in electron transfer fromDPPU’H to cytochrome-c, or (ii) it is a transformation artefact derived from ahypothetical “ DPN-cytochrome-c reductase ” similar to 1 the TPN-cytochrome-c reductase, its ability to reduce cytochrome-c having beendestroyed in the isolation process, or (iii) it reacts with cytochrome-c via anintermediary electron-carrier. Slater 27* 150 brings evidence to show thatalternative (iii) is correct, and that the intermediary carrier involved is thesame “ BAL-labile factor ” as that involved in the reduction of cytochrome-cby cytochrome-b (see scheme on p. 354); in other words, the hypothetical“ DPN-cytochrome-c reductase ” is equated with diaphorase-plus-factor.He also confirms that aerobic oxidation of DPNH by heart-muscle prepar-ations proceeds via cytochromes c, a, and a,, and shows that the rate ofoxidation of DPNH by diaphorase in the preparation (with methylene-blueas H-acceptor) is 3-4 times as fast as the overall oxidation of DPNH byoxygen.A brief note by Heppel lS1 describes the preparation of (‘ DPN-cyto-chrome-c reductase ” of ox liver in soluble form, removal from insolubleparticles being effected by digestion with steapsin (cf.ref. 87). Hogeboomand Schneider have also obtained soluble preparations by ultrasonic treatmentof rat-liver rnit0~hondria.l~~ Until these preparations have been purified it isimpossible to say whether or not this work fits Slater’s views.Electron Transport between Cytochrome-c and Oxygen. Slater 27 describesexperiments supporting the scheme on p.354, and confirming Keilin’sobservations on cytochrome-a, and its CO-compound and the probableidentity of a, with cytochrome oxidase; he also shows that cytochrome-bis not concerned in this part of the system. Wainio et aZ.1499153 claim thepreparation of cytochrome oxidase in “ soluble ” form by fractionation ofheart-muscle preparations with deoxycholate. The reduced pigment hadbands at 440 and 601 mp., and was separated almost completely from cyto-chrome-b and -c. These workers do not admit the existence of a separatecytochrome-a ; it appears to the Reporter, however, that the pigment whoseElectron Transport between Coenzymes and Oxygek150 Biochem.J . , 1950,46,499.152 Nature, 1950,166, 302.151 Fed. Proc., 1949, 8, 205.153 J . Biol. Chrm., 1949,173, 145HERBERT : OXIDISING ENZYMES. 357. spectra they describe could in fact be a mixture of cytochrome-a and-a3. Hogeboom and Schneider 152 obtained a small fraction of the cyto-chrome oxidase of rat liver mitochondria in soluble form by ultrasonic treat-ment. In their preparations, which are not sedimented a t 148,000 g., theoxidase would appear to be in true solution; the QO,, however, is only abouthalf that of the mitochondria.Rawlinson and Hale 154 found that the haematins extracted with acidacetone from Corynebact. diphtheriae and from heart-muscle preparationsconsisted in both cases of protohaematin (derived from cytochrome-b),and " haematin-a " (probably derived from cytochrome-a + -a3).Thelatter is a dichroic haem and contains a t least one aldehyde group. Lemberget ~ 1 . l ~ ~ compared the properties of haematin-a and porphyrin-a with varioussynthetic porphyrin derivatives ; the one showing closest resemblance wasoxyrhodoporphyrin.Stannard and Horecker 156 studied the inhibition of cytochrome oxidaseby cyanide and azide a t different pH values, and conclude that the oxidasecombines exclusively with undissociated hydrocyanic and hydrazoic acids.Ferricytochrome-c, methaemoglobin, and metmyoglobin on the other handcombine exclusively with the cyanide and azide ions. These workers deter-mine cytochrome-oxidase activity by following the aerobic oxidation ofreduced cytochrome-c spectrophotometrically.Smith and Stotz 15' describea colorimetric method with dichlorphenol-indophenol, while Slater 158 usesa manometric method with ascorbic acid as substratb.Coupling of Phosphorylution with Oxidations. During the oxidation ofmany intermediary metabolites by tissue homogenates and particulatepreparations in the presence of inorganic phosphate and a, phosphate acceptorsuch as AMP or ADP, about three atoms of inorganic phosphate may beesterified for each atom of oxygen consumed. The esterified phosphateappears as ATP, if precautions are taken to prevent its breakdown. Themechanism of this process remains largely obscure. I n Green's " cyclo-phorase " preparations, not only is orthophosphate esterified during theoxidation of all intermediary compounds of the citric acid cycle, but thepresence of phosphate increases their rates of 0xidati0n.l~~ In the absenceof an acceptor, orthophosphate disappearing is transformed into inorganicpyrophosphate. It is suggested that phosphorylation is linked to theprimary oxidation of the substrates and results in the formation of reducedcoenzyme pyrophosphates, which can either transfer phosphate to anacceptor system (such as glucose + hexokinase) via adenylic acid, or in itsabsence break down to inorganic pyrophosphate.Studies with 32P showedthat cyclophorase contains a very labile form of phosphate (" gel P "),possibly identical with the labile coenzyme pyrophosphates.160Friedkin and Lehninger,lG1 using rat-liver mitochondria, found thatlS4 Biochem.J . , 1949, 45, 247. ls5 Abstr. 1st Intern. Congr. Biochern., 1 9 4 9 , ~ . 351.lS6 J . BioLChem., 1948,172,599. 15' Ibid., 1949, 179, 891.Biochem. J . , 1949, 44, 305. 159 Cross et al., J . Biol. Chem., 1949, 177, 655.l 6 0 Green et al., Arch. Biochem., 1949, 24, 359; Albaum, ibid., p. 375; Tepley, ibid.,161 J . Biol. Chem., 1949,178, 611. p. 383358 BIOCHEMISTRY.esterification of orthophosphate into adenine nucleotides accompanies theaerobic oxidation of DPNH ; studies of the oxidation of P-hydroxybutyrate 162suggested that phosphate uptake is coupled with oxidation of DPNH viathe cytochrome system, and not with primary oxidation of the substrate assuggested by Green. In the Reporter’s view it is still too early to decidebetween these possibilities, which may not be mutually exclusive. Thelocation of the phosphorylation reaction has been further narrowed bySlater’s recent work,163 in which cytochrome-c was used as h a 1 acceptor ofelectrons from the substrate, functioning of cytochrome oxidase beingprevented by cyanide or anaerobiosis.Oxidation of cc-ketoglutarate by aheart-muscle preparation gave about the same phosphate uptake (P : 0>2 : 1) whether oxygen or cytochrome-c was the final electron-acceptor.Enzyme Complexes and Cell StrUctnre.-Keilin’s concept of the succin-oxidase system as a colloidal complex or mosaic of enzymes has already beenmentioned. Recent work has extended this concept to a wider range ofenzymes, while parallel cytochemical studies have located these enzymecomplexes in definite structural elements of the cell, notably the mitochondria.Methodologically, the subject has been approached from two angles.Someworkers (e.g., Green, Lehninger) have set out to isolate particular enzymecomplexes in a functionally unaltered state, while others (e,g., Claude, Dounce,Schneider) have set out to isolate cell structures such as nuclei or mito-chondria in a morphologically unaltered state. These two approaches haveon the whole given fairly similar results.Green (ref. 2, chapter X) has reviewed hisresearches on this system. Cyclophorase preparations l6* are obtained fromrabbit kidney or liver by differential centrifugation after the tissues have beenminced in 0.9 yo potassium chloride solution in a Waring blendor ; the productis a viscous gel containing much nucleic acid.Fresh kidney preparationswithout any additions but phosphate buffer catalyse the complete oxidationto carbon dioxide and water of (a) all the substrates of the citric acid cycle,(b) the fatty acids from acetic to tridecanoic and derivatives such asacetoacetic acid 165 and (c) certain amino-acids.ls6 Their activity rapidlyfalls but is restored for group ( a ) substrates by adding Mgf+ and AMP orATP; group (b) and (c) substrates require in addition a small amount of agroup (a) substrate to be added as a co-oxidant or “ sparker,” indicating thattheir oxidation is linked with the citric acid cycle.167The individual dehydrogenases of cyclophorase preparations (e .g., lactic,malic, isocitric) have different properties from the previously known solubleforms of these enzymes.They do not require the addition of DPN or TPN,which are present in cyclophorase preparations in firmly bound form, notremoved by washing; also their functioning requires phosphate and isaccompanied by phosphate esterification. In Green’s view, the dehydro-162 Friedkin and Lehninger, J . Biol. Chem., 1949,178,625. lES Nature, 1950,166,982.16* Green, Loomis, and Auerbach, J. Biol. Chem., 1948,172, 399.1 e 5 Grafflin and Green, ibid., 1948, 176, 95; Atchley, ibid., p. 123.1 e 6 Taggart and Krakur, ibid., 1949,177,641 ; Still, Buell, and Green, Arch. Biochem.,le7 Knox, Noyce, and Auerbach, J .Biol. Chem., 1948, 176, 117.The Cyclophorase System.1950, 26, 406, 413HERBERT : OXIDISING ENZYMES. 359genases exist in cyclophorase preparations as “ pyridinoproteins ” withfirmly bound DPN or TPN prosthetic groups, and in this state possesscertain firoperties that are lacking in the soluble dehydrogenases.168 Onsubjecting cyclophoraRe preparations to various treatments the bound co-enzymes are split off,168 and such preparations need to be supplemented withDPN or TPN, which untreated cyclophorase does not, and the dehydrogenasesthen behave as dissociating pyridinoproteins. On more drastic treatmentmost of the dehydrogenases are released in soluble form, and are thenapparently identical with the known soluble dehydrogenases. To whatextent the classical dehydrogenases must be regarded as transformationartefacts is not yet clear, but is obviously of great importance.In thisconnection the fact that glyceraldehyde-3 phosphate dehydrogenase has beencrystallised in the form of a protein containing firmly bound DPN 46 is ofconsiderable interest. Green’s conception is that all the enzymes in thecyclophorase system, as in the intact cell, occur as an organised mosaic ofproteins with non-dissociating prosthetic groups.Intracelluhr Locution of Respiratory Enzymes. This field has been fairlyrecently reviewed by Schneider (ref. 2, Chapter XIV) and by Claude; 169only a few selected topics will therefore be discussed. The technique thathas led to’the major advances of the last five years or so is that of isolatingparticulate fractions from disrupted cells by differential centrifugation.Cells are broken mechanically, usually with a Potter-Elvej hem homogeniseror a Waring blendor ; the resulting suspension can then be separated by theapplication of increasing centrifugal fields into successive fractions consistingof nuclei, mitochondria, submicroscopic particles or “ microsomes,” and asupernatant liquid containing the soluble cell components.The initial celldisruption must not be too violent or fragments of broken-up nuclei andmitochondria will appear in other fractions. The suspending fluid is alsoimportant. Hogeboom et uZ.l7O found that the use of salt solutions causesswelling and agglutination of mitochondria which then appear as “ largegranules ” in the nuclear fraction.If the tissue is homogenised in 30%sucrose solution, however, the mitochondria retain their normal morphologyand vital staining characteristics and do not agglutinate. In isotonic (8.5%)sucrose the mitochondria appear spherical instead of rod-shaped, but theirother properties remain unchanged, and this medium is more convenient.Analysis of such fractions shows that the glycolytic enzymes appear almostentirely in the soluble form in the final supernatant liquid,170 while the mainoxidation systems are concentrated in the mitochondria and to a lesser extentin the “ microsomes,” only m. 5% appearing in the nuclei. Thus, up to 80%of the total succinoxidase appears in the mitochondria,170* 171 and also mostof the enzyme systems responsible for the citric acid cycle 172* 173 and for the168 Huennekens and Green, Arch.Biochem., 1950, 27, 418, 428.169 Adv. Protein Ghem., 1949, 5, 423.170 Hogeboom, Schneider, and Pallade, J . Biol. Chem., 1948, 172, 619.171 LePage and Schneider, ibid., 1948, 176, 1021.Kennedy and Lehninger, ibid., 1948,179, 957.173 Schneider and Potter, ibid., 1949, 177, 893360 BIOCHEMISTRY.oxidation of fatty 17* Cytochrome oxidase,171 cytochrorne-c,176DPN-cytochrome-c reductase, 176 and TPN-cytochrome-c reductase 1 7 7 havealso been found to be concentrated in the mitochondria, which seem indeedto be the main seat of intracellular oxidations. Ribonucleic acid is found inall the fractions, but deoxyribonucleic acid appears only in the nuclei.Green’s “ cyclophorase ” preparations are not cytologically homogeneous,containing a considerable proportion of nuclei and nuclear fragments, andsome “ microsomes.” However, Harman 178 found that on fractionatingsuch preparations by differential centrifugation in sucrose solutions, almostall the cyclophorase activity was obtained in the mitochondrial fraction.The ‘‘ microsomes ” contained succinoxidase and most of the individualenzymes of the cyclophorase system, but could not bring about completeoxidation of citric acid cycle intermediates or esterification of inorganicphosphate.Most workers have found that enzyme systems concentrated inthe mitochondria are found also to some extent in the “ microsomes ” ; itseems possible that an appreciable proportion of the “ microsomes ” are infact disintegrated mitochondria. Prolonged homogenising in the Waringblendor leads to break-up of mitochondrial or cyclophorase preparations, andtransfer of oxidase activity to the “ microsome ” fraction and the super-natant At the same time the prosthetic groups of the non-dissociating dehydrogenases of the cyclophorase system are split off, so thatthey appear as ordinary dissociating dehydr0gena~es.l~~The manner in which the various enzyme systems are integrated in themitochondrial structure is unknown.Cytological studies 180 have apparentlyshown mitochondria to possess well-defined membranes, and a large portionof their total nitrogen represents soluble proteins that are released when themembranes are disrupted by ultrasonic treatment.ls2 Green,e.g.s 168 however,does not accept this view, and Harman 178 believes that mitochondria have agel-like structure, without a limiting membrane.The point is of considerableimportance in connection with Green’s conception of the cyclophorasedehydrogenases as “ non-dissociating pyridino-proteins,” but it cannot beconsidered settled.D. H.6. HORMONES OF THE ANTERIOR-PXTUI!l’ARY GLAND.This subject has not been reviewed in these Reports since 1940, so it mayThreeSix well-authenticated hormones are known to be secreted by the anterior-often be necessary to include work done as much as ten years ago.comprehensive reviews have more recently appeared elsewhere. l* 2r174 Schneider, J.Biol. Chem., 1948, 176, 259.1 7 5 Schneider, Claude, and Hogeboom, ibid., 1948,172, 451.176 Hogeboom, ibid., 1949, 177, 847.177 Hogeboom and Schneider, ibid., 1950,186, 417.178 Harman, Exp. Cell Research, 1950,1,382,394.180 Dalton et al., J. Nat. Cancer Inst., 1949, 9, 439.178 Still and Kaplan, ibid., p. 403.White, Phyeiol. Reviews, 1946, 26, 574.3 Li and Evans, in “The Hormones,” Vol. I, 1948 (G. Pincus and K. V.Chow, Adv. Protein Chem., 1944,1, 163.Thimann ed.), New York, Chap. 14FOLLEY : HORMONES OF THE ANTERIOR-PITUITARY GLAND. 361pituitary (A.P.) gland; there may be others but none has yet been satis-factorily characterised. The known A.P. hormones can conveniently beclassified as gonad-stimulating hormones or gonadotrophins, and “ meta-bolic ” hormones.The first group comprises the follicle-stimulatinghormone (FSH, thylakentrin), the luteinising or interstitial-cell-stimulatinghormone (LH, ICSH, metakentrin), and the lactogenic hormone (prolactin,mammotrophin, luteotrophin) ; the second comprises the growth hormone(GH, somatotrophin), the adrenocorticotrophic hormone or hormone complex(ACTH), and thyrotrophin. All appear to be proteins or protein derivatives ;prolactin, ICSH, GH, and possibly FSH have now been prepared fromanterior-pituitary tissue as proteins satisfying the currently accepted physico-chemical criteria of molecular homogeneity and in addition exhibiting ahigh degree of biological purity.Follicle-stimulating Hormone (FSH).-Several reviews of the chemistry ofFSH have been p~blished.l-~Puri3cation. FSH, together with ICSH, may easily be extracted fromanterior-pituitary tissue, fresh or acetone-dried, by dilute acid or alkali,saline or aqueous alcohol.The richest sources are human or horse pituitaries,but the most abundant and therefore the best and usual sources in practiceare sheep and pig glands ; the FSH activity of ox glands is low. The chiefproblem has been to free FSH from ICSH and this has mostly been accom-plished by utilising the greater solubility of FSH under most conditions.Thus it is the only known A.P. protein-hormone soluble in half-saturatedammonium sulphate solution. Only recently has FSH been obtainedas a pure protein ; earlier procedures gave preparations which, thoughsensibly free from ICSH and perhaps other A.P.hormones, containedinactive contaminants or, though relatively highly purified, were con-taminated with traces of ICSH.Procedures based on ammonium sulphate fractionation were used byFevold who first employed aqueous pyridine extraction of the tissuefollowed by adsorption of the gonadotrophins on benzoic acid, and later 7extraction with dilute aqueous ammonia. Fraenkel-Conrat et aL8 extractedacetone-dried sheep pituitaries with 40 yo alcohol and precipitated thegonadotrophins by increasing the alcohol concentration. Further purific-ation by ammonium sulphate fractionation gave a product showing someICSH contamination. Greep et aL9 obtained a product free from ICSH fromfresh pig pituitaries by a method based on the fact that FSH is soluble inacetate buffer (pH 444) containing 20.5y0 of sodium sulphate while ICSHis insoluble.This preparation however proved to be molecularly poly-disperse.1° The fact that FSH is more resistant to tryptic digestion thanChow, in “The Chemistry and Physiology of Hormones,” Amer. Assoc. Adv.Sci., 1944, p. 26.Endocrinology, 1939, 24, 435.Fevold, Lee, Hisaw, and Cohn, ibid., 1940, 26, 999.Li, Vitaminsand Hormones, 1949, 7 , 224.* H. Fraenkel-Conrat, Simpson, and Evans, Ann. Fac. Med., Montevideo, 1940,25, 617. Greep, van Dyke, and Chow, J. Biol. Chem., 1940,133,289.lo Chow, Ann. N . Y . Acad. Xci., 1943,43, 309362 BIOCHEMISTRY.ICSH has been utilised l1 to obtain preparations of FSH often apparentlyfree from ICSH ; these preparations contain inactive contaminants butare reported to be free from a toxic substance, present in earlier preparations l2and causing local reactions, and also to be free from lactogenic and thyro-trophic hormones.Li et ~ 1 .~ 1 ~ ~ have prepared FSH behaving as a single substance in theultra-centrifuge and on electrophoresis (solubility studies were not done)by extraction of sheep pituitaries with calcium hydroxide solution, followedby alternate fractionation with ammonium sulphate and cold aqueousethanol. This preparation in a total dose of 0-05 mg. initiated ovarianfollicular development in hypophysectomised rats, but in a total dose of2.0 mg. over four days showed no ICSH, ACTH, GH, or thyrotrophicactivities. Koenig and King l4 have recently described a method ofextracting FSH (with ICSH and prolactin) from sheep pituitaries, novelin that the extraction is carried out with solutions of high ionic strengtha t the pH of minimum solubility of the gonadotrophins.The method issuit able for large- s cale use.Chemical Properties. So far most data have been obtained with(slightly) impure preparations ; amplification of the few results for thepure protein The isoelectric point of puresheep hormone has been given as pH 4.5; its molecular weight estimatedfrom a single ultra-centrifugal run is 70,000.5 The hormone is in generalvery soluble in aqueous solutions (e.g., ref. 12) and is not precipitated by0.6 saturated ammonium sulphate solution or by 2.5 yo trichloroaceticacid.Since it contains considerable amounts of carbohydrate (sheep :carbohydrate by orcinol method 10-13y0,16 glucose by carbazole method22.0-24~6%,~~ hexosamine 8% ; l5 pig : mannose 46%, hexosamine4.4%16), from which the activity has hitherto proved inseparable andmoreover is destroyed by amy1ase,l7 the hormone is considered to be aglycoprotein. Lower values than those quoted above have been given 5for the carbohydrate content of the pure sheep hormone as follows : hexose(orcinol method) 1.3% ; hexosamine 006%.The activity is relatively resistant (as compared with ICSH) to digestionby commercipl trypsin 12*18 so long as no more than 48% of protein isdigested, which may mean that as with ACTH the activity is a propertyof a portion only of the protein molecule and suggests the possibility ofsplitting off an active carbohydrate-peptide.Indeed, Li 19 has recentlyreported the preparation of a biologically active and totally dialysablewill be awaited with interest.11 McShan and Meyer, Proc. SOC. Exp. Biol., 1946, 61, 57.12 McShan and Meyer, J . Biol. Chem., 1940, 135, 473.13 Li, Simpson, and Evans, Science, 1949,109, 445.14 Koenig and King, Arch. Biochem., 1950, 26, 219.1 5 Evans, H. L. Fraenkel-Conrat, Simpson, and Li, Science, 1939, 89, 249.1 6 Gurin, Proc. SOC. Exp. Biol., 1942,49, 48.1 7 McShan and Meyer, ibid., 1939,40, 701 ; J . B i d . Chem., 1938,126, 361.18 Chow, Greep, and van Dyke, J. Endowinol., 1939,1, 440.19 J . Amer. Chem. Soc., 1950, 72, 2815FOLLEY: HORMONES OF THE UTERIOR-PITUITARY GLAND.363material by peptic digestion of FSH protein. Moreover the hormone hasalso been detected in trichloroacetic acid extracts of fresh sheep pituitary,but this material was not ultra-filtrable.20 The activity of an impuresheep-gland preparation remained after 30 minutes at 75" (pH 7-8),12but a pure preparation was more heat-labile.6 The hormone was destroyedby acetylation with keten after 30 minutes but not 5 minutea2l and bytreatment with alkaline cysteine.l2* 22 It seems possible therefore that theactivity is bound up with the presence of amino-groups not immediatelyaccessible t o keten and of disulphide linkages. The hormone contains 4*4y0of tyrosine and 0.6% of tryptophan; cystine is present but not ~ysteine.~Interstitial-cell-stimulating Hormone (ICSH).-The chemistry of ICSHhas been reviewed several time~.l-~Puri$cation.ICSH has been isolated as a biologically and chemicallypure protein in two laboratories; a third group 23 made highly purifiedpreparations. Li, Simpson, and Evans 24s 2s extracted the hormone fromacetone-dried sheep gland with 40 yo alcohol, the gonadotrophins beingprecipitated by raising the alcohol concentration and separated by ammoniumsulphate fractionation. Tested in hypophysectomised rats the hormonewas free from FSH (total dose 3 mg.), thyrotrophin (2 mg.), ACTH (10 mg.),GH (10 mg.), and prolactin (10 mg.), and showed ovarian interstitial cellrepair in hypophysectomised rats in doses of 0.0054.01 mg.Solubility,ultra-centrifugal, and electrophoretic studies showed it to be a homogeneousprotein. Shedlovsky, Rothen, Greep, van Dyke, and Chow 26* 27 obtainedthe hormone from fresh pig gland by extraction with cold saline and thenammonium sulphate fractionation. The protein was homogeneous byelectrophoretic, ultra-centrifugal, and solubility criteria. A significantincrease in the weight of the anterior prostate lobe of the hypophysectomisedmale rat was caused by a minimum of 6.7 pg. of hormone.Chemical Properties. Consideration of the following physico-chemicalproperties shows ICSH from sheep and pig pituitaries to be different proteins :molecular weight (sheep,25 by osmotic pressure : 40,000 ; by ultra-centrifuge : 90,000) ; sedimentation constant (S,,) (sheep : 25 3.6 xcm./sec./dyne ; pig : 27 6.8 x cm./sec./dyne) ; electrophoretic mobility(sheep,26 in buffer at pH 7-40, ionic strength 0.1 : 6.0 x 10-5 cm.2/sec./v.;pig,26 in buffer a t pH 7.86, ionic strength 0.05 : 0.66 x 10-5 cm.2/sec./v.) ;isoelectric point (sheep : 25 pH 4-6 ; pig : 26 pH 7.45).The hormones alsodiffer in carbohydrate content (sheep : 24 mannose 4-45%, hexosamine5-85 % ; pig : 28 mannose 2.8 %, hexosamine 2.2 %) and tryptophan content(sheep : 29 1.0% ; pig : 25 3.8y0). Immunological differences have also2o Geschwind, Hess, Condliffe, Evans, and Simpson, Science, 1950,112, 436.21 Li, Simpson, and Evans, J . BioE. Chem., 1939,131, 259.a2 H. L. Fraenkel-Conrat, Simpson, and Evans, ibid., 1939,130, 243 ; Science, 1940,a4 Endocrinology, 1940, 27, 803.O 8 Gurin, Proc.SOC. Exp. Biol., 1942, 59, 48.em Li, Simpson, and Evans, Science, 1940,92,365.91, 363. 23 Fevold, Ann. N . Y . Acad. Sci., 1943,48,321.J . Amer. Chem. SOC., 1942, 54, 367.Endocrinology, 1942, 80, 650. Science, 1940, 92, 178364 BIOCHEMISTRY.been reported; sheep hormone does not react with specific antibodies topig hormone raised in the rabbit.30 Li and Evans3 report differencesin biological potency of the two proteins in female hypophysectomisedrats (ovarian interstitial tissue repair test) and intact rabbits (ovulationtest), but not in hypophysectomised male rats (ventral prostate repairtest).Sheep ICSH is more readily inactivated by keten 24 (5 minutes) than isFSH, and also by treatment with alkaline cysteine; 22 the activity seemstherefore to depend on amino-groups and disulphide linkages.ICSHdiffers from FSH in that its activity is rapidly destroyed by trypticdigestion l2, l8 but is relatively resistant to arny1a~e.l~Its biological specificity can be summarised thus : it repairs or maintainsthe ovarian interstitial cells in hypophysectomised female rats and willcause ripe follicles to ovulate and luteinise ; in hypophysectomised male ratsit will stimulate the interstitial tissue of the testes and through the resultingincrease in androgen production it will indirectly ‘bring about growth ofthe accessory reproductive organs.Lactogenic Hormone (Prolactin).-The biochemistry of prolactin has beenreviewed many times in the last decade.l* 2* 31 31-34 It was the first A.P.hormone to be obtained pure and has been extensively studied chemically.The biological potency (pigeon crop-gland test) of the pure hormone is30-35 i.u./mg.It seems that Lyons35 must have obtained almost purepreparations as long ago as 1937, since preparations later made by essentiallyhis procedure were found to exhibit this potency 36 and to be homogeneousas judged by solubility and electrophoretic studies.36* 37 The most potentand favoured sources are ox and sheep pituitaries; the activity of pigglands is much less.38 The hormone is less soluble in aqueous solventsthan FSH, ICSH, and thyrotrophin, and extraction with alkaline ethanolgives rather a complex mixture of proteins. Extraction by acid acetone 35or by chloroform39 avoids much of this contamination, but ACTH is stilla major hormonal contaminant though its separation from prolactin presentsno difficulty.Prolactin exhibiting molecular homogeneity as judgedby electrophoretic and solubility criteria was prepared by Li, Lyons, andEvans 361 27 by a slight modification of Lyons’s original method.3s Thisinvolved acid acetone extraction of fresh whole sheep pituitaries and repeatedprecipitation a t the isoelectric point (pH 5.5) in the cold, ACTH beingPurijcation.30 Chow, Endocrinology, 1942, 30, 657.32 White, Ann.N.Y. Acad. Sci., 1943, 43, 341.33 Idem, Vitamins and Hormones, 1949, 7, 253.34 Folley, in ‘‘ Marshall’s Physiology of Reproduction,” 3rd edn. (A. S.Parkes, ed.),35 Proc. SOC. Exp. Biol., 1937, 35, 645; Cold Spring Harbor. Symp., 1937, 5 ,38 Li, Lyons, and Evens, J . Cfen. Physiol., 1941, 24, 303.38 Chance, Rowlands, and Young, J . Endocrinol., 1939, 1, 239.39 Schwenk, Fleischer, and Tolksdorf, J . Biol. Chem., 1943, 147, 535.3l Voss, Ergebn. Physiol., 1941, 44, 96.London, chap. 20 (in the press).198.37 Ibid., 1940, 23, 433FOLLEY : HORMONES OF THE ANTERIOR-PITUITARY GLAND. 365removed by precipitation at pH 6.5. Li, Simpson, and EvansM havedescribed an alternative method of working up the crude acid acetone extract,in which ACTH is removed by utilising the fact that it is soluble in 0.36~-sodium chloride at pH 3.0, while prolactin is not. White, Bonsnes, andLong41 have also prepared the pure hormone from a crude acid acetoneextract.Their preparations were homogeneous by the usual physico-chemicalcriteria. These authors,41 following up an earlier preliminary report>2have reported further on two alternative methods of crystallising prolactinin small yield. Success with this method has been reported from anotherlaboratory but others have faiIed.32*= A novel method of obtaining highlyactive prolactin (30 i.u./mg.) in good yield from fresh sheep pituitarieshas been described by Schwenk, Fleischer, and Tolk~dorf.~~ This involvesextraction with chloroform, and removal of the prolactin from the chloroformgel containing prolactin, ACTH, and inactive proteins by dissolution in acidmethanol. Koenigand King have described a new method of extracting prolactin from sheeppituitaries, useful for large-scale work.14Prolactin, the protein nature of which was suspected longago on account of its solubility relations, exhibits the same physico-chemicalproperties whether prepared from ox or sheep glands except in two, probablyrelated, respects : (a) in NaC1-HCl solutions ox prolactin is less solublethan sheep hormone, though more soluble in citrate buffer a t pH 6.36; 3 6 w 4 3( b ) ox hormone contains more tyrosinc (5.7%) than does sheep hormone(4.5 yo) .439 44 They can therefore be considered as different proteins eventhough it has not been possible to distinguish them immunologically.45The molecular weight has been given as 26,500 (osmotic 22,000(diffusion and vis~osity),~~ 32,000-35,000 (by ultra-centrifuge, on the basisof a spherical molecule),41 and 33,300 (from analytical values for five amino-acids).47 Other physico-chemical data have been given as follows : iso-electric point ; pH 5.73 (electrophoresis, moving boundary method),48* 49pH 5.65 (cataphoresis of coated quartz particles) ; 41 sedimentation constantIS'^^) : 2.65-2-80 x 10-13 cm./sec./dyne ; 41 diffusion constant (D20) : 7-57 xlo-' cm.2/sec.(free diff~sion),~~ 9.0 x 10-7 cm.2/sec. (glass-membranedifbsion) ; 46 partial specific volume : 0.721 ; 46 disymmetry constant (fit,) : 1.29-1.37 ; 46 specific rotation : -40-5" at 25°.46The elementary composition of prolactin is typical of a protein and itcontains no carbohydrate, thiol groups, or phosphor~s.~~ A completeamino-acid analysis of' sheep prolactin has recently been reported by Li 47accounting for 99.9% of the nitrogen and for all the sulphur.60 TheThe degree of purity of this preparation is not known.Properties.40 J .Biol. Chem., 1942, 146, 627.4 2 White, Catchpole, and Long, Science, 1937, 86, 82.O3 Li, Lyons, and Evans, J . Biol. Chem., 1941,140, 43.4 5 Bischoff and Lyons, Endocrinology, 1939, 25, 17.46 Li, J . Biol. Chem., 1942,146, 633.Li, Lyons, and Evans, Science, 1939, 90, 622.4s Idem, J . Amer. Chem. SOC., 1940,62, 2925.50 Li, J . Biol. Chem., 1943,148, 289.41 Ibid., 1942, 143, 447.44 Ibid., 1940, 136, 709.4 7 Ibid., 1949,178, 459366 BIOCIHEMISTRY.biological activity is destroyed or diminished by treatment with keten,61nitrous acid, 62 and phenyl isocyanate,63 indicating the essentiality of freeamino-groups, by iodination of the tyrosine residues, 64 by esterification ofthe free carboxyl groups with methyl alcohol,66 and by treatment withthiol compound^.^^ No evidence that the activity is bound up with aprosthetic group has been obtained; it rather seems from the foregoingthat it depends on the integrity of the whole molecule.Prolactin is a heat-lebile protein though it will survive a certain amountof heating without loss of activity under some conditions of Proteasesdestroy the activity before all material precipitable by trichloroacetic acidis cleaved ; the activity is also destroyed by acid hydr~lysis.~~ The viscosityof prolactin is reversibly increased in presence of urea, indicating de-naturati~n,~' but osmotic pressures indicate no molecular dissociation 43and the biological activity was unchanged after removal of the urea.Treat-ment with another denaturing agent, trichloroacetic acid, also left thebiological activity unchanged.43 Detergents also increase the viscosityof prolactin ; bioassay of the protein-detergent complex indicated someloss of biological potency. 57The best known biological action of prolactin in mammals is the initiationof lactation (lactogenesis), its action on the mammary epithelium apparentlybeing direct,58 though it seems probable that other A.P. hormones are alsoIt also influences the function of the corpus luteum (seeref. 33). No direct metabolic effects in mammals are known; 6o theobservation that prolactin can reduce the fat content of peripheral tissues 61has not yet been confirmed.60Adrenocorticotrophin (ACTH).--lsolation of a " Pure " ProteinExhibiting ACTH Activity.By virtue mainly of dramatic developmentsin the therapy of rheumatoid arthritis and related disorders, ACTH haslately come to the forefront as currently the most important of the A.P.hormones ; its chemistry has several times been reviewed.'* 2* 3* 62s 63 Therichest practicable source appears to be pig pituitaries 64 though it has alsobeen isolated from the pituitaries of sheep and ox. Two groups of workersalmost simultaneously reported the isolation of an ACTH-active proteinsatisfying the currently available criteria of biological and chemical purity.5 1 Li and Kalman, J .Amer. Chem. SOC., 1946, 68, 285.52 Li, Lyons, Simpson, and Evans, Science, 1939, 90, 373.53 Bottomley and Folley, Nature, 1939, 145, 304.54 Li, Lyons, and Evans, J . Biol. Chem., 1941,139,43.5 5 Li and Fraenkel-Conrat, ibid., 1947, 107, 495.6 6 Fraenkel-Conrat, Simpson, and Evans, ibid., 1942, 142, 107.5 7 Li, ibid., 1944, 155, 45.59 Folley and Young, Lancet, 1941, I, 380.60 Li, Ingle, Prestrud, and Negamis, Endocrinology, 1949, 44, 454.61 Reisa, ibid., 1947, 40, 294.62 White in " The Chemistry and Physiology of Hormones," h e r . Assoc. Adv.Lyons, Proc. SOC. Exp. Biol., 1942, 51, 308.Sci., 1944, p. 1.Li and Evans, Vitamin8 and Hw?none8,1947, 5, 198.44 Astwood and Tyelowitz, Fed.Proc., 1942, 1 (Part 2), 4FOLLEY : HORMONES OF THE ANTERIOR-PITUITARY GLAND. 367Li, Evans, and Simpson 65 isolated it from fresh sheep glands by acidacetone extraction followed by fractionation with ammonium sulphate andsodium chloride. The protein behaved as a single substance by ultra-centrifugal, electrophoretic, and solubility criteria and was free from otherA.P. hormones within the limits of sensitivity of the tests used. Later,G. Sayers, White, and Long a6 isolated a protein satisfying similar criteria andexhibiting ACTH activity from pig pituitaries by isoelectric precipitation.The electrophoretic data given in this paper were later corrected.67 Amodification of the latter technique in which sodium chloride fractionationis used instead of isoelectric precipitation at the final step has been described ;the protein so obtained was homogeneous by electrophoretic and solubilitycriteria and as active biologically as that obtained by the method of G. Sayerset al.Physico-chemical data indicate that theACTH-active proteins as isolated from pig and sheep pituitaries are identical.The data are as follows : isoelectric point by moving-boundary electro-phoresis, pH 4.654.70 (sheep 65), pH 4074.8 (pig 66) ; S,, : 2.08 x 10-13cm./sec./dyne (sheep 9, 24M-2-11 x 10-13 cm./sec./dyne (pig 66) ; D,, :10.4 x cm?/sec.(sheep 69) ; disymmetry constant (filf,) : 1.1 (sheep 3* 69),giving an axial ratio of 3 : 1 for an ellipsoid of rotation; molecular weight :20,000 (sheep 69) from sedimentation and diffusion data, 20,000 (pig 66)from sedimentation data but without diffusion data.*The protein contains no carbohydrate, phosphorus, or ~ y s t e i n e .~ ~ Itcontains tryptophan 1 -0 tyrosine 4.5%: methionine 1.93 %, 70 andcystine 7019%,~O the last two acids accounting for all the sulphur.70 Itis very soluble but is precipitated by 2.5% trichloroacetic acid,66 207,sulphosalicylic acid, and 5 % lead acetate.66 The biological activityassociated with it is abolished or diminished by treatment with keten (someamino- and phenolic hydroxyl groups acetylated), 71 nitrous form-Properties of the Protein.6 5 Science, 1942, 96, 450; J. Biol. Chem., 1943, 149, 413.G6 Proc. SOC. Exp. Biol., 1943, 52, 199; J . Biol. Chem., 1943, 149, 425.g8 Fishman, ibid., 1947, 167, 425.69 Burtner, J.Amer. Chern. SOC., 1943, 65, 1238.70 Li, Fed. Proc., 1946,5 (Part 2 ) , 144.71 Li, Simpson, and Evans, Arch. Biochem., 1946, 9, 259.* [Added in proof.] Ghosh, Richards, Merkin, Burns, Brown, G. Sayers, andSmith [Fed. Proc., 1950, 9 (Part I), 1761 have found pig ACTH-protein to be morelabile at high pH than at moderate pH (acid to the isoelectric point). This prompteda re-investigation of its physico-chemical properties by Smith, Brown, Ghosh, andG. Sayers (J. Biol. Chern., 1950, 187, 631). The isoelectric point was pH 4.6, S,, :2.16 x 10-3 cm./sec./dyne, and D,,: 10.8 x sq. cm./sec., giving a molecularweight of 19,400. In electrophoresis experiments on the alkaline side of the isoelectricpoint the main (biologically active) component amounted to 90-94%; in acetatebuffer at pH 4, however, a number of components were present and it was suggestedthat the protein interacts specifically with acetic ion in such a way that primaryparticles are reversibly aggregated.Evidence of rapid dissociation of the hormonewithout loss of biological activity in strongly acid solutions was obtained.Wilhelmi and G. Sayers, ibid., 1948, 176, 175368 BIOCHEMISTRY.aldehyde,7f acetic anhydride,72 or iodine,71 and by esterification of thefree carboxyl groups with methyl alcoh01.~~ An interesting property ofACTH is its remarkable heat stability, a property utilised by Collip 73 formaking partly purified ACTH. Sheep hormone was stable to boiling atpH 7-5 or even for 60 minutes in 0.1M-hydrochloric acid but was inactivatedafter 30 minutes a t 100" in O*lM-sodium hydroxide.65Enzymic and acid hydrolysis of the ACTH-active protein has given results of great interest, showing in confirmationof earlier indications (see below) that the biological activity is associatedwith a molecule much smaller than a protein of molecular weight 20,000.This would accord with its remarkable thermo-stability.Sheep protein-hormone could be digested to a considerable extent by commercial trypsin(18% cleavage, but not 26%) and pepsin (37% cleavage) without loss of'activity.65 Later, Li 72 reported that if the protein were 50% hydrolysed bycrystalline pepsin the nitrogenous fraction soluble in trichloroacetic acid ordialysable was biologically active. Hydrolysis with hydrochloric acid a t37.4", causing 30 yo protein cleavage, also yielded an active non-proteinnitrogen fraction.The average chain length of the biologically activepeptide fraction which evidently was split off by these procedures wasstated to be 7-9 amino-acid residues,72* 74-76 and the average molecularweight determined in the ultra-centrifuge < 1200.75* 76 Fractionation ofthe active peptide mixture by paper chromatography gave a t least six distinctninhydrin-positive spots, one of which was biologically active to theextent of about 13 times the original protein.75* 76 Li 77 has since statedthat his ACTH peptides can be activated 2 or 3 times by boiling them withhydrochloric acid.Fractions showing very high biological activity-up to 120 times thatof an electrophoretically inhomogeneous Armour preparation no.La- 1 -a,itself slightly more active than a " pure '' protein-hormone preparation 78-have been isolated by Lesh, Fisher, Bunding, Kocsis, Waleszek, White,and Hays 79 by subjecting trichloroacetic acid-soluble fractions from pepticdigests of partly purified pig ACTH preparations to counter-current dis-tribution. Ultra-centrifugal studies indicated LL higher molecular weight(2500--10,000) than the average given for the much less biologically activefraction obtained by Li and P e d e r ~ e n . ~ ~ Lesh et ~ 1 . ~ ~ suggest that thelatter type of preparation might consist of low-molecular material containinga small proportion of a very active fraction of higher molecular weight.Raben, Payne, and Astwood 80 have also reported the preparation of ACTHup to 100 times as active as La-l-a.Dialysable material isolated from aConf. Metab. Aspects Convalescence, Josiah Macy Jnr. Foundn., 1948, p. 114." Low-molecular ACTH."79 Li. Abstr. Commun. 1st Int. Congr. Biochem., 1949, p. 386; Trans. 17th Meeting73 J . Amer. Med. Assoc., 1940, 115, 2073.74 Li, Fed. Proc., 1949,8, 219.76 Li and Pedersen, Arkiv Kemi, 1950,1,533.78 M. A. Sayers, G. Sayers, and Woodbury, Endocrinology, 1948, 42, 379.70 Science, 1950, 112, 43.80 Abstr. Papers, Amer. Chem. SOC. 118th Meeting, 1950, l l c .7 6 Li, J . Endocrinol., 1950, 6, xl.7 7 J . Amer. Chem. SOC., 1950,72,2815FOLLEY : HORMONES OF THE ANTERIOR-PITUITARY GLAND.369peptic digest of ACTH protein has been found to be active clinically aswell as by the adrenal ascorbic acid depletion test.81The idea that ACTH may be of relatively small molecular size is by nomeans new; Anselmino, Hoffmann, and Herold 82 long ago claimed thatit was ultra-filtrable through collodion membranes, a contention confirmedsome years later by Tyslowitz,83 using a more specific bioassay method fortesting his ultra-filtrates. More recently, Crooke, C. J. 0. R. Morris, andtheir colleagues g4--86 have prepared ACTH-active ultra-filtrates withoutpreliminary hydrolysis from crude ox ACTH preparations using membranesimpermeable to proteins of mol. wt. 17,500 and 13,700 but passing salmine(mol. wt. 8000). The activity of the total ultra-filtrable solids at theoptimum pH for ultra-filtration (approx.pH 2) was about 1-18 times thatof preparation La-l-a which has now been adopted as the provisionalinternational standard. Later, P. Morris and C. J. 0. R. Morris 87 reportedthe isolation, from the polypeptide mixture. so obtained, of what appears tobe a homogeneous peptide about 8.5 times as active as La-l-a. Furthercharacterisation of this peptide will be awaited with interest. Geschwind,Hess, Condliffe, and Williams 88 have also obtained low-molecularbiologically active material from ACTH-protein by dialysis and also bytreatment with trichloroacetic acid. In the latter case trichloroaceticacid-soluble fractions showing more than ten times the activity of theprotein (calculated per mg.of nitrogen) have been obtained, the activityof the insoluble material being reduced. Moreover, they were able toseparate active from inactive moieties by subjecting the protein to paperchromatography, the active spots exhibiting fluorescence. Similar resultshave since been obtained with trichloroacetic acid extracts of fresh sheeppituitaries .2OThe problem of designing relatively simple chemical procedures capableof giving maximum yields of ACTH in a form suitable for clinical use haslately received attention. Raben, Payne, and Astwood 8o described aprocedure involving extraction of dried pig pituitary powder with glacialacetic acid which gave preparations more active than La-l-a.* Reiss and81 Brink, Meisinger, and Folkers, J. Amer. Chem. SOC., 1950, 72, 1040.82 Klin. Woch., 1934, 13, 209; Arch. Bynaek., 1934, 157, 86.83 Science, 1943, 98, 225.84 Crooke, Henly, and C. J. 0. R. Morris, Abstr. Commun. XVIIth Int. Physiol.Congr., 1947, p. 139.85 Henly, P. Morris, and C. J. 0. R. Morris, Abstr. Commun. 1st Int. Congr. Biochem.,1949, p. 384.Cortis-Jones, Crooke, Henly, P. Morris, and C. J. 0. R. Morris, Biochem. J.,1950, 46, 173. Lancet, 1950, T, 117.Payne, Raben, and Astwood ( J . BioE. Chem., 1950, 187, 719),have given details of methods of preparing highly active ACTH fractions from acetone-dried pig pituitaries. Extraction with glacial acetic acid at 70" is followed by frac-tionation with acetone and ether. The activity of the material precipitated with etherwas increased by adsorption on powdered cellulose, followed by elution with hydro-chloric acid, and solvent partition in presence of picric, benzenesulphonic, or o-mer-captobeneoic acid, gave preparations assaying up to 80 times the potency of theprovisional international standard.Science, 1950, 111, 625.* [Added in proof.370 BIOCHEMISTRY.Halkerston 89 extracted fresh pig glands with acid acetone and, duringfurther purification to remove posterior lobe principles, obtained threeapparently chemically different active fractions. It is not yet knownwhether these represent molecularly distinct hormones or different “ carrier ”proteins associated with the same active moiety.The fact that fractions of enhanced biological activity can be separatedfrom the protein or from A.P. extracts by procedures such as dialysis,ultra-filtration 839 86 (ultra-filtration of the whole activity of the proteinhas been claimed 86), trichloroacetic acid fractionation, and paper chromat o-graphy 88 which would probably not hydrolyse peptide linkages may meanthat the hormone is a relatively low-molecular polypeptide adsorbed onan inactive protein molecule, though the possibility that the ACTH-proteinis a “ mother molecule ” consisting of a dissociation-association systemincorporating an active moiety, cannot yet be excluded. Peptide linkagesare essential for the biological activity since it is destroyed by t r y p ~ i n , ~ ~carbo~ypeptidase,~~ papain,86 and acid hydroly~is.~~The subject at the time of writing is in a state of flux and it is to behoped that tbe results of the extensive experiments now actively proceedingin various countries will soon clarify it. Speculation about the form inwhich ACTH is elaborated by the A.P. or exists in the circulation is clearlypremature at the present stage when we are still awaiting the isolation ofthe pure hormone as it appears in A.P. extracts. If the active moiety isof the size suggested by Lesh et U Z . , ’ ~ then the earlier hopes of preparing itsynthetically will have receded somewhat.Growth Hormone (GH).-A number of reviews of the chemistry of GHhave appeared in the last ten years.l, 2* 3* 62* 63Isolation. GH has been isolated as a molecularly homogeneous protein(by criteria of diffusion, electrophoretic mobility, and solubility), showingno significant contamination with other known A.P. hormones, by Li, Evans,and SimpsonM using salt fractionation of an aqueous calcium hydroxideextract of acetone-dried ox A.P. Later, Wilhelmi, Fishman, and Russell 91described a less laborious method, based on low-temperature ethanolfractionation, by which high yields (stated tc be ca. 3 g./kg. of fresh gland)of crystalline GH were obtained from fresh ox A.P. Li, Evans, andSimpson 92 subsequently crystallised their amorphous protein, bioassayindicating that the process had effected no concentration or fractionationof biological activity.Properties. Physico- chemical data for amorphous GH have beenobtained as follows : l-isoelectric point : pH 6.85; S,, (determined incrn.2lsec. ; 94 disymmetry constant (f/f,) (hydration being neglected andalkaline solution) : 3.1 x 10-13 cm./sec./dyne; 93 D,, : 7.15 x 10-789 J . Pharrn. Pharmacol., 1950, 2, 236.90 Li and Evans, Science, 1944, 99, 183; Li, Evans, and Simpson, J . B i d . Chem.,92 Science, 1948,108, 624.01 Li, J. Phya. Colloid Chem., 1947, 51, 218.1945,159, 353. *l Ibid., 1948, 176, 735.g3 Li and Moskowitz, J . Biol. Chem., 1949, 178, 203FOLLEY : HORMONES OF THB: ANTERIOR-PITUITARY GLAND. 371a prolate ellipsoid assumed) : 1.31, indicating an elongated molecule ;intrinsic viscosity : 7.64 ; 94 molecular weight : 44,250 (osmotic pressure 90),43,600 (analytical data for five amino-acids and total sulphur 94), 46;800(from histidine content 95), 47,300 (analytical data for 14 amino-acids 96),39,300 (diffusion and viscosity determinations "), 44,000 (ultra-centrifuge 93).Data obtained for the crystalline hormone 97 are : D2, : 7.36 xcm.2/sec. ; Szp (determined in glycine buffer) : 3.60 x cm./sec./dyne;molecular weight (ultra-centrifuge) : 49,200.The hormone contains no phosph~rus,~ carbohydrate: or c y ~ t e i n e . ~ ~The total sulphur is accounted for by its content of cystine and methionine.'O, 94Analytical data for 15 amino-acids, which together with the amide-nitrogenaccount for 88.2% of the total nitrogen, corresponding to about 80% of thedry weight, have been determined.O5, 96 From considerations based on thenitrogen partition it is suggested that the GH molecule consists of 369amino-acid residues (26 of whiph are unknown) arranged in two sub-unitswith polar groups located in the outer faces and non-polar groups towardsthe inner planes.96 It is a thermolabile protein more stable in alkalinethan in acid solution.g0 Viscosity,94 osmotic pressure,g4 and sedimentationvelocity 97 determinations indicate that some aggregation of the proteinoccurs in acid solution (pH 4.0) though diffusion measurements at pH 4-0provided no evidence of molecular inhomogeneity.gO Denaturation inacetate buffer 94 or by urea does not destroy the biological activity, butthe activity is destroyed or diminished by treatment with keten,3 nitrousacid: or acetic anhydride?* by i o d i n a t i ~ n , ~ ~ and by proteases.w Theantigenicity of a crystalline growth hormone preparation has been foundto be poor.99Besides re-establishing body growth and influencing chondrogenesisand osteogenesis in hypophysectomised rats (responses used for bioassay 3),GH is diabetogenic in the intact cat,lo0 glycostatic in the hypophysectomisedrat,O1 and galactopoietic in the lactating c0w.1~1Thyrotrophh-Thyrotrophin has not yet been isolated in the pure state,though considerably purified and highly active preparations have beenmade. Reviews of the chemistry of thyrotrophin 2, 3, 62 emphasise itsprobable protein nature but there is some indication that its molecularweight is rather low.The richestcommon sources appear to be pig lo2 and ox lo3 pituitaries ; sheep pituitariesare less potent.lo3 Several methods of preparation of highly potent thyro-It is very soluble and easily extracted from pituitary tissue.95 Franklin, Li, and Dunn, J . Biol. Chem., 1947, 169, 515.B6 Li and Evans, Recent Progr. Hormone Research, 1948, 3, 3.B7 Smith, Brown, Fishman, and Wilhelmi, J . Biol. Chem., 1949, 177, 305.B* Li, Simpson, and Evans, ibid., 1948, 178, 843.BB EIberg end Li, Endocrinology, 1950, 47, 143.loo Cotes, Reid, and Young, Nature, 1949,164, 209.lol Cotes, Crichton, Folley, and Young, ibid., p. 992,loa Rowlands, J. Physiol., 1936, 88, 298.lo8 Jorgensen and Wade, Endocrinology, 1941, 28, 406BIOCHEMISTRY.trophic hormone preparations have appeared during the last decade. J.Fraenkel-Conrat, H. Fraenkel-Conrat, Simpson, and Evans 104 used saltfractionation methods to obtain a purified thyrotrophin preparation froman acetic acid-sodium chloride extract of acetone-dried ox pituitary. Thispreparation was contaminated principally by ICSH and by only smallamounts of FSH, prolactin, ACTH, and GH. Salt fractionation methodswere also used by Fevold, Lee, Hisaw, and Cohn lo5 to obtain a thyro-trophic fraction from an alkaline extract of whole sheep pituitary.Jorgensen and Wade 103 effected considerable purification and concentrationof the activi$y by methods involving adsorption on " Permutit " and pre-cipitation by uranium acetate. More recently Ciereszko lo6 has obtainedhighly purified thyrotrophin preparations from whole fresh ox pituitaryglands by fractionation with acetone, lead acetate, and trichloroaceticacid, the method being essentially a simplification of that previously describedby Bonsnes and White. lo' The preparations so obtained were substantiallyfree from prolactin, GH, and gonadotrophic hormones, and preliminaryelectrophoretic and ultra-centrifugal studies indicated the presence of onlyone protein component.The hormone is not precipitated by trichloroacetic acid,lo6 sulpho-salicyclic acid,lOG or lead acetate,106 and does not sediment easily in theultra-centrifuge,lOB all of which suggest that its molecular weight may berather low. It is very soluble,lM readily adsorbed on a variety of adsor-b e n t ~ , ~ ~ ~ but is precipitated by flavianic,lo3 picric,lo6 or phosphotungsticacid,lo6 uranium acetate,lo6 or mercuric chloride.lo6 Highly purifiedpreparations have been found to contain carbohydrate 104* lo6 but nophosphorus.lo6 The activity is destroyed by proteolysis l8 and by treatmentwith cysteine lo4 or keten.104S. J . F.E. P. ABRAHAM.E. BOYLAND.J. DUCKWORTH.S. J. FOLLEY.D. HERBERT.W. E. VAN HEYNINGEN.G. G. NEWTON.104 J . Biol. Chem., 1940, 135, 199.lo6 J . Biol. Chem., 1945, 160, 585.108 Severinghaus, Levin, and Chiles, ibid., 1938, 23, 285.lo5 Endocrinology, 1940,26,999.lo' Endocrinology, 1940, 26, 990.37

ISSN:0365-6217    

DOI:10.1039/AR9504700285

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

ANALYTICAL CHEMISTRY.1. INTRODUCTION.THIS is the first occasion on which this Report covers the advances made inall fields of Analytical Chemistry during the current year. The very largenumber of pages published annually on analytical topics (4200 abstractsappeared in British Abstracts, C, 1950) reflects activity in very manydirections, and the task of summarising the important advances in allbranches of the subject, for readers with varied and diverse interests, hasproved difficult owing to limitations of space; for this reason, it has beenassumed that fundamental aspects of each subject are well known. Thepublications appearing in a single year on a particular topic rarely constitutea major advance; such advances are usually recorded by a succession ofpapers over a period of many years (often by many different authors), andprogress of this nature is frequently influenced by factors other than thepurely scientific ones.This is particularly true of certain physical methodsof analysis, such as infra-red spectroscopy, which have only attained wide-spread importance since the production of commercially available instru-ments ; the pattern of this Report therefore differs in many respects from thatof previous Reports, which have consisted of periodic reviews of special topics.F. R. C.2. CHEMICAL METHODS (INORGANIC).Standardisation.-The scheme of standardisation bf volumetric solutionsused in the I.C.I. analytical laboratories is described.1 It is based on silveras the ultimate standard. The working standards referred directly tosilver are sodium carbonate, sodium chloride, and iodine, and arseniousoxide and potassium dichromate appear as secondary standards.Thepreparation of the working standards, and the reference of all the commonvolumetric solutions to one of the standards, are described in detail. Nutten 2lists numerous substances recently proposed as standards, and Underwood 3gives tables of the changes of pH with temperature between 0" and 60" forO.O5~-potassium hydrogen phthalate, O*OlM-borax, and O.O25~-phosphatesolution. McLellan * reports a comparison by eight chemists of commerciallypure potassium dichromate against Bureau of Standards samples, by reactionwith acidified potassium iodide and titration .with thiosulphate.Six ofthe eight analysts agreed to 1 part in 2000 or better, and four samples wereso pure that improvement by recrystallisation could not be detected. Anew standard substance has been proposed-propylenediamine ferroussulphate tetrah~drate.~ Its high equivalent weight, stability, and ease ofpreparation make it attractive.Metallurgia, 1949, 41, 111, 177, 237. 1 Strouts et al., Analyst, 1950, 75, 577.3 J . Ass. 08. Agric. Chem., 1950, 33, 225.6 Nutten, Analyt. Chim. Acta, 1949, 3, 433.6 Grossmann and Schuch, 2. anorg. Chem., 1906,50, 24.' Ibid., p. 224374 ANALYTIUAL UHEMISTRY.Reagents.-(When the emphasis is on the reagent, work is reviewed inthis Section; where the element determined is of greater importance, i t ismentioned under the appropriate heading.) The outstanding new reagentis Schwarzenbach's sodium ethylenediaminetetra-acetate. This salt (or thecorresponding acid) has great possibilities; it is outstanding as a complex-forming agent, and the complexes have in many cases widely differentproperties from the metal ions ; oxidation-reduction potentials are changedand precipitations with many reagents are profoundly modified ; some ofthe compounds have distinctive colours.Systematic investigations arebeing made by Pribil and his collaborators. The reagent alters the oxidationpotential of the Co++/Co+++ reaction, so that cobalt can be oxidised in acidsolution by means of ceric sulphate; ' other metals behave similarly, so thereaction cannot be always applied volumetrically, but Cr++ reduces thecomplex, and by potentiometric titration Co can be determined in presenceof many elements common in ferrous alloys.8 Chromium forms a wine-redcomplex, and this reaction may be used in the colorimetric determination ofthis meta1.O The complex with beryllium is one of the least Etable, andeven in presence of excess of reagent, ammonia precipitates the hydroxide :in this way beryllium can be quantitatively separated from many metals.10Similarly, the reagent prevents the precipitation by hydroxyquinoline inammonium acetate-acetic acid solution of all metals except W, V, Mo, Ti,and U : molybdenum, for instance, can be separated from a very largenumber of metals.ll Exactly analogous is its use combined with sodiumdiethyldithiocarbamate, which in its presence only combines with Hg, Pb,Cd, Cu, and Bi.Only the last two are coloured, and hence the colorimetricdetermination of copper can in many circumstances be much simplified .12The reagent combines with rare earths, and improved separations can bemade by adding just sufficient to keep the earths in solution, and fractionallyprecipitating with oxalic acid.l3 This reagent will also dissolve " insoluble "precipitates, e.g. barium sulphate, calcium oxalate, and lead iodate.14a-Benxoinosime l5 is applied to the photometric determination of copperin ferrous alloys, but nickel and cobalt interfere. a-Nitroso-p-naphthol isbeing studied as a volumetric reagent; l6 although the silver compoundadsorbs excess of reagent, a method of determination is possible, andpotentiometric and gravimetric methods for copper and iron are reported.The solubility and dissociation constants have been determined.Shorne l7aays that N-benzoylphenylhydroxylumine has advantages over cupferron : itis stable and soluble in water, and at pH -4 it quantitatively precipitatesCu, Fe, Al, and Ti. Bdsdimethyluminodiphenylmethune is recommended byPribil and Klubalova, ibid., p. 42.7 Pribil and Malicky, Coll. Czech. Chem. Comm., 1949, 14, 1413.* Pribil and Svestka, ibid., 1960, 15, 31.lo Pribil and Kucharsky, kbid., p. 132.l2 Sedevic and V&Ak, ibid., p. 260.l4 Budde and Patempa, AnaZyt. Chem., 1950, 22, 1072.l6 Dunleary, Wiberley, and Harley, ibid., p. 170.l6 Wenger, Monnier, and Jaccard, Helv.Chim. Acta, 1950, 33, 1154, 1458.1 7 Shorne, Ana7yst, 1960, 75, 27.l1 Pribil and Malat, ibid., p. 120.l8 Marsh, J., 1960, 1819WILSON : CHEMICAL METHODS (INORGANIC). 375Cornfield and Pollard 18 for determination of traces of manganese in planttissues, etc.; 0-5 pg. in 25 ml. of extract can be determined; potassiumperiodate in the cold oxidises the manganese, which in turn oxidises thereagent to form the colour. For cobalt and nickel, P-nitrososalicylic acid isrecommended as a colorimetric reagent.19 I n absence of iron and copper thetwo elements can be determined simultaneously, the brown cobalt complexbeing soluble in light petroleum, and the red nickel complex remaining in theaqueous phase. Copper can be determined by a reagent made by dissolvingsalicylic acid in pyridine.20 The copper complex dissolves in chloroform togive a green solution : Williams21recommends tetraethylenepentamine for determination of copper, particularlyin light alloys : i t is intermediate in sensitivity and reasonably free frominterference.Hoste22 finds that compounds with two pyridyl groups or apyridyl and a quinolyl or two quinolyl groups linked in the 2 : 2’-positionform intensely coloured compounds with Cu+, insoluble in water but solublein organic liquids. 2 : 2’-DiquinoZyZ is absolutely specific for Cu+ and verysensitive. The reagent dissolved in amyl alcohol extracts the copper froman aqueous solution at pH -3 and containing hydroxylamine. Extractionis quantitative and at 540 my.Beer’s law is obeyed. Antimony can bedetected by an acetone solution of gossipol, which, with a dilute hydro-chloric acid solution containing phosphate, gives a red colour. The reagentis stable, selective, and sensitive (limit of detection 1 : 100,000).23 Thio-acetumide is said to have advantages over hydrogen sulphide for the precipi-tation of bismuth,24 m~lybdenum,~~ antimony,26 copper,27 and arsenic.28For cadmium in micro-quantities, Dwyer and Gibson 29 recommend methyl-triphenylarsonium chzoride, 100-fold excess of zinc not interfering ; 1-10 yg.are determined turbidimetrically, but larger amounts are precipitated.Sodium mphthiomte 30 at pH 2-3 can be used to separate thorium from therare earths, and zirconium can be separated from thorium by means oft ~ n n i n .~ l Gordon et review the reactions of some dibasic organic acidswith thorium; tetruchlorophthalic acid is recommended; at 70-85” and apH of 1-1.2, a dense crystalline precipitate is formed ; double precipitationgives complete separation from the rare earths. Oesper and Klingenberg 33tested a number of glycollic acid derivatives ; substituted mandelic acidsproved useful reagents for zirconium, p-bromo-, p-chloro-, and unsubstitutedFe, Co, Ni, V, and Ag interfere.l 8 Cornfield and Pollard, J . SOC. Food Agric., 1950, 1, 107.2o Gordieyeff, ibid., p. 1166.22 Hoste, Analyt. Chim. Acta, 1950, 4, 23.23 West and Conrad, Analyt. Chem., 1950, 22, 1336.2* Flaschka and Jacobljevich, Analyt. Chim. Acta, 1950, 4, 351.25 Idem, ibid., p.356.27 Idem, ibid., p. 482.28 Analyst, 1950, 75, 201.30’ Venkataramaniah and Raghava Rao, ibid., p. 553.31 Purushottam and Raghava Rao, ibid., p. 558.32 Gordon, Vanselow, and Willard, Analyl. Chem., 1950, 22, 1323.a3 Ibid., 1949, 21, 1609.Perry and Serfass, Analyt. Chem., 1950, 22, 565.21 Analyet, 1950, 75, 425.26 Idem, ibid., p. 247.28 Idem, ibid., p. 486376 ANALYTICAL CHEMISTRY.mandelic acid being suitable. Hahri 3* also uses mandelic acid for zirconium.The reagent is almost specific.8-Hydroxyquinoline (“ oxine ”) is used in photometric determinations, ad-vantage being taken of the solubility of the chelate compounds in chloroform.Gallium and thallium solutions in chloroform are somewhat photosensitive ;the absorption spectra are given,35 but the thallium compound is too photo-sensitive to be of use.The same authors 36 deal with the ultra-violet spectraof chloroform solutions of the oxinates of Group IIIB elements. Photochemicalinstability increases with increasing size of the central metal ion. Gentryand Sherrington 37 determined the pH ranges for which a chloroform solutionof the reagent completely extracted Al, Cu, Fe(m), Mn, Nb, Ni, and Sn(Iv).For Sn(Iv), extraction a t pH 2-5-55 and photometry (max. extinction390 mp.) is a valuable procedure. 5 : 7-Dibromo-8-hydroxyquinoline is avaluable reagent for gallium, and enables it to be separated from aluminium 38by precipitation from a faintly acid solution. Although the complex issoluble in chloroform, has intense absorption a t 410 mp., and obeys Beer’slaw, direct extraction from the aqueous phase is not quite complete; itmust be precipitated first.Xodium diethytdithiocurbamate can be used as avolumetric reagent, in neutral or ammoniacal solution, with a silver wire asindicating electrode, and a calomel half-cell. It may be an advantage totitrate in presence of ether, which removes the heavy-metal salt as formed.Copper and cadmium, or copper and lead, can be titrated successively in thesame solution.39 Glen and Schwab have prepared seven “ carbates,”i.e., disubstituted dithiocarbamates, of which the piperazine, morpholine,thiazine, and-most promising-pyrollidine derivatives are new. Manymetals give insoluble compounds which are readily filtered off, are intenselycoloured, soluble in esters and-in contrast to xanthates-are stable inneutral or alkaline solutions.By varying the pH in presence of ammonia orRochelle salt, a new group separation is possible. Though the reagents havea family resemblance, they differ in speed of precipitation and solubility ofprecipitates. One or two new separations are possible, e.g., Ni++ can becompletely separated from GO++ by the pyrollidine compound in presence ofhydrazine, and tellurium can be separated from selenium. A specific testfor molybdenum is also possible. Excess of organic reagents in a solutioncan often be completely destroyed by persulphate catalysed by a littlesilver, by which, e.g., masking agents such as tartaric acid and precipitantssuch as oxine, can be oxidised, after which metals can be precipitated ashydroxides, etc.Some reagents are more resistant and mercapto-groupsare oxidised to elementary sulphur.41 Three papersg2 deal with thedistribution of ferric iron between hydrochloric acid and isopropyl ether.34 Hahn, Analyt. Chem., 1949, 21, 1579. 35 Moeller andCohen,ibid., 1950,22,686.36 Idem, J . Amer.Chem. Xoc., 1950,72,3547. 3 7 Analyst, 1950, 75, 17.3 8 Moeller and Cohen, Analyt. Chim. Acta, 1950, 4, 316.39 Sedivec and Vasak, Coll. Czech. Chem. Comm., 1950, 15, 52.4O Angew. Chem., 1950, 62, 320.41 Feigl and Schaeffer, Analyt. Chim. Acta, 1950, 4, 458.Myers, Metzler, and Swift, J . Arner. Chem. SOC., 1950, 72, 3767, 3772, 3776WILSON : CHEMICAL METHODS (INORUANIC).377The effect of changes of acidity, and the ultra-violet spectra of the ethereallayer under various conditions are discussed. Salicyli&enethiosemicarbazoneis proposed as a reagent; several metal salts are disc~ssed,*~ and a gravi-metric method for cadmium is put forward, but other metals interfere.44 Ithas also potential applications in colorimetry, e.g., for mangane~e.~~ Twonew oxidising agents for volumetric analysis are potassium fe~rate,*~ said tobe stable in solutions more alkaline than 5M. with respect to sodium hydroxide,used in conjunction with sodium arsenite solution, and potassium cupri-periodate K , C U ( I O ~ ) ~ . ~ ~ Lang** advocates the use of ruthenium as acatalyst in the titration of tellurous acid with permanganate.If cericsulphate is used, with chromate as catalyst, Te(rv) can be oxidised in presenceof Se(Iv), which is unchanged. Starke 49 finds that molten chlorides of somearomatic amines, e.g., pyridinium hydrochloride, dissolve metals and someoxides readily at convenient temperatures, and indicates possible analyticalapplications. Belcher 50 reports that tartrazine can be used as a reversibleindicator in the titration of arsenite with hypochlorite. For oxidationreactions there is a novel suggestion to use aiZ~xene,~l for when a minuteexcess of oxidising agent is added to a solution containing this substance thewhole solution emits light.Qualitative Analysis.-A review of tests for 24 anions has appeared62with comments on sensitivity and interferences.By heating small samplesin micro-burner flames, oxidising or reducing, asbestos fibres being used assupports, flame colours are noted, and by allowing the tip of the flame toimpinge on a test-tube full of cold water, sublimates of metal or oxide canbe collected from 18 metals, and identified by recommended microchemicalreactions.% k semimicro-scheme of analysis, including several of the" rarer " elements, which does not use hydrogen sulphide, is described.54Holness and Trewick 55 recommend a solution containing 1% of lithiumhydroxide and 5% of potassium nitrate as giving a cleaner separationbetween the copper and the arsenic sub-group than the older reagents.Potassium xanthate can be used to separate copper and cadmium, which issubsequently identified as sulphide.66 Nutten 57 describes a method ofremoving phosphate ion by a nitric acid solution of titanic hydroxide; heindicates the amount of common ions which remain in solution after theseparation.By extraction of a hydrochloric acid solution with ethylacetate, chloroauric acid is extracted : by evaporating the solvent, addingsodium metaphosphate solution, and applying a test-paper impregnated4a Hovorka and Holtzbecker, Coll. Czech. Chem. Comm., 1960, 15, 267.44 Idem, ibid., p. 275.O 6 Schreyer, Thompson, and Oekerman, Analyt. Chem., 1950, 22, 691.4 7 Beck, Mikrochem., 1950, 35, 169.40 Canadian J . Ree., 1950, €3, 28, 225.s1 Kenny and Kurtz, Anulyt. Chem., 1950, 22, 693.52 Odekerken, 2. anal. Chem., 1950,131, 165.63 Geilmann and Isermeyer, ibid., p.249.54 Badry, McDonnell, and Wilson, Analyt. Chim. Acta, 1950, 4, 440.O S Analyst, 1950, 75, 276.s 7 Nutten, Anulyt. Chim. A&, 1950, 4, 340.4 5 Idem, ibid., p. 280.Lang, 2. anal. Chem., 1949,128, 484.Belcher, Analyt. Chim. Acta, 1950, 4, 468.56 Rahn, 2. anal. Chem., 1950,181, 263378 ANALYTICAL CHEMISTRY.with p-rosaniline hydrochloride, a specific test for gold is obtained (limit5 pg.).68 Trich6 59 identifies calcium by coprecipitation of its carbonatewith silver chromate. Because of mixed-crystal formation, the latter saltdissolves much less readily in dilute ammonia. This phenomenon does notoccur with strontium or barium. Neu 6o reports that long-chain quaternarysalts, such as dodecylpyridinium bromide, precipitate metaphosphates froman acetic acid solution, but not ortho- or pyro-phosphates.A thoroughinvestigation of the etching of glass by hydrogen fluoride has shown thatthe reaction is in part catalytic, the silicon tetrafluoride formed reacting withmoisture to form more hydrogen fluoride. Though the reaction is notquantitative, yet by use of special apparatus, 1 pg. of fluorine can be detected.Freytag 62 detects hydrogen peroxide by its bleaching action on paperimpregnated with lead sulphide : as little as 1 in 5 x lo6 can be detected.Percarbonates are discussed by Partington and Fathallsh,63 who point outthat Riesenfeld's test, which attempts to discriminate between true per-salts(e.g., K2S20,) and salts containing hydrogen peroxide of crystallisation, byreaction with neutral potassium iodide solution (the former liberate iodine,the latter oxygen), is not sound.General Methods of Analysis.-In determining small amounts of aluminiumWillard and Dean 64 polarograph in presence of Pontachrom-Violet-SW(colour index 169) at pH 4.6 the half-wave potential being about -0-5 v.Many metals which interfere can be removed by electrolysis over a mercurycathode; the only anions present should be acetate and perchlorate.Thereaction of aluminium with alizarin-S has been reinvestigated. 65 BelowpH 3-9 one atom of aluminium reacts with one mol. of the reagent ; a t higherpH values, more complex compounds are formed. Calcium combines withthe aluminium alizarinsulphonates to form a new compound, between pH 3.9and 4-55, which is utilised in an improved colorimetric method.Berylliumis discussed in papers by Seguin and Gramme.66t 6 7 9 68 The first is general,and describes the chemical analysis of beryllium-copper alloys, the seconddeals with colorimetric methods, reporting that 1 : 2 : 5 : S-tetrahydroxy-anthraquinone is not a satisfactory reagent, and the third describes in detailemission spectroscopic analysis of copper alloys for beryllium. Accordingto Ellis, Zook, and B a u d i s ~ h , ~ ~ 1 : 1'-dianthrimide was the best colorimetricreagent for boron of forty tested. Azides can be titrated with silver nitrateby using daylight or ultra-violet light and the usual adsorption indicators,e.g., rh0damine-6B.~~ Lang and Annis 71 recommend the reaction withpotassium iodide and acid in the absence of air for the semimicro-determin-58 West and Carlton, Analyt.Chem., 1950, 22, 1055.6n Analyt,Chim. Acta, 1950, 4, 12.61 Williama,Anulyst, 1950,75,510.69 J., 1950, 1934.as Parker and Goddard, Analyt. Chim. Acta, 1950, 4, 517.66 SBguin and Gramme, Bull. SOC. chim., 1950, 17, 375.67 Idem, ibid., p. 384. 6B Idem, ibid., p. 388.69 Anolyt. Chem., 1949, 21, 1345. ' 0 Haul and Uhlen, 2. a d . Chem., 1949,129,21.7 1 Compt. rend., 1950, 230, 208.6o 2. anal. Chem., 1950, 131, 102.6a 2. anal. Chem., 1950, 131, 77.6* Analyt. Chem., 1950, 22, 1264WESON : CHJMICAL METHODS (INORGANIC). 379ation of nitrim; in determination of nitrates with m-4-xyleno1, Barnes 72avoids distillation by extracting the acid solution containing the nitroxylenolwith toluene.To determine free nitric acid in presence of aluminium nitrate,the use of potassium oxalate instead of fluoride is recommended as a, complex-ing reagent.73 Following Cotte and Kahane,74 who quantitatively reducenitrates to ammonia by ferrous hydroxide in presence of silver as catalyst, itis now proposed to determine the nitrate by titration of the excess of ferrousiron, after reaction is complete.75.MonojlwopJwq&ate. ion, FPO," , is rapidly hydrolysed in acid solution,7sand if the bleaching of peroxytihnate solution is measured a t 3-minuteintervals, by extrapolation back fo zero time both free F' and FPO," can beestimated. Fluoride ion can be accurately determined by its bleachingaction on solutions containing a ferrisulphosalicylate complex.A generallyapplicable method of analysis is described, which is applied after separatingthe fluorine from aluminium, etc., by di~tillation.~~ Sodium can bedetermined in calcined alumina after dissolution of the sample by heating ina sealed narrow-bore tube to 200" with excem of hydrochloric acid.78 Shell 7Ddetermines sodium in presence of lithium and phosphate by precipitating itwith magnesium uranyl acetate, and treating the precipitate with a solutionof hydrogen chloride in butanol ; lithium and phosphate dissolve, leavingsodium chloride insoluble. SiEiwn is determined 80 by diEtillation from alead still with sulphuric acid and fluoride, the distillate being hydrolysed byammonia.After addition of boric acid, silica is determined calorimetrically.Jewsbury 81 makes a similar use of boric acid in the analysis of hydrogenfluoride, etc., for silica. For determination of free sulphur trioxide in chloro-sulphonic acid, Seaman et a l . 8 2 measure the heat of reaction with hydrochloricacid. Traces of chlorides can be determined 83 by centrifuging out the silverchloride, reducing it with alkali and hydrazine, dissolving in nitric acid, andextractive titration with dithizone (diphenylthiocarbazone). The analyticalchemistry of chlorites is discussed.84 The similarity of C10, to NO, is pointedout ; e.g., there is an insoluble copper-lead-potassium chlorite analogous tothe well-known triple nitrite : many other reactions are described.Gale and Mosher B5 determine milligram quantities of vanadium inpresence of large amounts of uranium and other metals by titration withferrous sulphate, polarised platinum electrodes being used fo detect the end-point.In the determination of manganese by oxidation with persulphste,Analyst, 1950, 75, 388.73 Blaedel and Panos, Anulyt. Chem., 1950, t22, 910.74 Cotte and Kahane, Bull. SOC. chim., 1946, 13, 541.7 5 Szabo and Bartha, Nature, 1950, 166, 309.7 6 Hill and Reynolds, Andyt. Chem., 1950, 22, 448.7 7 Lacroix and LEabalade, AnaZyt. Chim. Acta, 1950, 4, 68.7 8 Jackson, Analyst, 1950, 75, 415. 70 Analyt. Chem., 1950, 22, 574.81 Analyst, 1950, 75, 257. Bonnier, Bull. SOC. chim., 1950, 17, 365.Seaman, Woods, and Bank, Ancrlyt.Chem., 1950,22, 549.Iwantscheff, Angew. Chem., 1950,62, 361.84 Morandot and Duval, Mikochm., 1950, 85, 202.Gale and Mosher, Analyt. Chm., 1950, 22, 942380 ANALYTICAL CHEMISTRY.metaphosphoric acid is used to stabilise permanganic acid.86 Iron isadvantageously precipitated as basic ferric formate, the hydrolysis of ureain the boiling neutral solution being used to adjust the pH ; good separationsfrom some other metals can be a~hieved.~' Cobalt is determined by thepolarograph, after oxidation to CO(III) with ammonia and perborate.Manganese and iron, precipitated as hydrated oxides, carry down cobaltand cause low results.88 Yardley 89 recommends potentiometric titration ofcobalt with ferricyanide as an accurate process. Norwitz says that withsuitable precautions copper and tin 91 can be safely electrodeposited fromhydrochloric acid solutions.The photometric determination of molybdenumis thoroughly discussed by Rasin-Straden and Popoff-As~toff.~~ Althoughthe maximum absorption of the thiocyanate colour is -470 mp., a t longerwave-lengths, changes with time and the influence of iron are less. Accuratercsults can be obtained even with high percentage of molybdenum, providedthat pure reagents are used, the iron concentration is constant, and a stricttime cycle is followed. Ellisg3 uses acetone as reducing agent in the thio-cyanate method for molybdenum. Also, this metal can be concentratedfrom very dilute solutions by being adsorbed on active alumina : a t pH5-5-6 adsorption is practically complete.It is recovered by washing withdilute ammonia.94 Traces of molybdate catalyse the reaction betweeniodides and hydrogen peroxide in neutral solution. This reaction is moresensitive than the well-known test with titanic sulphate : the peroxide canbe determined by measuring the extinction of the solution a t 353 mp.95Rhenium is discussed by Geilmann and Bode.g6e97 They do notrecommend precipitation in alkaline solution as the sulphide Re2S7, as this ismarkedly soluble, but distil from 80% sulphuric acid at 200" in a stream ofhydrogen chloride ; 97--98y0 of the rhenium is removed in 1* hours ; Re207is itself volatile, but more slowly. The same authors 98 have recommendedprecipitation of Re2S7 from dilute sulphuric and hydrochloric acid solutionwith thiosulphate : nitrates interfere. Tribalat 99 determined the distri-bu tion coefficient between water and chloroform of tetraphenylphosphoniumper-rhenate, and showed that traces of rhenium can be isolated.Phosphonium or arsonium chloride serves as a reagent. Gob! is separatedfrom copper, silver, and platinum by means of morpholine oxalate,lW whichreduces it to metal in slightly acid solution.The precipitate of platinumsulphide is shown lol to approximate in composition to PtS, if excess ofhydrogen sulphide is used, and it is more or less contaminated by sodium86 Jean, AnaZyt. Chim. Acta, 1950, 4, 360.87 Willard and Sheldon, AnaZyt. Chem., 1950, 22, 1162.88 Watters and Kolthoff, ibid., p. 1467.90 Norwitz, ibid., p.551.B2 OeSteTT. Chm. Ztg., 1950, 51, 1.9' Kulm, 2. anal. Chem., 1950, 130, 210.g5 Ovenston and Rees, Analyst, 1950, 75, 204.96 Geilmann and Bode, 2. anal. Chem., 1950,130, 320.97 Idem, ibid., p. 323.99 Analyt. Chim. Acta, 1950, 4, 228.89 Analyst, 1950, 75, 156.Dl Idem, 2. anal. Chem., 1950,131, 266.D3 Analyt. Chem., 1950, 22, 328.98 Idem, ibid., p. 232.loo Malorvan, Mikrochem., 1950, 76, 104.101 Jackson and Beamish, AnaZyt. Chem., 1950, 22, 813WILSON : CHEMICAL METHODS (INORGANIC). 381chloride, if present. For bringing iridosmine into solution, roasting of thesample mixed with sodium chloride in an atmosphere of chlorine isrecommended; lo2 provision must be made to trap any volatile compounds.For the precipitation of iridium, hydrolysis of the bromate is preferred.Osmium is determined colorimetricdly with thiourea, and transmission datafor the stable rose-red solution are given; lo3 of the platinum metals onlypalladium and ruthenium interfere in moderate concentration.Rutheniumcan be photometrically determined at 465 mp. in the red solution obtained byfusion with potassium nitrate and hydroxide; Beer's law is obeyed, andother platinum metals do not interfere.lo4 Ryan recommends Z-mercapto-4 : 5-dimethylthiazole for the colorimetric determination of rhdium, theoptical density being measured a t 430 mp.lo5 Platinum and gold areprecipitated by the reagent, palladium is removed by glyoxime, and iridiuminterferes somewhat. Ryan also discusses 2-mercaptobenzoxazole lo5 as acolorimetric reagent for rhodium in the presence of iridium, but platinum andpalladium interfere somewhat.Ayres and Young 106 have made spectro-photometric studies of the blue complexes of ruthenium with thiourea andwith dithio-oxamide ; spectral transmission curves, rate of colour develop-ment, and other particulars are given. Of the other platinum metals,osmium and palladium interfere with the thiourea method, and osmium withthe dithio-oxamide procedure.The precipitation of the cerium group of rare earths by sodium sulphatecan be extended by salting out. Brine is of great value in fractionatingmiddle (gadolinium) and heavy (ytterbium) rare earths.lo7 Followingwork lo8 on the stability of complexes of titanium, zirconium, and thorium,oxalate, citrate, and tartrate complexes of tantalum and niobium arediscussed.Tridot presents a thorough study on the precipitation of uranium fromaqueous solutions by alkaline hydroxides and sulphides ; besides a reviewof past work, some new determinations of the solubilities of uranium andthorium nitrates have been published,lll and the influence of bicarbonateon the colorimetric determination of uranium is shown to be negligible a t awave-length of 445 mp.l12 Hecht and Gerhold 113 describe the determinationof uranium in phosphates by removal of the phosphate with molybdate,evaporation almost to dryness to remove most of the MOO,, and extractionof uranyl nitrate from the filtrate with ether.The determination of brominein brines is fully described by Haslam and who also give data onthe bromine content of salt deposits and in-shore waters.The analysis ofbromine is also described.l15 For the potentiometric determination oflo2 Hill and Beamish, Analyt. Chew&., 1950,22,574. lo3 Ayres and Wells, ibid., p. 317.lo4 Marshall and Rickard, ibid., p. 795. lo5 Ryan, ibid., p. 599.lo6 fbid., pp. 1277, 1281. lo' Marsh, Nature, 1949,163,998.lo* Haissinsky and Jeny-Isong, Analyt. Chim. Acta, 1949, 3, 422.lo@ Idem, ibid., 1950, 4, 329. I1O Ann. Chim., 1950, 5, 358.ll1 Templeton and Hall, Canadian J . Res., 1950, B, 28, 166.112 Scott, Anulyat, 1950, 75, 100. 113 Mikrochem., 1950, 35, 359.11' Analyet, 1950, 75, 343. 115 Haslam, ibid., p. 371.Resistance to hydrolysis decreases in the order given.lo382 ANALYTICAL CHEMISTRY.chlorine and bromine in presence of one another, an improved form ofMuller's retarded auxiliary electrode system 116 is used.The determinationof rubidium in carnallite is described by D'Ans.117 After precipitation asperchlorate along with potassium chlorate, the rubidium content is calculatedfrom the ratio of chlorine to metals, after reduction of the perchlorate tochloride. Mercurous salts can be titrated with potassium mercurithio-cyanate, and mercurous and mercuric salts can be determined in the samesolution.lls I n the absence of other metals, cadmium can be titratedpotentiometrically with sodium sulphide solution in presence of a protectivecoll0id.1~~Analysis of Metsls.-Th&y l20 determines tin in aluminium alloys bydissolution in sodium hydroxide, followed by evaporation with nitric andperchloric acid, to render stannic oxide insoluble.The residue is dissolvedin sulphuric acid, and tin determined electrolytically, or by turbidimetrywith cupferron as reagent. Por determination of silicon in aluminium,following amalgamation attack with dilute hydrochloric .acid rapidly dissolvesaluminium and leaves silicon in solution as a colloid. The solution isunstable and silicon rapidly flocculates. It is filtered off, and ignited tosilica.121 Aluminium in zinc is determined by measuring the colour which itproduces a t pH 5.8 with solochrome-cyanine. The results quoted showsatisfactory accuracy.122 Traces of lead in zinc can be determined colori-metrically as colloidal sulphide, since in a strongly ammoniacal solution smallquantities of hydrogen sulphide do not precipitate zinc sulphide, but leadsulphide is readily formed.123 Short determines aluminium in iron byremoving most of the iron as trichloride with ether, and the remainder byprecipitation with cupferron and extraction with chloroform from a solutionof pH -0.2 ; aluminium is finally determined oolorimetrically.Chromiumin steel can be determined by direct measurement of the green colour of itssolution in sulphuric and phosphoric acids.12s Bacon 126 discusses thephotometric determination of phosphorus in low-alloy steels. A new systemof absorptiometric analysis for steel 12' is analogous to the " internal-standard " technique of emission spectroscopy.On various aliquots of thesample solution, optical-density measurements are made for the variouscomponents and for iron, after adding appropriate reagents. The com-ponents are determined with reference to 100 parts of iron and, the iron beingdetermined, can be calculated to percentages. By this internal-standardtechnique errors due to varying cell thicknesses and apparent deviationsfrom Beer's law due to non-monochromatic or stray light are eliminated.116 Muller, 2. physikal. Chem., 1928, 135, 102.118 Burriel-Marti and Lucena-Conde, Analyt. Chim. Actu, 1950, 4, 344.f19 Silver, Kethely, and Kriety, ibid., p. 389.121 Berthier, Bull. SOC. chim., 1950, 17, 363.122 Pollak and Pellowe, Mebllurgia, 1950, 41, 281.Hahn, Analyt.Chim. Acta, 1950, 4, 453.12s Vredenburg and Sackter, Cunad. Chem., 1950, 34, 119.146 Analyst, 1950, 75, 321.127 Berger, Pirotte, Muylle, and Juliard, Bull. SOC. chim. Belge, 1960, 59, 465.117 Angew. Chem., 1950, 02, 118.lZo Chim. unalyt., 1950, 32, 19.lZ4 Analyst, 1950, 76, 420WILSON : CHEMICAL METHODS (INORGANIC). 383The determination of very small amounts of carbon in steels engages attention.The first step is combustion; the carbon dioxide after absorption in barytasolution may be estimated turbidimetrically 12* or by the change in electricalconductivity of the solution.12Q Wells, however,130 after drying the exitgases from the tube furnace and removing sulphur dioxide, condenses thecarbon dioxide in a trap cooled by liquid oxygen, then transfers the carbondioxide to a McLeod gauge measuring system.A determination can becompleted in 20 minutes. Beeghly 131 determines aluminium nitride insteels after solution of the iron by bromine in methyl acetate; aluminiumnitride is unattacked, and nitrogen is determined on the insoluble matter.Two reports 1329 133 have been issued, by the " Methods of Analysis Committeeof the British Iron and Steel Research Association," on the determination oftin in highly alloyed steels and on the determination of silicon; referencemust be made to the original for details of these referee methods. Oxygenin chromium, after annealing a t 800" in vacuo, is present as chromic oxide,and is insoluble in hydrochloric acid.13* Oxygen in titanium metal isdetermined by heating in vacuo in a special apparatus with powdered graphiteand metallic tin to 1900".Induction heating is used, and the evolved gasesare collected and ana1y~ed.l~~ Oxygen (present as oxide) in metallic sodiumis determined by dissolving the metal in mercury in a special apparatus,leaving the oxide as an insoluble residue which can be dissolved andtitrated.136 Silver in silver solder, after solution in acid, is reduced to metalwith ascorbic acid; 137 the ascorbic acid in the filtrate does not interferewith the determination of cadmium, copper, zinc, etc. Minute traces ofmercury in copper alloys are determined by dissolving the alloy in dilutesulphuric acid with the aid of peroxide and distilling off the mercury (chloridesmust be absent) ; it distils in steam, is collected in permanganate-sulphuricacid solution, and determined by extractive titration with dithizone.la8Minerals a d Rocks.--In the analysis of sedimentary rocks the ignitedsample is dissolved in hydrochloric acid and treated with gelatin to removesilicon, then on aliquots of the solution Fe, Ni, Mn, P, V, and Ti are determined~olorimetrically.~3~ Koritnig l40 fuses the rock with sodium hydroxide,removes silicon, alumina, etc., with ammonium carbonate solution, and thenuses Steiger's method for determination of 0*001--0*1 yo of fluorine.Biffen 141determines alkalis in refractory materials by the flame photometer after aLawrence Smith fusion, with added lithium as internal standard. Resultsare as accurate as by the classical method, and quicker.Shell 142 says that128 Agassaut and Andrieux, Bull. SOC. chim., 1950,17, 253.120 Gardner, Rowland, and Thomas, Analyst, 1950, 75, 173.130 J . Iron Steel Inst., 1950, 166, 113.132 Anon., J . IronSteelInst., 1950,165,190.134 Short, Analyst, 1950, 75, 335.136 Yepkowitz and Judd, ibid., p. 1283.138 Miller and Wachter, Analyt. Chem., 1950, 22, 1316.la0 Blumer and Erlenmeyer, Helv. Chim. Actu, 1950, 38, 45.140 2. anal. Chem., 1950, 131, 1.141 Analyt. Chem., 1950, 22, 1014.131 Analyt. Chem., 1949, 21, 1513.133 Anon., ibid., p. 430.135 Walter, Analyt. Chem., 1950,22,297.lS7 Goldberg, Metallurgiu, 1950,41, 174.142 Ibid., p. 326384 ANALYTICAL CHEMISTRY.platinum or platinum-rhodium crucibles should not be used for fusingsilicates, etc., with borax and sodium carbonates, before determination oftraces of iron, as they retain iron.Silver crucibles are recommended.Mott 143 prescribes the concentration of reagents, etc., to be used in therapid determination of various forms of sulphur in coals. Allen andBeamish 144 discuss fire assay for rhodium. Losses on the fire assay are notconstant, and can be minimised by reworking the slags. Cupellation is liablet o lead to losses, and the lead buttons must be parted by wet methods.Removal of lead as sulphate leads to losses, but thiobarbituric acid precipitatesrhodium in presence of lead, or perchloric acid may be used to dissolve thelead. I n determination of uranium in rocks 145 the final measurement ismade after fusion with sodium fluoride and comparison of fluorescence inultra-violet light with standards.After removal of silica the uranium iscollected in Group 111, and separated by ammonium hydrogen carbonatesolution. Feherand Heuer 146 point out that 30% hydrogen peroxide has advantages overmany oxidising reagents since excess can be completely destroyed by boilingand no foreign ions are introduced; pH and temperature control areimportant. Zinc blende, for example, can be completely oxidised by per-hydrol, a little saturated oxalic acid solution being added as well.Fertilizer Analysis.-Hanson 14' obtains excellent results in the rapidroutine determination of phosphoric oxide by the stable yellow colourproduced with molybdic and vanadic acid, measuring the extinction a t470 mp.Epps 148 applies the same method to citrate-soluble phosphoricoxide. Frey 149 discusses the precipitation of ammonium phosphomolybdate,but his interesting paper has few numerical results. Iodine in phosphaterock can be determined by distilling with acid, and collecting the distillatein bromine water; all the iodine is converted into iodate and there are nointerferences : Morocco rock contains 8-104 parts per mi1lion.l" Potassiumin fertilisers is quickly and conveniently determined by flame photometry.151Hamence l52 measures the relative rates of nitrification in soils of manureswhich contain organic nitrogen. A standard soil containing added calciumcarbonate is used; the samples are compared against dried blood and thereal value of nitrogenous wastes can be assessed.Water Analysis.-The application of disodium ethylenediaminetetra-acetate to the determination of hardness is the most interesting advance inrecent years.Schwarzenbach first applied it by determining the pH changeon reaction. Later it was shown that a t a pH -10 certain dyes (e.g.,solochrom black WDFA) would act as indicators, and calcium and magnesiumcould be titrated by a dilute solution of the reagent, as excess is indicatedThe uranium content of a large number of rocks is given.lP3 Fuel, 1950, 29, 53.145 Erlenmeyer, Oppliger, Stier, and Blumer, Helv. Chim. Acta, 1950, 33, 25.146 Angew. Chem., 1950, 62, 162.148 Analyt. Chem., 1950, 22, 1062.l50 Kahane and Rasch, ibid., p.147.151 Brearly, Chem. T~ades J., 1950, 137, 724.114 Analyt. Chem., 1950, 22, 451.147 J.Soc. Food Agric., 1950, 1, 172.14s Bull. SOC. chim., 1950, 17, 685.15= J . SOC. Food Agrk., 1950, 1, 92WILSON : CHEMICAL METRODS (INORGANIC). 385by a colour change from wine-red to blue; excellent summarising articleshave appeared.154 There is no agreement yet aa to the “ best ” method,and reasonably good results are obtained in a wide range of conditions;other bivalent metals must be masked, e.g., by sodium sulphide, and byslight modifications from <1 to 2000 p.p.m. of hardness can be determined.Excessive amounts of peaty matter must be removed by active charcoal.Calcium alone can be titrated by using purpurin as indicator. Variousuncouth trivial names have unfortunately already appeared for the reagent ;the reporter most strongly advocatea the initials E.D.T.A.if “ Schwarzen-bach’s reagent ” is thought to be beyond English chemists’ pens or lips.Traces of lithium in water are determined 155 by adding sodium hydroxideand carbonate until the pH is 12.5, allowing the precipitate to settle, andpercolating the solution down a column of alumina, pretreated with alkalisolution of the mme pH. Other alkalis pass through, but lithium remains,is eluted with a 2N-solution of hydrochloric acid in dioxan, and finallyconverted into lithium borate and titrated. Various elements (particularlyK, Na, and Ca) can be very rapidly determined by flame photometry : toovercome the effects of other ions, a “ radiation buffer ” solution is added.156The bleaching of the aluminium lake of eriochrom-cyanine is recommendedfor the determination of jiwrine; the usual components of potable waterdo not interfere; l5’ another paper 158 discusses interferences with thezirconium-alizarin43 method.Clark 169 recommends titrating chloride withmercuric nitrate solution, using diphenylcarbazone as indicator. Houghton 160reviews current practice in the determination of “ residual chlorine.”Gas An&sis.-Shepherd, in three interesting papers,161 discusses theaccuracy of analysis of carburetted water gas by mass spectrometer andchemical methods. Kilday 162 presents a thorough study of the evolutionof carbon monoxide when oxygen is absorbed in pyrogallol.A specialsolution in 50% potassium hydroxide solution gives off less carbon monoxidethan other reagents, and bubbler pipettes are superior to the older Orsatpipette. The evolution of carbon monoxide markedly increases with thevolume of oxygen absorbed. Reed l63 describes a portable constant-volumegas analysis apparatus, and Towler 164 recommends sulphuric acid activatedwith silver sulphate or chromium trioxide for determining “ unsats.” inilluminating gas : he points out that most reagents for “unsats.” alsodissolve carbon monoxide.153 Schwarzenbach et al., Helv. Chim. Ada, 1949, 32, 839, 1046, 1175, 1324, 1484,1543, 1682 ; Biedermann and Schwarzenbach, Chimia, 1948, 2, 56.154 Connors, J . Amer. Water WorksAssoc., 1950,42,34; Diehl,Goetz,andHach,ibid.,p.40 ; Betz and Noll, ibid., p. 49 ; Heald, Coates, and Edwards, I d . Chem., 1950,26,428.lS5 Balkzo and Sinabell, Mikrochem., 1950, 24? 178.156 West, Folse, and Montgomery, Analyt. Chern., 1950, 22, 667.15’ Thinn, ibid., p. 918.168 Taras, ASCO, and Garnell, J . Amer. Water Works Assoc., 1950, 42, 583.150 Analyt. Chem., 1950, 22, 553.161 Shepherd,J.Ree. Nat.Bur.Stund., 1960,44,509; Analyt.Chern., 1950,22,881,885.laa J.Re8. Nat. Bur.Stan&., 1950,45,43. le4 Ibid.,p. 159.Analyst, 1950, 75, 180.163 Fuel, 1950,29, 166.REP.-VOL . XLVII . 386 ANALYTICAL CHEMISTRY.An apparatus for determining nitrous oxide on samples as small as0=2--0*4 ml. by reduction with hydrogen over a heated filament isdescribed.ls6 Another micro-apparatus (for binary mixtures and volumesas small as 10 mm.3) is described.ls6 I n the determination of sulphurdioxide and trioxide in flue gases, errors arise through catalytic oxidationof this oxide to the trioxide by traces of copper, when the gases are absorbedin O*B~-sodium hydroxide solution containing benzyl alcohol.Satisfactoryresults are achieved if a little benzaldehyde is added, and, just beforeabsorption is started, 30 mg. of p-aminophenol hydroch10ride.l~~Toxic GCMS a d Htu~ds.-A new procedure for estimating the carbonmonoxide content in air is given by Griffon and Capus,16* who measure thetime which elapses before a grey spot appears when air is drawn through afilter paper impregnated with palladous chloride. For nitrogen peroxide inair a method is based 169 on the absorption of dinitrogen tetroxide, but notnitric oxide, on silica gel.Fluorine in the atmosphere (<40 p.p.m.) isestimated 170 by aspiration through a paper impregnated with zirconiump-dimethylaminoazobenzenearsonate, wetted with hydrochloric acid justbefore the determination : the brown or yellow zirconium compound isdecomposed, and the red free acid liberated ; the amount of fluorine presentis deduced from the volume which gives a pink stain. At 2 p.p.m. the erroris &O-2 p.p.m., but increases a t higher concentrations.An apparatus which continuously records the sulphur dioxide content ofair in the range 0-01-1 p.p.m. is described by Katz; 171 the bleaching ofvery dilute starch-iodine solution is measured photoelectrically.It has beenfound that, if air is drawn through a filter-paper disc at a speed of 60 c. ft./hr.,from 90 to 97% of any sulphuric acid present is retained; it can then beextracted with water and titrate~I.l'~ Clark describes the testing of theatmosphere in theatres and cinemas.Recent Apparatus, etc.-An ingenious electrostatic precipitator is designedfor the continuous sampling of the atmosphere and the recording of theconcentration of air-borne electrolytes. The testing of the apparatus isdescribed ; its efficiency is about 85% a t 3 c. ft./min. and 100% a t 1 c. ft./min.Full details are given; the anode is a slowly rotating steinless-steel disc,which dips into a bath of water so that precipitated material is continuallywashed A portable small-scale Venturi scrubber washes very largevolumes of air with small volumes of water, and is efficient a t high rates;the total volume of circulating liquid is about 150le5 Burke, Mikrochem., 1950, 35, 135.166 Tompkins and Young, J .Sci. Inatr., 1950, 27, 224.16' Berk and Burdick, U.S. Bur. Mines, 1950, Rep. Invest. 4168.188 Chem. Abe., 1950, 44, 8823 (from Ann. Med. Legal Crim., 1950, 30, 187).168 Wade, Elkin, and Ruotob, Arch. Ind. Hyg. OCCU~. Med., 1950, 1, 81.1 7 0 Harold and Hurlburt, AnaZyt. Chem., 1949, 21, 1504.171 Ibid., 1950, 22, 1040.173 Mader, Hamming, and Baker, ibid., p. 1181.17* Schadt, Magill, Cadle, and Ney, Arch. I d . Hyg. Occup. Med., 1950, 1, 566.175 Magill, Rolston, McLeod, and Cadle, Analyt.Chem., 1950, 22, 1174.Analyst, 1950, 75, 525WILSON : CHEMICAL METHODS (INORGANIC). 387Haslam and Williamson 176 have devised an apparatus for the quantitativemeasurement and manipulation of liquids (e.g., bromine) of high vapourpressure. The filling of a pyknometer and subsequent quantitative transferof the liquid to a reagent solution are described.A vacuum fusion furnace for investigating the dissolved gas content ofmetals has a glass envelope, air-cooled; apart from this it is constructedof quartz, with a graphite crucible, and the sample is heated by induction.Wurzschmitt 178 describes a new type of bomb for combustions or fusionswith sodium peroxide. Rafter 179 states that fusion with sodium peroxidecan be safely and expeditiously carried out in platinum crucibles at atemperatdre of 340-540".Duval and his collaborators continue their researches with the Chevenardthermobalance into the drying and combustion of precipitates.Duvallmdescribes the balance with which changes in the weight of an object underincreasing temperature may be recorded, and mentions his previous researchesinto numerous gravimetric methods 181 which show the exact temperaturesa t which precipitates become constant in weight. He now examines thecombustion of filter-paper, the drying of asbestos, and the behaviour ofplatinum, which is shown to gain in weight owing to oxidation above 538",but to return to its original weight at 800". Filters of glass fibres remainconstant to 520". With his collaborators he has described the behaviour onheating of various precipitates of phosphates, arsenates, " oxinates,' ' 182silver salts,l= cadmium,lB4 gadolinium,18s titanium,ls6 germanium,l87 tin,188lead,lB9 molybdenum,190 vanadium, lgl and antimony.lQ2 The thermalbehaviour of beryllium oxide is described by Dupuis.lg3Accuracy of Sampling and Analysis.-Scheur and Smith lg4 discuss theaccuracy of spectrographic and chemical methods of analysis of alloys.Onplotting the coefficient of variation against the amount present, for allelements curves of similar characters are obtained for chemical results, adifferent family of curves being given by spectrographic results. The latterare the more accurate when less than 0.1 yo of an element is present. Furtherdata are quoted by van Someron.lg5 Riley,lB6 in an account of the routinespectrographic analysis of cast iron, includes a table of standard deviationsfor Si, Mn, Ni, Cr, Cu, and Mo, giving figures founded upon several years'observations.The accuracy of sampling and analysing fertilisers and feeds1'16 Analyst, 1950, 75, 383.17' Guldner and Beach, Anulyt. Chem., 1950, 22, 366.178 Chem.-Ztg., 1950, 74, 356.180 Mikrochem., 1950, 35, 242.l a 1 Analyt. Chim. Acta, 1947, 1, 408; 1948, 2, 92, 103; 1950, 4, 159.lS2 Duval and Dupuis, ibid., p. 256. lE3 Duval and Marin, ibid., p. 393.Duval, ibid., p. 190. lEs Duval and Dupuis, ibid., 1949, 3, 438.lE6 Idem, ibid., 1950, 4, 180. 1 8 7 Idem, ibid., p. 186.lE8 Idem, ibid., p. 201. lag Duval, ibid., p.159.lQo Duval and Dupuis, ibid., p. 173. lgl Duval and Morette, ibid., p. 490.lQ2 Duval and Morandat, ibid., p. 494.Compt. rend., 1950, 230, 937. lQ4 Metallurgia, 1949, 41, 44.Io5 Ibid., 1950, 42, 52. l o 6 Spectrochim. Acta, 1950, 4, 93.179 Analyst, 1950, 75, 485388 ANALYTICAL CHEMISTRY.has been investigated.lg7 It is stated that with normal fertilisers it isunnecessary to sample more than 20 bags however large the consignment.On a consignment of 1000 bags, the standard deviation (for P,O, analysis)on a 20-bag sample was 0-11%, on a 60-bag sample O*OS%, and by samplingevery bag was 0.06%. In the ordinary case it makes no difference howwidely spaced the samples are. The importance and utility of automaticsampling is discussed by Visman.lg8 The need for large numbers ofincrements of a minimal weight and the calculation of attainable accuracy inadvance are discussed, and an automatic sampler (for coal, from conveyerbelts) is described.H. N.W.3. CHEMICAL I16ETHODS (ORGANIC).This section comprises the reports on ultimate organic microanalysis andon functional organic analysis, and although convention has often relegatedmicroanalysis to the end of comprehensive reviews it has been consideredmore logical than this report should deal with the determination of elementsfirst. The published papers on functional analysis necessarily cover a wideand scattered field, and a glance at the list of references will show that avery high proportion of the work is of American origin. It is, in fact,apparent that in this country and in Europe, comparatively little attentionis being given to functional organic analysis. Considerable impetus has, ofcourse, been given to the elucidation of complex problems by the elegantpartition chromatographic technique developed by Martin and his co-workers at Leeds in 1944, and as a result considerable activity has beennoticeable in recent years in the fields of protein and sugar analysis. Paperson these topics have been rather less numerous in 1950 than in former years,and those which have appeared have been logically included in the sectionon chromatography.Ultimate Organic Microanalysis.Several recent reviews cover the microchemical field up to the end of1949.Collaborative results for various methods for carbon and hydrogen,and for Kjeldahl and Dumas methods for nitrogen, have been reported.2Specifications have been recommended by the American Chemical Societyfor the standardisation of microchemical apparatus and by the BritishStandards In~titution.~ The numerous papers on microchemistry read atthe Graz Congress in July 1950 5 are due to appear in Mikrochemie (formerlyMikrochimica Acta).Specially noteworthy is the fact that British Carbon and Hydrogen.lo7 Miles and Quackenbuah, J .A88. Off. Agric. Chem., 1950, 33, 424.lsS Fuel, 1950, 29, 101.1 Willits, Analyt. Chem., 1949, 21, 132; Willits and Ogg, ibid., 1950, 22, 268;Kahane, Bull. SOC. chim., 1950, 17, D 1; Ingram, Ann. Reports, 1949, 46, 280.Willits and Ogg, J .Assoc. 08. Agric. Chem., 1949, 32, 561.Steyermark et al., Analyt. Chem., 1949, 21, 1555.British Standard 1428, Parts A1 and D3, 1950. Besterr. Chem.-Ztg., 1950,51,94STAGG : CHEMICAL METHODS (ORGANIC). 389practice has not followed the German lead and the American trend towardsuse of automatic combustion techniques ; 7 Unterzaucher 8 has now developedan automatic procedure based on the method for oxygen : the combustionproducts traverse successive packings of copper oxide, copper, bariumchloride to remove water, and carbon to reduce carbon dioxide to carbonmonoxide, which is then determined by reaction with iodine pentoxide.Most recent publications concern the rapid combustion method with anunpacked tube, following the work by Belcher and Sp~oner,~ Co1son,l0 andIngram.11 Belcher and Ingram 12 have designed a new apparatus in whichthe combustion zone consists of a vertical double-surface chamber heatedby a special type of split furnace.The purification system has been con-structed to allow replacements to be readily effected. These authors l3have also reported a study of absorbents for nitrogen oxides, and selectedmanganese dioxide as being the most suitable ; in slow-combustion proce-dures, special care must be taken to avoid condensation of moisture in thebeak of the combustion tube or in the entrance to the water absorption tube;in rapid combustion methods particularly, sufficient time (or space in thetrain) must be allowed for complete formation of nitrogen peroxide fromnitric oxide and oxygen.Kirsten l4 has given a detailed account of a new rapid technique whichinvolves thermal cracking by heating to 1000" in a platinum capsule, andoxidation in a quartz combustion tube containing an inner nickel sleeve.The sample, in the capsule, is pushed into the furnace by means of anautomatically-travelling electromagnet ; halogens and sulphur oxides areremoved by silver in a nickel roll at goo", and nitrogen peroxide is retainedin a special sulphuric acid-chromic acid absorber.Among the manydetailed points considered are the design of the absorption tubes, and theneed for avoiding condensation of water a t the entrance to the waterabsorption tube (cf. Ingram). Naughton and Frodyma l5 have described amethod involving combustion of the sample in a modified Pregl apparatus,collection of the resulting water vapour and carbon dioxide in a solid carbondioxide trap and a liquid-air trap, respectively, and, after pumping out theexcess of oxygen, determination of the water vapour and carbon dioxidemanometrically at constant volume.The automatic combustion procedure for the Dumas method,16which is widely used in Germany and has been recommended by Bussmann,"has now been adapted by Manganey l* so that controlled combustions canbe done without recharging the tube after each test and without altering theBelcher and Phillips, B.I.O.S.Report No. 1606.Steyermark, I d . Eng. Chem. Anal., 1945, 17, 523; Clark and Stillson, Anulyt.Chem. Ing.-Tech., 1950, 22, 39.J . , 1943, 313; Fuel, 1941, 20, 130; Ind.Ghem., 1943, 19, 653.Nitrogen.Chem., 1947, 19, 423; Fischer, ibid., 1949, 21, 827.lo Analy8t, 1948,73,541. l1 Ibid.,p. 648. la Analyt. C1.h. Acta, 1950, 4, 118.l3 Idem, ibid., p. 401. Mikrochem., 1960, 56, 217.l5 Analyt. Chern., 1950, 22, 711. 16 Zimmermann, Mikrochem., 1943,31,42.l7 Helv. Chim. Ada, 1949, 32, 995. 1e Bull. SOC. chim., 1960, 17, 74390 ANALYTICAL CHEMISTRY.temperature. Kirsten l9 has.recommended a nickel-nickel oxide permanent,filling a t 1000" for use with all types of compound, with a quartz apparatuswhich allows a backward sweep with cakbon dioxide to save time and tolengthen the life of the filling. Modifications have been introduced 20 whichgive greater flexibility or strength; by increasing the size of the part of thetube in the hot furnace, more analyses can be carried out on one filling, andvery small amounts of nitrogen (0.03-0*007 yo) can be determined becauseof the greater oxidative capacity, which allows use of larger samples (-50 mg.).A rapid combustion method has been described by Colson; 21 thecombustion is carried out in a slow stream of carbon dioxide, and the nitrogenproduced is then swept out by a much faster stream of carbon dioxide thanis used in the normal Pregl-Dumas method; complete reduction of thenitrogen oxides during the combustion is effected by a four-fold increase inthe amount of hot metallic copper.I n a review of the manifold variations which have been proposed uponthe original Kjeldahl method, Kirk 22 emphasised the difiiculty of choosing aset of digestion conditions which can be applied to all nitrogenous compounds,and stressed particularly the circumspection which must be used in employingoxidising agents for speeding digestion.In 1949 Willitts, Coe, and Ogg,Z3in a study of the digestion of nicotinic acid, demonstrated the danger of lossof nitrogen when selenium catalysis (the mechanism of which is discussed bySchwab and Schwab-Agallidis 24) is employed in the digestion of refractorysubstances, and recommended mercuric oxide in conjunction with fixedamounts of sulphuric acid and potassium sulphate. Subsequent collaborativestudy of this procedure gave disappointing results in some cases, and areason for this has been found 25 in the lower temperature of digestion whichis obtained if the liquid is not boiled vigorously.In studying the digestion of compounds which require previous reduction,Secor, Long, Kilpatrick, and White 26 have shown that volatility of nitrogenis increased by pretreatment with hydriodic acid.Prolonged digestion anda high potassium aulphate-sulphuric acid ratio are especially dangerous inthese circumstances.For absorption of ammonia during distillation, boric acid continues tobe popular, although it is clear that care is necessary if loss of ammonia is tobe negligible in micro- and semimicro-work,27 and Silverstein and Perthell 28state that losses are reduced if the boric acid is contained in a Goessmantrap.29 Blom and Schwarz30 claim that if the ammonia is absorbed innickel ammonium sulphate sharper end-points are obtained in titration withacid.l9 Analyt.Chem., 1947, 19, 925.z1 Analyst, 1950, 75, 264.23 J . Assoc. Ofl. Agric. Chem., 1949, 32, 118.26 Ogg and Willits, J . Aasoc. Off. Agric. Chem., 1950, 33, 100.28 Analyt. Chern., 1950, 23, 949.as Massachmetts Agric. Exptl. Sta., Bulletin 54, 1898.so Acta Chem. Scand., 1949, 8, 1439.*O Ibid., 1950, 22, 358.29 Analyt. Chem., 1950, 23, 354.24 NatUTWiSS., 1949, 56, 254.Ibid., p. 872. 07 Machemer and McNabb, Anal. Chim. Acta, 1949, 3, 428STAGG : CHEMICAL METHODS (ORGANIC). 391Habgens. In the potentiometric titration of halogens by silver (followinga Parr-bomb fusion for decomposing the organic material) LBvy31 hasopposed the e.m.f.of the cell to that of a potentiometer standardised to thee.m.f. of a reference electrode containing the same quantity of sodiumsulphate and equivalent quantities of halogen and silver ions. White andSecor 32 have determined iodine by Carius digestion with nitric acid-mercuricnitrate mixture, followed by conversion into iodate and subsequent titrationof the iodine liberated on addition of iodide; a similar method has beendevelopedRickson34 has reported that low results obtained by thorium nitratetitration after separation of fluorine from interfering impurities by distillation,may be caused by some of the fluorine being present as the fluorosilicate ion,Sip,", which does not form an un-ionised compound with thorium. Thiserror can be avoided by carrying out the titration in a 50% alcoholic systembuffered a t pH 5.3, with gallocyanine as indicator.A considerable numberof different indicators for the thorium nitrate titration have been studied byWillard and H0rt0n,3~ who also report 36 a procedure for photofluorometrictitration of fluoride, using quercitin as fluorescent indicator.Neudorffer 37 has developed a semimicro-method for the simultaneousdetermination of sulphur and fluorine, in which the vapour of the compoundis mixed with hydrogen and burned in an atmosphere of oxygen; theproducts of combustion were determined by conventional means.Neudorffer's method for sulphur and fluorine simultaneouslyhas been mentioned above. Zimmermann 38 has described a simplifiedversion of his method ; 39 this involves decomposition by potassium,distillation of the hydrogen sulphide into cadmium acetate, and iodometrictitration.In a similar method40 the organic compound was decomposedby heating it with oxalic acid and metallic calcium. Kirsten 41 has given apreliminary report on a new method for sulphur, based on combustion ofthe sample in oxygen, reduction in an oxy-hydrogen flame a t 1100" tohydrogen sulphide, absorption in strong alkali, and determination oxidi-metrically with hypochlorite.Maybott and Lewis42 have compared the ter Meulen, Liebig,and Unterzaucher methods for determining oxygen in organic compoundsand have concluded that the last 43 is the most reliable. Oxidation catalystsused in preference to iodine pentoxide have included mercuric oxide,44 andsilica impregnated with iodine pentoxide and sulphuric acid.46 Harris,for bromine in organic compounds.SuZphur.Oxygen.31 Compi?.rend., 1950, 230, 1958.33 White and Kilpatrick, ibid:, p. 1049.3 5 Analyt. Chem., 1950, 22, 1190.Compt. rend., 1950, 230, 750.38 Ibid., 1943, 31, 15; 1947, 33, 122.4O Chernyi and Podoinikova, Biokhim., 1950, 15, 134.45 Ber., 1940, 73, 391 ; Aluise et al., Analyt. Chem., 1947, 19, 347.44 Deinum and Schouten, Analyt. Chim. Acta, 1950, 4, 288.45 Beseet and Pokier, Bull. SOC. chim., 1949, 16, D, 539.3a Analyt. Chern., 1950, 22, 1047.34 Analyst, 1950, 15, 84.36 Ibid., p. 1194.38 Mikrochem., 1950, 35, 80.Mikrochem., 1950, 55, 174. 4x Analyt. Chem., 1950, 32, 1051392 ANALYTIUAL CHEMISTRY.Smith, and Mitchell 46 have described a recording thermal conductivitymethod for determination of carbon monoxide in the resultant helium-carbonmonoxide mixture.Functional Organic Analysis.Hydroxyl Group.-AZcohols and GZycob.A new application of thepowerful reagent lithium aluminium hydride is described by Lintner, Schleif,and Higu~hi.~' Previously, this substance had been employed as analternative to methylmagnesium iodide in Zerewitinow determinations ofactive hydr0gen.~8 I n this new method the reduction potential producedon a silver or platinum electrode is used to detect an added excess of thehydride during titration of saturated alcohols in tetrahydrofuran as solventand to indicate the end-point in back titration with standard alcoholsolution.Use of p-aminoazobenzene as a colorimetric indicator is alsoenvisaged. The recoveries quoted are from 1% to 5% higher than theory,but in a later note Higuchi49 states that if oxygen is not rigidly excludedfrom the titration vessel, some hydride will be lost by oxidation.Determination of small amounts of ethyl alcohol in the Conway diffusionunit, using potassium carbonate to expel the alcohol, and alkaline per-manganate to absorb it, is described by M ~ L e o d , ~ ~ and successful applicationof ceric sulphate oxidation to the determination of tetrahydrofurfurylalcohol is reported by Haslam and Ruddle.61 Middleton and Stuckey 52describe the application of a cloud-point technique to determination ofethylene and dipropylene glycol in propylene glycol.Water must first beremoved. The method is claimed to detect O*lyo of either impurity, but asethylene glycol raises and propylene glycol lowers the cloud point, thepresence of both simultaneously can only be detected by examination ofdistillation fractions. A rapid method of determining glycerol with anerror of -+2% in fermentation solutions containing 2-8 pap.m. of glycerol isgiven by Lambert and N e i ~ h . ~ ~Three advances in the selective determination of phenols byiodination are reported by Willard and Wooten : for determining o-substituted in p-substituted monohydric phenols, coloured iodoquinones areformed by treatment with iodine and alkali.54 The formation of thequinone is dependent on linkage through the free p-position, thus preventinginterference by phenols substituted in that position.Resorcinol isdetermined by iodination a t pH 5-0 a t which most phenols do not interfere ; 55o- and m-dihydric phenols interfere, but catechol may be prevented fromdoing so by removing it with lead acetate. o-.and m-Dihydric phenols aredetermined by iodinating a mixture of the two, and adding acetone. A bluecolour is formed whose intensity is proportional to the amount of the lesser46 Analyt. Chem., 1950, 22, 1297.4 8 Zaugg and Harrom, ibid., 1948, 20, 1026; Krynitsky, Johnson, and Carhart, J .Amer. Chem. SOC., 1948, 70, 486; Hochstein, ibid., 1949, 71, 305.49 Analyt. Chem., 1960, 22, 955.I1 Analyst, 1949, 74, 569.63 Canadian J . Res., 1950,28, B, 83.Phenols.4 7 Ibid., p.534..w J . Biol. Chem., 1949, 181, 323.5* Ibid., 1960,75, 406.54 Analyt. Chem., 1950,22,423. Ibid.,p. 585STAGQ : CHEMICAL ME’l’HODS (ORGANIC). 393c~mponent.~~ Of 25 phenols tested only o- and Ira-dihydric phenols gave thereaction.Attention has been given by Siggia, Hanna,and Kervenski to the problem of determining primary, secondary, andtertiary amines in mixtures containing all three, and a scheme is described 67which has given good resulfs with known mixtures of aniline, monomethyl-aniline, and dimethylaniline ; aniline and mono- and di-ethylaniline ; anda-naphthylamine and its mono- and di-ethyl derivatives. In this scheme,three determinations of total bases are made by titrating with hydrochloricacid in a solvent consisting of equal parts of ethylene glycol and isopropylalcohol to accentuate ionisation of the bases : the first titration is made onthe sample itself, the second on the sample after condensing it withbenzaldehyde to remove secondary amine, and the third on the sampleafter primary and secondary amines have been rendered neutral byacetylation.Primary, secondary, and tertiary bases can then be obtainedby calculation.The titration of organic bases in non-aqueous solvents has been extendedby Fritz 58 to the determination of heterocyclic bases by titration withperchloric acid in dioxan. Pyridine, 2 : 6-lutidine, 2 : 2’-dipyridyl, 1 : 10-phenanthroline, and brucine have been determined ; an important featureof the method appears to be the formation of a precipitate of base per-chlorate. The same author shows 59 that Bandel and Blumrich’s method oftitrating weak bases in anhydrous acetic acid with perchloric acid in aceticacid 6o can be employed when the bases are dissolved in less polar solventssuch as nitrobenzene, hydrocarbons, chlorobenzene, etc.Another methodof determining pyridine in presence of ammonium salts 61 involves distillationof a solution adjusted to pH 4.8; pyridine is completely volatile butammonia is not. Ballard describes two cdorimetric methods, one dependingon the diazo-reaction and the other on condensation with p-dimethylamino-benzaldehyde, for determining p-aminophenol in metol a2 and primaryamines occurring as impurities in succinylsulphathiazole, Carbarsone, andGlycarsamideAmides and Amino-acids. The reaction of a mixture of nitric andhydrochloric acids with amides and amino-acids to yield nitrogen andnitrous oxide 64 has been applied to the determination of a number of simpleamides and amino-carboxylic acids,65 and Kay and Mills 66 have shown, in are-investigation of Pope and Stevens’s copper ealt method 67 for amino-acids, that reproducibility is improved by washing the copper phosphate.free from phosphate with borax buffer.Moubasher, Sina, Awad, andAmino-~~p.-Amines.6 6 Analyt. Chem., 1950, 22, 670.m Idem, ibid., p. 1028.61 Ashmore and Thickens, Coke and Gas, 1949,11, 307.aa Anulyet, 1950, 75, 430.64 Renard, Bull. Acad. roy. Belg., Chase mi., 1946, 81, 219.6 5 Renard and MBdart, Bull.SOC. roy. Sci. Lidge, 1949, 18, 409;66 Anarlyt. Chem., 1980, 22, 760.Ibid., p. 1296. 5 8 Ibid., p. 578.ae Angew. Chem., 1941, 64, 374.J. Pharrn. Plaarmaeol., 1950, 2, 98.Renard andDeschamps, oaterr. Chern.-Ztg., 1950, 51, 112.Bhchem. J., 1939, 88, 1070394 ANALYTICAL CHEMISTRY,Othman 68 have based a method of determining amino-acids on the observ-ation that many of them yield their amino-nitrogen quantitatively ontreatment with perinaphthindanetrione hydrate or its nitro-derivative.The formation of non-ionic silver complexes affords the basis of a methodfor cysteine and ~ y s t i n e , ~ ~ and a specific colorimetric method for hydroxy-proline 70 depends on oxidation with sodium peroxide and condensing theoxidation products with p-dimethylaminobenzaldehyde to form an intensered colour. An extensive study of the stability of tryptophan duringalkaline hydrolysis of proteins has been carried out by Spies and chamber^,^'and conditions are described for hydrolysis a t temperatures up to 185"without destruction of tryptophan ; a fluorimetric method for determiningtryptophan 72 depends upon the formation of a fluorescent product onreaction with perchloric acid.Carbons1 Group.-Little advance has been made recently in the generalmethods for determining carbonyl groups.Wanka, Jurevek, and Holanek 73in reviewing the phenylhydrazine, hydroxylamine, and semicarbazidemethods conclude that they share a common disadvantage in the difficultyof obtaining a sharp end-point in the titration of the liberated acid.How-ever, Maltby and Primavasi 74 claim that an alcoholic solution of hydroxyl-amine hydrochloride can be used satisfactorily to determine many commonand unfamiliar carbonyl compounds, bromophenol- blue being used asindicator. Lieb and Schoniger 75 studied the phenylhydrazine method uponbenzoin and vanillin and found that condensation does not proceedquantitatively or uniformly. Useful data on the rate of oximation offourteen aryl alkyl ketones in pyridine-methanol solution are given bySuratt, Proffitt, and Lester.76Aldehydes are determined in the presence of ketones by oxidising thealdehyde to carboxylic acid with silver oxide and titrating with alkali,77 andaldehydes in the presence of carboxylic acids by oximation followed bytitration of the liberated hydrogen chloride to pH 2-50.78Determination of formaldehyde by reaction with ammonium chloride 79and of formaldehyde and acetaldehyde in admixture by formation of theSchiff colour first a t pH 0.7 and then a t pH 2.7 8o are described, and theapplicability of the well-known chromotropic acid method for formaldehydehas been extended by Bricker and Vail's observation *l that the complexcan be evaporated to dryness without destruction, the purple colour beingreadily developed by treating the residue with sulphuric acid.68 J .Biol. Chem., 1950, 184, 693.139 Kolthoff and Stricks, J . Amer. Chem. SOC., 1950, 72, 1952.' 0 Neuman and Logan, J . Bio2. Chem., 1950, 184, 299.7 1 Analyt. Chem., 1949, 21, 1249.72 Gordon and Mitchell, J .Biol. Chem., 1949, 180, 1065.7 3 Coll. Czech. Chem. Comm., 1949, 14, 162.7 b Mikrochem., 1950, 35, 407.7 7 Mitchell and Smith, Analyt. Chem., 1950, 22, 746.7 8 Idem, ibid., p. 750.79 Cassini, Ann. Chim. appl., 1949, 89, 600.74 Analyst, 1949, 74, 498.'13 J . Amer. Chem.Soc., 1950,72,1561.Velrsler, J . Anal. Chem. U.S.S.R., 1950,5,32. 81 A d y t . Chem., 1950, 22, 720STAGG : CHEMICAL METHODS (ORGANIC). 396Colorimetric methods have also been described for glycollic aldehyde, 82pyruvaldehyde,m and acet01.~~Carboxylic Acids.-Advances in the analysis of carboxylic acids havebeen confined to individual cases, no new techniques for the generaldetermining of the carboxyl group having been reported.Conditions for the determination of acetic acid present as an impurityin refined formic acid have been described by Arthur and Struthers; 86 theformic acid is oxidised to carbon dioxide with mercuric oxide and the residualacetic acid is titrated with alkali.For determining acetic anhydride inglacial acetic acid, Benson and Kitchen s6 add an excess of aniline, and aftercondensation with acetic anhydride has taken place the remaining aniline isdetermined colorimetrically with furfuraldehyde : it is stated that smallamounts of ethylidene diacetate, copper acetate, acetaldehyde, and water donot interfere, but the possibility of the acetic acid itself interfering is notdiscussed. Determination of minute amounts of acetic acid in the Conwaydiffusion unit is described by Conway and D~wney.~'Two procedures have been described for the determination of oxalicacid : one, due to Burrows,88 depends on the fading effect of oxalate upon thegreen iron complex of 8-hydroxy-7-iodoquinoline-5-sulphonic acid ( " ferron ")and is in effect a modification of Lange's method 89 using ferric thiocyanate.The other method depends on reduction of the oxalic acid with magnesiumand sulphuric acid to glycollic acid, which is determined by Eegriwe's test.Wohnlich 91 gives a method for determining lactic acid colorimetricallyafter reaction with a-naphthol in concentrated sulphuric acid, and Troupeand Kobeg2 analyse lactic acid-lactic ester mixtures by studying thehydrolysis rates of the components.In the field of long-chain fatty acids, work leading to the development ofa method for determining saturated acids in the presence of unsaturatedacids has been described by Fitelson and Weber; 93 this involves non-disruptive oxidation of the unsaturated acids with performic acid a t 70-75"to form hydroxy- and formoxy-acids, which can be separated from thesaturated acids by a process of extraction and chromatography.Hydroxamates of long-chain fatty acids have been determined byhydrolysis to the acid and hydroxylamine with standard hydrochloric acid,followed by titration of the excess mineral acid.Q4Nitro-group.-A novel method for gravimetric determination of nitro-groups, based on reduction to amino-groups with tin and hydrochloric acid,.82 Dische and Borenfreund, J .Biol. Chem., 1949,180, 1297.84 Forist and Speck, ibid., p. 902.Thornton and Speck, Analyt. Chem., 1950, 22, 899.Canadian J . Res., 1949, 27, 266.Biochem. J . , 1950, 47, iv.Lange, " Kolorimetrische Analyse," 1941.Pereira, &err. Chem.-Ztg., 1950, 51, 111.8b Ibid., 1949, 21, 1209.8 8 Analyst, 1950, 75, 80.91 2. phy8iOl. Chem., 1950, 286, 138.O3 J . Amer. Oil Chm. Soc., 1950, 27, 1.O4 Roe and Swern, Analyt. Chem., 1950, 22, 1160.O2 Analyt. Chern., 1950, 22, 646396 ANALYTICAL CHEMISTRY.has been described by Vanderzee and Edge11,95 who have shown that, ifatmospheric oxidation is prevented, and the evolution of gaseous hydrogenis minimised by controlling the acid concentration, the consumption of tin isproportional to the nitro-group.After reaction, the excess of tin is weighed.Recoveries within &0-5y0 of theory are reported for several simple aromaticnitro-compounds, though interference was experienced if iodo- or aldehydegroups were present.In a study of the reduction of nitroguanidine, nitroaminoguanidine, andnitrourea with titanous chloride, Zimmermann and Lieber 96 found that thenitroguanidines gave inconsistent results except in the presence of ferrousion; by adjusting the concentration of ferrous ion to an amount dependingon the particular nitramide, reduction equivalents of 6H per nitro-groupwere obtained with errors of the order of &2%. Nitrourea on the other handconsumed only 2H per nitro-group whether ferrous ions were present or not.Sugars.-Heidt and Southam 97 have established conditions for determin-ation of reducing sugars with cupritartrate reagent buffered to low pH, andstate that the pH should be controlled to 0.01 unit, the optimum valuebeing 8-70.Bevenue and Washauer 913 in studying the effect of clarificationand dealcoholation of extracts before determination of reducing sugars,found that in many cases these steps are unnecessary and in many others asimple carbon treatment is all that is required. Thomas, Melin, and Moore ggalso found that clarification with lead can be omitted from the procedurewhen determining sugars in many forage plants, and suggested that anyonewho has to perform large numbers of sugar determinations will be welladvised to ascertain whether this step is necessary.A new colorimetric method for determining reducing sugars loo dependson reduction of triphenyltetrazolium chloride to triphenylformazan inalkaline solution.The amount of the red insoluble formazan is proportionalto the reducing sugar present, and it can be dissolved in pyridine and acidto give a red colour suitable for absorptiometric measurement.A scheme for determining glucose, galactose, and rhamnose in mixturesobtained by hydrolysis of flavonol glycosides is described by Porter andFenske; lo1 this employs Schoorl's copper reduction before and afterselective destruction of (a) glucose and ( b ) glucose plus galactose by ferment-ation. Sedoheptulose (D-aho-D-fructoheptose) can be determined, accordingto Nordal and Klevstrand,l02 by extracting the blue-green colour obtainedwith orcinol and hydrochloric acid into amyl alcohol and measuring it ; anda new method for hexosamines, based on deamination with nitrous acid anddevelopment of the indole colour from the resulting hexose 2 : 5-anhydrides,has been described by Dische and Borenfreund.lo3 This test is claimed tobe more sensitive than the Elson-Morgan test with acetylacetone andO 5 Analyt.Chem., 1950, 22, 572.O 7 J . Amer. Chem. SOC., 1950, 72, 589.O' Ibid., p. 1151.J . Assoc. Off. Agrie. Chem., 1950,33, 122.100 Mattson and Jensen, ibid., 1950, 22, 182.lo3 J . Biol. Cilem., 1950, 184, 517,Anulyt. Ch.em., 1949, 21, 1363.lol J . Assoc. Ofl. Agric. Chem., 1949,82, 714.102 Analyt. Chim. Acta, 1950, 4, 411STAGG : CHEMICAL METHODS (ORGANIC).397p-dimethylaminobenzaldehyde lo* and may be of special interest in view ofthe interference experienced in the latter from sugars and amino-acids.lO5Unsaturation.-The advantages of catalysed hydrogenation as a means ofmeasuring unmturation have again been emphasised in papers dealing withthe technique of microhydrogenation ; lo6 the interference of side reactionsin halogenation methods has also received attention : Braae 10' has shownthat the mercury-catalysed addition of bromine to isolated double bondsproceeds so rapidly that direct titration can be used in conjunction with anelectrometric end-point, the oxidising influence of an excess of bromine beingthus eliminated; and Lee, Kolthoff, and Johnson lo* employ a graphicalmethod to differentiate between the true iodine chloride addition and theabsorption in excess of theory which occurs as a result of decomposition inolefins which are branched in the vicinity of the double bond.For the specific determination of terminal unsaturation, Bricker andRoberts lo9 have combined th6 Malaprade reaction (whereby the terminalunsaturated groups are converted by permanganate into 1 : 2-glycols andthese are split by periodic acid into formaldehyde and a higher aldehyde)with the chromotropic acid method for determining formaldehyde, while forterminal unsaturation in styrene derivatives, especially those containinghalogen in the ring, Marquardt and Luce 110 recommend mercuric acetate inmethanol as a reagent.In the acidimetric determination of acetylene derivatives by reactionwith silver nitrate and titration of the nitric acid formed, advantage isclaimed 111 for the use of sparingly soluble silver salts (in particular silverbenzoate) in place of silver nitrate : insoluble complexes are formed withmany acetylides which do not give a precipitate with silver nitrate.Whereinterference is likely to arise in argentometric procedures from the presenceof silver reductants, precipitants, or complexing agents, Hanna and Siggia 112recommend a procedure based on reaction with potassium mercuri-iodide.Compounds of Biological Signiflcance.--AZkaloids. The effect of am-monium salts on the gravimetric determination of nicotine by precipitationwith silicotungstic acid has been studied by Ogg, Willits, and Ricciuti,113who showed that ammonium and other inorganic salts delay the formationof the precipitate; attempts to remove the ammonia, failed but conditionswhich minimise the salt-effect were found.However, in a later communi-cation, Willits, Swain, and Connelly 114 give a method for determiningnicotine spectrophotometrically at 259 mp. which is not affected by am-monium salts. A method for determining nicotinic acid in nicotinamide 115depends for its success upon the establishment of conditions for benzylatinglo4 Biochem. J . , 1933, 27, 1825.l o 5 Horowitz, Ikawa, and Fling, Arch. Biochem., 1960, 26, 226.106 Ogg and Cooper, AnaEyt. Chem., 1949, 21, 1400; Mead and Howton, ibid., 1950,107 Ibid., 1949, 21, 1469. log Ibid., 1949, 21, 1331.110 Ibid., p.1194.lI2 Analyt. Chem., 1949, 21, 1469. llS Ibid., 1950, 22, 336114 Ibid., p. 430.22, 1204.lo8 Ibid., 1950, 22, 995.ll1 Marszak and Koulkes, Bull. SOC. chim., 1950, 17, 364.us Cuke, Mikrochem., 1950, 35, 20398 ANALYTICAL CHEMISTRY.the tertiary nitrogen of the amide without benzylating that of the acid,which can then be determined by the Konig reaction. Koszegi and Salgo ll6note the formation of precipitates between potassium mercurithiocyanateand a number of purine bases, alkaloids, and primary, secondary, tertiary,and quaternary amines, and describe the volumetric determination ofstrychnine nitrate and quinidine sulphate. Preliminary results of a quali-tative study of the chromatographic separation of morphine, codeine, andheroin from each other and from barbiturates are given by Stolman andStewart .l17Pyrimidines, Purines, and Urea Derivatives. Soodak, Pircio, andCerecedo 118 have extended the WheelerJohnson test for uracil andcytosine 119 to give quantitative results and give conditions whereby cytosinecan be removed quantitatively from mixtures of these two pyrimidines byadsorption on Decalso ; Holt and Mattson 120 have obtained quantitativeresults within 1-2% of theory for uracil, thiouracil, and related compoundscontaining the -CO*NH-CO- and -CO*NH*CS- groupings by the colorimetriccobalt method described by Dille and Koppanyi.l21 Spinks l Z 2 determines'' Antrycide " in biological fluids by extracting the intensely fluorescent eosinsalt into chloroform-butanol solution and measuring the fluorescence.Alloxan has been determined in commercial samples by condensing it with2-amino4 : 5-dimethyl-1 -D-ribitylaminobenzene to give riboflavin andmeasuring the flu0re~cence.l~~ Pedley 124 prefers neutral mercuric per-chlorate to silver nitrate as a precipitant for the gravimetric determinationof barbiturates.Haurowitz and Lisie 126 have applied Kitamura's observ-ation 126 that thiourea is oxidised to urea by alkaline hydrogen peroxide, tothe determination of thiourea obtained by the action of ammonium thio-cyanate on proteins.Methods have also been given for the determination of amidines,12'methylaminoacridine,128 P-naphthoxyethanol in blood,129 a-pyridyl-carbinol,l3O and sulph~namides.~~~Hanna and Siggia 132 determine chloroform and bromoform bythe carbylamine reaction with aniline and caustic alkali and claim thatrecoveries approach theory more closely than those obtained by simplehydrolysis with alcoholic potassium hydroxide.Brain and Helliwell,l33dealing with determination of trichloroethylene in blood, give improvedconditions for the quantitative production of the colour from its reactionHalides.1l6 2. anal. Chem., 1950, 130, 403.118 J . Biol. Chem., 1949, 181, 713.120 Analyt. Chem., 1949, 21, 1389.122 Biochem. J . , 1950, 47, 299.123 Tipson and Cretcher, Analyt. Chem., 1950, 22, 823.124 J . Pharm. Pharmacol., 1950,2, 39.12E J . Phurm. Soc. Japan, 1935, 55, 300.127 Trought, Ashton, and Baker, Analyst, 1950, 75, 437.ll8 Anderson and Lederer, ibid.,p.318.130 Wollich, Kuhris, and Price, Anulyt. Chem., 1949, 21, 1412.131 Schaefer and Wilde, 2. anal. Chem., 1950, 130, 396.182 Analyt. Chem., 1950, 22, 569.117 Analyst, 1949, 74, 536, 543.119 Ibid., 1945, 159, 333.lal J . Amer. Pharm. ASSOC., 1934, 23, 1079.la5 Analyt. Chim. Acta, 1950, 4, 43.12s Spinks, Biochem. J., 1950, 46, 178.lS5 Riochem. J . , 1949, 45, 75CROPPER : PHYSICAL AND PHYSICOCHEMICAL METHODS. 399with pyridine and alkali. The specific determination of the y-isomercontent of technical benzene hexachloride (hexachlorocyclohexane) continuesto attract interest ; Davidow and Woodward l3* have co-ordinated theobservations of a number of other workers in developing a method based onhydrolysis to trichlorobenzenes and meaaurement of optical density ah threewave-lengths in the ultra-violet, while Monnier, Roesgen, and Monnier 136find that only the y-isomer and heptachlorocyclohexane are reduced polaro-graphically, and that the latter compound is destroyed a t pH 13 whereasy-benzene hexachloride is stable.H.E. S.4. PHYSICAL AND PHYSICOCHEWICAL METHODS.provide excellentsummaries of progress, to late 1949, in almost all the subjects discussedbelow. A comprehensive review on the determination of organic function-ality by molecular spectra covers applications of ultra-violet, infra-red, andRaman spectroscopy, micro-wave spectroscopy, and mass spectrometry ;a review on the same theme by Lykken deals with electrometric methods.Limitations of space make it impossible to review the 1950 literature in suchan exhaustive manner; attention has been given to advances in principlesand general techniques, although some applications have been noted whenconcerning important products, new features in technique, or improvedways of eliminating difficulties. Subjects in which there have been onlyminor advances, or which are still a t an early stage (such as micro-wavespectroscopy, acoustical methods) have been left for consideration insubsequent Reports ; radioactivation analysis was reviewed in detail in lastyear's Report .4The division of subject matter has been made on conventional lines, withno attempt to follow the more logical but unfamiliar classification given bySerfass, Steinhardt, and S t r ~ n g .~Further efforts have beenmade to improve the stability of the light source in spectrochemical analysis.Rouse has described the use of an induction heater to evaporate the samplefrom a carbon cup, and excitation of the vapour by a spark, so that the twomain functions of a source (evaporation and excitation) are controlledseparately ; a d.c. arc source with an automatic ~ontroller,~ and an improvedintermittent arc generator for steel analysisThe direct-reading instrument constructed by Crosswhite for measuringrelative spectral intensities employed two photomultiplier tubes with anelectronic pen-and-ink ratio recorder ; the fixed phototube served as alS5 Analyt. Chim. Acta, 1950, 4, 309.The recent Annual Reviews in Analytical ChemistryOptical Methods.-Emission Spectroscopy.have been designed.134 J .Assoc. Off. Agric. Chem., 1949, 32, 751.1 Analyt. Chem., 1949, 21, 1-173; 19.50, 22, 1-126.Coggeshall, ibid., p. 381.Smales, Ann, Reporter, 1949, 46, 285.6 J . Opt. SOC. Amer., 1950, 40, 82.8 Marti, Spectrochim. Acta, 1950, 4, 43.Ibid., p. 396.Analyt. Chem., 1950, 82, 966.Ibid., p. 122.' Fetterley and Hazel, ibid., p. 76400 AXAIJYTIOAL UHEIWSTRY.monitor while the moving phototube scanned the spectrum, so that intensityfluctuations of the source were cancelled out. Direct quantitative studies ofligh t-source properties were possible by this means, and the relativeintensities of over 1000 lines from a standard d.c.iron arc have been recorded.Sinclair lo has investigated the variations of spectroscopically significantproperties in pulsed-arc discharges produced by a condensed-arc source unit,particularly with respect to analysis of zinc alloys. A useful report has beenmade l1 on the effects of Variations in cathode dimensions and sampleweights on the intensities of trace element and internal standard lines, andon the intensity ratios; variations in electrode dimensions can alter lineintensities and intensity ratios, so that accurate determinations of traceelements will only be obtained if the dimenaions, and especially the boringdepth, are kept constant. Brode and Timma l2 have studied the effect ofvarying amounts of extraneous elements on the line intensity of differentelements, and claim that Slavin’s total-energy method makes possible a moreexact correlation than has been achieved by using the internal standardmethod; the effect varies with the amount of extraneous element, but notin a simple linear fashion.A survey of light sources has been made byKaiser .13A rapid method of assessing intensities of blackening of the photographicplate has been based l4 on visual comparison with standard density lines on aHilger Spectrum Projector ; the difficulties in detecting and determiningtrace elements in complex spectra, however, led Davis and Webb l5 todevelop an apparatus for presenting a picture of the blackening contour of aportion of a spectrogram on the screen of a cathode ray tube.The variability of density differences on different plates, and theconsequences of such variations on routine analyses, have been studied; l6statistical treatment of spectrochemical computation methods has shown l7that the inherent precision closely approximates to its maximum value whencomputations are carried out with y values which are the mean of fourindependent determinations.Kaiser 18 has reviewed methods for thecalibration of step filters, and has given methods for calculating filter factorsa t various wave-lengths with a minimum number of observations.Spectrochemical methods have been described for alkali metals in ironcatalysts,19 for cast iron,20 for boronY21 for impurities in berylliumin titanium,23 and in for trace elements in oil 26 and serum,26 and10 J .Opt. Soc. Amr., 1949, 39, 958.l 2 J . Opt. Soc. Amer., 1949, 89, 478.l4 Addink, Spectrochim. Acta, 1950,4,36.16 Dehio, Eggert, HonerjBger-Sohm, Hormann, and Kaiser, ibid., 1949, 8, 488.l7 Schmidt, ibid., p. 538.21 Roux and Husson, Compt. rend., 1950, 230, 1068.28 Smith and Fassel, Analyt. Chem., 1949, 21, 1095.*3 Peterson, ibid., 1950, 22, 1398.9 5 Carlson and Gunn, AwZyt. Chem., 1950, 22, 1118.11 Scott, Spectrochim. Acta, 1950, 4, 73.l8 Chimia, 1950, 4, 89.la Ibid., p. 13.1* Ibid., p. 518.20 Riley, Spectrochim. Acta, 1950, 4, 93. Fast, Analyt. Chem., 1950, 22, 320.L4 Walsh, Spectrochim. Acta, 1950, 4, 47,Pfeilsticker, Spectrochim Acta, 1950, 4, 100CROPPER : PHYSICAL AND PHYSICOCHEMICAL METHODS.40 1€or traces of thallium in muscular tissue; 27 bismuth has been determinedin its alloys after a preliminary concentration stage using cupferron,2* andberyllium in air dust has been measured by a procedure involving use ofoxine to remove certain metals.29 The use of photomultiplier tubes for thealmost instantaneous spectroscopic determination of carbon in steels hasbeen described by Breckpot,30 who has also reported a very rapid method fordetermining phosphorus in steels, using the 2 1 3 6 ~ . line and a photo-multiplier with an interposed fluorescent screen.31 A special chamber hasbeen designed to permit electrodes to be loaded with radioactive materialsbefore spark excitation, with minimum risk to operators.32Interest in flame photometry has been greatly stimulated in recent yearsby the production of the flame photometer attachment for the Beckmanspectrometer ; this photometer, now described in detail by Gilbert, Hawes,and BeckmanF5 has a simple detachable atomiser handling samples of 1 ml.or less, a heated spray chamber for evaporating the spray, and a versatileburner for oxygen-gas or other flames.Monvoisin and Mavrodineanu a4have improved the dispersion of the liquid particles in a flame source bybuilding an ultrasonic oscillator into the jet, and have stabilised the flameby heating the fuel inlet in the base of the burner. A simple, inexpensiveburner has been described35 for use with a medium spectrograph fordetermination of 15 elements in the dissolved ash of biological material; theBeckman flame photometer has also been used with a large Littrow spectro-graph for determination of sodium, potassium, and lithium in plant andanimal substances.36 Flame spectra, detection limits, and excitationcharacteristics have been given for several dozen elemenfsF3 and applicationsof flame photometry have been described for the analysis of refra~tories,~~1 37magnesite and b r ~ c i t e , ~ ~ water,a9 and blood ser~m.~OSchiiler 41 has described the use of a new type of discharge tube whichmakes it possible to observe the spectra of organic molecules instead of theusual glow discharge spectrum of molecular fragments ; results are illustratedby emission and absorption spectra of various benzene derivatives.The most important advance in absorptiometry (andalso in ultra-violet spectrophotometry) has been the use of high opticaldensity reference solutions as a means of attaining a high degree of precision.The early work of KortiimP2 has now been extended by Bastian, onAbsorptiometry.a 7 Jansch and Mayer, Mikrochem., 1950, 35, 310.28 Burriel-Marti and Ramirez-Muiioz, Analyt. Chim.Acta, 1950, 4, 428.29 Peterson, Welford, and Harley, Analyt. Chem., 1950, 22, 1197.3O Breckpot and Gobert, Bull. SOC. chim. Belg., 1950, 59, 102.31 Breckpot and Marzec, ibid., p. 280.3= Feldman, Hawkins, Murray, and Ward, Analyt. Chem., 1950, 22, 1400.33 Ibid., p. 772. 34 Spectrochim. Acta, 1950, 4, 152.36 Robinson, Newman, and Schoeb, Analyt. Chem., 1950,22, 1026.36 Schrenk and Smith, ibid., p.1023. 37 Biffen, ibid., p. 1014.38 Mosher, Bird, and Boyle, ibid., p. 716.39 West, Folse, and Montgomery, ibid., p. 667.40 Willebrand, Rec. Trav. chim., 1950, 69, 799.*l Specfrochim. Acta, 1950, 4, 86, 42 Angew. Chem., 1937, 60, 193402 ANALYTICAL CHEMISTRY.theoretical grounds, to copper in copper base alloys43 and to potassiumdichromate and potassium permanganate ; 44 a full theoretical treatment hasbeen given by H i ~ k e y . ~ ~ ' The mode of operation requires the measurementof the optical-density difference between the test solution and a referencesolution, both of high optical density, so that the concentration difference isevaluated; the error in the photometric measurement (and hence, in theconcentration difference) is the same as in ordinary absorptiometry orspectrophotometry, but when results are expressed as total concentration inthe test solution, a significantly improved precision (e.g., & 0-1-@2%) isobtained, far surpassing that given by any other photometric process andmaking light-absorption methods of this type comparable in accuracy withgravimetric and volumetric methods. The work published to date has beencarried out on a Beckman spectrometer, the slits of which can be widened toallow a larger section of the spectrum to fall on the photocell; with existingabsorptiometers, the fixed spectral intensity given by the source and filterresults in a greatly reduced light intensity falling on the photocell whenhigh optical density solutions are used, and this restricts the upper limit atwhich the work can be done.The Reporter has used this difference methodwith good results in the 290 mp. region on a Beckman spectrometer, and at434 mp. with a Spekker absorptiometer fitted with a mercury lamp; in thelatter case, the optical density of the reference solution could be no higherthan about 1-5. The need has now arisen for a commercial absorptiometerwhich will give adequate galvanometer response even when solutions areused with optical densities as high as 3.0.A detailed account of the improvements which have been made in thenew Spekker absorptiometer has been given by Isbell ; 46 photoelectricinstruments have been described for use in microanalysis 47 and for protein-bound iodine.48 A further contribution has been made 49 on the permanenceof glass standards of spectral transmittance, and a very useful study has beenreported on the isolation of the lines of the mercury arc by filters,50 so as togive more truly monochromatic light.Buc and Stearns 51 have given dataon the transmittance of interference filters.Although a considerable number of absorptiometric and spectrophoto-metric studies have been reported, mention can be made only of the work oncopper diethyldithiocarbamate, on cobalt-nitroso-R-salt complex and nickeldimethylglyoxime,52 and on methods for beryllium.53 Spectrophotometricdata have been given for 11 rare-earth elements in aqueous solutions as43 Bastian, Analyt. Chem., 1949, 21, 972.44 Bastian, Weberling, and Palilla, ibid., 1950, 22, 160.45 Ibid., 1949, 21, 1440.4g Analyst, 1949, 74, 618.4 7 Ellis and Brandt, AmEyt. Chem., 1949, 21, 1546. 48 Chaney, ibid., 1950,22,939.49 Gibson and Belknep, J . Res. Nut. Bur. Stand., 1950, 44, 463; J . Opt. SOC. Amer.,60 Nicholas and Pollak, Analyst, 1950, 75, 662.5 1 J . Opt. SOC. Amer., 1950, 40, 336.52 Ovenston and Parker, Analyt. Chim. Acta, 1950, 4, 135, 142.63 S6guin and Gramme, Bull. SOC. chim., 1950, 17, 384,1950, 40, 435CROPPER : PHYSICAL AXD PHYSICOCHEMICAL METHODS. 403chlorides, nitrates, acetates, and perch lor ate^,^^ and quantitative methodshave been outlined for mixtures of several of these elements.54* 5sWokes and Slaughter 56 have studied the performance ofthe new Spekker instrumenb with its fluorimetry attachment, and havestressed the difficulties encountered in obviating the errors induced by straylight in fluorimeters with large photoreceptive areas ; these have beenovercome, and sensitivity greatly increased, by replacing the barrier-layerphotocells by a photomultiplier tube.In a number of specific applicationsof the fluorimetric method, the effects of the many variables, the influenceof interfering substances, quenching, and decay, have been studied ; amongthese may be mentioned the work on pyru~aldehyde,~~ acet01,~~ alloxanmon0hydrate,5~ and traces of beryllium in biological material.60Considerable attention hasbeen directed towards avoiding the difficulties usually encountered in theanalysis of multicomponent mixtures, and in correcting for " background "absorption due to unknown impurities.Vaughn and Stearn 61 haveanalysed xylene mixtures by taking readings a t four wave-lengths, calculatingtwo pairs of differences between these measurements and using thesedifferences with a calibration chart (a ternary composition diagram) todeduce the required results, which are free from errors due to level back-ground absorption. A rapid and accurate method for xylenes has beendeveloped 62 in which only the mutual relationships of absorption co-efficients are determined instead of their absolute values. A method similarto that of Morton and Stubbs c3 has been used for determining anthracene inanthracene cakes,6* in which the background absorption was assumed to belinear over the spectral region used; the same principle has been applied inthe determination of vitamin A.65 Tunnicliff, Rasmussen, and Morse 66have presented an algebraic method for correcting for the effect of interferingbackground ; instead of assuming that the interference is linear over a smallspectral interval, it is assumed that the absorption curve of the interferingmaterial can be represented by a general expression which is a function ofwave-length.The function chosen to represent the interference must do soaccurately over the required spectral interval, and must not even approxi-mately represent the absorption curve of any one component or combinationof components to be determined.Results of a collaborative test on 28 Beckman spectrometers have beenreported ; 67 the individual results show a very wide variation (0*681-0*782)64 Moeller and Brantley, AnaZyt.Chem., 1950, 22, 433, 1393.5 5 Wylie, J. SOC. Chem. Ind., 1950, 69, 143.5 7 Thornton and Speck, AnaZyt. Chem., 1950,22, 899.5 8 Forist and Speck, ibid., p. 902.6o Klemperer and Martin, ibid., p. 828.6 z Perry, Sutherland, and Hadden, ibid., 1950, 22, 1122.63 Analyst, 1946, 71, 348.64 Hazlett, Hannan, and Wells, AnaZyt. Chem., 1950, 22, 1132.67 Photoelectric Spectrometry Group, Bulletin No. 1, April, 1949, and Bulletin No. 2,Fluorimetry .Ultra-violet A bsmption Spectrophtometry.5 6 Analyst, 1949, 74, 624.59 Tipaon and Cretcher, ibid., p. 822.61 Ibid., 1949, 21, 1361.McGillivray, ibid., p. 494. 6 8 Ibid., 1949, 21, 895.March, 1950404 ANALYTICAL CHEMISTRY.in for the maximum for potassium nitrate, the mean value (0-713)being about 2% higher than the generally accepted figure based on previouswork.The standard error of the mean of duplicate tests on an " average "instrument was found to be 1-69; the difference between the mean resultsof duplicate tests on two instruments would be greater than 2.8% on one-third of the occasions, and greater than 5.5% in one case in twenty. Thiswork, therefore, emphasises the need, when maximum accuracy is required,for careful checking of instrument performance by measurements on standardsubstances. The errors introduced in spectrophotometry by the use of slitsof finite width have been considered by Eberhardt,68 who gives theoretically-derived expressions to allow an estimate to be made of the magnitude ofthese errors.The method for increasing the accuracy of spectrophotometricwork by using test and reference solutions of high optical density has beenreferred to under Absorptiometry (see p. 401).The glycol saponification/ultra-violet spectroscopic method for analysisof polyene fatty acids, which was reviewed and improved by Hilditch,Morton, and Riley G9 five years ago, has been examined by eight collaborators,who tested four oil samples for conjugated diene, arachidonic, linolenic, andlinoleic acids; 7O the results were satisfactory, and a detailed procedure hasbeen recommended for adoption. The method has also been used on themicro-scale 71 but, to avoid interference due to background absorption, achange has had to be made from glycol-potassium hydroxide to aqueousalkali at high temperature and pressure.Analytical methods have been described for mixtures of quinine (orquinidine) and cinchonine (or ~inchonidine),~~ for pyridine in hydrocarbonsof the kerosene-naphtha range,73 for nicotine,74 and for chlorinated solvents.75A review of the techniques of microspectroscopy, particularly in theultra-violet region, has been given by Loofbourow ; 76 the various combin-ations of microscope and spectroscope optics for examining the spectra ofsmall samples are discussed (for emission spectroscopy and for absorptionmeasurements in the ultra-vioIet, visible, and infra-red regions) and examplesare given of the performance of particular instruments.The increasing popularity ofdouble-beam instruments, with percentage transmission recording devices,is reflected in the number of papers dealing with modifications to, and/orperformance of, commercial models, or with the building of new instruments.Hales 77 has described an improved Hilger D.209 instrument with a double-beam system, using twin thermopiles of high sensitivity and fast response,and has reviewed the various problems which arise with this type ofapparatus. Simple modifications have been made to a Perkin ElmerInfra-red Absorption Xpectrophotometry .68 J .Opt. SOC. Amer., 1950, 40, 172.7O Anon., J . Amer. Oil Chem. Soc., 1949, 28, 399.7 1 Berk, Kretchner, Holman, and Burr, Analyt. Chewt., 1950, 22, 718.7 2 Grant and Jones, ibid., p.679.74 Willits, Swain, Connelly, and Brice, ibid., 1950, 22, 430.7 5 Berton, Bull. SOC. chim., 1949, 18, 858.76 J . Opt. SOC. Amer., 1950, 40, 317.69 Analyst, 1945, 70, 68.73 Le Rosen and Wiley, ibid., 1949, 21, 1175.7 7 J . Sci. Instr., 1949, 26, 359CROPPER : PHYSICAL AND PRYSICOCHEMICAL METHODS. 405instrument 78 to convert it for double-beam operation, and to make itportable to facilitate rapid analyses at the site of laboratory experiment^.^^White and Liston 80 have given a very detailed account of the construction,performance, and applications of the new Perkin Elmer Model 21 double-beam instrument ; these authors have also modified a Perkin Elmer spectro-meter for the continuous determination of six components in a samplestream.a1 The recording spectrophotometer constructed by Brownlie,82which is basically similar to that of Baird Associates, used a Schwartzthermopile in place of the bolometer, slits of a new design, and a d.c.servosystem for the recorder. A simple scheme has been described 83 by whichany spectrometer is readily converted to a direct ratio-recording instrument ;a double-beam " slit illuminator " passes light from a, common source areaalternately over two identical paths to the spectrometer entrance slit, andthe signals corresponding to the two beams are amplified, " sorted," andrectified by synchronous switches, compared potentiometrically, and theratio recorded. The energy in the comparison beam is kept constant at anypredetermined level while a spectrum is scanned, by means of a simpleservo slit width control.Menzies B p has given an account of the Hilger instrument with cathode-ray presentation of the spectrum; Bullock and Silverman 85 have describeda spectrometer with cathode-ray presentation, in which the detector is alead telluride cell, and which works with scanning rates of 120 cycles persecond and a scanned interval of better than 1.5 p.The ultimate limits ofsensitivity of photoconductive cells have been discussed by Moss.8sBlout, Bird, and Grey 8' have described an experimental infra-redmicrospectrometer, based on a commercial macro-instrument which can bechanged from macro- to micro-work in a few minutes; the performancecharacteristics are also discussed in terms of the cross-sectional area andminimum volume which can be observed with satisfactory signal to noiseratio, and five ways are given in which the ratio of measured absorption tospectrometer noise can be increased, Elliott, Ambrose, and Temple s8 havedesigned an apparatus for spectroscopy in the 3-p.region (with lead selenidephotocell) which is particularly suited for measurements on small specimensof oriented materials with polarised radiation.The large number of individual spectra, published each year contributesto a general fund of information, and each spectrum is potentially useful tothe analyst engaged in qualitative and quantitative infra-red work ; mentionof those published in 1950 is not possible in this Report.The paper byKuhn,89 however, records the spectra of 79 sugars and sugar derivatives and'13 Savitzky and Halford, Rev. Sci. Instr., 1950, 21, 203.79 Chapman and Torley, AnaZyt. Ghern., 1950, 22, 987.J . Opt. SOC. Amer., 1950, 40, 29, 36, 93; Analyt. Chem., 1950, 22, 768.White, Liston, and Simard, ibid., 1949, 21, 1166.(12 J . Sci. Instr., 1950, 27, 215.8a Hornig, Hyde, and Adcock, J . Opt. SOC. Amer., 1950, 40, 497.8 8 J . Sci. Instr., 1950, 27, 21.Ibid., 1949,39,1060. Ibid., 1950,40,608. 8 6 Ibid., p . 603. 137 Ibid., p . 304.Analyt. Chern., 1950, 22, 276406 ANALYTICAL CHEMISTRY.concludes that infra-red spectroscopy is an excellent method for theidentification of sugars and of functional groups in the carbohydrate molecule,including cellulose; an example of the analytical value of this method isgiven in connection with the determination of nitrate group in nitrocellulose.The analysis of natural and purified methane streams for small amountsof ethane, propane, and n- and iso-butanes has been achieved on a BeckmanI.R.2 spectrometer specially adapted (and calibrated) with a methanecompensation ~ell.~O O'Neal 91 has described the analysis of C,-C, paraffin-mono-olefin hydrocarbon mixtures (including differentiation of cis- andtrans-but-2-ene) without the usual low-temperature distillation, by acombination of mass spectrometer measurement of hydrocarbon groups, andinfra-red measurement of the butenes.trans-Octadecanoic acids and esterscan be determined 92 by measurements a t 10.36 p., a t which wave-length thetrans-isomers show strong characteristic absorption, whereas the cis-isomers,and saturated acids, do not.Brief mention may be made of the work ondi-tert. -butylcresols,93 and on methylphenoxyacetic acid and its chloro-derivatives ; O4 the application of infra-red spectroscopy in the analysis ofdistillation fractions of an and the methods used in the Esso laboratoriesfor the analysis of complex hydrocarbon mixtures, have been de~cribed.~~Opler 97 has advocated the use of punched-card machines for rapid calculationof results for ten-component mixtures. Colthup 98 has published chartssummarising spectra-structure correlations in the infra-red region.Few improvements in instrumentation or techniquehave been reported since the review in Annual Reports for 1949.99 A photo-electric instrument has been described, loo incorporating a low-pressuremercury source, a three-prism monochromator, and a refrigerated photo-multiplier detector which actuates a two-recorder system through a d.c.amplifier.Braun, Spooner, and Fenske 101 have reported the Raman spectra of 119different compounds, supplementing the data on 172 compounds givenpreviously; lo2 such compilations of data are of much potential value, andtheir growth should stimulate the much wider exploration of the value ofRaman spectroscopy in analytical work.The detection and determinationof isomeric hexachlorocyclohexanes has been described.lo3Detailed reviews of theseRamn Spectroscopy.X-Ray Absorption and Diffraction Methods.O0 Stroupe, Analyt.Chem., 1960, 22, 1125. O1 Ibid., p. 991.O3 Hales, Analyst, 1950, 75, 146.Swern, Knight, Shreve, and Heether, ibid., p. 1261; J . Amer. Oil Chem. SOC.,O4 Sjoberg, Acta Chem. Xcand., 1950, 4, 798; an ultra-violet method is given by85 Lecomte, La Lau, and Waterman, Bull. SOC. chim., 1950, 17, 141.96 Bell, Analyt. Chem., 1950, 22, 1005.J . Opt. SOC. Amer., 1950, 40, 397.Woodward, Ann. Reports, 1949, 46, 216.Ibid., p. 1074.1950, 27, 17.Grabe, ibid., p. 806.O 7 Ibid., p. 558.loo Heigl, Dudenbostel, Black, and Wilson, AmEyt. Chem., 1950, 22, 154.lo3 Luther, Lampe, Goubeau, and Rodewald, 2. Naturforsch., 1950, 5, a, 34.108 Ibid., 1947, 19, 700CROPPER : PHYSICAL AND PHYSICOCHEMICAL METHODS.407subjects, up to late 1949, have been p~b1ished.l~~ The General ElectricX-ray photometer has been used for quality control of petroleum products,lo5particularly for determination of sulphur, tetraethyl-lead, and metallicadditives; very detailed accounts have been given lo6 of the technique andresults of X-ray absorption spectrophotometric determination of tetraethyl-lead in petrol. Birks has described 107 apparatus for measuring X-rayfluorescence spectra and has now reported methods for determining smallamounts of hafnium in zirconium, and tantalum in columbium,fO* and fortetraethyl-lead and ethylene bromide in aviation petr01.l~~Advances in apparatus for diffraction work include construction of apoint-focus monochromator for low-angle diffraction,l1° an optical centeringsystem,lll a, Geiger counter spectrometer for single-crystal measurements,lf2and a camera to eliminate spottiness from X-ray photographs of coarse-grained material ; 113 apparatus for operation at high temperatures has beendesigned by Goldschmidt and Cunningham,l14 by Williams,l15 and bySteward.ll6 A camera for low-temperature work,117 and a low-temperaturesingle-crystal technique 11* have also been described.X-Ray diffraction studies (for identification purposes) have been madeon di-p-~ylylene,~l~ and on the itmides,l20 anilides, and silver salts of thesaturated fatty acids up to C22.Data on certain complex alkyl sulphideeand sulphones show that X-ray diffraction may be used for identifyingalkyl halides from which the sulphides and sulphones were prepared.122Susich 123 has discussed the difficulties which arise, owing to polymorphism,in the identification of organic dyes, and has described applications todifficult problems in dye chemistry.Electxometric Methods.-Potentimetric and Conduetometric Titrations.Gran 124 has shown, both theoretically and in practice, that the equivalentpoint in a potentiometric titration can be determined more accurately fromAV/ApH or AV/AE versus V , rather than the usual AEIAV versus V , curves.Potentiometric titration of functional groups has been referred to abovelo4 Liebhafsky, Analyt.Chem., 1949,21,17 ; 1950,22,15 ; Kaufman and Fankuchen.lo6 Vollmar, Petterson, and Petrizzelli, ibid., 1949, 21, 1491.lo6 Calingaert, Lamb, Miller, and Noakes, ibid., 1950, 22, 1238; Hughes andlo' Birks and Friedman, Rev.Sci. Instr., 1948, 19, 323.lo8 Birks and Brooks, Analyt. Chem., 1950, 22, 1017.lo* Birks, Brooks, Friedman, and Roe, ibid., p. 1258.110 DuMond, Rev. Sci. Instr., 1950,21,188.lla Cochran, Acta Cryst., 1950, 3, 268.114 Ibid., p. 177.1l6 Ibid., 1949, 26, 371.118 Kaufman and Fankuchen, ibid., 1949, 20, 733.11# Brown and Farthing, Nature, 1949, 164, 915.Wurz and Sharpless, Analyt. Chem., 1949, 21, 1446.lal Matthews, Warren, and Mitchell, ibid., 1950, 22, 514.la2 Merritt, Cutter, Golden, and Lanterman, ibid., p. 519.les Ibid., p. 425.(p. 399).ibid., 1949,21, 24; 1950,22, 16.Hochgesang, ibid., p. 1248.111 Perrine, ibid., p. 262.113 Thewlis, J .Sci. Instr., 1950, 27, 72.115 Ibid., p. 154.117 Clifton, Rev. Sci. Instr., 1950,21,339.184 Acta Chem. S a n d . , 1950, 4, 559408 ANALYTICAL CH.RMIS!CRY.Rapid progress is now being made in the development and application ofhigh-fi-equency titrimeters, which have been reviewed at length.la5 Instru-ments giving good sensitivity, stability, speed and ease of operation havebeen built (operating up fa 40 Mc./sec.) ; their use has been exemplified byacid-base neutralisations, precipitation reactions, formation of complexes,and redox titrations.126 Blaedel and Malmstadt 12' have compared thepotentiometric, conductometric, and high-frequency titration procedures forthe determination of chloride by mercuric nitrate, and show that the high-frequency end-point is superior, although it is still inferior to end-points inargentometric titrations.A new titrimeter, operating a t 350 Mc. persecond, has also been described 12* by which it is possible to carry out accuratetitrations in presence of a considerable quantity of foreign electrolyte.The revival of interest in electroanalysis or coulometricanalysis has continued ; Lingane 129 has described an improved apparatus(" potentiostat ") for maintaining constant cathode potential, and anelectromechanical integrator which integrates the curve drawn by a recofdingp0tentiometer.l3~ Lamphere and Rogers 131 and Allen 132 have alsodescribed stable instruments which give constant cathode potential.Maxwell and Graham 133 have reviewed the applications of the mercurycathode and point out that the technique is especially useful in removinglarge concentrations of one or more elements before polarographic determin-ations of minor components.A review has been given of the advantagesand disadvantages of electroanalysis as a method of separation; 134 theproblems which arise in devising a quantitative separation by electro-analysis on the submicrogram scale have been discussed by Rogers.135The technique known as coulometric titration with amperometric end-point has received further attention ; this process involves timing a reactionoccurring with 100% current efficiency at the electrode, followed by a rapidincrease in current after the end-point has been reached. Farrington andhis collaborators have continued their work on the use of electrolytically-generated bromine1a6 and have extended the method to determination oftervalent arsenic by means of iodine and ch10rine.l~~ Cooke andFurman have described a similar technique for titration of ceric sulphateand potassium dichromate by means of ferrous iron generated a t a platinumelectrode.The methods of derivative and differential polarographyElectroanalysis.Polarography.lZ5 Analyt.Chem., 1950, 22, 734.lZ6 Anderson, Bettis, and Revinson, ibid., p. 743; Anderson and Revinson, ibid.,p. 1272; West, Burkhalter, and Broussard, ibid., p. 469 ; Blake,AnaEyst, 1950,75,32,689.127 Analyt. Chem., 1950, 22, 1410.lZg Ibid., 1949, 21, 497; 1950, 22, 1169.lS1 Ibid., p. 463.133 Chem.Reviews, 1950, 46, 471.la4 Ashley, Analyt. Ghem., 1950, 22, 1379.136 Wooster, Farrington, and Swift, ibid., 1949, 21, 1457.137 Ramsey, Farrington, and Swift, ibid., 1950, 22, 332.138 Farrington and Swift, ibid., p. 889.128 Idem, ibid., p. 1413.130 Lingane and Jones, ibid., p. 1220.13% Ibid., p. 804.135 Ibid., p. 1386.lS9 Ibid., p. 896CROPPER : PHYSICAL AND PHYSICOCHEMICAL METHODS. 409devised by Heyrovsky 14* and by Semerano and Riccoboni 141 have presenteddifficulties which have delayed more general adoption. Derivative polaro-graphy automatically gives curves representing A i l A E versus E , and id ofvalue in dealing with close half-wave potentials (e.g., for sodium and lithium)and for giving specific waves; differential polarography, on the other hand,compares two similar solutions so that (a) the effect of a major and moreeasily-reduced component is eliminated or ( 6 ) the concentrations of twosolutions can be compared more accurately.These techniques, potentiallyof great analytical value, require synchronisation of the drop formations toprevent appearance of a succession of nodes in the recorded polarogram.The use of capillaries with very short drop time, and Heyrovsky's streaming-mercury electrode, have not proved the complete answer to this problem.Airey and Smales 142 have given a very detailed account of their studies oncontrolled disengagement of a mercury drop, by electrostatic or * electro-mechanical means, and show that two capillaries can be accurately syn-chronised.Circuits are also given for modifying a Cambridge polarographfor work on derivative or differential polarography, and the performance ofthe modified instrument, with synchronised drops, has been described forboth methods ; this work will undoubtedly stimulate further research in thisfield, and publications on particular analytical problems can be expected.Snowden and Page 143 have given a detailed description of an improvedcathode ray polarograph, with results on inorganic and organic mixtures andalso on the use of the instrument for following rapid reactions; Delahay 144has constructed an improved apparatus and procedure for recording wavesa t high rates of potential variation, and also a portable electronic instrumentfor general purposes.145From a comparison of potentiometric and polarographic measurements,Stone 146 has concluded that pH effects may not be identical if reduced speciesare stabilised by resonance, and that buffer constituents play a vital r6le inthe polarographic reduction if a relatively stable species is formed betweenthe buffer anion and either the starting material or its reduction products.The value of Trilon B (ethylenediaminetetra-acetic acid) as a base solutionhas been emphasised; 14' it strongly displaces the reduction potentials ofnumerous elements, so that cobalt, for example, is reduced at a very lownegative potential and preliminary separation from other metals is notnecessary in the analysis of steel.A polarographic study has been made ofthe stability of the complexes formed between heavy metals and the' complexones " (ethylenediaminetetra-acetic acid and nitrilotriaceticacid) .I48The determination of small concentrations of zinc has been studied,149140 Chem.&sty, 1946, 40, 222 ; Analyst, 1947, 72, 229.141 cfazzettu, 1942, 72, 297.143 Anctlyt. Cilem., 1950, 22, 969.145 Anulyt. Chem., 1949, 21, 1425.14' Souchay and Foucherre, Analyt. Chim. Acta, 1949, 3, 252.148 Koryta and Kossler, CoEL Czech. Chem. Comm., 1950, 15, 241.142 Analyst, 1950, 75, 287.144 J . Phys. Colloid Chem., 1950, 54, 402.146 J . Electrochem. SOC., 1950, 97, 63.Champa and Wallach, Analyt. Chem., 1950, 22, 727410 ANALYTICAL CHEMISTRY.and methods have been given for determination of zinc in zinc ores,150 incompounded rubber,l51 in and in waters ; 153 polarographic methodshave been described for common impurities in refined lead,154 in calcium,155and in high-quality indium,l56 for cobalt in presence of excess of copper,iron, and nicke1,157 for trace metals in gelatins,15* for titanium in paintpigments (after removal of copper and antimony),159 and for uranium inpresence of iron, copper, and phosphates.ls0 Polarographic determinationof aluminium has been based on reduction of an aluminium di-o-hydroxyazo-complex.l6l Furness 162 has discussed the application of polarography tothe analysis of dithionites, and has outlined procedures for the determinationof thiosulphate, sulphide, and trithionate in presence of dithionite.Elving 163 has given recently a review on the polarographic behaviour oforganic compounds, adding to those already given by W a w ~ o n e k , ~ ~ ~ andmention is, therefore, made only of subsequent papers.The value ofpolarographic methods for the analysis of fine chemicals, for both inorganicand organic impurities, has been emphasised by Osb0rn.1~~ Carbon tetra-chloride gives two reduction waves corresponding to reduction to chloroformand to methylene chloride respectively, whereas chloroform only gives theone wave,166 so that analysis of mixtures of chloroform and carbon tetra-chloride can be done polarographically . The polarographic reduction ofdiazotised arylamines has been studied,ls7 and it is claimed that the measure-ment of concentrations of diazonium solutions by this means is moregenerally applicable than the gasometric or colorimetric methods.Sartori and Liberti le8 have shown that, in the polarography of mercapto-benzothiazole, the anodic reaction does not correspond to formation ofdisulphide, but to formation of the mercury compound, and also that theheight of the cathodic wave is proportional to [H+] of the buffer.Studieson quantitative polarographic methods have been made on g l ~ t a t h i o n e , ~ ~ Chloromycetin,l70 " Parathion ",171 n i n h ~ d r i n , l ~ ~ and cholesterol.173 Hall 17*150 Semerano and Gagliardo, Analyt. Chim. Acta, 1950, 4, 422.lS1 Poulton and Tarrant, Trans. Inst. Rubber Ind., 1950, 25, 328.lS2 Wiley, Deloney, and Winstead, Analyt. Chem., 1950, 22, 201.153 De Salas and Graells, Anal.Asoc. Quim. Argentina, 1949, 37, 208.lS4 Cozzi, Analyt. Chim. Acta, 1950, 4, 204.lS6 Haupt, Olbrich, and Nause, 2. Electrochem., 1950, 54, 67.15? Kolthoff and Watters, Analyt. Chem., 1950, 22, 1422.lS8 Michel and Maron, Analyt. Chim. Acta, 1950, 4, 542.15s Potts, Canadian J . Res., 1950, 28, F , 128.160 Block, Bull. SOC. chim., 1949, 16, 831.181 Willard and Dean, Analyt. Chem., 1950, 22, 1264.16z J . SOC. Dyers Col., 1950, 66, 270.164 Ibid., 1949, 21, 61; 1950, 22, 30.1~ Kolthoff, Lee, Stocesova, and Parry, Analyt. Chern., 1950, 22, 521.167 Elofson, Edsberg, and Mecherly, J. Electrochem. SOC., 1950, 97, 116.188 Ibid., p. 20.16@ Coulson, Crowell, and Friess, Analyt. Chern., 1950, 22, 525.170 Hess, ibid., p.649.172 UlEak, Spalek, and KrStkf, Coll. Czech. Chem. Comm., 1950, 15, 340.173 Talafant, ibid., p. 232.155 Stage and Banks, ibid., p. 551.163 Analyt. Chem., 1950, 22, 482.185 Analyst, 1950, 75, 671.171 Bowen and Edwards, ibid., p. 706.174 Analyt. Chem., 1960, a, 1137CROPPER : PHYSICAL AND PHYSICOCHEMICAL METHODS. 41 1has described the determination of traces of elemental sulphur in petroleumfractions, using methanol and pyridinium hydrochloride as an electrolyticsolvent miscible with hydrocarbons, and claims that the method is rapid,sensitive, and free from interference from organic sulphides, disulphides, andthiophen. The polarographic determination of tetraethyl-lead in petrolhas received attention; a rapid method has been described by Hansen,Parks, and Lykken,l75 and a direct-reading instrument, based on the use ofantimony as a pilot ion, has been developed by Offutt and S ~ r g .l ~ ~ Parksand Lykken 177 have reviewed the applications of potentiometric, ampero-metric, and polarographic methods for determination of common constituentsin petroleum products.The use of ethylene glycol monoalkyl ethers (Cellosolves) as non-aqueoussolvents for organic polarography has been a d ~ 0 c a t e d . l ~ ~ Gordon andJones 179 have reviewed previous work involving distribution between twoimmiscible solvents, followed by polarographic measurement on one of thephases; the name " partition polarography " is proposed, and formulae arededuced which predict the accuracy of the method and permit selection ofoptimum experimental conditions for each system with a minimum ofexperimental work.An improved amperometric titration cell, foruse with a dropping-mercury electrode, has been described 180 which usescontinuous gas flow for oxygen removal and solution mixing.A furtherreport has been made by Kolthoff and Liberti 181 on the titration of copperand ferric iron with cupferron ; conditions for the amperometric titration(dropping-mercury electrode) of zinc and indium with ferrocyanide havebeen studied.182 The rotating platinum electrode has been used for titrationof zinc by ferrocyanide 183 and for chromium and vanadium (after conversioninto chromate and vanadate) by means of ferrous ammonium sulphate.ls4It has been shown 185 that amperometric titration of thiols under theconditions described by Kolthoff and Harris 186 gives low results due topresence of oxidising agents in ammoniacal ethanol and dissolved air in thesilver nitrate solution.A modification of the method for thiol groups hasbeen developed for use with microgram quantities (in biological material),in which a vibrating platinum electrode is used as a combination electrode-stirrer.MIUS Spectrometry.-The mass spectrometer is rapidly growing inAmperometric Titrations.175 Analyt. Chem., 1950, 22, 1232.178 Parks and Hansen, ibid., p. 1268.lao Laitenen and Burdett, ibid., p. 833.lS1 Analyst, 1949, 74, 635; see J. Amer. Chem. SOC., 1948, 70, 1879.Nimer, H a m , and Lee, Analyt. Chem., 1950, 22, 790.lS3 Butenko and Rynskaya, Z h w .Anal. Khim., 1950,5,145; Chem. Abs., 1950,44,lE4 Parks and Agazzi, Analyt. Chem., 1950, 22, 1178.lS6 Strafford, Cropper, and Hamer, Analyst, 1950, 76, 55.17t1 Ibid., p, 1234.179 Ibid., p. 981.177 Ibid., p. 1444.6345b.I n d . Eng. Chem. Anal., 1946, 18, 161.Rosenberg, Perrone, and Kirk, AnaZyt. Chem., 1950, 22, 1186412 ANALYTICAL CHEMISTRY.importance as an analytical tool ; its applications have now expanded beyondthe limited fields of gas analysis and hydrocarbon analysis, and with improvedtechniques in handling samples of very low vapour pressure, the possibilitiesare immense. In addition to the reviews in Anulyticul Chemistry,l thearticle by Washburn 188 provides general information of special value tonewcomers to the subject.New instruments have been described byK e r ~ i n , ~ ~ ~ and by Duckworth,lg0 and an instrument has been developedfor the French Petroleum 1 n ~ t i t u t e . l ~ ~ Special interest must be given to thenew three-stage non-magnetic instrument, employing the principle of velocityselection, which has been developed by Bennett lg2 and which is small,simple in operation, and of high sensitivity; currents are of a magnitudewhich makes recording comparatively simple, and resolution is relativelyindependent of source slit width. Adaptation is being made to rapidscanning of the mass spectra, with display on a cathode-ray oscilloscope ;the instrument has been described as “ the poor man’s mass spectrometer ’’and although of more limited range and accuracy than the highly developedconventional instrument, it opens up innumerable possibilities for analyticalapplications.Improved methods of introducing liquid samples have been de-scribed,l939 lg4* lg5* 196 mainly in connection with the analysis of oxygen-containing samples ; results for synthetic mixtures of methanol, form-aldehyde, formic acid, methyl formate, and methylal were accurate only toabout 5%,lg4 whereas accuracy to within 2% is claimed in the analysis of3-, 4-, and 5-component mixtures of alcohols and other oxygenated deriva-tives containing 6 carbon atoms.lQ6 The “ internal standard ” method ofHindin, Grosse, and Kirshenbaum lg7 has been applied to the analysis ofliquid oxygenated C1-C5 mixtures, benzene being used as internal standard.lg5Several methods of introducing liquid samples of neohexane and of styrenehave been compared.lg8Satisfactory means of preparing deuterium-hydrogen mixtures in knownproportion, for calibration purposes, involved reducing deuterium oxide-water mixtures over zinc; 199 the deuterium content of water has beendetermined on a mass spectrometer after reaction with methylmagnesiumiodide to yield a mixture of methane and deuteriomethane.200Mohler has reported the mass-spectra data for ten C,H8 isomers 201 and188 “ Physical Methods in Chemical Analysis,” edited by W, G.Berl, Acad. PresslSg Rev. Sci. Inetr., 1950, 21, 96.lgl Bertein, Vastel, Reis, Buzon, and Nief, Rev. Inst.fran9. Pdtrole, 1950, 5, 59.lg2 Rev.Sci. Instr., 1950, 21, 578; J . Appl. Physice, 1950, 21, 143.lg3 Friedel, Sharkey, and Humbert, Analyt. Chem., 1949, 21, 1572.lo* Langer and Fox, ibid., p. 1032.lg8 Gifford, Rock, and Comaford, ibid., p. 1026.lg8 Wise, Reese, Dibeler, and Mohler, J . Res. Nat. Bur. Stand., 1950, 44, 215.lgB Alfh-Slater, Rock, and Swislocki, AnaEyt. Chem., 1950, 22, 421.201 Mohler, Bloom, Williamson, Wise, and Wells, J . Res. Nut. Bur. Stand.,Inc., 1950.190 Ibid., 1950, 21, 54.lg5 Thomas and Seyfried, ibid., p. 1022.lg7 Ibid., p. 386.Orchin, Wender, and Friedel, ibid., 1949, 21, 1072.1949,43, 533CROPPER : PHYSICAL AND PHYSICOCHEMICAL METHODS. 413for 35 nonanes,202 and points out that analysis of mixtures of nonanes willgenerally be impossible unless the material is a narrow cut containingcomparatively few components.Results have been given 203 showing the reproducibility and accuracy ofanalyses of a standard sample of carburetted water gas, by laboratories co-operating with a Sub-Committee d the American Society for TestingMaterials; low concentrations of solvents in air have been determined aftercondensation by liquid nitrogen.204 O’Neal 91 has described a combinationof mass-spectrometer measurement and infra-red measurement for theanalysis of C,-C, paraffins and mono-olefin mixtures.Radioactive Tracer Methods.-In addition to the general review articleon ‘‘ nucleonics ” by Gordon,2*5 monitoring instruments have been reviewedby Taylor 206 and Curtiss; 207 several reports have been given on thebehaviour of, or improvements to, counters,208 and an ionisation chamberhas been described by Tompkins, Wish, and B~rnett.~O~ Beamer andAtchison 210 have described quantitative techniques for 14C in compoundsof high specific activity, involving conversion into carbon dioxide and thenceinto barium carbonate, which is deposited on an aluminium plate ; measure-ment as carbon dioxide, admixed with carbon disulphide vapour, has beenstudied by EidenofK211 Radio-phosphorus has been used212 to show thatthe zinc and cadmium methods are equally eficient in separating ortho- frompyro-phosphate.Very small amounts of iodide have been determined 213 byconversion into iodate, reaction with excess of radioactive iodide of knownspecific activity, extraction of the radioactive iodine, and measurement byy-ray counting.A study of the precision attainable in P-activity counting,with special reference to the determination of cerium, has been made,214 andradioactive assays involving calcium 215 and elementary sulphur 216 havebeen reported. The use of radioactive silver in a radiometric titration hasbeen described by Langer.217Methods involving Separations.-Little new work has been published onmethods involving separation except in the fields of solvent extraction andchromatography. Rose 218 has reviewed the theory and practice of analyticaldistillation, and Starr, Anderson, and Davidson 219 have given it second1950, 44, 291.2O2 Mohler, Williamson, Wise, Wells, Dean, and Bloom, J. Res. Nut.Bur. Stand.,2os Shepherd, ibid., p. 509; Analyt. Chem., 1950, 22, 885.204 Happ, Stewart, and Brockmyre, ibid., p. 1224.a05 Ibid., 1949, 21, 96. 2 0 ~ J . Sci. Instr., 1950, 27, 82.207 U.S. Nat. Bur. Stand., Circular No. 490, 1950.208 Brown and Maroni, Rev. Sci. Instr., 1950, 21, 241 ; Laufer, ibid., p. 244; Bern-209 Anulyt. Chem., 1950, 22, 672.210 Ibid., p. 303.212 van der Straaten and Aten, Rec. Truv. chim., 1950, 69, 561.213 Raben, Analyt. Chem., 1950, 22, 480.a15 Shirley, Owens, and Davis, ibid., p. 1003.416 Kirshenbaum and Grosse, ibid., p. 613.217 Ibid., p. 1288. 218 Ibid., p. 1369.stein and Ballentine, ibid., p. 158 ; Wilkinson, J. Sci. Instr., 1950, 27, 36.211 Ibid., p. 529.a14 Freedman and Hume, ibid., p. 932.21D Ibid., 1949, 21, 1197414 ANALYTICAL CHEMISTRY.paper in continuation of their studies 220 on charging rates, distillation rates,and fraction cut-points.A review has been given 221 on the theory, scope,and methods of recrystallisation, and an apparatus has been designed 222 forseparations by sublimation under reduced pressure, the sublimate beingdeposited on a removable transparent plastic film. Mellon 223 has discussedsome general aspects of separations in analytical chemistry.The apparatus for counter-current liquid-liquid extraction, described by Craig and Post,224 has now been greatlyimproved ; 225 simple apparatus for similar purposes are described byRaymond,226 by Lochte and M e ~ e r , ~ ~ ' and by Nolan.228 Recent studies havebeen made of the distribution characteristics of 18 organic mono- and p l y -carboxylic acids between water and numerous organic solvents,229 and of 18polynuclear hydrocarbons between cyclohexane and 80% ethan01,~3O andapplications in analysis of mixtures within these classes have been given;the use of the Craig machine for determination of 2 : 4-dichlorophenoxyaceticacid has been described by Warskowsky and S ~ h a n t z .~ ~ ' Nichols 232 hasderived expressions for the prediction and evaluation of results from counter-current distribution experiments, including a method for determining thenumber of transfers necessary for a given degree of separation and pair ofpartition coefficients. Theoretical aspects of the simple extraction ofinorganic ions or complexes have been discussed by Sande11.233If the number of papers published be taken as a guideto the importance of a particular analytical technique, there is no doubt thatchromatography now ranks as foremost.The reviews which have appearedfrequently 234 should be consulted for earlier references in each particularfield of application.The detection and measurement of colourless bands as they leave thechromatograph column has continued to receive attention ; Holman 236 hasextended the range of concentrations dealt with by the Tiselius-Claessoninterferometric apparatus by introducing the effluent liquid, when itsconcentration is constant at each step, into the comparison cell. Automaticrecording flow refractometer apparatus for adsorption analysis have beendescribed 236 which, while not as sensitive as the interferometer, open upXolvent Extraction Methods.Chromatography.aao Analyt.Chm., 1947, 19, 409.222 Gettler, Umberger, and Goldbaum, ibid., p. 600.a23 Ibid., p. 1342.225 Craig, ibid., 1950, 22, 1346.227 Ibid., 1950, 22, 1064.229 Marvel and Richards, ibid., p. 1480.a31 Ibid., p. 460.z34 Martin, Ann. Reports, 1948,45, 267; Ann. Rev. Biochem., 1950,19,517; Lederer," ProgTBs RBcents de la Chromatographie," Part 1, Hermann et Cie, Paris, 1949 ; Cook,Nature, 1949, 164, 300; Strain, Analyt. Chem., 1949, 21, 75; 1950, 22, 41; Clegg, ibid.,p. 48 ; Biochemical Society Symposium, No. 3, Partition Chromatography, 1949 ;Discussions of the Faraday Society, No. 7, Chromatographic Analysis, 1949.am Tipson, ibid., 1950, 82, 628.224 Ibid., 1949, 21, 500.itas Ibid., 1949, 21, 1292.228 Ibid., 1949, 21, 1116.230 Golumbic, ibid., 1950, 22, 579.aa3 Analyt.Chim. Acta, 1950, 4, 504. Ibid., p. 915.e35 Analyt. Chem., 1950, 22, 832.as6 Hellstrom and Borgiel, Acta Ckm. Xcand., 1949, 3, 401 ; Thomas, O'Konski, andHurd, Anulyt. Chem., 1950, 22, 1221CROPPER PHYSICAL AND PHYSICOCHEMICAL METHODS. 415possibilities for extensive application. The polarograph, set a t the requiredvoltage, has been used237 for following concentrations of proteins in theeffluent from columns, and an ultra-violet sensitive photomultiplier tube hasbeen used to locate zones of methyl benzoate, benzaldehyde, and anisaldehydeon silicic acid and alumina.238 Harvalik 239 has studied the illumination ofcolumns by infra-red radiation, and has developed an electronic imageconverter to convert the infra-red radiation into visible light, so that colour-less zones can be detected by a process analogous to the use of ultra-violetfluorescence.A very sensitive micro-electrode, consisting of a smallplatinum electrode bearing a thin film of silica gel containing a trace ofquinhydrone, has been fitted to a chromatograph column to provide potentio-metric indication of change of concentration in the emerging effluent.240The “ carrier displacement ” method suggested by Tiselius 241 has beenutilised by Tiselius and Hagdahl 242 with promising results in the separationof amino-acids and peptides ; this method involves interposing between thezones to be separated a number of substances of intermediate adsorptionaffinities which would form part of the chromatogram and afterwards couldbe removed by evaporation or extraction.Apparatus has been described 243 for chromatography on narrow filter-paper strips or strips of mercerised cotton, glass, wool, or thin asbestos withboth ascending and descending solvent flows.Howard and Martin 244 claimto have widened the scope of partition chromatography for higher fatty acidsby using ‘‘ unwettable ” kieselguhr, obtained by treating it with dichloro-dimethylsilane vapour, as the support fir the static phase, which can be theless polar of the two phases. Boldingh 245 has used natural and syntheticelastomers to hold the static phase, e.g., benzene absorbed in vulcanisedrubber, with a strong polar solvent as flowing phase, for the separation of thesaturated fatty acids c($-c,8 and for separation of these from hydroxy-acids.Rutter 246 has described a useful improvement in paper chromatographyby the capillary run-out technique ; apparatus has been constructed for themass production of two-dimension paper chromatograms 247 and for applyingseveral ml.of solution to a sheet of paper as a narrow band, in work onmacro-q~antities.~~~ The separation of micro-quantities of mixed pigmentshas been carried out by chromatography on a restricted channel of paper,through which passed a beam of monochromatic light directed on to a photo-multiplier tube; this, in turn, operated a pen-and-ink recorder so thattime-transmittancy graphs were obtained.249 Silica-impregnated paper hasa37 Drake, Acta Chem.Scand., 1950, 4, 448.236 Sease, AnaEyt. Chem., 1949, 21, 1430.240 Kamienski, Compt. rend. MBd. Acad. polon., 1949, 1-2, 7 ; Compt. rend. Sci.241 Kolloid Z . , 1943, 105, 101. 24a Acta Chem. Scand., 1950, 4, 394.a43 Longenecker, Analyt. Chem., 1949,21,1402. a44 Biochem. J., 1950, 46, 532.a45 Rec. Trav. chirn., 1950, 69, 247. a46 Analyst, 1950, 75, 37.a47 Datta, Dent, and Harris, Biochem. J . , 1950, 46, Proc, xiii.a48 Yanofsky, Wasserman, and Bonner, Science, 1950, S, 61.24* Muller and Clegg, Analyt. CFem., 1949, 21, 1123.a39 Ibid., 1950, 22, 1149.math. nat. Acad. polon., 1949, 3-5, 11, 13416 ANALYTICAL CHEMISTRY.been used to achieve separations which could not be done on ordinary filterpaper, such as separation of the 2 : 4-dinitrophenylhydrazones of ethylmethyl, methyl propyl, and methyl isopropyl ketones.2mThe outstanding feature of the chromatographic techniques now availableis their wide applicability ; past reviews 234 have shown that chromatographyhas been applied to almost all subjects of great importance (e.g., fissionproducts of uranium, fatty acids, amino-acids, sugars, penicillins, carotenoids,to mention but a few), and there have been many recent refinements andimprovements in these applications; it is not possible, however, to reviewthem all in this Report (though each in its own way may present an advancein technique for dealing with particular mixtures) and mention can be madeonly of selected items published in 1950.Several important papers have appeared on the separation of the hydro-carbons in cracked petroleum ; Fink, Lewis, and Meiss 251 have made adetailed study of adsorption characteristics (and regeneration techniques) ofsilica gel for fractionating paraffins, olefins, and aromatic compounds by" frontal analysis " a t -40°, and have assessed the influence of the importantvariables affecting the efficiency of the process.Clerc, Kincannon, andWeir 252 have used " displacement development " and elution procedures onsilica gel for separating paraffins and mono-, di-, and tri-cyclic aromaticcompounds from petroleum oils. Petroleum fractions have also beenanalysed by chromatographic separation on silica into 4 parts, each of whichis then tested by conventional m e a n ~ .~ ~ 3 Adsorption on Florisil (syntheticmagnesium silicate) and elution by'pentane has been used 254 to separatehydrocarbons from nitrogenous and other heterocyclic compounds in shale-oil distillates ; the chromatographic method for analysis of shale oilnaphthas 255 has been improved 256 and consists of " displacement develop-ment " by octanol or cyclohexanol, at 70°, to separate paraffins, olefins, andaromatic CompoundB, followed by boiling-point and refractive-index measure-ments for determining the percentage of paraffins and cycloparaffins. All theabove methods use refractive index of the percolate to follow progress ofcolumn development.Frontal analysis and displacement development analysis of fatty acidshave been improved 257 by decreasing the solubility in alcohol by addition ofwater, or by lowering the temperature; adsorption on alumina has beenused to separate the 2 : 4-dinitrophenylhydrazones of aldehydes arising fromamino-acids treated with ninhydrin, as part of a method for determiningis0leucine.~5* Separation of water-soluble organic acids on a partitionKirchner and Keller, J.Amer. Chem. SOC., 1950, 72, 1867.251 Analyt. Chem., 1950, 22, 850, 858.253 Ibid., p. 864.252 Spakonski, Evans, and Hibbard, ibid., p. 1419.e54 J. R. Smith, C. R. Smith, and Dinneen, ibid., p. 867.355 Dinneen, Bailey, Smith, and Ball, ibid., 1947, 19, 992.256 Dinneen, Thompson, Smith, and Ball, ibid., 1960, 22, 871.z5' Hagdahl end Holman, J.Amer. Chem. SOC., 1950, 72, 701.258 Lohr, Biochem. Z., 1950, 320, 115CROPPER : PHYSICAL AND PHYSICOCHEMICAL METHODS. 417chromatographic column has been carried out 259 by making the developingliquid progressively more polar by adding increasing amounts of butanol tochloroform. A collaborative study of the partition method for volatilefatty acids has been reported.260The paper partition method attracts much attention. Since theappearance of the extensive general review by Clegg 261 further advanceshave been reported in some important applications ; further general inform-ation on inorganic separations has been given by Burstall, Davies, Linstead,and Wells,262 and conditions have been described for the separation of thecopper and the tin gr0up,~63 and nickel and ~ o b a l t .~ 6 ~ A method for deter-mination of potassium 2G5 depends on development with sodium leadcobaltous hexanitrite solution, and measurement by planimetry. For workon 1-2 ml. of solution, strips of paper pulp 6 mm. thick have been used, fordetermination of thallium. 266Block 2G7 has given detailed directions for the separation and determin-ation of all the common a-amino-acids on one- and two-dimensional paperchromatograms; reports have been made on the suitability of differentpapers 268 and water-miscible solvents 269 for use in amino-acid separations.The method has been used for the identification of the amino-acids fromphenylthiocarbamyl peptides,270 and for the determination (by planimeter)of the amino-acid residues of insulin.271 Paper chromatography has beenapplied 272 to the separation of p-iodobenzenesulphonyl derivatives formedfrom amino-acids with a reagent containing radioactive 1311 ; the recoverywas measured by adding known amounts of known p-iodobenzenesulphonylderivatives containing 35S, immediately after forming the 1311 derivatives ofthe amino-acids. The bands were located by radio-autographs, andconstancy of l311/355 ratios in successive portions of the bands provided atest of the validity of the analysis.Chloroacetic acid and a-chloropropionic acid have been separated onpaper with use of indi~ators,~~3 and zones containing keto-acids have beenmade visible by conversion into the semicarbazones and viewing under ultra-violet light; 274 the method has been applied to small amounts of hydroxy-259 Marvel and Rands, J . Amer. Chem. SOC., 1950, 72, 2642.260 Ramsey and Hess, J . Assoc. 08. Agric. Chem., 1950, 33, 848.261 Analyt. Chem., 1950, 22, 48.262 J., 1950, 516.263 Lederer, Analyt. Chim. Acta, 1949, 3, 476.264 Lacourt, Mikrochena., 1950, 35, 262.265 Beerstecher, Analyt. Chem., 1950, 22, 1200.266 Anderson and Lederer, Analyt. Chim. Acta, 1950, 4, 513.2 6 7 Analyt. Chem., 1950, 22, 1327.268 Kowkabany and Cassidy, ibid., p. 817.268 Bentley and Whitehead, Biochem. J . , 1950, 46, 341.270 Edman, Acta Chem. Scand., 1950, 4, 283.271 Archer, Fromageot, and Justisz, Biochim. Biophys. Acta, 1950, 5, 81.272 Keston, Udenfriend, and Levy, J . Amer. Chem. Soc., 1950, 72, 748.273 Renard, Bull. SOC. chim. Belg., 1950, 59, 34.274 Magasanik and Umbarger, J. Amer. Chern. Soc., 1950,72, 2308.REP.-VOL. XLVII. 41 8 ANALYTICAL CHEMISTRY.benzoic acids and amides 275 and to p-aminobenzoic acid derivatives.276The behaviour of numerous phenolic compounds has been studied277 andapplied to analysis of tea catechins 278 and wood extracts; 279 in a veryelegant analysis of certain flavonol 3-glycosides, the pigment zones werelocated under ultra-violet light, leached from the paper with aluminiumchloride, and the ultra-violet absorption intensities of the flavonoid-aluminium complexes were measured on a Beckman spectrometer.280Halogenated aliphatic and aromatic hydrocarbons, and n-octanol havebeen held as the static phase on acetylated paper, for the separation of thedinitrophenylhydrazones of the carbonyl fission products of sugars.281 Themethod of Goodall and Levi 282 for penicillins has been improved, by usingmixtures of pure penicillins as standards,283 or by conversion into thchydroxamic acid derivatives, separation on paper strips, development withferric chloride, and extraction of the iron complexes for colorimetric measure-ment ; 284 application of the latter procedure in penicillin production controlhas been reported.285 Progesterone has been determined 286 in commerciallyprepared oils, by paper chromatography, location by the m-dinitrobenzene-potassium hydroxide reaction, extraction, and measurement by its ultra-violet absorption.Chromatography on ion-exchange resins, applied in elegant ways byteams of American investigators on rare earths, and by several independentworkers on amino-acids, has been reviewed in detail quite recently byTompkins 287 and Schubert,288 and little can be added in this Report.Miscellaneous.Numerous papers have appeared on subjects outside the broad classific-ations given in this Report, but few can be mentioned. The use ofthermistors for cryoscopic measurements 289 and apparatus for micro-determination of melting, transition, and segregation points down to -40"have been described; 290 Siggia and Hanna 291 have devised a method foranalysis of one-phase ternary mixtures by titration with one of thecomponents (immiscible with the others) until a turbidity results. Anempirical relationship between refractive index and wave-length has been2 7 5 Bray, Thorpe, and White, Biochem. J., 1950, 46, 271.276 Keleman, Tanos, and Halmagyi, ibid., 47, 138.277 Bate Smith and Westall, Biochim. Biophys. Acta, 1950, 4, 427.278 Bradfield and Bate Smith, ibid., p. 441.279 Lindstedt, Acta Chem. Scand., 1950, 4, 448.280 Gage and Wender, Analyt. Chem., 1950, 22, 708.281 KoBtii and Slavik, Coll. Czech. Chem. Comm., 1950, 15, 17.282 Analyst, 1947, 72, 277.281 Baker, Dobeon, and Martin, ibid., p. 651.285 Albans and Baker, ibid., p. 657.286 Haskine, jun., Sherman, and Allen, J . Biol. Chem., 1950, 182, 429.287 Analyt. Chem., 1950, 22, 1352.2e8 Ibid., p. 1359.290 Tschamber, Mikrochem., 1950, 55, 353,291 Analyt. Chem., 1949, 21, 1086.283 Glister and Grainger, ibid., 1950, 75, 310.289 Zeffert and Hormats, ibid., 1949, 21, 1430CROPPER : PHYSICAL AND PHYSICOCHEMICAL METHODS, 419worked out 292 for calculation of one dispersion from any other, for use inhydrocarbon analysis ; the use of refractive index-density charts has beenextended to the analysis of liquid halogen and oxygen compo~nds.~~3 A newform of dew point-bubble point apparatus, for molecular weights and foranalysis of binary liquid and apparatus for precision pH measure-ment with a glass electrode295 have been noted. Tadayon, Nissan, andGarner 296 have described apparatus for determination of magneto-opticalrotation and its application to analysis of ternary hydrocarbon mixtures. Adetailed review has been given 297 on automatic operations in quantitativeanalysis from the point of view of the individual unit operations involved inany general method of analysis.F. R. C.P. R. CROPPER.H. E. STAOG.H. N. WILSON.Sankin, Martin, and Lipkin, AnuZyt. Chem., 1950, 22, 643.298 Gilmore, Menaul, and Schneider, ibid., p. 892.204 Feller and McDonald, ibid., p. 338.295 Kraus, Holmberg, and Borkowski, ibid., p. 341,Ibid., 1949, 21, 1532.Patterson and Mellon, ibid., 1950, 22, 136

ISSN:0365-6217    

DOI:10.1039/AR9504700373

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

CRYSTALLOGRAPHY.1. INTRODUCTION.AGAIN this report is far from an annual report in scope-it now has tocover papers published over a period of four years. In its arrangement, wehave followed somewhat the plan we adopted last year. The first sectiondeals with one aspect of crystallographic technique, that of neutron crystal-lography, while the second section describes the crystal structures of organiccompounds studied in the four years, 1947-50.We have again found it impossible to deal with many interesting researcheswhich have by common consent fallen in past years within the scope ofcrystallographic reports-particularly X-ray scattering in liquids and parti-ally ordered systems, such as many high polymers. There are also interestingdevelopments of technique, such as the use of polarised infra-red and ultra-violet radiation in crystal analysis, to which a whole section of the reportmight well be devoted another year.In connection with crystal optics, theappearance of the new edition of “ Crystals and the Polarising Microscope,’’by Hartshorne and Stuart, is particularly welcome.2. NEUTRON CRYSTALLOGRAPHY.Introduction.-A new development has arisen in crystallography withthe advent of nuclear piles, namely the application of neutron diffraction toproblems of crystal structure. This was briefly mentioned in last year’sreport on crystallography but otherwise the topic has not been referred toin these pages. It is therefore proposed to survey the subject as a whole.As long ago as1936, Elsasser pointed out the tbeoretical possibility of the diffraction ofslow neutrons by crystalline materials, and in the same year the existence ofthe phenomenon was shown experimentally by Halban and Preiswerk,2and by Mitchell and power^.^ Monochromatic neutrons were, of course, notavailable and the experimental results were not such as to permit of anyuseful practical application, but nevertheless about a dozen papers on thesubject appeared up to 1940, mostly in Physical Review.With the building of nuclear piles the possibility arose of obtainingmonochromatic neutron beams of sufficient intensity to put neutron diffrac-tion on an entirely new basis, and since 1946 a steady stream of papers hasappeared.Reference will be made at this stage only to some of the moregeneral papers on the s u b j e ~ t .~ - ~The idea of neutron diffraction is, of course, not new.Elsassor, Compt. rend., 1936, 202, 1029.Halban and Preiswerk, ibid., 1936, 203, 73.Mitchell and Powers, Phys. Review, 1936, 50, 486.* Wollan and Shull, Nucleonics, 1948, 3, 8 , 17.Shull and Wollan, Science, 1948,108, 69.Wollan and Shull, Phys. Review, 1948, 73, 830.Bacon and Thewlis, PTOC. Roy. SOC., 1949, A , 196, 50.* Lonsdale, Nature, 1949,164, 205THEWLIS : NEUTRON CRYSTALLOGRAPHY. 421The first neutron spectrometer to be built was erected at the ArgonneNational Laboratory in 1945, and was used principally for nuclear-physicalexperiments requiring a monochromatic beam of neutrons. Serious attentionwas nevertheless also given at the outset to neutron scattering by crystals,and Zinn,g Fermi and Marshall,lo and Goldberger and Seitz l1 publishedimportant papers on this question.The main body of work on neutroncrystallography has, however, come from Oak Ridge where Shull and Wollanand their co-workers have published a series of' papers which will be referredto in more detail later in the report. Work of this type is also being carriedout at Chalk River in Canada, and at Harwell in this country.With this brief introduction a more detailed account of neutron diffractionwill now be attempted and a comparison with X-ray diffraction given.Some idea of the experimental techniques involved will be presented andapplications to crystallography described. A final section will be devoted tofuture developments.The Wave-length of Neutrons.-In a conventional nuclear pile the fastneutrons produced by fission are slowed down by repeated collisions withina " moderator " of heavy water or graphite until they are slow enough toprovide further fission.The favourable energy for the occurrence of suchfission is obtained when the neutrons are in thermal equilibrium with theirsurroundings a t or about room temperature. These thermal neutrons giverise to further thermal neutrons by the successive processes of fission andslowing-down, and if a collimator is inserted in the pile some of these neutronswill emerge from it in the form of a beam which may be used in neutron-diffraction experiments.The equivalent wave-length h of a beam of neutrons of energy E is givenby the expressionA = 2/(0.081/E) x lod8 cm.where E is expressed in electron-volts ; and the equilibrium temperature in apile, which is normally in the range 0" to loo", corresponds to a peak energyof several hundredths of an electron-volt ; so that the wave-lengths concernedare of the order of l ~ ., i.e. they are very like the X-ray wave-lengths usedfor crystal analysis and are, of course, similar to the inter-atomic distancesin crystals. For example, the peak wave-lengths corresponding to 0" and100" are 1.9 and 1 - 6 3 ~ . , re~pectively.~It is therefore to be expected that thermal neutrons will be diffracted bycrystals; but, since the distribution of energy among the neutrons in thebeam follows approximately the Maxwellian curve appropriate to theequilibrium temperature, there is nothing to correspond to characteristicradiation, and the beam is, in effect, "white." Fig.1, due to Bacon,12gives, for example, the variation of neutron counting rate with wave-lengthfor a typical equilibrium temperature of 50". It refers to the thermalcolumn of the Harwell pile, in which a mass of graphite ensures that theenergies of the neutrons present are, for the most part, in the thermal region.Zinn, Phye. Review, 1947,71, 752.I1 Goldberger and Seitz, ibid., p. 294.lo Fermi and Marshall, ibid., p. 666.l2 Bacon, unpublished data422 CRYSTALLOORAPHY .In view of this energy spread it is customary to monochromatise theneutron beam for use in neutron crystallography, usually by reflection at aflat crystal surface, although a bent-crystal monochromator has been usedfor neutron cross-section measurements.13 Fig.1 shows the band of wave--Bud sejectedat 8 = 175"6g monochromatorWave-length, A.FIG. 1.Spectrum of thermal neutrons as diflracted by calcium fluoride.lengths selected by a fluorite monochromator in use at Harwell. It isdeliberately chosen to be on the short wave-length side of the peak to avoid thecomplications that would arise from the presence of an appreciable second-order component in the monochromatised beam.X-Ray and Neutron Dieaction.-It has been seen that a neutron beam, asit emerges from a pile, is analogous to a beam of '' white " X-rays and that itis usually monochromatised for use in neutron crystallography.Only aboutone thousandth of the neutrons in a collimated pile beam are reflected from themonochromator. Photographic detection of neutrons presents considerabledifficulties for neutron crystallography is not generally applicable. In orderto achieve a sufficiently high counting rate in the boron trifluoride detectorsusually employed, neutron beams of 106-107 neutrons per second arecommonly used. To obtain neutrons in these numbers from the nuclearpiles at present available, very wide beams must be utilised, the cross sectionsof which are measured in square. inches, whereas those of X-ray beams are,of course, measured in square millimetres. As a result, neutron spectro-meters have had to be constructed on a massive sca1e.6v9*14*16 Theyresemble gargantuan X-ray spectrometers, may weigh several tons and arecorrespondingly expensive. Recent developments at Harwell suggest thatthis massive equipment may not always be necessary, but more will be saidlater about techniques in general. At this stage it is sufficient to point outthat neutron diffraction demands different techniques from X-ray diffraction.Sawyer, Wollan, Bernstein, and Peterson, Phys.Review, 1947, 72, 109.Hurst, Pressesky, and Tunnicliffe, Rev. Sci. Instr., 1950, 21, 705.l5 Bacon, Smith, and Whitehead, J . Sci. I'nstr., 1950,27, 330THEWLIS : NEUTRON CRYSTALLOGRAPHY. 423Not only, however, does neutron diffraction differ from X-ray diffractionin the techniques employed, it differs fundamentally in certain underlyingphenomena.Perhaps the most obvious difference is in the extremely lowabsorption of most elements for thermal neutrons. Table I gives the massabsorption coefficients Bf a number of common elements for thermal neutronsand for X-rays (Fe K J , together with those for lithium, boron, cadmium, andgadolinium which are among the most highly absorbent elements for thermalneutrons. It will be seen that even these elements have mass absorptioncoefficients which are of only the same order as that of most elements for acomparable X-ray wave-length. For other elements, the neutron absorptionis very much less indeed.TABLE I.X-Ray and Neutron Absorption Coeficients.Mass absorption coefficient.X-Rays NeutronsElement. At.NO. (A = 1.934.). (A = 1-84.).Lithium ............... 3 1.5 5.8Boron .................. 5 5.8 38.4Carbon ............... 6 10.7Aluminium ............ 13 92.8 0.005Iron ..................... 26 72.8 0.026Bromine ............... 35 169 0.057Silver ............... 47 402 0.32Cadmium ............ 48 417 13.0Gadolinium ......... 64 199 183.0Gold .................. 79 390 0.29Lead .................. 82 429 0.00060-00023Copper ............... 29 98.8 0.032Another difference in absorption is that, whereas for X-rays the massabsorption coefficient varies in a regular fashion with atomic number, forneutrons there is no such regularity. The latter fact has no particularbearing on problems of neutron crystallography, but the extremely low valueof neutron absorption that occurs in general means that multiple scatteringis possible, with a consequent increase in background intensity; and that,for imperfect crystals, extinction plays a much more important part thanabsorption, with striking effects on the integrated intensity.The latter wasfirst pointed out by Bacon and Thewlis,’ and the theory was developed indetail by Bacon and Lowde.16 It will be recalled that, for XLrays and perfectcrystals, the integrated reflection is proportional to the structure factor F,“ true ” absorption playing no part : this is also true for neutrons. I npractice, however, crystals are usually ‘‘ imperfect,” and the integratedX-ray reflection is then proportional, apart from small extinction effects,to F2.In the case of neutron diffraction, on the other hand, the integratedreflection may be controlled almost entirely by secondary extinction.Indeed, for a thick mosaic crystal it is, in general, independent ofstructure factor, increasing with the degree of mosaic spread. The theorypredicts however that, for very thin crystals, the integrated reflection willbe proportional to F2, and also suggests that there will be a range ofl6 Acta C y s t . , 1948, 1, 303424 CRYSTALLOGRAPHY.somewhat greater thicknesses where it will be proportional to F. As pointedout by Bacon and Lowde,ls the latter result explains the observations madeby Fermi and Mar~hall,~ who found in a number of cases that their measuredintensities were proportional to P rather than F2. Recent quantitativeresults obtained by Bacon l2 confirm the theory, and it seems fair to concludethat, if single crystals are to be used successfully in neutron crystallography,they will have to be much thinner than those used by Fermi and Marshall.So far, use has been made of transmission through blocks of powdered crystal,where the situation is not complicated by extinction, and where diffractedbeams of adequate power may be obtained by using large specimens andneutron beams of large cross-section.The reflecting power of such a blockis proportional to F2 just as for X-rays.There are other differences between X-ray and neutron diffraction thatmust be taken into account to complete the picture. For example, neutronscattering is in general spherically isotropic, since such scattering is a nuclearphenomenon.In other words the f-curve takes the form of a horizontalstraight line. Lonsdale has suggested * that this means that only " trialand error " methods of structure analysis are possible. In the Reporter'sopinion, however, it should be possible in principle to use the usual Fouriermethods in neutron crystallography, for example by using the device of anartificial temperature factor. Even so it would appear to be foolish toexamine an unknown crystal by neutron diffraction until all possible inform-ation had been obtained by X-ray difhaction.Another difference is concerned with the intensity of scattering per atom.For X-rays, of course, this increases with atomic number and is proportional,other things being equal, to the square of the atomic-form factor; but forneutrons, where the scattering is by the nucleus, it appears to be quiteunpredictable. Not only does the scattering power per atom vary a t randomfrom atom to atom, but also from isotope to isotope of the same atom;moreover the sign of the scattering amplitude may also vary in the sameway.For X-rays the scattered wave from an atom is 180" out of phasewith the incident wave; and whereas for neutrons this is also true in mostcases, the scattered and incident waves are in phase for certain isotopes, andhence for certain elements. By convention the scattering amplitude isregarded as negative in the latter cases, and it is so shown in Table 11, inwhich X-ray- and neutron-scattering amplitudes are given for a number ofelements and individual isotopes.It will be noticed that different isotopesof the same element (e.g. lithium and nickel) can have scattering amplitudesof different signs.In neutron diffraction there is the possibility of much more " background "scatter than in X-ray diffraction. In the latter case some background scatteralways results from the temperature effect and from the Compton effect;the excitation of fluorescent radiation and the presence of a disordered ordeformed structure in the material under investigation will also contributeappreciably in appropriate cases. Of these possible causes, only three canoperate in neutron diffraction, namely the temperature effect and the disordeTFIEWLIS : NEUTRON CRYSTALLOGRAPHY. 425and deformation effects, but the absence of the others may be more thancompensated for by new effects which have no parallel in X-ray diffraction.One of these, multiple scattering, has already been noted ; but the main effectarises from the fact that almost all structures are disordered from the point ofview of neutron scattering.A disordered structure is essentially a structurein which the atomic scattering power varies randomly from site to site in thestructure. In X-ray diffraction, this arises in a disordered alloy, for example,TABLE 11.Ordered scattering amplitudes * *for X-rays and thermal neutrons.Scattering amplitude.1' Scattering amplitude."Element.HDLiBeCN0FNa 3 8c1KCaTiVCrMnFeAt.NO.11346789111213161719202223242526X-Rays (forsin ep=o.s)x 10-12 cm.0.020.020.28 { 6Li'Li0.390.480.540.620.751-141.351-551.932.042-232-37 { 40Ca44Ca2.682.852.993-13rThermalneutronsx lo-" cm.- 0.40-0.18- 0.250.640.70.780-640.850.580.550.350.440.350.3 10.990.350.490-490.18- 0.38(0.09 -f0.370.960.421.000.23- 0.33Element.coNicuZnGeAsseBrRbSrZrNbMoPd4 3SnSbI csTaWPtAuT1PbBiAt.No.2728293032333435373840414246475051535573747879818283X-Ra s(forx lo-" cm.3.42sin e:K=o.b>r3.58 {::;:62Ni3.753-924.234.404.544.7 15-055.195.565-705-87 :::: { l07Ag7-227.427.758.0911.2511.4212.1712.3712.7012-9013-10lo9AgThermalneutronsx 10-1* cm.0.281.031.480.280-760.590-840.630.890.670.550.570.620.690.640.630.610.830.430-610.540.520.490.700.5 10.950.770.75 l80.960.89- 0.85* For meaning of ordered amplitude see preceding paragraph.t Sign of amplitude in doubt owing to small magnitude.from the random occurrence at the various atomic sites of different kinds G fatom. In neutron diffraction it can actually arise for a single element, sincethe nuclear scattering power may vary in a random fashion from site to siteon account of the random distribution of isotopes and sometimes of nuclearspins. It has already been seen that different isotopes of the same elementhave, in general, different scattering powers, and the existence of isotope-disorder should be a t once apparent.The existence of spin-disorder also[Added in proof:These figures have now been published-Shull and Wollan, Phys. Review, 1951,81,527.]1' Figures except those for T1, kindly supplied by Dr. C. G. Shull.I* Winsberg, Meneghetti, and Sidhu, ibid., 1949,76, 975426 CRYSTAILOGRAPHY.follows when it is realised that the scattering amplitude of a nucleus will bedifferent according as its spin is parallel or antiparallel to that of the neutron,and that spins, like isotopes, are randomly distributed. Where the nucleushas no spin, as in the case of isotopes of even atomic mass above 14N,there will, of course, be no spin-disorder.In some cases, e.g. hydrogen,the disordered scattering is so great that the " Bragg " or ordered scatteringamplitude (Le. the amplitude scattered into the Bragg peaks) is very muchless than the total scattering amplitude, but in many others10 there isevidence that the dependence of scattering amplitude on spin orientation andisotopic constitution is not so marked. With hydrogen the total scatteringamplitude is 2.5 x cm. whereas the ordered scattering amplitude isonly 0.4 x 10-l2 cm., a difference that arises largely from spin-disorder. Indeuterium on the other hand, although the ordered amplitude is not muchgreater, namely 0.63 x cm., the spin-disorder is relatively low, and it isoften advisable, therefore, to carry out neutron-structure determinations ondeuterated compounds if possible.This has been done for sodium hydridc 19and ice 2o with results to be described below.There are four other ways in which neutron diffraction differs fromX-ray diffraction. The first is concerned with the binding of the nuclei,and the second with inelastic scattering of the neutrons. In addition, furtherscattering (both elastic and inelastic) occurs for ferromagnetic * and stronglyparamagnetic substances on account of an interaction between the magneticmoment of the neutrons and the orbital magnetic moments of' the atomicelectrons. Finally there is the fact that neutrons are not, in general,polarised on scattering, except in a few cases where polarisation arises fromthe effect of magnetic fields.Theneutron-scattering amplitude for an element is different according as theatoms are free (as in a gas) or bound (as in a liquid or solid).The ratio ofthese two amplitudes is A / ( A + 1) where A is the mass number of the isotopeconcerned, and it will be seen that this ratio is effectively unity for all butlight atoms.The possibility of appreciable inelastic scattering arises from the fact thatthe frequency of vibration of the thermal neutrons is of the same order as thatof the atomic vibrations (about 1013 per second). In consequence a neutronwill lose an appreciable fraction of its energy in exciting a quantum ofvibrational energy ; in other words appreciable inelast.ic scattering (i.e.scattering with a change of ware-length) will occur, which will again manifestitself as an increase in background scattering.Weinstock 21 has calculated,for a " free " atom of polycrystalline iron, that 0.6% of the scattering will beinelastic at absolute zero and that this will rise to 19% at 1000"~. Cassels 22We will now revert to binding and inelastic scattering effects.19 Shull, Wollan, Morton, and Davidson, Phys. Review, 1948, 73, 842.2o Wollan, Davidson, and Shull, ibid., 1949, 75, 1348.2 1 Weinstock, ibid., 1944, 65, 1.22 Cassels, " Progress in Nuclear Physics," (Frisch) , Butterworth-Springer, London,* Antiferromagnetic substances are referred to later.19.50, Ch.8THEWLIS : NEUTRON CRYSTALLOGRAPHY. 427has more recently treated the problem in some detail. I n the correspondingX-ray case the loss of energy is too small to be noticed, since the frequency ofvibration of X-rays is about 1018 per sec. and the quantum energy is thereforeof an entirely different order of magnitude.To summarise, then, the main differences between X-ray and neutrondiffraction may be stated as follows :Phenomenon. X-Rays.Absorption Regular variation with in-creasing atomic number.Extinction Subordinate to absorption.Bragg scattering Electronic atomic-form factorexists. Scattering power peratom increases regularlywith atomic number.Scattered wave 180' out ofphase with incident wave.Effect of thermal Intensity reduction accordingvibrations to Debye-Waller formula.Diffuse scattering.Nomeasurable change in wave-length.Polarisation Scattered wave is polarised.All isotopes of same elementhave same scattering power.Isotope effectSpin effectNeutrons.Irregular variation. Absorptionnearly always small. Back-ground increased by multiplescattering.Generally outweighs absorption sothat large single crystals im-practicable for much structurework. Powders must be usedin these cases.Nuclear scattering sphericallyisotropic. Scattering powervaries at random.Scattered wave usually 180' out ofphase, but eometimes in phase.Similar phenomena to be expected,but also appreciable amount ofinelastic scattering with largechange in wave-length and in-creased background intensity.Scattered wave is not polarisedexcept for magnetic effects'Isotopes may have different scat-tering powers, resulting in iso-tope-disorder and increase inbackground intensity .Scattering power is different, forfinite spin, according as this isparallel or anti-parallel.Re-sults in spin-disorder, withincrease in background scatter - - ing.Experimental Techniques.-As already indicated, measurements inneutron crystallography are usually made with the aid of wide neutron beamsand large spectrometers in which provision is made for monochromating thebeam, and the neutrons are detected by means of a boron trifluoride counter.Laue photographs have been taken by Wollan, Shull, and Marney23 by amethod involving the use of an " intensifying screen " of indium foil, whichemits (3-rays when bombarded by neutrons; but, apart from this one type ofapplication, photographic methods have not been used in neutron-diffractionwork up to the present.The reason for this is obvious when one considersthat the exposure time for even a Laue photograph is about 10 hours.The boron trifluoride counter depends on the reactionliB + in --+ iLi + :He.ms Wollan, Shull, and Marney, Phys. Review, 1948,73,627428 CRYSTALLOGRAPHY.It is arranged to record the a-particles emitted in this reaction, but is insensi-tive to y-rays. Ordinary boron contains only about one part in five of thel0B isotope, so that whenever possible separated loB is used to make theboron trifluoride gas with which the counter is filled; the efficiency of thecounter may then be as high as 80 or 90%.Great care is necessary to shield the counter from unwanted radiation,and it is also necessary to keep a watch on variations in the intensity of theincident neutron beam by some form of a monitor, for example a fissionchamber may be inserted in some convenient place in the pile.The mono-chromating crystal may take the form of the usual flat plate or the crystalmay be cut obliquely, as first recommended by Stephen and Barnes z4 andlater used by Fankuchen 25 for X-ray work, so as to " foreshorten '' thereflected beam with consequent increase of intensity. The type of equipmentin use a t Oak Ridge is a restricted double-crystal instrument, using sodiumchloride or a metal single crystal as the monochromating crystal, capable ofemploying a single wave-length only.* A true double-crystal instrument is inuse a t Harwel1,l53n which both monochromating crystal and specimen maybe rotated a t will.This spectrometer is also mobile. As in most of theneutron spectrometers so far used the counter arm is arranged to turn a ttwice the angular speed of the specimen table, so as to enable the counter toreceive the reflected beam for single crystal work and to maintain the sym-metrical transmission position for powder work.A rather different type of technique is also being developed a t Harwell 26in which an attempt is being made to reduce the dimensions of the neutronspectrometer to the X-ray scale, and to use small single crystals, again as inX-ray techniques.The use of such crystals should, of course, overcome theextinction problems already referred to, and the lack of neutrons is beingovercome by not monochromatising the beam (thus gaining a very largefactor), setting up the specimen in the reflecting position for a given plane andwave-length, and placing a miniature counter (made of multiple boron-coatedfoils) 27 in the correct position to receive the reflected beam. In this way, asingle measurement of counting rate gives the required integrated reflection.Difficulties will undoubtedly arise from the presence of unwanted high-ordercomponents in the beam, and, of course, reflections must not overlap; butpreliminary results have been very encouraging, and the ratio of peak count-ing rate to background appears to be such that hydrogenous crystals may beexamined directly without the need for deuteration.The possibility ofusing orthodox methods is being examined, and the first results suggest that,given an increase in neutron flux of only ten or twenty over that availablein the Harwell pile,? monochromatic beams might be used which wouldpermit rotating-crystal techniques exactly analogous to those used ineveryday X-ray analysis.24 Stephen and Barnes, Nature, 1935,136,793.26 Lowde, ibid., 1951, 167, 243.* [Added in proof.]t Such as the flux provided by the heavy-water pile at Chalk River.2s Fankuchen, ibid., 1937, 139, 193.e7 Idem, Rev. Sci. Instr., 1950,21,835.A true double-crystal instrument is now in operation atOak RidgeT H E W S : NEUTRON CRYSTALLOGRAPHY.429Applications of Neutron Diffraction to CrystaUography.-The firstpossibility that suggests itself, when the phenomenon of neutron diffractionby crystals is considered, is that of the determination of the positions oflight atoms in a structure. It is well known that it is very difficult, andoften impossible, to determine by X-ray analysis the positions of light atomsin a structure in which heavy atoms are also present, since the contributionsof the latter swamp those of the former. I n the case of neutron diffraction,however, since the scattering powers of all atoms are roughly of the sameorder, it should clearly be possible to determine the positions of light atomsin the circumstances considered.In particular it should be possible to deter-mine the positions of hydrogen atoms, although, as already explained, therelatively high background scattering from hydrogen makes i t advisable tosubstitute deuterium if possible. The first work of this kind was done byShull, Wollan, Morton and Davidson l9 on sodium hydride and sodiumdeuteride. They showed that the structure is the sodium chloride structureand found, as expected, a much greater amount of background scatter withsodium hydride than with the deuteride. Also, owing to the fact thatsodium and deuterium scatter neutrons with a positive scattering amplitudeand hydrogen scatters with a negative scattering amplitude, a marked reversalof the relative intensities of the 111 and 200 reflections was observed insodium hydride and sodium deuteride.Further work on the structural positions of hydrogen atoms, this time byWollan, Davidson, and ShuIl,20 has been carried out on ice.Ice made fromheavy water (D,O) was used and maintained at -90" during the experi-ments; from the results i t was possible to decide between the various struc-tures that had been proposed, although the resolution was not sufficient toenable all the lines to be separated. Intensity measurements were made onan absolute scale, and supported the model proposed by Pauling on thegrounds of the existence of residual entropy a t low temperatures, in whichthe hydrogen molecules possess some randomness, one hydrogen atom andone only lying on each of the lines joining neighbouring oxygen atoms.Since the oxygen atoms are arranged tetrahedrally this means, in effect, thatthere are, on the average, four half-atoms of hydrogen arranged tetrahedrallyaround each oxygen atom, as shown in Fig.2.Such a random type of structure should give rise to disordered scatteringof its own, and such disordered scattering was indeed observed, the measuredand calculated values agreeing quite well.A second type of application, which also utilises the difference betweenthe relative scattering powers of different elements for X-rays and neutrons,is the demonstration by Shull and Siegel 28 of the existence of superlatticelines in ordered samples of FeCo and Ni,Mn. In these two alloys the X-ray-scattering powers of the elements involved are practically identical, whereasthe neutron-scattering powers, as can be seen from Table 11, are sensiblydifferent, indeed manganese has a negative scattering amplitude.I nconsequence Shull and Siegel were able to reveal the existence of superlattice28 Shull and Siegel, Phys. Review, 1949,75, 1008430 CRYYTAIJAOORAPHY.lines which could not be obtained by X-ray methods. Conversely, as mightbe expected, they were unable to show the existence of such lines in Cu3Auby neutron diffraction, although X-rays show them up quite clearly.Shull and Siegel, in this connection, make sn interesting suggestion, basedon the fact that the neutron scattering amplitudes of various isotopes mayvary not only in magnitude but in sign.They take the case of nickel, forwhich the scattering amplitude is +0.3 for and + l o 4 1 for 58Ni, andcompare it with manganese, for which the elemental scattering amplitude is-0-32; and suggest making up an alloy Mn60 Ni, for which the intensitiesof the usual diffraction lines should be vanishingly small, while the super-lattice lines should be quite strong.Other applications of a metallographic nature have been carried out bySidhu,2Q who showed, by transmitting monochromatic neutrons throughsolid solutions and mixtures of titanium carbide and tungsten carbide, thatthe scattering power was related in a definite way to the degree of solid 8Fra. 2.solution obtaining; and by Arnold and Weber,30 who showed that theeffects of the preferential orientation of aluminium on the observed intensitiesof neutron reflection agreed, for various orientations, with those expected.Naturally a considerable amount of work has been devoted to measure-ments of scattering amplitude, since these are essential before neutroncrystallography can be developed. As will be seen from Table I1 values formany elements and some separated isotopes are now known.Work has been carried out on liquids by Chamberlain:' and on gases byAlcock and Hurst 32 and Spiers.= Chamberlain's results on sulphur, lead,and bismuth agree well with those obtained by X-ray examination.Alcockand Hurst, and Spiers, working on oxygen, carbon dioxide, and deuteriummade the interesting discovery that the correct but complex and laboriousquantum-mechanical method of calculating the neutron scattering by a gasgives a result not very different from a semi-classical calculation in whichSidhu, J.Appl. Phys., 1948, 19, 639.Arnoldand Weber,Phys. Review, 1948,73,1385. s1 Chamberlain,ibid., 1950,77,305.s2 Alcock and Hurst, &bid., 1949,76, 1609. aa Spiers, W., 1949, 75, 1766T H E W S : NEUTRON CRYSTALLOGRAPHY. 431the neutron is represented by a wave and the molecule by a rigid system ofpoint scatterers, the normal procedure for X-ray scattering being used.An application of a somewhat different kind has recently been reportedby Bacon,= who has studied the electron distribution in graphite by acomparison of X-ray and neutron intensities. The X-ray intensity of the1010 line relative to that of the 1120 is found to be about 85% too high, andthat of the 1011 relative to the 1122 to be about 80% too high, and Franklin 35has suggested that this can be explained if the L-electrons are not distributedspherically about the carbon nucleus but are concentrated about the centresof the C-C bonds.If the explanation is indeed concerned with electrondistribution and not with atomic positions the corresponding intensitiesobtained by neutron diffraction should show no anomalies since the scatteringcentres are the nuclei and the electrons play no part. In fact Bacon finds noanomalies, although he restricts his comparison to the ratios of the theoreticaland measured intensities of the pairs of lines 1010 and 1011, and 1120 and 1122,owing to lack of resolution.Another novel application of neutron diffraction is t o the detection ofantiferromagnetism.36 An antiferromagnetic material is one in which belowa certain temperature, the Curie temperature, the magnetisation directionsof neighbouring pairs of atoms are opposed, so that no net spontaneousmagnetisation exists.Above the Curie temperature the thermal energy issufficient to overcome the tendency of the atoms to set themselves in anti-parallel pairs and the behaviour is that of a normal paramagnetic substance.Since, as already mentioned, there is magnetic scattering of neutrons in thecase of certain magnetic atoms, the possiblity arises of the existence ofsuperlattice lines in the antiferromagnetic state, owing to the differentscattering amplitudes of the parallel and antiparallel atoms.In manganousoxide, which has the sodium chloride structure, Shull and Smart have shownvery beautifully the presence of magnetic superlattice lines below theCurie point (122°K.) of this material. At these temperatures the chemical(and X-ray) unit cell has a side of 4.43 A. whereas the magnetic unit cell sideis 8.85 A. Similar effects have also been found for a-ferric oxide. Neutrondiffraction has thus provided a direct way of detecting the orientation ofmagnetic moments.Future Developments.-Future developments in such a new subject asneutron crystallography are difficult to forecast, but it seems reasonable toassume that work will continue on the determination of the positions ofhydrogen and other light atoms in simple substances; and that this will beextended to more complex structures as suitabJe methods are developed andsingle-crystal techniques are perfected, as they must be if adequate progressis to be maintained.This stage of the development will call for the closestco-operation between X-ray and neutron diffraction to make full use of thefact that, in effect, we can alter the relative scattering powers of the atoms ina structure, and sometimes the scathering phases, although the atoms them-34 Bacon, Nature, 1950, 166, 794.a6 Shull and Smart, Phy8. Review, 1949, 76, 1256.a6 Franklin, ibid., 1950,165, 71432 CRYSTAIXOGRAPHY.selves remain in exactly the same configuration. Work on metallic order-disorder will also no doubt proceed where the scattering powers of the metalsconcerned warrant it, as, for example, in the brasses, and there is undoubtedlyscope for the determination of structures containing atoms which are closeneighbours in the Periodic Table, and for which the neutron-scattering ampli-tudes may differ more than the X-ray-scattering amplitudes.New types ofapplication, such as to antiferromagnetism and to problems of electronicstructure, will presumably continue to arise. One such application, suggestedby C a s ~ e l s , ~ ~ is to the study of atomic vibrations by the investigation of thechanges of wave-length that occur when inelastic scattering takes place.Problems concerned with the determination of the nature or degree ofpreferential orientation may well present a wide scope for neutron crystal-lography, since the whole body of a specimen may be examined, and not onlyits surface.Indeed this possibility of examination in depth may well turnout to be of great importance for many types of problem. It should bepossible to make measurements on large pieces of material and average theresults over a considerable volume, which would be quite impossible by X-rayexamination owing to the large absorption involved. It should, for example,be possible to determine the orientation of large single crystals which couldnot otherwise be examined.Another possible type of application is to phenomena which are difficultto deal with by X-rays on account of the falling off in scattering power withincreasing angle.For example problems could be attacked which areconcerned with the effects of temperature or other disturbing influences onthe stability or nature of the atomic arrangement. I n much of this work,as already indicated, it will be advantageous if not essential to combine theresults of X-ray and neutron diffraction ; partly because they will in so manyrespects turn out to be complementary, and partly because the pressure ofwork on any one neutron spectrometer is likely to be heavy. It seemsdesirable indeed to restrict the use of neutron dift'raction to critical experi-ments as much as possible, so as to prevent the overloading of the facilitiesavailable, and it is clearly undesirable to carry out experiments by neutrondiffraction that can be done just as well by X-rays.One should obtain allthe information possible by X-ray examination and turn to neutron diffrac-tion only when necessary. In this way it should be possible to make thebest use of this powerful new crystallographic tool.J. T.3. ORGANIC COMPOUNDS.I n early Reports on crystallography, the organic compounds on which adetailed X-ray analysis was carried out were rather few, and these wereoften grouped with similar-sized inorganic molecules as a combined group-molecular crystals. Any measurements that led fo a set of proposed atomicpositions within the crystal of an organic compound appeared a notableachievement. Now we recognise that an approximate structure analysis,Caeseb, private communicationHODGKIN AND PITT : ORQBMC COMPOUNDS.433giving atomic positions based even on calculated electron densities can becarried out relatively easily by present methods for most types of organiccompound, crystalline at ordinary temperatures, and with molecularweights of less than about 200. We have about 120 such analyses to reportthis year; these provide a variety of interesting evidence on the way in.\ ' .J2 3I J A. 0 1SaleFIG. 3.A section through the plane of the athracene molecule. Electron-densitycontours are at intervals of + 8. A.-~, the Erst one dotted.[Reproduced, by permission, from Acta Cryst., 1950, 3, 254.1which molecules pack in crystals and on the factors determining the dis-position in space of non-bonded atoms, while as many as twenty of thetotal number lead to the direct deduction of the correct chemical or stereo-chemical formulation of molecules of imperfectly known structure.But itis clear that the conditions under which the exact measurement of bondlengths and electron densities can be achieved through X-ray analysis haveto be carefully considered in relation to each individual structure. Inonly a handful of the structure analyses described can %he bond lengths b434 CRYSTALLOGRAPHY.considered to be accurate to within rt0.02 A., limitations being imposed inmost cases either by the nature of the available experimental data or byincomplete refinement of the electron-density series.I n the most careful structure analyses reported, such as those oft h r e ~ n i n e , ~ ~ na~hthalene,~~ and anthra~ene,~~ the electron-density dis-tribution can be calculated sufficiently accurately to provide evidence ofthe positions of hydrogen atoms (compare Fig.3), but only very tentativeconclusions can yet be drawn, even in cases such as these, on the characteror shape of the chemical bonds. Brill*l has re-examined the structure ofdiamond from this point of view, and has shown that the differences betweenobserved and calculated structure factors for diamond, summed as a Fourierseries, do give an electron distribution which is most marked along the linejoining the two atoms-the covalent bond; here it corresponds to a densityof about &-+ electron per bond. In graphite, too, as mentioned above,deviations between calculated and observed structure factors can becorrelated with an asymmetric electron-density distribution.I n both ofthese structures there have been interesting new measurements of latticeconstants. Lonsdale finds that the carbon-carbon distance varies a littlein different diamonds from 1.54465 to 1.54444 A . , ~ ~ figures which agree wellwith the mean value given by Riley, 1.54453 A j 3 I n graphite the c dimen-sion varies with crystallite size-it appears to be an almost linear functionof the reciprocal of the number of carbon layers.44 In certain carbons,produced by pyrolysis of polydichloroethylene, highly perfect graphiticlayers about 16 A. across have been shown to be present, usually arrangedin pairs a t a mean distance of about 3.7 A.45 Such graphitic layers are notmuch larger than the largest aromatic molecules of which detailed X-rayanalyses exist.Some of the general principles determining the ways in which moleculespack in organic crystals have been the subject of a number of recentreviews,46 and were also discussed in an earlier Report.47 As more crystalstructures are analysed, we can examine the situation in greater detail.It is possible to collect groups of compounds that fall into well-definedcrystal-structure types and then to examine the deviations that occur inindividual cases on account of the actual peculiarities of molecular shapeor inter-atomic attractive forces.A number of such structure types havebeen recognised for a long time.For example, small roughly sphericalmolecules such as tetranitromethane 48 or quinuclidine 49 pack in crystal38 Shoemaker, Donohue, Schomaker, and Corey, J . Amer. Chem. Soc., 1950,72,2328.40 Mathieson, Robertson, and Sinclair, ibid., 1950, 3, 245, 251.41 Ibid., p. 137.43 Nature, 1944, 153, 587.44 Bacon, Acta Cryet., 1950, 3, 137.d6 Macgillavry, Chem. Weekbkzd, 1948, 44, 169; Kitaigorodski, Uepekhi Fiz. Nauk.4 7 Powell, Ann. Reports, 1946, 45, 88.4i1 Nowacki, Helv. Chim. Acta, 1946, 29, 1798.Abrahems, Robertson, and White, Acla Cryst., 1949, 2, 233, 238.42 Phil. Trans., 1947, 240, A , 219.4 6 Franklin, ibid., p. 107.1948, 34, 122; Uspekhi Khim., 1948,17, 287.Oda and Matsuba, X-Rays, 1950,6, 27HODCfKIN AJ5iD PIT" : ORGANIC COMPOUNDS.435structures based on body-centred or face-centred cubic close packing. Itis characteristic that at ordinary temperatures these crystals have toohigh a symmetry corresponding to molecular disorder or rotation. Recentstructure-factor calculations, however, indicate that disorder is not complete-there are usually certain preferred orientations of the molecular axes inthe crystals. In hexamethylenetetramine one can see the situation atthe other end of the order-disorder scale. Calculations by Schaffer M,show that the best agreement with the observed structure factors can beobtained by assuming that the molecule is performing anisotropic oscillations-the calculated root mean square amplitude normal to the radius joiningan atom to the molecular centre is 0.26 A.With short-chain molecules,somewhat similar conditions are found. The chain axes may take uproughly criss-cross positions in relation to one another, and rotation oroscillation is usual a t high temperatures. As the chain length increases,parallel chain packing in at least one layer is the rule, though in succeedinglayers various crossed arrangements are being discovered in the structuresstudied. With small aromatic molecules two varieties of packing arecommon-either parallel disc packing, with an interval between layersof about 3.5 A,, similar to graphite, or a criss-cross arrangement with thedisc centres in nearly hexagonal close packing. The first is shown byhexaniethylbenzene, several pyrimidines, 61 and coronene with one celldimension 3 .5 4 4 A., the second by durene, aniline hydrochloride,62dibenzyl, and many others, with the shortest cell dimension between 5.3and 6.5 A.The condition of close packing may be disturbed by the existence ofparticular active groups in the molecule, each of which favours certaintypes of arrangement of neighbouring atoms. Regular co-ordinationpolyhedra about ions can seldom be established in organic crystals, butthere is a marked tendency for the co-ordination numbers and interionicdistances found among inorganic crystals to be preserved. Thus sodiumhas six oxygen neighbours in sodium ben~ylpenicillin,~~ potassium six inpotassium decanoate (capr~ate),~* seven in potassium benzylpenicillin,53calcium eight 5s and strontium eleven 66 in calcium and strontium formates, theform of the co-ordination polyhedra being in all cases quite irregular.Inthe case of negative ions, such as the halogens, the co-ordination polyhedraare smaller-usually only three or four atoms are involved s2-and theposition of these often suggests hydrogen bonding. When halogens occurin the un-ionised state, the molecules are frequently so arranged that thehalogen atoms of neighbouring molecules are in contact-presumably owingto the greater contribution they make to van der Waals interaction. Aparticularly striking example is provided by the three varieties of p-iodo-bo J . Arner. Chem. SOC., 1947, 60, 1557.sa Crowfoot, Bunn, Rogem-Low, and Tumer- Jones, " The Chemistry of Penicillin,"64 Vand, Lomer, and Lang, Actcr Cryst., 1949, 2, 214.ss Nitte and Osaka, X-Rays, 1948, 5, 37.Clewsand Cochran, Acta Cryst., 1948,1,4.b* Brown, dbid., 1949, 2, 228.Princeton Univ. Press, 1949, p. 310.Nitta and Sailo, ibdd., 1949, 5, 89436 CRYSTALLOGRAPHY.N-picrylaniline, in all of which the iodine atoms of neighbouring moleculesare within the van der Waals distance of one another.67 Again, whereverhydroxyl or amino-groups are present hydrogen- bonding systems can betraced. Some of these form particularly stable types which are repeatedwith variations, e.g., substitution of nitrogen or chlorine for oxygen, in anumber of crystal structures. One might expect, for example, a closerelation to exist between the system found in phloroglucitol dihydrate mand diammoniate where the water or ammonia molecules link threeneighbouring phloroglucitol molecules together and are themselves linkedthrough a fourth bond.It is more surprising that a similar system is foundin 2-hydroxy-4 : 6-dimethylpyrimidine dihydrate 6o where a planar moleculeis substituted for the puckered cyclohexane ring.Distances described by different authors as hydrogen bonds range fromall lengths between 2.51 A. in oxalic acid dihydrate to 3.2 or 3.3 A. incompounds involving nitrogen (the term has even been used for theN . . . . HC distances in hexamethylenetetramine which are 3.88 A. long !).It is clear that very varying degrees and probably also types of interactionare involved in different structures.Within the range of " hydrogen-bonded " distances there are also now observed a number of short distancesinvolving carbon to which the term is not usually applied. Some of these,as for example, the short contact CH . . . 0, 3.28 A., in threonine, seem tobe forced on the molecule by packing considerations. Others, such as thedistance, 2.66 A. in p-nitroaniline,61 between one oxygen and the benzenering carbon atom, appear to have a more specific character. A particularlyinteresting example is provided by the silver perchlorate-benzene complex .62Here each silver ion is a t a distance of about 2.6 A. from two carbon atomsof each of two benzene rings, an arrangement which suggests x-bonding.The perchlorate ion is pushed away from one side of the silver to makeroom for the benzene rings.Once normal stereochemical standards of molecular shape have beenestablished, it becomes interesting to examine the conditions under whichdistortions from these occur.The necessity for a special packing, or theformation of a particularly stable hydrogen-bond system, appears sufficientto deflect the bond direction between an atom and attached benzene ringfrom the normal position directed to the ring centre. In tetraphenylcyclo-butane,63 for example, the deflection of one such bond is 7' from the planeof the ring. The distortion of an aliphatic chain from the planar zig-zagform would be expected to be much easier; so far, one or two examplesonly have been observed. A distortion from the planar form of the benzenering itself must, on the other hand, be much more difficult.In the only6 7 Grison, Actu Cryst., 1949, 2, 410.1.9 Anderson and Hassel, Acta Chem. Scad., 1948, 2, 527.Idem, Nature, 1949, 163, 721.6 1 Abrahams and Robertson, ibid., p. 252.62 Rundle and Goring, J . Amer. Chem. SOC., 1950, 72, 6337.6s Dunitz, Actu Cryst., 1949, 2, 1.6o Pitt, Actu Cryst., 1948, 1, 168HODGKIN AND PITT : ORGANIC COMPOUNDS. 437example, di-p-xylylene,a the distortion is forced by covalent bonds withinthe molecule.Another aspect of the X-ray crystallographic investigation of organiccompounds is the use of crystallographic data as a means of identification.This subject has been reviewed recently by Franzen 6s and by Bannister 66who have described both morphological and X-ray methods.The forth-coming publication of the first volume of the Barker index should encouragesome return to the first method. The body of data in the powder indexon organic compounds is not so far very great, and the problem of decidingwhich compounds should be included in it is one of considerable interestand importance. I n many cases an identification of an organic compoundis undertaken with reference to one particular investigation, e.g., to dis-tinguish different penicillins or to identify their degradation products withsynthetic specimens 53*67 or to show that gramicidin yields on hydrolysisa mixture of D-valyl-L-valine and L-vaIyl-D-valine.6s Data on many ofthese compounds would be doubtfully useful in a general index, while otherdata collected, e.g., on a series of constituents of explosive^,^^ ought to beincorporated.There are also certain special warnings that have to beattached to the use of crystallographic data in general. More complicatedmolecules may often crystallise in polymorphic modifications which showvery little tendency to be converted one into the other, for example, thethree forms of glycylglycine. Often also the crystal structure may beeasily deformed or affected by the exact conditions of crystallisation andthis, in turn, affects the appearance of the powder photographs. Stosick 70has pointed out that a number of the ‘ polymorphic ’ modifications of soapscan be explained in terms of crystals with varying degrees of disorderedstructure and not in terms of phase change.One interesting point is the occasional possibility of effecting theidentification, by crystallographic means, of a natural, optically activeisomer with molecules in a synthetic DL-preparation.The vast majorityof DL-preparations crystallise as racemic crystals containing both D- andL-molecules and often having crystal structures very different from thoseof the separated D- and L-isomers. Direct identification of the molecularspecies present by crystallographic means is then, of course, impossible,Not infrequently, however, the crystal structures of the racemic crystalsbear a marked resemblance, allowing for the presence of different symmetryoperations, to those of the optically active crystais-some examples are D- andDL-alanine, D- and DL-leucine, D- and DL-penillamine hydrochlorides. Itis relatively seldom that the D- and the L-crystals separate, as in Pasteur’soriginal experiments, but it happens occasionally, and has recently beenBrown and Farthing, Nature, 1949, 164, 9156 5 Chem.Weekblad, 1948, 44, 217.6 7 Clark, Kaye, Pipenberg, and Schisltz, “ The Chemistry of Penicillin,” Princeton66 Ibid., p. 220.Univ. Press, 1949, p. 367.Hinman, Caron, Louis, and Christenson, J . Amr. Chem. SOC., 1950, 72, 1620.Bg Soldate and Noyes, Anulyt. Chem., 1947, 19, 442.70 J . Chem. Phyeic8, 1950, 18, 757, 1035438 CRYSTALLOGRAPHY.observed in the case of one form of p-phenylglyceric acid, tetrachlorocycb-hexane, m. p. 174", and threonine. Crystals of natural L-threonine are,for example, indistinguishable by X-ray methods from those in preparationsof synthetic DL-threonine.The number of organic compounds which we have to consider this yeartempts us to draft this Report in the form of an outline text-book of organicchemistry, although sections of our text are still absent or very incomplete.Few measurements on organic compounds have yet been made a t lowtemperatures, and the first members of most of the aliphatic homologousseries are, therefore, still missing.These have, for the most part, beenalready examined by electron diffraction, and the series here described maybe completed, so far as molecular structure is concerned, by reference tothe very useful summary of electron-diffraction data given by Allen andSutton in, significantly enough, Acta Crysdallographica.72D. C. H.Aliphatic Compounds.-Although they raise a number of other problemsas well, the crystal structures in the first six classification groups below areconcerned principally with the character and influence of long hydrocarbonchains. The way in which long-chain molecules fit into crystals has beenapproximately known for some time. But the accurate knowledge nowobtained introduces detail that could not have been guessed, both in thearrangement and in the structure of the chains. The interest of thecrystal structures in the lat,er classification groups is rather different, partly~t~ereochemical and partly concerned with the effect of active groups oncrystal and molecular structure.Hydrocarbons. The smallest hydrocarbon we have to mention isdimethyltriacetylene (octa-2 : 4 : 6 - t r i ~ n e ) , ~ ~ which has a very simplecrystal structure based on rod close packing, with one molecule in therhombohedra1 unit cell.The bond lengths have been measured with goodaccuracy and are given in Fig. 4. They agree well with those in otheracetylenes that have been recorded.The zig-zag paraffin chain introduces crystallographic complications ;the hydrocarbon C,,H3, for example, of which preliminary measurementlsare This is the low-temperature form. The higher-temperature modification, which is ortho-rhombic, has been re-examined for several hydrocarbons by Mazee 76 whofinds that the a and b axes do not begin to change appreciably when thecrystals are heated until a certain definite temperature is reached and thattheir ratio never quite reaches the hexagonal value that might be expectedfor fully rotating chains.The mean atomic positions in very long hydrocarbons have beeninvestigated in a new and most interesting way by Pinsker and others using71 Furberg and Hassd, Acta Chem.Scund., 1950,4, 1020.75 Jeffrey and Rollett, Nature, 1950, 166, 476.74 Muller and Lonsdale, Acta Cryst., 1948,1,129.crystallises in a one-molecule triclinic cell.Bid., 1950,8, 46.70 Rec. Trav. chim., 1948,67,197HODGKIN AND PITT : ORGANIC COMPOUNDS. 439electron diffraction of the crystal^.'^ A Fourier series formed, using theelectron-structure amplitudes, gives a density function in which the maximashow the nuclear positions.Small peaks due to hydrogen atoms appearclearly visible and the mean bond dimensions are given as C-C 1.52, C-H1.17 A., L C-C-C 110", L H-C-H 105".The very remarkable complexes formed by shaking straight-chaincompounds with urea in the presence of a little solvent have been investigatedby Smith who has proved that the complexes are of the clathrate type."I 1-19, I 1-45CH31.44C1.13N164 hFFFFFIU. 4.Interatomic distances i n (a) dimethyltriacetylene (octa-2 : 4 : 6-triyne), (b)diucetylenedicurboxylic (butadienediuarboxylic) acid, ( c ) methyl cyanide-boron trifluoride, and (d) methylamine-boron triJuoride.The urea molecules are arranged in a hexagonal unit cell on a spiral frame-work held together by hydrogen bonds.In the centre of the spiral lie thehydrocarbon chains, the plane of the zig-zag having a random distributionabout three directions at 120" to one another. The parameters of thecarbon atoms in the chain-length direction are not fixed unless it happensthat this length is a near multiple of the urea repeating unit. Similarstructures are formed with thiourea-the channels are wider hereand branched-chain hydrocarbons or cyclohexane derivatives can beaccommodated.X-Ray diffraction effects observed for liquid ethylalcohol suggest a gradual change as the temperature is lowered betweenassociation of the molecules in pairs at -75" and in chains in the super-7 6 Vainshtein and Pinsker, Dokl. Akad. Nauk., S.S.S.R., 1950, 72, 53; cf.Weinsteinand Pinsker, ibid., 1949, 64, 49.Alcohols und Ei&rs.7 7 J . Chem. Physics, 1960, 18, 150cooled liquid at -150".78 No detailed structure anal$& of a simple alcoholhas, however, been reportsd and only preliminary measurements are givenon higher-chain alcohols, such as the cetyl alcohols 79 and 9 : 10-epoxy-octadecanols.sOAn important ester structure described is that of pentaerythritoltetranitrate.*l The bond lengths found are recorded in Fig. 5a and arenot as expected, particularly the short CH,-0 bond of 1.37 A. Dipolemeasurements suggest that this bond has little double-bond character, asfar as effect on restriction of rotation is concerned.82 It is possible that theX-ray data are not yet sufficiently refined for the inter-atomic distancesto be completely established, but it is at least suggestive that CH,-0 shows0AFIG. 6 .and (c) chain form i n di-(2-iodoethyZ) trisulphide.a progressive change from 1-46 A.in pentaerythritol 83 to 1.41 A . in thetetra-acetate 84 and 1.37 A. here; the explosive character of the compounda h suggests that some abnormality might be present.From among the huge volume of preliminary data recorded on long-chain esters and particularly glycerides, one may select for mention the factthat X-ray data have been used to show the identity of (-)-cc-(dipalmitoy1)-lecithin and the natural (dipalmitoyl)lecithin.86The most interesting feature ofsulphur-containing chains appears to be their tendency-shown in plasticInterdomic distance8 in (a) pentaerythritol tetranitrate, (b) bisnitraminoethne,Alkyl Sulphides and Polysulphides.Jagodzinaki, 2.Natzsrforsch., 1947, 2u, 465.Sano and Ktakiuchi, J . Phys. SOC., Japan, 1949,4, 178.Witnauer and Swern, J . Amr. Chem. SOC., 1950,72, 3365.Booth and Llewellyn, J . , 1947,837.Springall and Spedding, Research. 1949, 29.5.Llewellyn, Cox, and Hardy, J., 1937, 887.Goodwin and Hardy, Proc. Roy. SOC., 1938, A , 164,369.B a r and Rates, J . Amer. Chem. Soc., 1950,72,942HODQKIN AND PITT : ORGANIC COMPOUNDS. 441sulphur and S, itself-mt to conform to the planar zig-zag hydrocarbontype. Dawson and Robertson 86 were able to show that in di-(2-iodo-ethyl) trisulphide the sulphur atoms form an unbranched chain.Theirarrangement of the carbon atoms has, however, been revised by Donohuewho has given reasonably convincing evidence that the molecule as a wholehas a twisted structure (Fig. 5c) with the dihedral angles S-S-S-C andS-S-C-C both close to 90" (cf. dimethyl trisulphide and the groupS-C-C-I coplanar and trans.88A mines, A1 kyhmmonium Sa Its, and Nit roarnines .-Meth ylamine itselfmay be taken as represented by the compound methylamine-boron tri-fluoride,8a although the main interest here lies in the comparison of thiscompound with methyl cyanide-boron trifluoride and the characteristicsof the boron-nitrogen link. The bond distances in the methylamine andmethyl cyanide parts of the molecules appear to be quite normal. Butthe B-N bond, the co-ordinate link, is long in both compounds, and theadditional lengthening in the methyl cyanide compound is correlated bothwith a shortening of the B-F distance and increase in the angle F-B-F,and also with the decreased stability of the substance (Fig.4c and d). Thegeneral arrangement of the atoms conforms to staggered packing, and thehydrogen atons can be placed in the crystal.With the next amine on the list, n-propylamine as hydrochloride, wehave a crystal structure which caused some controversy many years agountil the possibility of chain rotation was rec~gnised.~~ I n the room-temperature structure of n-propylammonium chloride, the cations have adisordered arrangement in relation to the tetragonal axis. They are takingpart either in rotation, or in hindered rotation, about the chain axis as awhole and there seems also rotation about the central carbon-carbon bondwith production of some of the " gauche" or twisted form of chain.At-80" to -90" there is an arrest of the cooling curve of the salt suggesting asecond-order transition, disorder-order, in the solid. The diffraction spotsbecome doubled, indicating break up of the crystal into domains of lowersymmetry. Here the cations are fixed in the planar zig-zag form and liein the (1 TO) planes of the room-temperature structure, with consequent,small changes in lattice constants and symmetry.we reachstable room-temperature structures with an intricately fitted together criss-cross arrangement of the chains. The molecules have a planar zig-zagform with the exception of the terminal C-NH bonds which lie 7-10"from the plane of the other atoms.This is almost certainly caused bythe necessities of packing round the C1 ions, and hydrogen-bond formationwith them, since in the very beautiful structure of hexamethylenediamineWith hexamethylenediamine dihydrochloride 92 and86 J . , 1948, 1256.89 Geller and Hoard, Acta C y s t . , 1950, 3, 121.90 Hoard, Owen, Buzzell, and Salmon, ibid., p. 130.S1 King and Lipscomb, ibid., pp. 222, 227.*a Binnie and Robertson, ibid., 1949, 2, 180.Donohue and Schomaker, J . Chem. Physics, 1948,16, 92.J . Amer. Chem SOC., 1950, 72, 2701.OS Idem, ibid., p. 116442 CRYSTALLOGRAPHY.itself the atoms are coplanar throughout.M In both structures there appearsto be an alternation in the bond lengths along the chain (Fig.6). suggestinghyperconjugation, and the angles within the chain are generally slightlygreater than the tetrahedral. Again hydrogen atoms appear in the electron-density projection.With amine salts having much longer chains, such as the stearyl- andpalmityl-choline salts, of which very approximate electron-density pro-jections have been cal~ulated,~5 the molecular arrangement conforms moreclosely to the ionic double layer-parallel chain type.The two nitramines studied have little relation to the other compoundsin this group. The molecular structures are dominated by the nitro-group.In dimethylnitramine, all the atoms lie in one plane; g6 in s-bisnitramino-114' & 4150 122FIG.6.Interatomic distance8 in (a) hezamethylenediamine, (b) adipic acid, and( c ) glutaric acid.ethane,Q7 the half molecule CH,*NH*NO, is nearly planar. In both, theN-N bond is considerably shorter than the normal single bond distance(Fig. 5b).I n view of the enormous volume of workpublished on long-chain acids and their salts, one is particularly grateful forthe first detailed analyses of some of these compounds, potassium hexanoate(caproate) 51 and strontium l a ~ r a t e . ~ s In both compounds the aliphaticchains have the planar zig-zag structure and the slightly long carbon-carbon repeat interval 2-59,-2.60, A. along the chain agrees well with thatin other structures where the L C-C-C is about 115" and C-C about 1.54 A.I n potassium decanoate, the potassium ions are arranged in a double layer,each surrounded by six carboxyl oxygens, four from two chains on one side,Monobasic Acids and their Salts.94 Binnie and Robertson, Acta Cryst., 1949,2, 424.06 Stora, Compt.rend., 1949, 228, 324; 229, 874; 1950,230, 1675.Costain and Cox, Nature, 1947,160, 826.97 Llewellyn and Whitmore, J . , 1948, 1316.98 Morley and Vand, Nature, 1949, 163, 284HODGKIN AND PITT : ORGANIC COMPOUNDS. 443two from one chain on the other. But within a single decanoate layer,bounded by the ions, the molecules form further layers in which the chaindirections are crossed to one another at angles of about 60". In such astructure, it is not surprising that frequent faults occur in the regulararrangement of the molecules.Measurements of unit cell dimensions have been reported for a numberof other fatty acid salts and acids 99 and it is to be hoped that some of theother structure types reported will soon be known in detail.One interestingpossibility has been proposed for aluminium monolaurate 100-tliat thereare oxygen octahedra present similar to those in alumina, joined by sharingof two corners, the carboxyl oxygen atoms occupying the remaining fourcorners of each octahedron.The appearance, in natural products, of a number of long-chain com-pounds with small branched chains, such as tuberculostearic acid, (-)-lo-methyloctadecanoic acid lol and phthiocerane, which is mainly ( -J-)-4-methyltristriacontane,102 has led to a good many X-ray measurements oftheir different crystalline forms.Usually the compounds show markedsimilarities with the normal straight-chain derivatives; in the case of oneseries of amides, Velick lo3 noticed marked deviations, which he suggestedmight be correlated with the appearance of a spiral-chain configuration.However, later observations indicate the occurrence of different crystallinemodifications here.10P While therefore a spiral carbon-chain structureremains still to be found, definite distortions from the planar zig-zag-chainform do occur in the next group of compounds, the dibasic acids.As a result, principally, of two independent series ofresearches carried out in Holland and at Glasgow, we can now considerdetails of the crystal structures of six different straight-chain dibasic acidsin the interval oxalic to sebacic acid.These are particularly interestingin relation to the marked alternation in physical properties which occursbetween acids of the odd and the even series.As might be expected, the early members of both series, oxalic, malonic,and succinic acids, show deviations from the types of crystal structurecharacteristics of the higher members. Oxalic acid itself crystallises inthree different modifications, a- and p-oxalic acid which are both anhydrous,l06and the dihydrate. Of these, the a-form is unique in that the moleculesare linked in sheets by hydrogen bonds between the oxygen atoms of onecarboxyl group and two other carboxyl groups. Malonic acid has not yetDibasic Acids.Iball, Nature, 1947, 159, 95; Vand, Acta Cryst., 1948, 1, 109; Vand, Aitken, andCampbell, a i d ., 1949, 1, 398; Lingafelter and Jensen, ibid., 1950, 3, 257; Minor andLingefelter, J . Amer. Chem. Soc., 1949, 71, 1145; Witnauer, Lee, and Senti, ibid., 1950,72, 283 ; Kohlhaas, Ber., 1949, 82, 487.loo McGee, J . Amer. Chem. SOC., 1949, 71, 278.Iol S. Stallberg-Stenhagen, Arkiv Kemi, Min., Geol., 1948, 26, A , No. 12.lo2 S . Stallberg-Stenhagen and E. Stenhagen, J . Biol. Chem., 1948, 173, 383; 1950,Io4 Arosenius, Stallberg, E. Stenhagen, and B. Tagstrom-Eketorp, Arkiv Kemi, Min.,183, 223.Geol., 1948, 26, A , No. 19.lo3 J . Amer. Chem. SOC., 1947, 69, 2317.lo6 Hendricke, 2. Krist., 1936, 91, 48444 CRYSTALLOGRAPHY.been studied in detail; only cell dimensions are recorded for the differentvarieties. In all the other crystal structures found, the molecules arelinked in chains with the carboxyl groups of succeeding molecules joinedend to end through hydrogen bonds.I n the dihydrates, the water mole-cules are inserted into the hydrogen-bond system; in all the others, theform of the group -C C- is remarkably constant, whateverthe rest of the crystal structure may be like, with O * * * . O contacts of2.64-2-69 A.The even acids, adipic lo6 and sebacic acid,lo7 are characterised by asimple monoclinic crystal structure in which the molecules are centro-symmetrical. The atoms have a nearly planar zig-zag arrangementthroughout the length of the molecule: in adipic acid the atoms of thecarboxyl group lie in a plane tilted about 6" from the plane of the otheratoms; in sebacic acid this tilt is only 3", in p-succinic acid 9O.108 Thea- and the p-form of succinic acid differ from the higher members chieflyin the relative arrangement of the molecules normal to the chain, thetriclinic or-form approximating most closely to the packing shown by thehigher numbers.lo9The most obvious difference in the crystal structures of the acids of theodd series, glutaric no* 111 and pimelic acid ll1 is that the molecules are nolonger nearly planar, but have a twisted form; even the carbon atoms donot lie strictly in one plane, and the terminal carboxyl groups lie in twoplanes a t about 60" to one another and 30" to the plane of the centre threecarbon atoms.As a whole, the molecules have a, two-fold axis of symmetry,a centre of symmetry being, of course, impossible. It seems very probable,from calculations carried out by MacGillavry et al., that the twisted formof the molecule is responsible for the extra energy content of crystals inthe odd series. The potential barrier to rotation in acetone, m. 1 kcal.,suggests that the extra energy in the odd series molecules would be of theorder of 2 kcals., a difference which corresponds in magnitude to observeddifferences in the heats of combustion. Morrison and Robertson make twoobservations : first, that there is a tendency to alternation in the carbon-carbon bond lengths along the hydrocarbon chains in the even series andno such effect in glutaric acid (Fig.6 ) ; this may be correlated with themolecular twisting; and, secondly, that one very short contact, 3.29 A.,occurs between an oxygen and a carbon atom of neighbouring moleculesin adipic and sebacic acids and not in glutaric acid; this may also affectthe melting point and hardness of the crystals. We have the impressionthat the hydrogen-bond system is the dominating factor in determiningthe crystal structures, molecular compression or distortion occurring asnecessary to fit this with particular conditions of molecular shape.106 MacGillavry,Rec. Trav.chim., 1941,60,605 ; Morrison andRoberteon,J., 1949,987.10' Idem, ibid., p. 993. 108 Idem, ibid., p, 980.109 Riech,Rec. Trav. chim., 1944,63,170. 110 MorrisonandRobertson,J., 1949,1001.111 MacGillavry, Hoogachagen, and Sixma.Rec. Trav. chim., 1948,67, 869./ / O * * * * H O\\O H . . . . O / HODGKIN AND PITT : ORGANIC COMPOUNDS. 445Another series of acids is the series formed by oxalic acid dihydrate 112 andacetylene- 113 and butadiyne (&acetylene) -dicarboxylic acid dihydrates.l14The system by which two succeeding carboxyl groups are linked in a chainthrough the water molecules is the same in all three crystals; all show thevery short hydrogen bond, 2051-2.56 A., between one carboxyl-oxygenatom and the water molecule, here probably an oxonium ion. The systemholds in spite of the fact that oxalic and acetylenedicarboxylic acid areessentially planar and centrosymmetrical molecules, while butadiyne-dicarboxylic acid has, like the odd acid series above, a two-fold axis ofsymmetry and carboxyl groups turned in two planes a t an angle of 57"to one another.The evidence on the bond distances within the oxalicacid molecule itself has been reviewed by Dunitz and Robertson ; 112 theirpreferred values may still have to be modified further after three-dimensionalanalysis. The figures for bond lengths in the acetylenic chains ought to beless liable to change; the most remarkable bond length here is the centralbond length in the diacetylenic acid, the shortest formal single carbon-carbon link so far recorded, 1-33 & 0-02 A. In this molecule, however, thecontraction of the bond lengths does not appear to be correlated withdecreased freedom of rotation, if the relative arrangement is taken asevidence-compare the situation in pentaerythritol tetranitrate discussedabove.Hydroxy-acids : the Tartrates.To tho crystal structure of Rochellesalt,l15 sodium potassium tartrate tetrahydrate, we can now add those ofsodium potassium DL-tartrate tetrahydrate,ll6 D,-tartaric acid,l17 andracemic acid,li8 a group of crystals interesting on account both of Pasteur'soriginal work on molecular asymmetry and also of the electrical propertiesof Rochelle salt.In all four crystal structures the asymmetric tartaric acid moleculemaintains essentially the same relative arrangement of the atoms present :the two groups HO'C-CRo are both closely planar and are arrangedwith their planes a t an angle of approximately 60" to one another. Thereis a certain similarity between the packing of the molecules in the two salts,which is partly controlled by the requirements of the ions.The differencesintroduced by the centre of symmetry and the changed hydroxyl-grouprelations in the crystal of the racemate are however enough to destroy theunidirectional series of hydrogen bonds found in Rochelle salt, and hencethe development of abnormal electric properties. The relation between theexact atomic positions and these properties in the Rochelle salt crystal isin any case very sensitive, as illustrated by the correlation found byUbbelohde and Woodward between the thermal expansion and the dielectric\OH112 Dunitz and Robertson, J., 1947, 142.113 Idem, ibid., p.148.115 Beevers and Hughes, Proc. Roy. SOC., 1941, A , 177, 251.116 Sadanaga, Acta Cryst., 1950, 3, 416.11' Stern and Beevers, ibid., p. 341.11* Idem, ibid., p. 1145.11* Parry, Nature, 1949,164, 885446 CRYSTALLOGRAPHY.constant of the crystal,llg and also by the effect of altering the proportionsof the ions in the crystals.120 The view that the crystal between the twoCurie points consists of domains of lower symmetry has received someadditional confirmation from measurements on the integrated intensityof X-ray reflections.121 These are higher between the Curie points thanabove them, as might be expected from the imperfect kind of crystalformed. Changes in the size and nature of the domains might account forthe marked effect that certain impurities such as cupric ion and boric acidalso have on the electric properties of crystals grown in their presence.The differences between the crystal structures of D-tartariC and racemicacid are pronounced.In the racemic acid crystal unit there are only twomolecules related by a crystallographic centre of symmetry. But, as ex-pected, there is no definite pair association of D- and L-units, no racemic acidmolecule present, but an over-all hydrogen- bond system holding the moleculesin the crystal. Parallel to the a axis, columns of D- and L-molecules are con-nected by a square system of hydrogen bonds between the hydroxyl groups.End to end, D- and L-molecules are also linked through one pair of carboxylgroups as in the other dibasic acids described.The second carboxyl groupin each case makes contacts with both carboxyl- and hydroxyl-oxygenatoms of neighbouring molecules. In D-tartaric acid, the system of hydrogenbonding is necessarily different from that in racemic acid. The two moleculesin the unit are related by a screw axis of symmetry and in this crystalstructure there is no pairing of carboxyl groups of neighbouring moleculesof the dibasic acid type. Instead a more complex linking of carboxyl groupswith hydroxyl groups is present.One particularly interesting point about the D-tartaric acid crystalstructure is that correlation of the molecular arrangement found with theface development of the crystals would permit a determination of the absoluteconfiguration of the molecule.This has been attempted by Waser,122but it is doubtful whether our knowledge of the factors affecting crystalgrowth is sufficient to feel complete confidence in his assignment 123-~hi~his the exact opposite of Fischer’s assumed arrangement.Amino-acids and Peptides. The X-ray analysis of the amino-acidL,-threonine 38 establishes unambiguously the stereochemical relationbetween the sugar-lactic acid and the amino-acid series.124 The relationis the one derived as most probable by the original observations of Meyer andRose,126 following the first isolation of threonine, and recently independentlyestablished through kinetically-controlled chemical reactions.12s It is shown inFig. 7 by a perspective drawing of the threonine molecule, which illustratesboth the arrangement of the atoms and the bond lengths within the molecule.119 proc.ROY. soc., 1946, A , 185,448.110 Thorp and Buckley, Acta Cryst., 1949, 2, 333.121 Mujake, ibid., p. 192.123 Turner and Lonsdale, J. Chem. Physics, 1950,18, 166.12* Cf. Neuberger, Adv. Protein Chem., 1948, 4, 297.126 J . Biol. Chem., 1936, 115, 721.126 Brewster, Hughes, Ingold, and Rm, Nature, 1950, 166, 178.laa J. Chm. P h y e h , 1949, 17, 498HODGKIN AND PITT : ORGANIC COMPOUNDS. 447The crystallographic investigation of threonine constitutes the mostcomprehensive and thorough study yet undertaken of an asymmetricmolecule and provides a fund of useful information on the technique ofcrystal analysis. The full, three-dimensional electron-density distributionhas been calculated six times in the course of refinement-until recentlya single such calculation would have been considered to involve an impossibleamount of labour.The electron-density distribution shows the positionsof hydrogen atoms as separate, but not very precise, maxima, and enablesthe positions of all the remaining atoms to be fixed with a high degree of?0 0I L.540 0NH1.48o--- - 0 q 2 1.29 C 1-31 1.510 <:2 C 1.27(di O(c) ?&-56FIG. 7 .Interatomic distances in (a) threonine, (b) alanine,( c ) acetylglycine, and (d) 8-glycylglycine.precision. The C-N distance, 1.49 A., is close to that expected from thesum of the covalent radii, and it is clear that the short values reportedearlier for glycine and alanine 12’ were based on insufficient refinement ofthe data.Both have been found to be normal from recent three-dimensionalanalyses.Within the threonine molecule it is noticeable that the staggered arrange-ment of the atoms is very precisely observed, so that the contacts betweennon-bonded atoms are of the most favourable form-even to placing thehydrogen of C(3) in the gap between NH3+ and C0,- rather than the largerOH and CH, groups. At the same time, a high density of packing of themolecules in the crystal is achieved with all the hydrogen atoms of the1-2’ Donohue, J. Amer. Chent. SOC., 1950, 72, 949448 CRYSTALLOGRAPHY.NH3+ and OH groups involved in hydrogen bonding. It is clear from thedistribution found for the hydrogen atoms that the molecule has thezwitter-ion structure.There is one comparatively short distance, 3.28 A . ,between one carbon atom and the hydroxyl group.The analyses of other amino-acids reported as begun during these years-glycylglycine hydrobromide,12* DL- and ~-leucine,l~~ and L-proline 130-have not yet reached the stage at which interatomic distances can be given.But two analyses, those of acetylglycine 131 and of @-gIycylgly~ine,~~~provide a useful basis for the theoretical construction of extended peptidechains.133 Both molecules are planar or very nearly so; in glycylglycinethe terminal nitrogen atom only is out of the plane of the other atoms. Itis noticeable that the C-0 distances in the acetylglycine carboxyl group,which has no zwitter-ion character, are markedly asymmetric, correspondingto not greatly modified C=O and C-OH distances.Apart from this:the crystal structures have considerable similarity ; in both, the moleculesare linked in sheets by hydrogen bonds between carboxyl, keto-, amino-,or imino-groups. In both, one can see a formal relationship to the extended@-form of peptide-chain structure, though the sideways linking in one caseinvolves C0,H and NH groups, in the other case C=O and NH,, and inneither case the postulated C=O * * NH bonds of the @-keratin structure.NH linking occurs are, however,well established by the work of Bunn and Garner and others on certainpo1yamides.la These are fibres and give only limited X-ray diffractiondata; 135 consequently the position of the atoms cannot be very preciselyfixed.But they are sufficiently clear to prove that here too the moleculesare held in sheets, the oxygen-nitrogen separation of neighbouring moleculeswithin the sheets being 2-8 A. The sheet thickness, 4.4 A., compares verywell with the backbone spacing of the @-keratin type of fibrous protein.Alicyclic Ring Systems. With the alicyclic ring compounds stereo-chemical problems are again dominant. Few of the X-ray analyses in thisseries are sufficiently accurate for the interatomic distances found to beworth reporting, but there are one or two interesting exceptions.No cycbpropane derivative has yet been analysed in detail by X-raymethods. For completeness, we may quote Skinner's conclusion 136 thatthe ring-carbon atoms are arranged in a regular equilateral triangle of siderather smaller than 1.54 A., in accordance with certain quantum-mechanicalcalculations.In all the cyclobutane derivatives studied on the other hand, the carbon-carbon distance has proved, unexpectedly, greater than normal.The mostcareful analysis is that of the centrosymmetrical isomer of 1 : 2 : 3 : 4-Structures in which the C=O * - -128 Barney, Amer. Min,, 1947, 685.130 Wright and Cole, Acta Cryst., 1949, 2, 129.131 Carpenter and Donohue, J . Amer. Chem. SOC., 1950,72, 2315.132 Hughes and Moore, ibid., 1949, 71, 2618.133 Corey and Donohue, ibid., 1950,72, 2899.135 Beauvalet, Champetier, and Tertian, Compt. rend., 1949, 228, 2028.136 Nature, 1947,160, 902.ltO Moller, Acta Chem.Scand., 1949, 3, 1326.13' Proc. Roy. SOC., 1947, A , 189, 39HODGKIN AND PIl'T: ORGANIO COMPOUNDS. 449tetraphenylcycZobutane,m which is formed by the photochemical dimerisationof stilbene. The analysis of this compound was undertaken, in the firstinstance, to establish the nature of the toxic irradiation-product of the drug" Stilbrtmidine," from which it had been obtained. The crystal structureshowed both that the four-membered ring was present, and that the phenylgroups were disposed centrosymmetrically round it. As a result, this arrange-ment is different in relation to the two adjacent bonds in the four-memberedring alternately, cis and trans, which may account for the fact that thesebonds appear to be of unequal length, 1.585 and 1-555 A.respectively.The other X-ray analysis of a four-membered ring derivative, that ofthe centrosymmetrical dimer of acenaphthylene,l3' is not complete, but ittoo shows bonds in the ring which are longer than 1.54 A . Bond iengtheningalso appears very markedly from the electron-diffraction data on octafluoro-cyclob~tane,l~~ for which the carbon-carbon distance in the ring is given&s 1.57-1-62 A. and the ring appears to be non-planar. The lengtheningof the bond is probably correlated with a greater proportion of p-bondcharacter, the angle between the C-C valencies in the ring being nearly90". Repulsion between non-bonded atoms must also have an efrect.Certainly with five- and six-membered rings, it is clear that the packingof non-bonded atoms plays a dominant part in determining their overallarrangement.The five-membered carbon rings so far observed are allnon-planar, usually with one atom out of a plane formed by the other four.They include the five-membered rings in bromo-, chloro-, and cyano-camphor 139 and ring D of cholesteryl iodide 140 and calciferol 4-iodo-5-nitr0ben~oate.l~~ In almost all cyclohexane derivatives so far studied,including the cis-decalins, the ring has the staggered or chair form. Onlyin the camphor derivatives mentioned does the boat form appear, imposedby the fusion with five-membered rings. In 1 : 2-epoxycy~Zohexane,~~~ring fusion again imposes a constraint but the atomic arrangement foundby electron diffraction is still approximately staggered.The simpler derivatives of cyclohexane, the hydrocarbon itself,143 cyclo-hexanol,lqq and dodecafluoracy~lohexane,~~~ crystallise in cubic cells inwhich the molecular arrangement is considerably disordered. The mole-cules appear not to be freely rotating, but to have certain preferred orient-ations in relation to their neighb0~rs.l~~ With additional hydroxyl groupsattached, the cyclohexane molecule can be readily held in a fixed position.Particularly good examples are provided by a-phloroglucitol dihydmte 58and diarnm~niate.~~ The molecules have trigonal symmetry, which13' Dunitz and Weissman, Acta Cryst., 1949,2, 62.13* Lemaire and Livingston, J.Chem. Physics, 1950,18, 569.13D Wiebenga and Krom, Rec. Trav. chim., 1946,65, 663.140 Carlisle and Crowfoot, Proc.Roy. SOC., 1945, A , 184, 64.141 Crowfoot and Dunitz, Nature, 1948,162, 608.I r a Otter, Acta Chem. Scand., 1947, 1, 283.143 Oda, X-Rays, 1948, 5, 26.145 Christoffers, Lingafelter, and Cady, J. Amer. Chem. SOC., 1947, 89, 2502.14* King and Lipscomb, Acta Cryst., 1950,3, 155.144 Oda, ibid., 1949, 5, 95.REP.-VOL. XLVII. 450 CRYSTGOGRAPHY .establishes their stereochemical form (Fig. 8), and are linked in threes roundwater or ammonia molecules which form a fourth bond between one another.It is dficult to summarise adequately the crystal analyses of the veryinteresting group of different halogenated cyclohexanes-all of which involvethe determination of the nature of the stereochemical isomer present, andin two cases, those of the hexachlorocyclohexane, m.p. 145",14' and tetra-chlorocyclohexane, m. p. 174", the carbon atoms to which the chlorineatoms were attached. " Gammexane," 148 as an insecticide, is the mostimportant of these compounds. The molecule proves to have the structureshown in Fig. 8, which was unexpected since it had been supposed thatsteric hindrance might prevent altogether the formation of the isomer withchlorine atoms a t 1 and 3 both in the erect position. There is a slightdistortion of the molecule as a result-the bonds C,l,-Cl and C,3,-CI are notquite parallel, but the distortion is small. The same problem does not arise inthe case of the other isomers studied, vix., 6- lg9 and ~-hexachlorocycZohexane,150though all show small deviations from the quite regular form of the moleculesindicated in Fig.8, which all should have planes of symmetry. 1 : 2: 4: 5-Tetrachlorocyclohexane, m. p. 174", and 1 : 2 : 3 : 4-tetrabromocycZohexane,m. p. 142", both have two-fold axes of syrnmetry;l5l in the first case i tshould be possible to separate the crystals by hand into (+)- and(-)-forms. Actually one polymorphic modification of 1 : 2 : 4 : 5-tetra-bromocyclohexane, m. p. 218", was picked out by hand from a mixture ofcrystals and the molecule proved by X-ray analysis to have the structuregiven in Fig.Rings in which methylene groups have been replaced by oxygen atomsor imino-groups have not been so fully investigated but appear from thefew known examples to have similar stereochemical characteristics.Theseexamples include di-iodomethyloxacycZobutane,153 the furanose and pyranoserings of the sugars discussed in the next section, and 1 : 4-dichloropipera~ine.~~It should be possible to get really good measurements from di-1 : 3-dioxa-~yclopentyl,~~~ the chemical structure of which was found by the calculationof a Fourier projection from data on crystals which had been supposedpreviously to be a cis-decalin type of isomer of naphthodioxan.The crystal structures of three sugars have recentlybeen analysed or partly analysed : r~-D-ghCOSe,l~~ difructose strontiumCarbohydrates.Ellefsen, Hassel, and Wang Lund, Acta Chem. Scand., 1950, 4, 1145.148 van Vloten, Kruissink, Strijk, and Bijvoet, Acta Cryet., 1950, 3, 139; Nature,149 van Bommel, Strijk, and Bijvoet, ?roc.K . Akud. Wetensch., 1950, 53, 50.l50 Norman, Acta Chem. Scand., 1950,4, 251.151 Hassel and Wang Lund, ibid., 1949, 3, 203; Acta Cryst., 1949, 2, 309 ; Mrang152 Haak, Thesis " De berciding en structure van cyclohexadieen-1, 4 en van enige153 Toussaint, Bull. SOC. roy. Sci. Likge, 1948,1, 18.154 Anderson and Hasel, Acta Chem. Scand., 1949, 3, 1180.155 Dano, Furberg, and Hasel, ibid., 1950, 4, 965.156 McDonald and Beevers, Actu Cryst., 1950, 3, 394.1948,162, 771 ; Rec. Truv. chim., 1948,67, 777.Lund, Acta Chem. Scand., 1950, 4, 1109.derivaten," P. Harte, Bergen op Zoom, 1948HODGKIN AND PITT : ORUANIC COMPOUNDS. 451chloride trihydrate,l57 and sucrose sodium bromide dihydrate.lS8 Thedetermination of the structure of a-D-ribofuranose is also involved in theanalysis of ~ y t i d i n e .l ~ ~ I n all cases the stereochemical arrangement ofthe hydroxyl groups, predicted on chemical grounds, is confirmed. Thisincludes the cis-arrangement of the 1- and the 2-hydroxyl group in the twoor-glucose units.Tetrachloroc yclohexane,m. p. 174".(a)a-Phloroglucitol.(b)&Tetrabromoc yclohexane,m. p. 218'.(4'' Qammexane." 8-Hexachloroc yclohexane.(4c-Hexachloroc yclohexane. Hexachloroc yclohexane,FIG. 8.Stereochemical form of some substituted cyclohexane derivatives.m. p. 145".Both the glucose ring and also the fructose ring in the strontium chloridecomplex are six-membered in the Sachse trans-configuration. In sucrosethe fructose ring is five-membered as expected, and non-planar, one carbonatom being removed from the plane of the other four.Bond lengths arenot accurately determined in any of these compounds, but the ct-D-g1uCOSecrystal structure is being further refined and should provide a good set ofmeasurements. All the hydroxyl groups in both this and the sucrosestructure are involved in hydrogen-bond formation. I n ct-D-ghCOSe, as1 5 7 Eiland and Pepinsky, Acta Cryst., 1950, 3, 160.1 5 8 Beevers and Cochran, Proc. Roy. SOC., 1947, A, 190. 257.150 Furberg, Acta Cryst., 1950, 3, 325.REP.-VOL. XLVIL P 452 CRYSTALLOGRAPHY.in cytidine, the ring-oxygen atom itself appears to be bonded to the hydroxylgroup of a neighbouring molecule.A consideration of the stereochemical conditions obtaining in pyranoseand in cyclohexane rings has led Hassel and Otter to suggest a new way oflinking P-glucose units in a chain with an 8-5-A.period 160 that may corre-spond to the arrangement in many natural polymers, such as alginic acid,which show periods of this kind. This arrangement seems more probablethan that put forward originally by Astbury.lG1 For cellulose itself, inspite of much controversy, the structure proposed by Meyer and Mischstill seems the most likely.162 A three-fold spiral arrangement is suggestedby the cell dimensions found for cyanoethyl cellulose. 163More complicated unit cells are found among starches, and varioustentative structures involving helical arrangements of the molecules havebeen proposed for different forms.16* The variety known as V-amylose,starch, or amylose precipitated from alcohol forms a lattice which suggeststhat the glucose residues are arranged in a six-fold helix of external diameterroughly 17 A.and height 7.9 A., and this idea receives some confirmationfrom an exceedingly rough Fourier projection derived by comparison withthe iodine ~omp1ex.l~~ The iodine fits into the column surrounded by thehelix, and observations by West suggest that the iodine molecules dis-sociate ; l 6 6 characteristic diffuse layer lines appear indicating a layerinterval of 3.1 A. in the direction of the iodine column.The very interesting Schardinger dextrins form true crystalline latticesfrom which the molecular weights of the units present may be calculatedin the usual way.They correspond to six, seven, and eight glucose residuesrespectively for a-, p-, and y-Schardinger dextrins; 16' more than onepolymorphic modification appears to exist for the a- and the p-dextrins.D. C. H.Aromatic Compounds.-The structures of aromatic compounds have forsome time been studied very largely because they provide a wide variety ofexamples of bonds the characters of which are intermediate between singleand double. This applies not only within the ring systems, but also in thebonds from the rings to substituting atoms or groups, and in the latterinstance in particular has a bearing on the chemical reactivity of the sub-stituents. While earlier work was of interest in indicating qualitatively thesevariations of bond length, it is only in the past few years that advances incrystallographic computing methods, and in the theoretical methods of calcu-160 Acta Chem.Scand., 1947, 1, 929.162 van der Wyk and Meyer, J . Polymer Sci., 1947, 2, 583.1e3 Happey and MacGregor, Nature, 1947, 160, 907.164 Kreger, ibid., p. 369; Rundle, ibid., 1948, 162, 107; Senti and Witnauer,166 J . Chem. Physics, 1947,15, 689; cf. Rundle, ibid., p. 880.1 6 7 Borchert, 2. Naturforsch., 1948, 3b, 464; cf. Gruenhut, Cushing, and Caesar,161 Nature, 1945, 155, 667.J . Amer. Chem. SOC., 1948, 70, 1438. 165 Rundle, ibid., 1947, 69, 1769.J . Amer. Chern. Soc., 1948, 70, 424HODGKIN AND PITT : ORGANIC COMPOUNDS. 453lating bond characters and bond lengths, have made possible a detailedcomparison of experiment and theory. The calculations on both sides areeven now very long and arduous, so that a full correlation can be expectedonly for a relatively small number of compounds. This correlation has beenachieved for a few of the compounds discussed below, but in many of the othercases there are fresh indications of the occurrence of bonds of intermediatecharacter.Naphthalene and anthra-cene were among the first organic crystals to be analysed by X-ray methods,and the recent full three-dimensional analyses (Fig.3) by Robertson and hisco-workers ~ 4 0 are of particular interest in confirming and extending theprevious findings. The bond lengths found are shown in Fig. 9, and thecorrelation between the chemical reactivity of the a-p-bonds and theirrelative shortness is immediately apparent.The authors claim that errors inthese bond lengths should not exceed 0.01 A., and they observe that bondsCurbocyclic Compounds.-(a) Hydrocarbons.1.359 1.420 1364 1.419 1.391(1.395) (1.450)FIG. 9.Aromatic hydrocarbons : (a) naphthalene ; (b) anthracene. Observedand calculated (parentheses) bond lengths are shown.which are crystallographically different but chemically identical have lengthswhich differ by about 0.01 A. This however raises the difficult question ofthe distortions caused by the packing of molecules in a crystal lattice, whichappear to be noticeable in certain cases where observations of both the crystaland the vapour have been made. It may be pointed out that no correctionhas as yet been applied for series-termination errors.Superposition of the simple Kekul6-type structures is insufficient in thecases of naphthalene and anthracene to explain the observed bond lengths,but it is now recognised that the contributions of the excited states mustbe included if a quantitative estimate is to be made.The bond lengths fromsome recent wave-mechanical calculations 16** 169 are shown in parentheses inFig. 9.Further investigations by two-dimensional Fourier methods have beenreported on pyrene (1),170 1 : 2-5 : 6-dibenzanthracene (11),171 1 : 12-benzperylene ovalene (octabenzonaphthalene) (IV),173 and triphenyl-16* Vroelant and Daudel, Compt. rend., 1949, 228, 399.lag Daudel and Daudel, J .Chem. Physics, 1948,16, 639.170 Robertson and White, J., 1947, 358.171 Idem, ibid., p. 1001.173 Donaldson and Robertson, Nature, 1949, 164, 1002.172 White, J . , 1948, 1398454 CRYSTALLOGRAPHY.ene (V),174 and in all cases bond lengths of intermediate character have beenfound. It would be unreasonable to expect very close correlation between(111.)these experimental measurements and the results of calculations based onthe simple non-excited Kekul6 structures, but in fact in a number of in-stances there is quite good qualitative agreement. This sometimes improvesslightly if the contributions of the various Kekul6 structures are weightedaccording to their benzenoid character, lending support to the Fries rule thatstructures with benzenoid rings are more important than those with quinoinoidrings.175Another modification of hexamethylbenzene has been reported, stablebetween 110" and the melting point (165°).176 It is more symmetrical thanthe form stable a t ordinary temperatures but very closely related to it.Thelattice energy and thermal expansion have been discussed by Seki andChihara.177( b ) Benzene derivatives : halogen compounds. The sequence of compounds,pdichloro-, p-bromochloro-, p-dibromo-benzene, was first shown by Hen-dricks to be isomorphous. More recent work 178 has verified that the bromo-chloro-compound has a statistical structure, the chlorine and bromine atomsoccupying two sets of crystallographically different positions a t random.The bonds from the benzene ring appear equal, 1.77 A., a value intermediatebetween that for C-C1 (1.69) found in the dichloro-compound and that forC-Br (1.84, 1-88) in the dibromo-compound. In p-chloroiodoxybenzene 179the 10, plane is almost a t right angles to the benzene ring, the 0-1-0 anglebeing 103".The 1-0 distances are shorter than usual (1.60, 1.65 A.) and theiodoxy-groups of neighbouring molecules approach one another very closely.The chlorine atom is displaced from the plane of the benzene ring, and theI-C bond does not make equal angles with the adjacent sides of the ring.17* Klug, Acta Cryst., 1950,3, 165.1 7 6 Watanctb6, Ssito, and Chihara, Sci. Papers Osaka Univ., 1949, No. 1, p. 9.177 Ibid., p. 1.1 7 ~ Archer, Acta Cryst., 1948, 1, 64.175 Robertson, ibid., 1948,1, 101.178 Klug, Nature, 1947, 160, 570HODGKIN AND PITT : ORGANIC COMPOUNDS. 455(c) Nitrogen compounds.The crystal structure of p-dinitrobenzene cannotyet be considered t o be entirely satisfactorily elucidated. The early work,based on Fourier projections, led James, King, and Horrocks to the conclusionthat the molecule was centrosymmetrical but distorted so that the benzenering was not regular, the nitro-groups not coplanar with it, and the N-0distances unequal. Criticism of these distortions by Pauling led to a repeti-tion of the analysis by Llewellyn lSo utilising three-dimensional methods forgreater accuracy. He obtained a set of atomic co-ordinates which gave a,disagreement factor of 0.24, and he stated that they corresponded to a planarmolecule, a regular benzene ring and equal N-0 distances, whereas in factthey require angles of l l + O between the planes of the NO, groups and the ben-zene ring.An independent investigation by Abrahams lS1 seems to indicatethat there is a small angle between these planes, but it is clear that morecareful three-dimensional analysis is required if the finer details of thestructure are to be elucidated. As reported in 1946, the structure of m-dinitrobenzene was reconsidered by Archer 18, and the reputed space-groupshown to be incorrect. A new trial structure was proposed and the principalFourier projection of the unit cell calculated; this showed the moleculealmost completely resolved, but gave no reliable evidence of any smalldeviations of the nitro-groups from the plane of the benzene ring; this hasbeen independently confirmed by Gregory and Lassettre.ls3 The bondlengths quoted in these two accounts are not identical but, since the structureis one in which there is adequate resolution in only one projection, it isunjustifiable to attach any quantitative significance to the deviations found.I n p-nitroaniline the evidence is in favour of a symmetrical nitro-groupcoplanar with the benzene ring, the N-0 distances being slightly greaterthan those found in the dinitrobenzenes, while the C-N distances appearto be somewhat short.Considerable interest centres on the remarkablyclose approach of one oxygen atom to the carbon atoms of the adjoiningmolecule : distances of 2-66, 2-99, and 3-03 A.are found, all much shorterthan the normal van der Waals value of 3-4 A. It is suggested that a powerfulattraction, possibly of an electrostatic nature, exists between the atomsconcerned, a self-complex being formed by the nitro-group of one moleculeacting as ‘‘ acceptor ” while the benzene ring of another molecule acts as“ donor.’’ Independent evidence of complex formation in p-nitroanilinecomes from the ultra-violet absorption spectrum and the entropy of vaporis-ation, and the authors incline to the view that this attraction may be thecause of some molecular complexes formed between aromatic nitro-compoundsand polycyclic aromatic hydrocarbons although no evidence of short inter-molecular distances has as yet been found.By analogy with the propertiesof structures involving short hydrogen bonds between molecules, it would beexpected that the thermal expansion parallel to the short intermolecularC-0 linkage would be anomalous. This has been confirmed by McKeown,lSo J., 1947, 884.lB2 PTOC. Roy. SOC., 1947, A , 188, 61.lS1 Acta Cryst., 1950,3, 194.lS3 J. Arner. Chem. SOC., 1947,69, 102456 CRYSTALLOGRAPHY.Ubbelohde, and Woodwardl84 who find for the total contraction from288" K. to 90" K. the coefficients :(at 55" to c axis, i.e., within 11" of direction of short" bond "),(parallel to 71)all : 2.95 xa22 : 0-24 xam : 0.45 xThe molecular complexes of 4 : 4'-dinitrodiphenyl with diphenyl, 4-bromo-and 4-iodo-diphenyl, benzidine, etc., have been shown to be all of the sametype as that previously described with 4-hydroxydiphenyl, the proportionsof the two components being determined by geometric factors.The com-plexes with 4-bromo- and 4-iodo-diphenyl show diffuse X-ray scatteringexplained in terms of random displacements of the latter component alongthe holes in the lattice of dinitrodiphenyl molecules. No close intermolecularapproaches appear to occur in these complexes.185* 186* 18'The mode of dimerisation of nitrosobenzene has previously been uncertain,but two independent investigations (of dimeric p-bromonitrosobenzene 188and dimeric tribromonitrosobenzene 189) have shown conclusively that themolecular formula is (VI) and not e.g. (VII), the positions of the formalR'charges not being definitely known owing to the approximate nature of thebond lengths so far determined.I n the p-bromo-compound the molecule iscentrosymmetrical, the planes of the benzene rings being parallel butstaggered (cf. dibenzyl), whereas the tribromo-molecule has a two-fold axisof symmetry, the benzene rings being twisted in opposite directions by about72" from the plane of the nitroso-groups. The difference is probably due tothe effect of the extra bromine atoms which have to be packed in.Aniline hydrochloride and hydrobromide form ionic structures of differingtypes. I n the former,s2 all the cations point in the same direction in thelattice and each nitrogen atom has three contacts with chlorine ions at3.17 A. ; the chlorine ions are more than 5 A.apart, and the determining factorin the packing is the size of the cation, In the hydrobromide lgo howeverthere are cations facing in opposite directions, lying on the two-fold axesin the space-group P2,22,. Each nitrogen atom makes contact with fourbromine ions at about 3-5 A., and the arrangement is somewhat similar tothat in some alkylammonium halide structures.la* Nature, 1950, 166, 69.18* James and Saunder, ibid., p. 518.la' van Niekerk and Saunder, Acta Cryst., 1948,1, 44.la8 Darwin and Hodgkin, Nature, 1950,166, 827.lBg Fenimore, J . Amer. Chem. SOC., 1950, 72, 3226.loo Nitta, Watanabe, and Taguchi, X-Rays, 1948,5, No. 1, p. 31.186 Saunder, Proc. Roy. Soc., 1947, A , 190, 508HODGKIN AND PITT : ORGANIU COMPOUNDS. 457The polymorphism of p-iodo-N-picrylaniline has been studied byGrison with some very interesting results.57 Three crystalline varieties(red, orange, and yellow) all appear together on recrystallisation of any one.The orange is the stable variety a t ordinary temperatures; the red form isstable at temperatures near the melting point ; and the yellow form is meta-stable.X-Ray analyses demonstrate that the molecule preserves itsstereochemical form almost unchanged in all three forms; only in the redform is there a slight reduction of the valency angle at the amino-nitrogenatom, the molecule becoming a little more compact. This together withother minor differences can be interpreted as the outcome of rotations aboutthe single C-N bonds according to the exigencies of packing; for example,steric interactions appear to be the governing factor in the inclination of thenitro-groups to the plane of the benzene ring. The polymorphism is thuscaused by the same molecules being packed in three different stable ways,The energies of the three forms must be nearly equal since they all appearNFIG.10.s p- p- Chlorobenzaldoxine molecule, with hydrogen bonds (broken).simultaneously from solution. A further form, obtained when a melt iscooled, and described as vitreous, clearly represents the result of moleculesbeing unable to take up one of the stable packing arrangements. The pres-ence of a hydrogen bond between the amino-nitrogen atom and one of theoxygen atoms of an o-nitro-group is thought to explain the absence of basicproperties in the picrylanilines.Some fairly short intermolecular distances(about 3 A.) occur in all three structures.with polarised infra-red radiation led topredictions of the directions of the C,H,-N, N-H, C=O, and C-CH, bondsin the structure of acetanilide which facilitated the early stages of determin-ation of the atomic co-ordinates. A short note by Brown and Corbridge 192gives the arrangement of the molecules in the unit cell, linked in chains byN-H-0 bonds, but does not claim sufficient accuracy to warrant discussionof the bond lengths.The structure of syn-p-chlorobenzaldoxime confirms the configurationwith the hydrogen atom attached to carbon and the oxygen atom attached tonitrogen on the same side of the C-N double bond.lg3 As shown in Fig.10,lD3 Jerslev, ibid., 1950,166, 741.Observations, by191 Nature, 1947,160, 17. lea Ibid., 1948,162, 72458 CRYSTALLOGRAPHY.the nitrogen and the oxygen atoms of adjacent molecules are engaged inhydrogen bond formation in a manner which makes it possible that thehydrogen atoms are at XX or YY. If it is accepted that the rules of stereo-chemistry apply with smaller force to the atom on the side of the hydrogenbond remote from the hydrogen atom (Pauling),lS4 then the hydrogen atomswould be placed at XX, and thus the molecular formula would be (VIII).While this is of interest in connection with N-ether formation by oximes, itis in conflict with generally accepted views that, if tautomerism occurs, theequilibrium is strongly on the side of (IX).A detailed investigation of thecrystal structure might settle this question in the way that the lactam-lactim controversy in isatin was settled (see below).(d) Salts and esters ; ethers. Zinc and magnesium benzenesulphonatesare isomorphous, crystallising with six water molecules per X(C,H5*S03),.195Both structures may be described as consisting of sheets of metal atomssurrounded by regular octahedra of water molecules and separated by sheetsof benzene rings; the oxygen atoms of the sulphonate groups are hydrogen-bonded to the water molecules at distances ranging from 2-72 to 2.86 A.The S-C and S-0 distances appear to be normal, the valency angle of thesulphur atom being tetrahedral. The isomorphous zinc and magnesiumtoluenesulphonates have closely similar structures,1s6 the a axis of the unit cellbeing lengthened to accommodate the extra methyl groups.I n potassium hydrogen bisphenylacetate 197 there are again planes ofmetal atoms separated by the aromatic rings, but in this case the oxygenatoms of the carboxyl groups make up the octahedral co-ordination about themetal atoms.The potassium and hydrogen atoms must occupy specialpositions in the unit cell, and it is found that the potanssium atoms lie on two-fold axes; the hydrogen atoms must therefore lie a t centres of symmetry,halfway between oxygen atoms 2.55 A. apart. This situation is similar tothat reported for trona (Na,C03,NaHC03,2H,0) .lS8Aromatic esters have not until recently been studied in detail by X-rayor electron-diffraction methods.Although the structure assigned todiethyl terephthalate lS9 is not claimed to have the highest accuracy itshows that the molecule is planar except for a tilt of the ethyl groups of about9", and that the C-0 bond to the ethyl group is unusually long (1.510.05 A.). The structure is built up of layers between which the ethyl groupsand ketonic oxygen atoms make contact at about 3.5 A.The structure of quinol dimethyl ether provides evidence that theralency angle of oxygen is here about 120", in general agreement with the" The Nature of the Chemical Bond," Cornell, 1939.ls5 Broomhead and Nicol, Acta Cryst., 1948, 1, 88.lS6 Hargreaves, Nature, 1946, 158, 620.lug Ann. Reports, 1949, 46, 82.lS7 Speakman, J., 1949, 3357.Bailey, Acta Cvyst., 1949, 2, 120HODGKIN AND PITT : ORGANIC COMPOUNDS.459expectation of Sutton and Hampson.200 In the crystal the molecules assumea planar tram-configuration, and there is no indication of rotation of themethoxy-groups. Although the molecule is centrospmetrical in the crystalit cannot be so in solution, where a dipole moment has been observed. TheC-O-C angle appears to have the same magnitude in di-p-iodophenyl ether.201There is considerableinterest in the question of the planarity or otherwise of diphenyl derivatives.Diphenyl itself is known to be planar in the crystalline state because thespace-group demands a centre of symmetry in the molecule; 202 electron-diffraction studies of its vapour 203 however indicate that the two benzenerings must be inclined to each other a t an angle of about 45".Ultra-violetabsorption measurements confirm that the molecule is planar in the crystal,and non-planar in the vapour, and also show it to be non-planar in solution,as confirmed by measurement of a dipole moment.204 The steric repulsionbetween the 2 : 2'-hydrogen atoms tends to make the free molecule take up anon-planar form in opposition to the tendency towards coplanarity due toconjugation ; in the crystal lattice the additional effect of intermolecularforces may be sufficient to make the planar configuration the more stable.(In 2 : 2'-dipyridyl 205 the effect of steric forces between the hydrogen atomsis eliminated if the nitrogen atoms are trans to the 1 : 1'-bond, and in thecrystal structure this configuration has in fact been proved, the moleculebeing again strictly planar. The fact that the ultra-violet absorption spec-trum of 2 : 2'-dipyridyl in solution is similar to that reported for diphenylhowever seems to indicate non-planarity in the free molecule.) 3 : 3'-Dichlorobenzidine, 3 : 3'-dibromodiphenyl, and 3 : 5 : 3' : 5'-tetrabromo-diphenyl deviate from the cis-planar form by about the same angle asdiphenyl in the vapour state,206 whereas in solid 3 : 3'-dichlorobenzidine thechlorine atoms are tram and the molecule is planar, or approximately ~0.207In gaseous 2 : 2'-dichloro-, 2 : 2'-dibromo-, and 2 : 2'-di-iodo-diphenyl thephenyl rings are rotated from the cis-planar position so that the anglebetween them is about 75°.206 A similar configuration is found in crystalline2 : 2'-dichlorobenzidine (72") 208 and m-tolidine dihydrochloride (71") ; 209 inthese structures the bond joining the phenyl rings is equal in length, withinexperimental error, to a single C-C bond, but the lengths of some of the bondsin the phenyl rings may be abnormally short.When the two benzene rings are linked not by a single bond but throughone or more atoms, the valency angles at the latter influence the molecularconfiguration. In diphenylmercury the valency angle at the mercury atomis 180", and the molecule is again planar and centrosymmetrical in the(e) Compounds containing unfwed benzene rings.Goodwin, Przybylska, and Robertson, Acta Cryst., 1950,3, 279.201 Pleith, 2.Naturforsch., 1947, 2a, 409.202 Dhar, Proc. Nat. Inst. Sci. India, 1949,15, 11.=03 Bastiensen, Acta Chem. Scand., 1949, 3, 408.204 Merkel and Wiegand, 2. Naturforsch., 1948, 3b, 93.205 Cagle, Acta Cryst., 1948,1,158.307 Toussaint, Acta Cryst., 1948, 1, 43.208 Smare, ibid., p. 150.206 Bastiensen, Acta Chem. Scand., 1950, 4, 926.209 Fowweather and Hargreaves, ibid., p. 81460 URYSTALLOGRAPHY .erystal.210 The angle between the two phenyl links a t the ketonic carbonatom in 4 : 4'-dichlorobenzophenone is 127", and the molecule now has two-fold symmetry about the C=O bond, the benzene rings being twisted by 30'about the line joining the chlorine atom to the keto-group.211 A correlationbetween the approximate bond-lengths, the dipole moment, and the possibleresonant structures has been drawn.Di-p-bromophenyl sulphide, disulphide,and sulphone also all have molecules possessing an exact two-fold axis ofsymmetry.212 In the sulphide Toussaint claims to have found the S-C bondto have 12% double-bond character, and he deduces that the C-S-C angle iaincreased from the theoretical value of 90" to l09$" owing to this partialdouble-bond character ; van der Waals repulsion between the o-hydrogenatoms prevents the adoption of a planar configuration, the benzene ringseach making an angle of 36" with the BrS-Br plane. In the disulphide theS-C and S-S bonds are thought to be single, and the inclination of thebenzene rings at 43" to the BrS*S-Br plane arises from steric repulsion ofthe hydrogen atoms, unaffected by resonance in the S-C bonds; the S-S-Cangle is 107", apparently owing to steric repulsion between the 1-carbon atomand the 1'-sulphur atom.The benzene rings are inclined at 90" to theBrS-Br plane in the sulphone, where the S-C bond is again single. Owingto the increased tilt of the molecule, the C-S-C angle falls to 100" withoutsteric hindrance. The isomorphous dichloride and dibromide of di-p-tolyl-selenium 213 have molecular structures essentially identical with thosepreviously found for the diphenylselenium d i h a l i d e ~ . ~ ~ ~ The C-Se-C angle(107") resembles the sulphur valency angle in the sulphide discussed above,while the halogen-Se-halogen angle is 177", representing a slight deviationfrom linearity so that the halogen atoms are bent away from the p-tolylgroups. Theselenium-halogen bond distances are approximately 0.23 A.longer thanthe sum of the accepted single-bond covalent radii.The tetraphenylmethane molecule has h symmetry, and has been studiedas a two-parameter problem : 216 the angle (0) of rotation of a phenyl groupabout the bond joining it to the central carbon atom (measured from a planeparallel to c), and the angle (+) of rotation of the whole molecule about thec axis (measured from the a plane). The best fit was obtained with 8 = 55", + = 7.5". The structure is an open one owing to the awkward shape of themolecules.Therelated compounds meso-ap-divinyldibenzyl-(3 : 4-diphenylhexa-1 : 5-diene)and meso-ap-diethyldibenzyL(3 : 4-diphenylhexane) (X ; R = CHXH, orC2H,) have been shown to exist in the staggered configuration of dibenzyl inwhich the plane of the central bonds is approximately perpendicular to theThe benzene rings are inclined a t 40" to the C-Se-C plane.The fully-refined structure of dibenzyl was reported last year.210 Kitaigorodski and Grdenic, Izv.Akad. Nauk. S.S.S.R., 1948, NO. 2, 262.811 Toussaint, Bull. SOC. roy. Sci. Lidge, 1948, No. 1, 10.212 Idem, Bull. SOC. chim. Belg., 1945, 54, 319.213 McCullough and Marsh, Acta Cryst., 1950, 3, 41.214 McCullough and Hamburger, J . Amer. Chem. SOC., 1942, 64, 508.216 Sumsion and McLachlan, Acta Cryst., 1950, 3, 217BODGKIN AND PITT : ORUANIC COMPOUNDS. 461benzene rings.21s a@-Diethylidenedibenzyl (3 : 4-diphenylhexa-2 : 4-diene)(XI) is concluded to have the bTcans-trcans-configuration from considerations.R'Meof space-group symmetry and steric interaction. None of these structureshas however been analysed in detail.Two other compounds may be mentioned here, in which the demands ofnormal bond lengths and valency angles conflict with the van der Waalsseparation of atoms in the molecule.The first of these is di-p-xylylene (XII),a compound so far obtainable only as a result of drastic low-pressurepyrolysis.6* The formula shown has been established by X-ray methods, andthe bond lengths have normal values, while the valency angle a t the CH,group is 114.3;". If the benzene rings remained planar under these conditions,the distance between them would be 2-83 A.instead of the usual van der Waals(Benzenerings aromatic.)value of about 3.5 A. While the substituted carbon atoms of the benzene ringsare in fact separated by this short distance, the benzene rings are distortedso that the remaining atoms have a separation of 3-09 A. The second com-pound,217 bisdiphenylene-ethylene (9 : 9'-difluorenylidene) (XIII), is anorn-alous from several points of view : it is chemically more reactive and absorbslight of wave-lengths further towards the red end of the spectrum than mightbe expected by comparison with other members of the homologous series.Steric interaction between the carbon atoms marked * may be expected ifthe molecule is completely planar since on the basis of customary bondlengths the separation of these carbon atoms will be only 2.5 A.Unfortun-ately the crystal structure is rather complex and will not readily yield accurateatomic co-ordinates, but evidence has been deduced that the molecule is infact centrosymmetrical and approximately planar and that the separationof the carbon atoms marked * is about 2.5 A. ; it is not however possible tosay whether there is any tendency to form a doubled radical with oppositecharges on adjacent carbon atoms marked *.216 Jeffrey, Koch, and Nyburg, J., 1948, 1118.217 Fenimore, Acto Cryst., 1948,1, 295.REP.-VOL. XLVII. 462 CRY STAUOGRAPHY .( f ) Naphthalene and anthracene derivatives. A number of naphthalenederivatives have been studied by Kitaigorodski and others, but detailedresults are in general not yet available.21s The methods of analysis seem notto present any novel features beyond a rather detailed consideration ofpacking efficiency where the molecules can be approximated in shape bytriaxial ellipsoids.Among other structures reported as under consideration,those of a- and @-naphthol are perhaps of greatest interest.219 In theformer the molecules form close-packed layers with six-fold co-ordination,and the hydroxyl groups are linked in chains by hydrogen bonds (2.54 A,).The @-naphthol structure is very similar to that of the parent naphthalene,the unit cell being doubled in the c direction. The molecules are linked inpairs by hydrogen bonds (2.60 A.) perpendicular to the c axis, and the separ-ation parallel to the c axis corresponds to van der Waals interaction.Considerations of packing have been used to solve the structure of theawkwardly shaped di-n-octylnaphthalene.220 Approximate results havealso been published for 1 : 5-dinitro-,,,l 2 : 6-diphenyl-, and 2 : 6-dicyclo-hexyl-naphthalene,21s all of which have centrosymmetrical molecules ; in the2 : 6-diphenyl compound, the angle between the phenyl ring and thenaphthalene plane is .23".Acenaphthene has been resurveyed, and foundto have a flat molecule, the bond joining the CH, groups being at least1.8 A . , ~ ~ ~ a surprising result requiring confirmation.The molecule of anthraquinone is also centrosymmetrical, and shows ageneral similarity to benzoquinone. As in the latter compound, the C=Obond is short (1.15 A.) while the adjoining C-C bonds are relatively long(1 6 0 A.) .223The stereochemistry of this interesting compound isstill unsettled : it remains questionable wheth& it should be classified asaromatic (if it is regular and highly resonant) or aliphatic (if an alternationof double and single bonds exists in the ring).The former view is supportedby electron-diffraction study of the vapour : according to the interpretationby Bastiensen and Hassel the crown form with angles 1213" fits the databest, the mean C-C distance being 1.425 A. with small alternations p0ssible.22~X-Ray crystallographic evidence (of a somewhat limited nature as yet) leadsKaufman, Fankuchen, and Mark to support the latter view : the tub formwith angles 125" and alternate single (1-54 A.) and double (1.33 A.) bondsis Infra-red absorption measurements also agree with a crownform .226Heterocyclic Compounds.-(a) Six-membered ring jused to jice-memberedring.The most important structural investigation in this group is that,cyclooctatetraene.218 Izv. Akad. Nauk. S.S.S.R., 1947, No. 6, 561.219 Kitaigorodski, Dokl. Akad. Nauk. S.S.S.R., 1945, 50, 319.220 Idem, Acta Phys. Chem. U.R.S.S., 1947, 22, 309.221 Sevastyanov, Zhdonov, and Umansky, J . Phys. Chem. Russia, 1948,22, 1153.2a2 Kitaigorodski, ibid., 1947,21, 1085.224 Acta Chem. Scand., 1949, 8, 209.226 Lippincott, Lord, and McDonald, ibid., 1950,166. 227.Sen, Indian J . Physics, 1948,22, 347.225 Nature, 1948,161, 165HODGEIN AND PITT : ORGANIC COMPOUNDS.463of isatin 227 which has been carried out with considerable accuracy in orderto distinguish between the lactam (XIV) and the lactim (XV) structure.H(XIV.)The bestassuming0 0agreement with the observed bond lengths can be obtained bythat the lactam and the lactim form contribute in a ratio of aboutsix to one. The molecule therefore exists in a state close to pure lactam.Molecules are related in pairs by a centre of symmetry and linked by N-H-0bonds of length 2.93 A. which do not lie exactly in the planes of the molecules.Considerable use was made of electron-density maps on the plane z = 22which show the outline of the whole molecule since the latter makes an angleof not more than 10" with (109); the authors claim that the apparentdecrease of the electron-density maxima with increasing distance from t4heorigin is probably due to the greater thermal motion at the benzenoid endof the molecule (a somewhat similar effect was found in geranylamine hydro-chloride).In the present case, however, it must be borne in mind that thetilt of the molecule out of the section plane x = 22 means that for atoms remotefrom the origin the electron density is being sampled on a section of the atomwhich does not pass through its centre ; a decrease of the maxima is thereforeonly to be expected.In piazthiole and piaselenole (XVI; X = S or Se) there is again aheterocyclic ring fused to a benzene ring, but owing to theabsence of hydrogen bonds the packing arrangement is x quite different.22* The isomorphous structures have been\\ NN/ solved in projections by the heavy-atom technique, and agenerous estimate of the probable errors in bond lengths hasbeen given.The N-S distances are 1.57 A., and the N-Sedistances 1.85 (-+ 0.08) A.(b) Two five-membered rings fused together. The accurate analysis ofthiophthen by three-dimensional Fourier methods 229 was mentioned brieflyin last year's Reports. This compound was examined in order to discoverwhether theoretical calculations gave better agreement with the deductionsfrom experimental than that in the case of thiophen (experimental data byelectron diffraction). The theoretical calculations for thiophthen are anextension by Evans and de Heer 230 of the molecular-orbital treatment ofthiophen by Longuet-Higgins in which it is assumed that the sulphur 3 p and3d atomic orbitals are hybridised and compounded with the carbon 2porbitals in the formation of molecular x orbitals (vide Schomaker and(xvI.) i"j""'227 Goldechmidt and Llewellyn, Acta Cyst., 1950, 3, 2?4.228 Luzzati, Compt.rend., 226, 738; 227, 210.22s Cox, Gillot, and Jeffrey, Ada. 1949, 2, 350. Ibid., p. 363464 CRYSTALLOGRAPHY.Pauling 231). I n the comparison of the bond lengths experimentallydetermined with those calculated in the above manner it is necessary toestablish a relation between bond order and bond length for C-S bonds, inaddition to that well established for C-C bonds. The available estimatesof the lengths of pure single and double C-S bonds seemed to the authors some-what unreliable, and they conclude that there is a possibility that thedifferences between the observed and calculated C-S bond lengths may bedue to incorrect standard bond lengths and not to inadequacy in themolecular-orbital calculations.The agreement in the case of the C-Cbond lengths was found to be very satisfactory except for the bond commonto the two rings which is found experimentally to be 0.05 A. shorter than thetheoretical value. Longuet-Higgins 232 has subsequently pointed out thatEvans and de Heer's calculations assume that the a-bond system is free fromstrain, whereas the results of the X-ray analysis indicate that the exteriorangles a t the tertiary carbon atoms are about 135", and hence the a-skeletoncannot be strain-free.When the effect of this strain is allowed for, the centralbond is shortened by 0.06 A., bringing the calculated length into excellentagreement with that determined experimentally.The stereochemistry of the amino-and hydroxy-pyrimidines has been established in some detail by Clews andCochran and Pitt. A reliable basis is thus available from which may beundertaken the investigation of some of the many interesting and importantcompounds containing these groupings which occur in biological systems.The isomorphous 2 -amino-4 : 6 -dichloro- and 2 -amino-6-chloro -4-methyl-pyrimidines first studied by Clews and Cochran s1 are related to each other ina manner similar to that in p-dibromo- and p-lpomochloro-benzene mentionedabove, the methyl groups and chlorine atoms in the second compoundoccupying at random the 4- and 6-positions which are both occupied bychlorine atoms in the first compound.Weak N-H-N bonds (3.21, 3-37 A.)link the molecules in sheets, all three nitrogen atoms in the molecule partici-pating; the amino-nitrogen atom forms two such bonds almost coplanarwith the molecule, while the nitrogen atoms in the ring each form one non-coplanar hydrogen bond. The chlorine atoms pack together in columns, onehaving contact with six near neighbours and the other with seven, some ofthe CI-C1 distances being unusually short. The C-Cl distances resemblethose found in aliphatic compounds more closely than those found in chloro-benzenes, and this is in accordance with the high chemical reactivity.Avery careful analysis of 4-amino-2 : 6-dichloropyrimidine shows it to existin the amino- rather than the dihydroimino-pyrimidine form.233 Thenitrogen atoms are again engaged in hydrogen-bond formation, and theauthors claim that there are low maxima in the electron density near thenitrogen atoms which could be explained as due to (i) hydrogen atoms nearthe amino-nitrogen atoms, and (ii) unshared electron pairs of the ring-nitrogen atoms, interacting with (i), on the basis of Pauling's theory of(c) Pyrimidine and purine derivatives.233 J. Amer. Chem. SOC., 1939,61, 1779.232 Acta Cryst., 1950.3, 76. *38 Clews and Cochran, ibid., 1949, 2, 46HODGKIN AND PITT : ORGANIC COMPOUNDS.465hydrogen-bond formation. The heights of the latter peaks are not muchabove the background undulation, and the interpretation seems notaltogether convincing to the Reporter who, having encountered similarsmall regions in electron-density measurements of another structure, recentlycalculated the corresponding " Fc-synthesis " baaed on the calculated struc-ture factors (without hydrogen-atom contributions) and found that theregions persisted and were thus due to termination-of-series errors. Althoughthis may not be the explanation in the previous case, some such confirmationwould carry more weight than the series-termination corrections actuallyapplied.In 2-hydroxy-4 : 6-dimethylpyrimidine the hydrogen-bond system iscomplicated by the presence of water of crystallisation.60 The structure ismade up of interlinked corrugated sheets of molecules united by hydrogenbonds of three types : (i) from 2-hydroxy- to water molecule(2.8,2-9 A.) (not coplanar with the ring) ; (ii) from 1- or 3-nitrogento water molecule (2.8, 2.9 A.) (coplanar with the ring) ; (iii) fromwater molecule to water molecule (2.8 A.).More recent workshows that there is a definite tendency towards a quinonoidtype of molecule, with the C=O bond short ; this may imply thatstructures of the type inset make an appreciable contribution tothe description of the molecule. If this is so, the negative charge on the5-position can be correlated with the chemical reactivity at this point.Adenine hydrochloride has been found by Broomhead234 to have aplanar molecule and bond lengths comparable to those in the amino-pyrimidines except for the C-C bond common to the two rings which is long(1.44 A.).The hydrogen bonding again forms a major factor in determiningthe arrangement of purine molecules, chlorine ions, and water molecules inthe lattice, and it is understood that the hydrogen atoms have all been locatedin more recent work involving the (F,-FJ-synthesis.The nucleic acids have molecules too large for detailed study by X-raymethods as yet developed, but chemical evidence shows that they arebuilt up from a relatively small number of nucleotides (composed of apyrimidine or purine with ribose or deoxyribose and a phosphate group),and X-ray methods will no doubt be very valuable in the determination ofthe steric relations in these compounds of rather unpredictable shape.As aprelude to the examination of one of the nucleotides, cytidilic acid, Furberghas investigated the nucleosides ~ytidine,~~5 and (in outline) uridine, adeno-sine, and g ~ a n o s i n e . ~ ~ ~ Direct confirmation is obtained that cytidine iscytosine-3 p-D-ribofuranoside. The pyrimidine ring has dimensions com-parable to those found in the structures mentioned above ; the C=O distanceis very similar to that in the hydroxypyrimidine, while the C,6,-N distanceresembles that in the aminopyrimidines. The C-N bond between the ringsis effectively a pure single bond. The D-ribose ring is approximately planarexcept for one carbon atom which is displaced by 0-5 A.; the hydroxyl group7' N/\N I] 11-234 Acta Cryst., 1950, 3, 324.,236 Acta Chem,. Bcnnd., 1950, 4, 751.a35 Ibid., p. 325466 CRYSTALLOGRAPHY.attached to this atom then falls in the plane of the ring, as was found byBeevers and Cochran in the case of fructofuranoside. The bond joining thetwo rings lies in the plane of the pyrimidine ring but makes approximatelytetrahedral angles with the adjacent sides of the ribose ring, so that the tworings are nearly perpendicular, contrary to Astbury’s prediction. All theactive groups are engaged in hydrogen-bond formation and, in addition, thedistance from C(4, of the pyrimidine ring to the 0(5) atom of the ribose ringis short (3.24 A , ) and may represent a weak linkage, possibly to be correlatedwith the chemical stability.G. J. P.Natural Products of Moderate Complexity.-The three compounds wehave to consider in this group, strychnine, penicillin, and calciferol, arechemically very different and are linked here by the crystallographic problemsinvolved in the determination of their structures. In all three cases, theX-ray analysis has been achieved largely through the use of heavy atomderivatives to assist in the process of phase determination.The structure of strychnine has been found independently by two groups,Bokhoven, Schoone, and Bijvoet in Utrecht 237 and Robertson and Beevers inThe analysis in both cases proceeded remarkably smoothly.The Dutch workers used the isomorphous series, strychnine sulphate andselenate, and calculated electron-density projections.Prom the first ofthese, a centrosymmetrical projection, they found it possible to select thecorrect formula and to confirm this by calculation of a second projection.Robertson and Beevers proceeded directly to the determination of theelectron density in three dimensions via the calculation of a three-dimensionalPatterson series for strychnine hydrobromide and the correct selection of theBr-light atom vectors. Curiously enough, although the crystal structuresstudied differ in symmetry and ionic content, the arrangement of themolecules is very similar in relation to the crystal axes and their appearancein the electron-density contours in the main projections, viewed down 7.58and 7.64 A.respectively, is almost identical. The large roughly boat-shapedmolecules are fitted together as closely as possible, the spaces between thembeing filled by the ions and a few odd water molecules. By a happycoincidence, the same structure was deduced on purely chemical groundsalmost simultaneously with the first X-ray analysis.239The structure analysis of penicillin was also based on the examination ofthree salts having two types of crystal structure-sodium, potassium, andrubidium benzylpenicillin-and these were investigated, to a large extentindependently, by two groups of research workers.53 Here, owing partly tothe low atomic weight of sodium and to the special positions occupied bypotassium and rubidium in the crystal, the direct phase determination wasvery incomplete. Extensive trial-and-error analysis by the use of opticaldiffraction and the direct comparison of rough electron-density patternsobtained in the two investigations played a part in the solution of the237 Proc.K . Ned. Alcad. Wet., 1947, 50, 8 2 5 ; 1948,51, 990; 1949, 52, 120.238 Nature, 1950,165, 690. 239 Robinson, ibid., 1947, 160, 18HODGKTN AND PITT : ORGANIC COMPOUNDS. 467structure. At the time that the atomic arrangement shown in Fig. 11 wasreached by calculation of the electron density in three dimensions, there wasstill considerable weight of chemical opinion against the formula found. Itis somewhat amusing now, five years later, to find the C-N and K-0 distancesin penicillin quoted as reasonable standards with which to compare distancesfound in such molecules as alanine and potassium hydrogen bisphenylacetate.Actually the accuracy of bond-length measurement in the penicillin structureas published is not high, though fully sufficient to establish the general arrange-ment of bonds within the molecule.A later refinement of the three-dimensional electron-density distribution in potassium benzylpenicillin hasprovided a series of more accurate bond lengths; 240 the greatest deviationof these from generally accepted values is now 0.06 A. instead of 0.13 A. inthe earlier refinement. The new bond lengths agree very well with those0 9CCH(a) (b )FIG. 11.(a) Bensylpenicillin. (b) Strychnine.to be expected from the straightforward p-lactam formula ; within theamide side chain the inter-atomic distances are, for example, similar to thosein acetamide within the limits of experimental error.I n the benzylpenicillin molecule, neither the thiazolidine five-memberednor the p-lactam four-membered ring is planar, in agreement with conclusionson other similar rings. The two are fused together in the cis-position andthe phenylacetamide group is attached to the p-lactam ring on the same sideas the thiazolidine-sulphur atom. This arrangement fixes the stereochemicalconfiguration at the two centres and C,,,, as opposite to one another;chemical degradation has established them as D and L respectively. I n bothcrystal structures the molecule has a compact semicircular form, with thethiazolidine and benzene rings not far from parallel to one another. This mustlargely be due to packing considerations ; it enables all the oxygen atoms, bothof the p-lactam and the amide groups, as well as the carboxyl groups, to bearranged around the ions to form an ionic layer in the structure, and, as inmany inorganic crystals, the change in crystal structure when passing from240 Pitt, p arsonal communication468 CRYSTALLOGRAPHY.the potassium to the sodium salt enables seven oxygen atoms to makecontacts with the potassium ion, while only six make contact with each sodiumion. Owing to the complex geometry of the molecule, however, the packingsituation is also very complicated-for example, the oxygen atoms of sixdifferent penicillin ions have to make contact with each potassium ion inthe potassium salt.The third crystal structure in this group, that of calciferol 4-iodo-5-nitrobenzoate, is not completely solved. But the single electron-densityprojection obtained is sufficient to show both the intricate way in which themolecules fit into the crystal and the general form of the molecule i t ~ e 1 f . l ~ ~Earlier preliminary crystallographic measurements on calciferol had suggestedthat the molecule should be not very unlike cholesterol and had thrown somedoubt on the chemical evidence for the breaking of ring B.241 However, thepresent more exact data show that, in this crystal, ring B is not only openbut wide open. The molecule is fully extended and the projected atomicpositions agree very well with those expected from chemical evidence.Certain stereochemical details can be added-for example, the arrangementa t the 22 : 23 double bond is clearly trans.The use of heavy atoms in all these structures to achieve a t least partialphase determination raises the question of how long the process can becontinued-what is the maximum size of molecule for which this processmight work. Calciferol 5-iodo-4-nitrobenzoate has 41 atoms * and is thelargest organic molecule for which an electron-density projection showingindividual atoms has yet been published. But there are a number of othersimilar-sized molecules under investigation, including chloromycetin anda u r e o m y ~ i n , ~ ~ ~ and a t least one larger, of which the crystal structure hasbeen essentially The possibility of structur? analysis, with orwithout a heavy atom, certainly does not end a t this order of complexity.I n a rough way this can be shown by the comparison of the average scatteringpower of a single heavy atom in the crystal unit to that of a number ofcarbon atoms, assuming as correct the deduction by Wilson 244 and others ofthe statistical distribution of the intensities of X-ray reflections in complexcrystals. It seems most probable that, in practice, the limit will be set bythe intensities of the observable reflections. As the molecules become morecomplex, reflections from planes of small spacing no longer appear on X-rayphotographs. For a molecule of the order of magnitude of vitamin BI2, forexample, for which the crystal asymmetric unit has a weight of about 1600,no reflections are visible from planes with spacings smaller than about1.1 A.245 At this limit, it should still be possible, at least theoretically, tocalculate an electron-density distribution showing resolved atoms. Butwhen we come to molecules of much greater weight, such as proteins, this is241 Bernal, Crowfoot, and Fankuchen, Phil. TTans., 1940, 239, A , 135.242 Dunitz and Leonard, J. Arne?-. Chem. Soc., 1950, 72, 4276.s13 Vend, Personal communication.mi Hodgkin, Porter, and Spiller, Proc. Roy. Soc., 1950, B, 136, 609.* Not counting hydrogen.244 Wilson, Acta Cryst., 1949,2, 318HODGKIN AND PITT : ORGANIC COMPOUNDS. 469no longer true, beautifully crystalline though many proteins may be. Theinterpretation of their crystal structures raises problems of an altogetherdifferent dimension from those discussed here and must be left for yetanother Report.D. C. H.We were very greatly assisted in covering the field of this Report by theuse of the classified bibliography issued by the American Society for X-Rayand Electron Diffraction and the American Crystallographic Society, and alsoof the abstracts prepared for forthcoming Structure Reports through theInternational Union for Crystallography. We are much indebted for theloan of these to Professor Lonsdale and Dr. A. J. C. Wilson. We alsoacknowledge the help we received in preparing the report from Dr. ClaraBrink and Dr. June Broomhead.DOROTHY CROWFOOT HODGKIN.G. J. PITT.J. THEWLIS

ISSN:0365-6217    

DOI:10.1039/AR9504700420

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

INDEX OF SUBJECTS.Absorptiometer, Spekkcr, 402.Absorptiometry, 401.Aconaphthylene, crystal structure of, 449.Acetaldehyde, low-temperature oxidationphotodecomposition rate constants of, 47.reaction of hydroxyl radicals with, 30.of, 25, 42.Acetaldehyde, trifluoro-, 177.Acetaldehyde dehydrogenase, 340.Acetanilide, structure of, 457.Acetic acid, aluminium salt, 103.determination of, 395.isopropenyl ester, as acetylating reagent,,thermal decomposition of, 38.2’4-dichlorophenoxy-, determination of,difluoro-, and trifluoro-, and its ethyl156.Acetic acid, chloro-, uranyl salts, 116.414.ester, 177.Acetone, photolysis of, 48.Acetone, hezafluoro-, 177.Acetophenone, cis-benzylidene-, 150-Acetyl phosphate, preparation of, 156.radical, heat of formation of, 23.Acetylation, 156.Acetylenes in organic synthesis, 166.Acetylenic compounds, halogen additionAcids, aliphatic carboxylic, 170.aromatic, salts and esters, crystalcarboxylic, determination of, 395.fatty, preparation of, 158.monobasic, and their salts, crystalAcrylic acids, a-substituted, preparationActinium, pure, preparation of, 104.Actinomyces, antibiotics from, 290, 294.prodigiosin-like pigment from, 278.Actinomycin, 294.acid hydrolysis of, 176.Actinorhodin, 295.Acylamino-acids, anhydrides of, 163.Addition reactions, 126.Adenine hydrochloride, crystal structurepreparative uses of, 159.to, 131.structure of, 458.preparation of, 160.structure of, 442.of, 159.of, 465.methylthiodeoxypentoside, 266.Adenosine, periodate oxidation of, 259.phosphates, 262, 267.synthesis of, 260, 261.Adipic acid, crystal structure of, 442, 444.Adrenocorticotrophin, 366.Adsorption at non-uniform surfaces, 54.multilayer, 58.Aerosporin, 296.4Bitioporphyrin I, 273, 283.Agenising of flour, 173.Agrocybe dura, antibiotic from, 289.Agrocybin, 289.Albumin, bovine-serum-, solut,ion of dyesserum-, light scattering of, 90.Alcohols, carbonation of, 161.crystal structure of, 439.determination of, 392.Alcohol dehydrogenase, 339.Aldehydes, determination of, 394.oxidation of, 42.photolysis of, 48.preparation of, 160.unsaturated, dipole moments of, 19.Alicyclic ring compounds, crystallographyAliphatic compounds, crystallography of,Alkaloids, determination of, 397.Alkyl halides, elimination from, 137, 138,sulphides and polysulphides, crystalAlkylammoniun salts, crystal structure of,Allene, vibrational-rotational spectrum of,Alloxan, determination of, 398.Alloys, analysis of, 387.Alterrulria sotani, antibiotic from, 290.Alternarine, 290.Aluminium borohydride, oxidationin, 82, 83.of, 448.438.141.structure of, 440.441.16.structure of, 429.initiated by, 41.compounds, 102, 103.determination of, 378, 382, 410.hydride, Raman spectrum and struc-nitride, determination of, in steel, 381.oxide, stability of suspensions of, 84.ture of, 11.Ambratriene, 203.Ambrein, 203.Americium, separation of, 118.Amides, determination of, 393.Amine picrates, conductivity of, in toluene,Amines, crystal structure of, 441.Amino-acids, 173.preparation of, 161.75.determination of, 393.preparation of, 161.crystal structure of, 446.determination of, 393.requirement of, by man, 330.by poultry, 314.by swine, 323.separation and determination of, 417.INDEX OF SUBJECTS.471Amino-groups, determination of, 393.protection of, in peptide syntheses, 162.Ammonia, mercury-photosensitised de-Ammonium nitrate, isotopic effect in de-permanganate, explosion of, in air, 123.phosphomolybdate, precipitation of,Amni vimaga, active principles from, 223.hnperometric titration, 41 1.a- and 8-Amyrins, 199.Analysis, reagents for, 374.Anhydrides, N-carboxy-, polymerisationof, 251.Anhydro-bases, 228.Aniline hydro-bromide and -chloride,crystal structure of, 456.Aniline, p-iodo-N-picryl-, polymorphismof, 457.Anionotropic rearrangements, 142.Anodic passivation, 73.Anthracene, crystal structure of, 433, 434,derivatives, crystal structure of, 462.determination of, 403.clinical uses of, 302.mode of action and acquired resistancecomposition of, 45.composition of, 27.384.453.Antibiotics, 285.to, 298.Antiferromagnetism, detection of, 431.Antimycin A, 294.Antrycide, determination of, 398.Aromadendrene, 196.Aromadendrone, 196.Aromatic compounds, bromination andiodination of, 159.crystallography of, 452.polyc yclic, 180.energy of, 13.pure, preparation of, 111.reagent for cadmium, 375.Arsenic trifluoride, vibrational-rotationalArsonium chloride, methyltriphenyl-, asAscaridole, 193.D-Aspartic acid oxidase, 345.Aspergillus, amino-acid oxidases of, 345.Atom-photosensitised reactions, 45.Aureomycin, 293.Auxin-b, 231.Ayfivin, 295.Azides, determination of, 378.Azobenzene, cis-truns-isomerisation of, 30.Azonitriles, decomposition rates of, 26.Azulenes, 187, 196.spectra of, 9.Azulenes, hydro-, 197.Baccatine A, 289.Bacillus prodigiosus, pigment from, 277.Bacitracin, 295.Bacteria, antibiotics from, 295.Gram-negative, somatic antigens from,toxins from, 303.313.Baikiaea plurijzsga, 175.Baikiain, 175.Basidiomycetes, antibiotics from, 289.Benzaldoxime, sym-p-chloro-, structure of,Benzene, emission spectrum of, 9.Benzene, chloro-, fluorescence spectrum of,fluoro-, fluorescence spectrum of, 10.halogen derivatives, crystal structure of,nitro-, dipole moment of, 18.m- and p-dinitro-, crystal structure of,nitroso-, dimerisation of, 456.Benzhydrazide, p-iodo-, as reagent forpreparation of ketone derivatives,160.Benzidine change, 145.Benziminazole, 5 : 6-dimethyl-, 240.Benzofurans, 222.Benzoic acid, photo-oxidation of, 51.Benzopyrans, 226.Benzopyranols, 228.Benzothiazole, mercapto-, polarographyBenzoyl peroxide, decomposition rate of,Benztropolones, synthesis of, 185.Berkelium, 1 18.Beryllium, determination of, 378.Betulin, 202.Blood, determination of protoporphyrinBlood cells, action of lecithinases on, 306.Blood-group A substance, 248.Bond-dissociation energies, 22, 26.Borohydrides, infra-red spectra of, 15.Boron compounds, 101.Botulinus toxins, 310.Brain, horse, acid from, 17 1.Brass, electrolytic, 72.Brassidic acid, synthesis of, 17 1.Brazilones, anhydrotrimethyl-, 225.Bromination of aromatic compounds, 159.Bromine, addition of, to olefins, 127, 128,457.10.454.hydroxylation of, by X-rays, 52.surface tension of solutions of, 76.455.of, 410.25.in, 276.determination of, 378.129.cations, existence of, 123.determination of, 381.trifluoride, absorption spectrum of, 7.as fluorinating agent, 120.Brornoform, determination of, 398.Burner cones, photographs of, 44.Butadiene, addition of 2-akylpyridinesButadienes, 8-substituted, syntheses with,Buta-1 : 3-diene, burning velocity of, 44.cycloButane, octafluoro-, structure of, 2 1,1 : 2 : 3 : 4-tetruphehyl-, crystal struc-Butenes, mercury-photosensitised hydro-to, 158.178.449.ture of, 449.genation of, 46472 INDEX OF SUBJECTS.isoButene, catalysed polymerisation of,tert.-Butyl chromate as oxidising agent,hydroperoxide, thermal decompositionn-Butyric acid, y-amino-, from beetroot,36.153.of, 25.173.heptafluoro-, 177.sulphate complexes with quinoline, 100.Cadmium, determination of, 382." Camdosin ", 348.Caesium, extraction of, 99.Calameon, 195.Calciferol, conf5guration of, 2 15.4-iodo-5-nitrobenzoate, crystal struc-Calcite, electrokinetics of, against electro-Calcium, determination of, in water, 385.Californium, 119.Carbinol, isobutylethylmethyl-, resolutionof, l48,.," Carbates , 376.Carbazoles, tetrahydro-, 239.Carbohydrates, crystallography of, 450.from nucleic acids, 258.requirement of, for cattle, 326.ture of, 468.lyte solutions, 81.dichromate hydrates, 100.for man, 332.for poultry, 316.for sheep, 328.388.Carbon arc as infra-red source, 13.determination of, in organic compounds,in steel, 383.tetrafluoride, photosensitised decom-heat of atomisation of, 22.monoxide, determination of, in air, 386.disulphide, absorption spectrum of, 8.Carbon-hydrogen bond, dipole moment of,Carbonyl compounds, preparation of, 160.Carnations, chromone constituents of,&Carotene, synthesis of, 166.cis9 : 9'-/3-Carotene, 169.Carotenoids, 166.Carotol, 195.Carragheenin, 247./3-Caryophyllene, 19 7.Caryophyllenic acid, 197.Catalases, 249, 348.Catalysts, hydrogenation, 154.Cattle, nutrition of, 324.Cells, Tiselius, 81.Cellulose, nitro-, molecular properties of,Cerium sulphides, 105.Chain extension, 156.Chemisorption, 66.Chitin, 248.position of, 46.oxidation of, by iodine, 105.14.groups, determination of, 394.229.89.Chloral hydrate solutions, action of X-raysChloramphenicol, 292.Chlorin, tetraphenyl-, zinc complex of, 283.Chlorins, 274.Chlorine, addition of, to olefins, 127, 130.trifluoride, absorption spectrum of, 8.oxides, 121.Chlorites, analytical chemistry of, 379.Chloroform, determination of, 398.photochlorination rate of, 60.Chlorogallates, 103.Chlorohydrins, preparation of, 159.Chloromycetin, 292.Chlorophyll series, 274.Chloroprene, dimer of, 189.Chromatography , 4 14.of antibiotics, 287.partition, with polysaccharides, 247.Chromium compounds, 113, 114.determination of, in steel, 382.electrodeposition of, 72.Chromobacterium iodinum, antibiotic from,Chromones, 229.Chromoproteins related to vision, 169.Cinerins, 204.Cinerolones, 205.Circulin, 296.isocitric acid dehydrogenase, 342.Citrinin, 228.Citromycetin, 227.Citromycin, 227.Citromycin, O-dimethyl-, 227.Citromycinol, O-dimethyl-, 227.Citrom ycinone , 0 -dime t h yl- , 2 2 7.Clathrate compounds, 98.Clay sols, stabilisation of, 84.Clitocybe itludens, antibiotics from, 290.Clostridium species, lecithinases from, 305.Coacervation, 84.Cobalt complexes, 124.Coenzymes, 337.Colchicine, 185.Colloids, physical chemistry of, 76.Columbium.See ypbium." Conchoporphyrin , 275.Conductometric titration, 408.Copaene, 195.Copolymerisation, 33.Copper, anodic polishing of, 73.chromite, 154.complexes with amines, 99.Copper-protein enzymes, 347.Cordycepin, 289.Cordyceps militaris, antibiotic from, 289.Corynebacterium diphtheriae, porphyrinfrom, 278.Cortisone, 211, 215.Coumarans, 222.Coumarins, 236.Coumarins, 4-hydroxy-, 236.on, 52.liquid, density of, 121.298.determination of, 380.electron transport between oxygen and,356INDEX OF SUBJECTS. 473isoCoumarins, synthesis of, 236.Coumarones, 222.Counters, boron trifluoride, 427.Cryptotethia, nucleoside from, 266.Crystals, organic, molecular packing in,Crystallography as means of identification,8- and y-Curcumenes, 194.Curium, separation of, 118.Cyclisation, 155.Cyclophorase system, 358.Cysteine, S-allyl-, S-oxide, 174.Cysthe, synthesis of, 173.Cytidine, 258, 259, 260.Cytidine-2’ phosphate, 262.Cytidylic acid, 257.Cytochromes, 353.Cytochrome-a, prosthetic group of, 277.Cytochrome-c, 352.434.437.electron transport between oxygen and,electron transport from succinate to, 355.Cytosine, determination of, 398.Decane, mercury-photosensitised decom-position of, 45.Decanoic acid, potassium salt, crystalstructure of, 442.Degradation, kinetics of, 34.Dehydrochlorination, 3 7.Dehydrogenases, 3 39.Deoxynucleosides, synthesis of, 262.Deoxypatulin, 235.Deoxypatulinic acid, 235.Deoxypentosenucleic acid, 254.Deoxypentosenucleic acids, structure of,2-Deoxy-~-ribose, 258.Deoxyribosides, 257.Depolymerisation, 34.Deuterioacetaldehyde, photolysis of, 48.Deuteriochloroform, photoahlorinationDeuteriodiborane, infra-red spectrum of,Deuteriohydrocarbons, optically active,Deuterium-hydrogen mixtures for calibra-Dextrins, crystal structure of, 452.Dialkyl chlorophosphonates, 157.Dialysis, 78.Diamond, crystal structure of, 434.1 : 2 : 5 : 6-DibenzocycZooctatetraene, 188.Dibenzyl, and its derivatives, crystalstructure of, 461.Diborane, structure of, 15.Dimes, preparative uses of, 158.Diene synthesis, 177.Dietary factors for poultry, 319.Diffusiometer, Gouy, 79.Diffusion of colloids, 78.9 : 9’-Difluorenylidene, crystal structure356.264.rate of, 50.15.148.tion purposes, 412.with steroids, 208.of, 461.Difructose strontium chloride dihydrate,crystal structure of, 450.“ Dihydronicotinamide-D-ribofurano-side,” 268.ay- and fly-Diketo-acids, preparation of,159.cis- and trans-Diols, acid dehydration of,184.Dioxans, 236.Diphenic acids, perhydro-, 151.Diphenyl, and its derivatives, crystalstructure of, 459.Diphosphopyridine nucleotide, 337.Diphospho thiamine, 337,Diphtheria toxin, 308.Dipole moments, 19.2 : 2’-Diquinolyl as reagent for copper,Disalicylide, 152.Dithionate ion, Raman spectrum andDithionates, analysis of, 410.Di-p-xylylene, crystal structure of, 461.Dodecanoic acid, aluminium salt, 103.Dracoic acid, 228.Draconol, 228.Dracorhodin, 228.Dracorubin, 228.“ Dragon’s blood ’’ resin, pigments from,375.structure of, 11.228.Earths, rare, analysis of, 381.Echinocystic acid, 200.Edestin, mol.wt. of, 249.Electrical double layer, 79.Electroanalysis, 408.Electrodes, mechanism of reactions at,Electrode systems, 74.Elec trokine tics, 7 9.Electrolytes, activity and osmotic co-Electrolytic conductivity, 75.polishing, 72.Electrometric analysis, 407.Electron capture, mechanism of, 53.diffraction, 19.transfer reactions, photo-excited, 51,Electronic spectra, 7.Electro-osmosis, 81.Electrophoresis, 79, 80.Electrostatic precipitator, 386.Eleutherin, 226, 232.+Eleutherin, 227.Eleutherina bulbosa, constituents of, 226,Eleutherol, 226, 232.Elimination rate, effect of constitution on,68.efficients of, 74.232.136.reactions, 134.Emission spectra, 7.Enzyme complexes, location of, in mito-substrates, electron transport betweeneffect of environment on, 140.chondria, 358.oxygen and, 354474 INDEX OF SUBJECTS.Enzymes, isolation and purification of,respiratory, intracellular location of,336.359.Epoxides, 219.bisEpoxides, 220.(+)-Equilenin, 217.Ergothioneine, synthesis of, 176.Erucic acid, synthesis of, 171.Esparto grass, xylan of, 246.Esters, crystal structure of, 439.Ethane, s-bisnitramino-, crystal structureEthanol, photo-oxidation of, 61.Ether, ally1 vinyl, gas-phase rearrange-Ethers, cyclic, preparation of, 165.Ethyl radicals, disproportionation of, 3 1.Ethylamines, 2-phenyl-, 158.Ethylene, induced polymerisation of, 30.oxidation of, 40.oxide, photolysis of, 49.oxides, 219.ultra-violet spectrum of, 8.of, 440, 442.ment of, 38.ultra-violet spectrum of, 8.Ethylene, trichloro-, determination of, indi- and tetra-fluoro-, molecular structureof, 20.tetrafluoro-, induced polymerisation of,49.trans-diiodo-, photoreaction of iodineatoms with, 47.Ethylenes, dinaphthyl-, oxidation of, 183.E thyleneimines, 2 38.Eucalyptus globulus oil, cyclopentenonefrom, 193.Eugenia species, chromone constituents of,229.Eugenin, 229.Eugenitin, 229.isoEugenitin, 229.isoEugenito1, 229.Extinction coefficients from vacuum ultra-blood, 398.violet spectra, 8.by poultry, 316.by sheep, 328.283.Fats, requirement of, by man, 332.Ferriporphyrins, dissociation constants of,Ferriprotoporphyrin, 283.Ferroprotoporphyrin, 283.Ferrous sulphate, oxidation of aqueousFertilisers, analysis of, 384.Films, adsorbed, 76.Flames, hydrogen atoms in, 43.Flavine adenine dinucleotide, 337.solutions of, by X - and yrays, 51.sampling and analysis of, 387,insoluble, 77.propagation of, 42.dinucleotide, 270.mononucleotide, 337.Flavones, 229.Flavonols, 229.Flavoprotein enzymes, 344.Flour, " agenised ", toxicity of, 173.Fluorescence spectra, 10.Fluorimetry, 403.Fluorine, absorption spectrum and dis-sociation energy of, 24.and its compounds, 119.determination of, in air, 386.in water, 385.organic compounds, 176.Fluorocarbons, emission spectra of, 7.Fluorophosphates, determination of, 379.Folinic acid, 242.Formaldehyde, determination of, 394.low-temperature oxidation of, 25.oxidation of, 42.reaction of hydroxyl radicals with, 30.ultra-violet absorption spectrum of, 8.Formamide, dimethyl-, as solvent inGabriel synthesis, 16 1.Formic acid, dehydrogenation rate of,over alloys, 65, 67.Formic acid, chloro-, benzyl ester, prepara-tion of, 162.Formic dehydrogenase, 342.Fradicin, 292.Frog-spawn mucin, 249.Fucoidin, 247.Fulvenes, reaction of, with maleic an-Fungi, antibiotics from, 287.Furans, 221.Furfuryl halides, tetrahydro-, ring fissionof, 222.Fusarium species, pigments from, 227.Fusarubin, 227.Gallium halides, 103.Gamabufotalin, 2 1 1.Gammexane, crystal structure of, 450,451.Gas analysis, 385.Gases, inert, clathrate compounds of, 98.Gelatin, adsorption of, by clays, 82.Germanium compounds, 107.Qibberella baccata, baccatine A from, 289.Ginketin, 230.a-D-GIucose, crystal structure of, 450.Glucose dehydrogenase, 342.Glucose oxidase, 345.Glutaric acid, crystal structure of, 442,D-Glyceraldehyde 3-phosphate dehydro-L-a-Glycerophosphate dehydrogenase, 341.Glycine, deamination of, by a-rays, 52.Glycine, acetyl-, and /3-glycyl-, crystalstructures of, 447, 448.Glycols, cyclisation of, 222.determination of, 392.Glyco-polypeptides, 248.Glycyl peptides, preparation of, 163.Gold tetrachloride ion, structure of, 11.determination of, 380.sols, coagulation of, on dialysis, 84.hydride, 178.toxic, determination of, 386.solutions, 84.444.genase, 340INDEX OF SUBJECTS. 475Gossip01 as reagent for antimony, 375.Gramicidins, 297.Graphite, crystal structure of, 434.electron distribution in, 431.Crrijola confluens, antibiotic from, 170, 290.Grifolin, 170, 290.Growth factors for poultry, 319.for swine nutrition, 323.naturally-occurring, 242.Guaiazulene, 196,Guanosine, 260.Guanosine, benzylidene, 263.Gums, polysaccharides of, 247.Haems, iron removal from, 274.Haem a, 277.Haematoporphyrin, 275.Haematoporphyrin, tetramethyl-, 283.Haemin, preparation of, 276.Haemochromogens, 283.Haemoglobin, crystallography of, 283.preparation of, 276.Haemolysins, 3 1 1, 3 12.Hafnium, separation of, from zirconium,Halides, determination of, 398.108.organic, Szilard-Chalmers effect in, 53.preparation of, 159.Halogen compounds, preparation of, 159.fluorides, 119.Halogens, addition of, in non-hydroxylicsolvents, 13 1.to acetylenic compounds, 130.to olefins in acetic acid, 127.391.in, 132.determination of, in organic compounds,Halogenation, electrophilic, intermediatesHalogeno-amines, rearrangement of, 146.Hardwickia pinnata oil, sesquiterpeneHeparin, 248.Heptane, vapour pressure of polythenewith, 85.Herculin, isomeride of, 172.Heteropolysilicomolybdic acid, 106.I l-Heteropolytungstic acids, 114.Hexamethylenediamine dihydro-bromideand -chloride, crystal structure of,441.cycZoHexane, tetra-bromo- and -chloro-derivatives, crystal structure of,450, 451.1 : %epoxy-, crystal structure of, 449.Hexanoic acid, potassium salt, crystalstructure of, 442.cycZoHexy1 hydroperoxide, dissociationrate of, 26.Hiptagenic acid, 176.Hofmann rule, 136.Hormones, anterior-pituitary, 360.administered to pigs and swine, 324.administered to poultry, 320.administered to sheep, 330.from, 195.effect of, administered to cattle, 327.follicle-stimulating, 36 1.Hormones, growth, 370.Hydrxarbons, aliphatic, 166.interstitial-cell-stimulating, 363.lactogenic, 364.aromatic carbocyclic, crystal structureatom-photosensitised reactions of, 45.crystallography of, 438.mixed, spectrometric analysis of, 24.oxidation of, 39.solubilisation of, in s0a.p solutions, 83.reaction of, with olefins, 46.bromide, effect of, on gas-phase oxida-tion, 41.cyanide, liquid anhydrous, propertiesof, 105.determination of, in organic compounds,388.deuteride, vibrational-rotational spec-trum of, 14.electron capture by, 53.overpotential, 68.peroxide, absorption spectrum of, 8.reaction of deuterium with, a-ray-induced, 53.vibrational-rotational spectrum of, 14.of, 453.polycyclic, oxidation of, 182.Hydrogen atoms in flames, 43.Hydroxy-acids, crystal structure of, 445.Hydroxyl, potential of, 71.Hydroxyl groups, determination of, 392.H.ydroxylamine, N-benzoylphenyl-, asanalytical reagent, 374.Hypoiodous acid, acid dissociation cons-tant of, 122.Ice, structure of, 429.Illudin, 290.Infra-red spectra, 12.Insulin, structure of, 250.Inulin, dahlia, 247.Iodides, organic, photolysis of, 49.Iodination of aromatic compounds, 159.Iodine, absorption spectrum of, 8.addition of, to olefins, 128.atoms and molecules, rate of exchangecations, existence of, 122.monochloride as ionising solvent, 122.cyanide, 123.,determination of, in phosphate rock,penta- and hepta-fluorides, structure of,heptafluoride, 12 I.of, 31.solutions, polarisation of, 18.384.15.Iodinin, 298.Ionic processes in non-aqueous solutions,74.Ionisation of macroelectrolytes, 95.y-Ionone, &hydro-, 204.Iridosmine, analysis of, 381.Iron compounds, 124.determination of, 380.Iron-porphyrin proteins, 346.Isatin, crystal structure of, 463476 INDEX OF SUBJECTS.Isotopic effects, 27.Javanicin, 227.( f)-Kawain, synthesis of, 230.Kessyl alcohol, 197.Keten, photolysis of, 48.a-Keto-lactones, properties of, 231.Ketones, cyclic, condensation of, withni tromalondialdehyde, 190.tracers in adsorption investigations, 57.photolysis of, 48.preparation of, 160.unsaturated, dipole moments of, 19.Ketones, diazo-, Wolff rearrangement of,161.Kevisin, 225.Khellin, 222, 223.Khellinone, 223.Kinetics of chemical reactions, 21.Lactams, 238.k!- actic acid, determination of, 395.Lactic dehydrogenase, 342.Lactobacillus arabinoms, acid from, 17 1.Lactobacillus casei fermentation factor,242.Lactones, 230.ap- and By-unsaturated, distinctionbetween, 232.Lactonic acids, formation of, from ketodi-carboxylic acids, 23 1.Laminaria clouatoni, polysaccharide from,247.Laminarin, 247.Lanceol, 194.Lanosterol, 203.Lanthanons, 104.Lauric acid, aluminium salt, crystalpotassium salt, micelles of, 83.strontium salt, crystal structure of, 442.determination of, in zinc, 382.monoxide as reducing agent, 122.dioxide as oxidising agent, 154.structure of, 443.Lead alunite, 102.“ Leaf alcohol,” stereochemistry of, 170.Lecithin, L- a-dimyristoyl-, -dipalmitoyl-,Lecithinases, 305, 307.Lenzites trabea, antibiotic from, 290.Licheniformin, 296.Light scattering by polymers in solution,Linoleic acid, synthesis of, 171.Lithium alkenyls, 157.and -distearoyl-, 172.88.aluminium hydride as preparative re-in alcohol determinations, 392.determination of, in water, 385.hydride as reducing agent, 99.tungsten bronzes, 115.agent, 154.Lupeol, 199.Lupulone, 205.Lutetium, purification of, 104.Lyogels, 84.Lysine, synthesis of, 174.Lysine, b-hydroxy-, 174.Malaria parasites, pigment of, 277.p l i c dehydrogezase, 342.Malic enzyme , 343.Malonic acid, crystal structure of, 443,444.isotopic effect in decarboxylation of, 27.Malonic acid, bromo-, isotopic effect indecarboxylation of, 27.Manganese compounds, 123.determination of, 379.Marcescin, 297.Mass spectrometry, 41 1.Medicine, antibiotics in, 301.Membranes, 77.Menthofuran, autoxidation of, 221.Mercuric bromide, molten, as electrolyticMercury atoms, reactions of, with cyclo-Mercury, dimethyl-, photolysis of, 29, 49.Mesoporphyrin, 274.Metals, analysis of, 382.tion of, 71.solvent, 101.pentane and cyclopropane, 46.determination of, 382, 383.cathodic deposition and anodic dissolu-electrolytic polishing of, 72.Metal alkyls, heats of reactions of, 23.Metal films as catalysts, 66.Metallic state concept applied to adsorp-Methacrylic acid, methyl ester, thermalMethane, oxidation of, 40.Methane, bisdimethylaminodiphenyl-, astion forces, 64.degradation of, 35.analytical reagent, 374.preparation of, 160.trifluoroiodo-, photolysis of, 49.tetranitro-, preparation of, 176.tetraphenyl-, crystal structure of, 460.Methanol, mercury-photosensitised de-Methionine sulphoximine, 173.Methyl iodide, photolysis of, 49.Methylamine, boron trifluoride compound,Micelles, 83.Microanalysis, organic, 388.Minerals, analysis of, 383.for man, 333.for poultry, 317.for sheep, 329.for swine, 323.359.composition of, 45.synthesis of, 173.radical, reactions of, 28.crystal structure of, 441.requirement of, for cattle, 326.Mitochondria, location of enzymes in, 358,Molecular rearrangements, 142.structure, 7.weight determination from osmoticweights of polymers, 89.fluorides, 120.pressure, 91.Molybdenum, determination of, 380INDEX OF SUBJECTS.477Molybdenum blues, 114.Monolayers, properties and structure of,Moulds, amino-acid oxidases from, 344.cis-trans-Muconic acid, 150.Muscle adenylic acid, 266.Mussel shells, porphyrin from, 276.Myoglobin, 283.Myristic acid, potassium salt, micelles of,77.83.synthesis of, 171.Naphthalene, crystal structure of, 453.derivatives, crystal structure of, 462.reaction of, with maleic anhydride,Naphthionic acid, sodium salt, for thoriumseparation, 375./3-Naphthol, a-nitroso-, as volumetricreagent, 374.a- and /%Naphthols, crystal structure of,462.1-Naphthy Iamine, N - 2-hydroxye thy1-N- 8-nitrobenzenesulphonyl-, 153.Nemotin, 289.Nemotinic acid, 289.Neomycin, 291.Neptunium, chemistry and physics of, 115.Neurospora crama, amino-acid oxidases of,Neurotoxins, 310.Neutron crystallography, 419.Neutrons, absorption coefficients of, 423.diffraction of, 422, 427, 429.scattering amplitudes for, 425.wave-length of, 421.compounds, 125.electrodeposited, structures of, 72.ferrite, 124.179.344.Nickel, catalytic, Raney, 154.Nickel-copper alloys, catalytic activity of,Nickel-iron alloys, catalytic activity of, 65.Nicotinamide glycosides, 268.Nicotine, determination of, 397.Nicotinic acid, determination of, 397.Niobium pentaiodide, 112.separation of, from tantalum, 11 1.Nisin, 297.Nitramine rearrangement, 145.Nitramine, dimethyl, crystal structure of,Nitramines, crystal structure of, 441.Nitrates, determination of, 379.solubility of, in ether, 116.Nitric acid, properties of aqueous solutionssolutions, Raman spectra and structure65.442.of, 74.of, 11.Nitriles, formation of, from halides, 161.Nitrites, determination of, 379.Nitrogen, determination of, in organictrifluoride, structure of, 20, 110.liquid, infra-red absorption spectrum of,compounds, 389.14.Nitrogen oxides, determination of, 386.Nitro-groups, determination of, 395.Nitronium ion, and its compounds, 11, 12,Nitrosonium ion, 110.Nitrosyl complex fluorides, 120.Nootkatin, 184.19-Nortestosterone, 210.‘‘ Notatin ”, 345.Nucleic acids, 263.crystal structure of, 465.properties of, 82.Nucleosides, 256, 258, 266.Nucleotides, 256, 262, 263, 266.Nutrition, human, 330.cis- Oc tadec- 1 1 -enoic acid, 1 7 1.cycZoOcta-1 : 5-diene, isomerism of, 188.bicycZoOctadiene , 187.Octanoic acid, (+)-6-methyl-, from poly-cycZoOctatetraene, 187.structure of, 16, 462.Octa-2 : 4 : 6-triyne, crystal structure of,438, 439.cycZoOctene, isomerism of, 188.cycZoOctyne, 189.(FJstrogens in poultry nutrition, 322.CEstrones, 205.Oleanolic acid, 200.Olefins, halogen addition to, in aceticacid, 127.ring systems, 238.109, 110.mixin, 170.hydrogen addition to, 30.oxidation kinetics of, 41.reaction of hydrogen atoms with, 46.preparative uses of, 158.Oligopeptides, synthesis of, 162.Onium salts, elimination from, 136, 138.“ Ophio-amino-acid oxidase ”, 344.Optical activity due to restricted rotation,Optically active compounds, crystallo-Organic analysis, 388, 392.Organo-metallic compounds, 157.Oscilloscope, cathode-ray, 412.Osmium complexes, 125.determination of, 381.Osmotic pressure, 91.Overpotential, hydrogen, 68.oxygen, 70.Oxalic acid, crystal structure of, 443.determination of, 395.isotopic effect in decomposition of,ytterbium salts, 105.152.isomerism, 148.graphic identification of, 437.compounds, .polarogaphy of, 410.27.Oxazolid-2 : 5-diones, preparation of, 162.Oxazolid-5-one, 2-thio-, 163.Oxidation, 153.in gas phase and in solution, 39.low-temperature, 40.“ Oxine ” as analytical reagent, 376.Oxirans, 219478 INDEX OFOxygen, determination of, in metals, 383.in organic compounds, 391.liquid, abso tion spectra of, 7, 114.overpotentg, 70.Ozone, infra-red spectrum of, 15.Palladium, hydrogen absorption by, 125.Palladium-gold alloys, catalytic activityParaffins, high mol.wt., 252.thermal decomposition of, 38.Paraldehyde, polarisation of, 19.Parasantonide, 233.Parthoniol, 195.Patchoulene, 197.Patchouli alcohol, dehydration of, 197.Patulin, 234, 287, 288.alloPatulin, 235, 288.Pavine, 188.Peach gum, 247.Pelletorine, isomeride of, 172.a- and 8-Peltatins, 232.Penicillic acid, 230, 287.Penicillin, 287, 288.of, 65.crystal structure of, 466.potassium and sodium salts, colloidalPenicillin, benzyl-, crystal structure of,5-phenyl-, 239.Penicillins, analysis of, 418.Penicillinase, 288.Penicillium, amino-acid oxidases of, 345.Penicillium citrinum, citrinin from, 228.Pentaerythritol tetranitrate, crystal struc-Pentane, 2-methyl-, oxidation kinetics of,Pentanes, 1 : 5-diamino-N-alkylated, 158.cycZoPentane, reaction of mercury atomsPentosenucleic acid, 254.neoPenty1 bromide, preparation of, 160.chloride, structure of, 20.spirocycloPenty1-+indoxyl, 239.Peptides, crystal structure of, 446.end-group removal from, 164.synthesis of, 162.Perchloric acid in acetic acid and aceticPerinaphthene, 182.Perinaphthenone, 182.Peroxidases, 351.Peroxides, bond-dissociation energies of,Peroxy-acids, and their salts, 112.Petrol, determination of tetraethyl-lead in,Petroleum, cracked, separation of hydroPhenanthrenes, perhydro-, and theirPhenols, determination of, 392.dihydric, oxidation of, 154.dipole moments of, 19.properties of, 82.467.ture of, 440.39.with, 46.anhydride, C-acetylation with, 156.25.411.carbons in, 416.derivatives, 152.products, analysis of, 407.IUBJECTS.Phenols, reaction of, with iodine mono-chloride, 122.Phenolases, 347.a-Phloroglucitol diammoniate and di-hydrate, crystal structure of, 449.6-Phosphogluconic acid dehydrogenase,343.Phosphorus compounds, 11 1.determination of, in steel, 382.trifluoride, vibrational-rotational energyoxidation of, by steam, 110.Phosphorus, pentaphenyl-, 149.Phosphorus acids, esters, monodebenzyl-ation of, 157.Phosphor yla t ion, 1 5 7.Photoconductive cells, 13.Photolysis, 48, 50.Photometer, flame, 401.Phthalic acid, butyl ester, aerosols, 83.Phthalic acid, tetrachloro-, as analyticalC,,-Phthienoic acid, 172.Phthiocerane, structure of, 166.Phthiocerol, structure of, 166.Phthioic acid, 171.Piaselenole, crystal structure of, 463.Piazthiole, crystal structure of, 463.Picropodophyllotoxin, 232.Pigs, nutrition of, 323.Pimelic acid, crystal structure of, 444.Piperazine, 1 : 4-diphenyl-, N-oxides of,Piperylenes, syntheses with, 178.Pituitary, anterior, hormones of, 360.Platinum complexes, 125.Plutonium, chemistry and physics of,Pneumolysins, 3 12.Podophyllotosin, 232.Podophyllum peltatum, 232.Polarographs, 409.Polarography, 408.Poly(acry1ic acid), viscosity of solutionsof, 96.Polyalanine, 252.Poly(N-n-butyl-4-vinylpyridinium brom-ide), conductivity and viscosity of, 96.Polyelectrolytes, 95.Poly-D- and L-glutamic acids, 252.Polymers, high, solutions of, 85.of, 13.coupled with oxidation, 357.reagent, 375.152.115.light scattering by, in solution, 88.osmotic pressure of, 91.surface-film behaviour of, 77.viscosity of, 93.Polymerisation, 3 1.cationic, inhibition of, 37.ionic, 36.Poly(methacry1ic acid), dissociation of, 95.viscosity of, 78.water absorption by, 97.Polymethylene, 252.Polymyxin, fatty acid from, 170.Polymyxins, 296.Polypeptides, 251INDEX OF SUBJECTS.479plypeptin, 296.Polyporic acid, 232.Polysaccharides, 246.Polysaccharides, amino-, 248.Poly(sodium acrylate), conductivity andPolystyrene, depolymerisation of, 35.light scattering of poly(methacry1ate)osmotic and vapour pressures of, 86.solutions, light scattering of, 89.virial coefficient of, 92.viscosity of, 94.Polyphosphoric acid ” as reagent forcyclodehydration, 155.viscosity of, 96.mixtures of, 90.Polythene, vapour pressure of heptanePoly(viny1 acetate), thermodynamics ofPolyvinyl alcohol, phosphorylation of, 253.Poly(vinylpyridine), ionisation of, 95.Poly-ynes, conjugated, preparation of,Porphobilin, 2 79.Porphobilinogen, 279.Porphyria, 278, 279, 282.Porphyrins, 271.bacterial, 278.biosynthesis of, 280.determination of, 276.physical chemistry of, 282.determination of, in fertilisers, 384.ferrate as volumetric reagent, 377.iodohezafluoride, 12 1.stannates, 108.Potential, reversible, 73.Potentiometric titration, 407.Potentiostat ”, 408.Poultry, nutrition of, 314.Praseodymium oxides, 104, 105.Precipitates, ageing of, 84.Pregnane, 17a : 20-dihydroxy-derivatives,Proactinomycin, 295.Prodigiosin, 2 7 7.Progesterone, determination of, 414.Prolactin, 364.Proline, synthesis of, 175.Prolines, hydroxy-, configuration of, 149.Promethium, absorption spectrum of, 105.Propaldehyde, fi@-trifluoro-, 177.Propane, mercury-photosensitised de-composition of, 45.Propane, hmafluoro-, structure of, 21.cycZoPropane, reaction of mercury atomswith, 46.Propionic acid, aluminium salt, 103.Proteins, 249.164.with, 85.solutions of, 86.virial coefficient of, 92.sulphonates, 252.166.Potassium cupriperiodate as volumetricreagent, 377.drying and combustion of, 387.213.adsorption of gases by, 81.arrangement of amino-group residues in,Proteins, boundary spreading and electro-phoresis of, 80.mol.wt. of, 82.physico-chemical properties of, 81.requirement of, for cattle, 324.for man, 330.for poultry, 314.for sheep, 327.for swine, 323.Protoporphyrin, 274, 275.Prototropic rearrangements, 142.Psoralene, 222.Psoralic acid, 223.Pteridine, 245.Pterins, 241.“ Pterin oxidase ’I, 346.Pteroic acid, 242.Puberulic acid, 287, 289.Puberulonic acid, 184, 287, 289.Pulvinic lactone, 232.Purine derivatives, crystal structure of,Purines, determination of, 398.Purpurogallin, synthesis of, 186.Pyrans, 226.Pyrethrins, 204.Pyrimidine derivatives, crystal structureof, 464.Pyrimidines, determination of, 398.from nucleic acids, 255.Pyrimidines, 5-thioformamido-, c yclisationPyrolysis in gas phase, 37.Pyrones, 226.Qualitative analysis, 377.Quinol, clathrate compounds of, 99 .Quinoline, 5 : 7-dibromo-8-hydroxy-, asQuinones, oxidation-reduction potentials464.nucleosides, synthesis of, 260.from nucleic acids, 255.of, 261.reagent for gallium, 376.8-hydroxy-. See “ Oxine ”.of, 73.Radiation chemistry, 51.Radical reactions, activation energy andRadioactive elements, electrodepositiontracer analysis, 413.Raman spectra, 10, 406.X-Ray absorption analysis, 406.X-Rays, absorption coefficients of, 423.diffraction of, 422, 427.scattering amplitudes for, 425.Reaction chains, life-times of, 47.kinetics, 21.Reagents, analytical, 374.Reduction, 154.Resonance lamps, 46.Rhenium, absorption spectra of, 123.Rhodium, determination of, 381.Rhodopsin, 166.isoRhodopsin, 170.frequency factors of, 27.of, 72.determination of, 380480 INDEX OF SUBJECTS.Riboflavin, phosphorylation of, 270.Riboflavin-5’ phosphate, 270.l-a-D-Ribofuranoside, 240.Ribonuclease, inactivation of, by X-rays,Ribonucleic acid, 254.Ribonucleosides, X-ray analysis of, 259.D-Ribose, 258.Rings, many-membered, 189.Rocks, sedimentary, analysis of, 383.Rotational isomerism in molecules, 17.Rubber, oxidation of, 42.Rubidium, determination of, 382.Ruthenium compounds, 125.determination of, 381.52.Salicylic acid, p-nitroso-, as colorimetricSalicylidenethiosemicarbazone as analyt-Sampling, accuracy of, 387.Santonic acid, 233.Santonide, 233.Santonin, 232.+-Santonin, 232.Sarcosine N-carboxy-anhydride, poly-merisation of, 251.Sarmentogenin, 211.Saytzeff rule, 137,138.Scrubber, Venturi, 386.Sebacic acid, crystal structure of, 444.Sedimentation of colloids, 78.Serratia marcescens, antibiotic from, 297.pigment from, 277.Sesquiterpenes, 193.Sheep, nutrition of, 327.Shiga neurotoxin, 311.Silicon compounds, 106.Silicon, chloromethyltrimethyl-, structureof, 20.Silk fibroin, 251.Siloxene in oxidation reactions, 377.Silver, determination of, in solder, 383.Snake venom, L-amino-acid oxidase of,Soap-like substances, 82.Sodium bismuthate as oxidising agent,determination of, 379.deuteride and hydride, structure of,ethylenediaminetetra-acetate as analyt-paratungstate, formula of, 114.silicates, 106.superoxide, 99.zincate, 100.reagent, 375.ical reagent, 377.determination of, 379, 382.perchlorate, 100.344.153.429.ical reagent, 374, 384.Sodium, benzyl-, 158.Soils, nitrification of manures in, 384.Solvent effect on elimination rate, 140:.extraction analysis, 414.Soyasapogenols, 203.Spectrochemical analysis, 399.Spectrometers, 336, 403, 405.infra-red prism, 12.mass, measurements with, 23.neutron, 421.photo-electric Raman, 10.Spectrophotometers, 405.Spectrophotometry, infra-red absorption,Spectroscopy, emission, analytical pro-Spirographis porphyrin, 272.Spongo t h ymidine , 2 6 6.Staphylococci, haemolysins from, 313.Starches, crystal structure of, 452.Statistical mechanics of adsorption, 61.Stearic acid, synthesis of, 171.Steel, analysis of, 382, 383.Sterculia setigera gum, 247.Stereochemical standards, relation of, 150.Steroids, 205.aromatisation of, 218.biogenesis of, 216.diene syntheses with, 208.natural, 210.oxides, use of, in preparing hydroxy-stereochemistry of, 217.404.ultra-violet absofption, 403.Raman, 406.cedure and apparatus for, 399.compounds, 218.Stibiafluorenes, optically active, 148.Stipitatic acid, 184.Streptolysin, 312.Streptomyces species, antibiotics from, 291.Streptomycin, 291.potassium and sodium salts, colloidalStreptomycin, hydroxy-, 291.Strychnine, crystal structure of, 466.Styrene, addition of 2-alkylpyridines to, 158.oxidation of, 42.polymerisation of, radiation-induced, 53.polymerisation rate of, 33.properties of, -82.Styrenes, /?-nitro-, reduction of, 155.Succinoxidase system, 354.Sucrose, diffusion coefficient of, 79.structure of, 451.Sugars, determination of, 396.Sulphides, ethylene, 236.Sulphinic acids, photosynthesis of, 50.Sulphoximine, dimethyl, 173.Sulphur compounds, 112, 113.sodium bromide dihydrate, crystalspectra of, 405.determination of, in organic compounds,hydrosols, 83.monoxide, structure of, 15.dioxide, determination of, in air, 386.trioxide ion, structure of, 11.oxides, determination of, in flue gases,ring systems, 236.Sulphuric acid as ionising solvent and forcryoscopy, 113.equilibria in formation of, 112.properties of aqueous solutions of, 74.391.386INDEX OF SUBJECTS.481Sulphuryl fluoride, 112.Surface area, determination of, 58.Surfaces, non-uniform, adsorption at,Swine, nutrition of, 373.TPN-c ytochrome-c reduc tam, 346.Tactoids, 84.Tantalum trifluoride, 121.Taraxasterol, 202.Tartaric acid, and its salts, crystal struc-&Tartaric acid, configuration of, 149.2-Tartrato-2-niobic acid, and its salts, 112.Temperature effect on elimination rate,Terephthalic acid, diethyl ester, crystalTernatin, 230.‘‘ Teropterin ”, 242.Terpenes, 190.acyclic, structure of, 166.mono- and bi-cyclic, 191.Terramycin, 294.Tetanus toxin, 311.Tetralin, autoxidation of, 42.Thallium, anodic oxidation of, 102.chlorides, 104.Thermistors for cryoscopy, 418.Thermobalance, Chevenard, 387.Therrnochemical measurements, 23.Thermodynamics of adsorption, 6 1.Thermophillin, 290.5-Thiazolidone, 2-thio-, 163.Thioacetamide as analytical reagent, 375.diThiocarbamic acid, diethyl-, sodiumsalt as volumetric reagent, 376.Thiols, preparation of, 165.Thiophen, 237.Thiophen-aldehydes, 237.Thiophen-3- thiol, 23 7.Thiophthen, crystal structure of, 463.Thiouracil in poultry nutrition, 321.Thiourea, complexes of, 165.Thixotropy, 84.Thorium bromide and sulphides? 109.tension, 76.54.pentuiodide , 1 12.separation of, from niobium, 1 11.ture of, 445.141.structure of, 458.compounds, behaviour of, in liquidextraction of, 109.Threonine, crystal structure of, 446, 447.resolution of, 147.synthesis of, 173.Thujaplicins, 186, 193.Thymidine, 258.Thyrotrophin, 371.Thyroxine in poultry nutrition, 321.Tin compounds, 108.Titrimeters, 408.Toxins, bacterial, 303.Transport numbers, measurement of, 75.Transmanic elements, 1 15.ammonia, 109, 117.determination of, in aluminium alloys,382.Triacetylene, 166.Tricosanoic acid, synthesis of, 171.Trilon B aa base solution in polarography,Triphosphopyridine nucleotide, 269, 337.Trisalicylide, 152.cyctoTrisiloxan, itemzmethyl-, structure of,Triterpenes, 199.Tropolone, synthesis of, 186.Tropolones, 184.Tropolonecarboxylic acid, 186.Tryptophan, synthesis of, 175.Tryptophan, ~~-2-hydroxy-, synthesis of,175.Tungsten dibromide, 121.Turacin, 278.Turaco feathers, pigment of, 278.Ultracentrifuge in sedimentation, 78.Ultra-violet, new light source for, 8.Ultra-violet absorption spectra of organiccompounds in solution, 9.Unsaturated compounds, addition ofhydrogen to, 30.catalysed polymerisation of, 36.Unsaturation, determination of, 397.Uracil, determination of, 398.Uracil deoxyriboside, 259.Uranium, and its compounds, 115.409.20.compounds, behaviour of, in liquiddetermination of, 381, 384.hemfluoride, structure of, 20.trioxide, pure, preparation of, 117.sulphides, 109.Urea, complexes of, 165.containing I5N, 325.Uridine, 258.diphosphate-glucose, 270.Uridylic acid, 257.Urine, determination of uroporphyrin in,porphyrin in, 278.Uroporphyrins, 278.“ Vaccenic acid ”, 171.Valines, valyl-, 176.Vanadium compounds, absorption spectraammonia, 109, 117.276.of, 111.determination of, 379.pentoxide sols, 84.Vibrational-rotational spectra, 13.Vinyl chloride, polymerisation of,catalysed, 33.a-ray induced, 53.fluoride, induced polymerisation of, 49.polymerisation, 31.polymers, depolymerisation of, 34.Vinylidene dicyanide, preparation of, 158.Virjal coefficient, 92.Viridin, 290.Viscosity of colloids, 78.of polyelectrolytes, 96.of polymers, 93.Visnagin, 222, 223482 INDEX OF SUBJECTS.Visnagone, 224.Vitamin BL2, 240.Volumetric analysis, standardisation ofsolutions for, 373.n- and iso-Vulpinic acids, 232.Wagner rearrangement, 143.Water, analysis of, 384.irradiation of, 52.photo-oxidation of, 51.photoreduction of, 51.Water gas, oerburetted, analysis of, 385,413.Xanthine oxidase, 346.Xanthosine, 260.Xylenes, analysis of, 403." Yeast adenylic acid ", 262.Yeast ribonucleic acid, 263.Zinc, determination of, 409.Zingiberene, 193.Zirconium compounds, 108.sulphate complexes with quinoline,100.separation of, from hafnium, 108

ISSN:0365-6217    

DOI:10.1039/AR9504700470

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

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Acad. Sci. Fenn..Ann. Biochem. Exp. Med. .Ann. Bot. .Ann. Chim. .Ann. Chim. snalyt. .Ann. Chim. appl. .Ann. Chim. Phys. .Ann. B'em.rwnt. .Ann. Inst. Pasteur .Ann. Intern. Med. .Ann. pharm. Franc. .Ann.Physik .FULL TITLE.Actualit& scientifiques et industrielles.Acta Brevia Neerlandica de Physiologia, Phannacologis,Acta Chemica et Physica.Acta Chemica Scandinavica.Acta Chimica Belgica.Acta Crystal lographica.Acta Medica Scandinavica.Acta Ophthalmologica Kjebenhavn.Acta pathologica et microbiologica Scandinavica.Ac ta Physicochimica U .R.S. S.Acta Phytochimica, Tokyo.Advances in Carbohydrate Chemistry.Advances in Catalysis.Advances in Colloid Chemistry.Advances in Enzymology.Advances in Protein Chemistry.Agricultural Chemicals.American Chemical Journal.American Institute of Mining and Metallurgical EngineersAmerican Journal of Botany.American Journal of Digestive Diseases.American Journal of Medical Sciences.American Journal of Pharmacy.American Journal of Physiology.American Journal of Public Health and the Nation'sThe American Journal of Roentgenology and RadiumAmerican Journal of Science.American Mathematical Society, Collective Publications.American Mineralogist.Anais da Associack Quimica do Brasil.Anales de la Asociacibn Quimica Argentina.Anales de la Sociedad Espaiiola Fisica y Quimica.The Analyst.Analytical Chemistry.Analytica Chimica Acta.Anatomical Record.Angewandte Chemie (formerly 2.angew. Chem.).Annales Academh Scientiarium Fennicae (SuomalaisenAnnals of Biochemistry and Experimental Medicine.Annals of Botany.Annales de Chimie.Annales de Chimie analytique et de Chimie appliqube.Annali di Chimica applicata.Annales de Chimie et de Physique (now divided: seeAnnales des Fermentations.Annales de L'Institut Pasteur.Annals of Internal Medicine.Annales Pharmaceutiques Franqaises.Annalen der Physik.Microbiologia, e.a.Publication.Health.Therapy.Tiedeakatemian Toimituksia) .Ann.Chim. and Ann. Physique).48484 PRINCIPAL REFERENCES USED IN CHEMICAL SOCIETY PUBLICATIONS.Abbreviated Title.Ann. Physique .Ann. Reports .Ann. Rev. Biochem. .Ann. Sci. .Ann. Sect. Anal. phys. chim.Ann. SOC. scd. Brux. .Annalen .Arch. Biochem. .Arch. Eisenhuttenw. .Arch. exp. Path. Pharmak. .Arch. klin. Chir. . .Arch. Mikrobiol. .Arch. Pharm.Arch. Sci. biol. (C.R.S:S.) 1Arch. Sci. phys. nat. .Arch. 2001.Arkiv Kemi, Min., Geol. .Atti R. Accad. Lincei .Apoth.-Ztg. .A t t i R. Accad. Sci. TorinoAustral. J . Exp. Biol. .Avh. mrske Vidensk.-Akad.Oslo, Mat.-nat. Kl.Ber. .Ber. deut. bot. Ges. .Ber. deut. keram. Qes. .Biochem. J . .Biochem. SOC. Symp. .Biochem. 2. .Biochim. .Biochim.Biophys. Act& .Biokhim. .Biol. Reviews .Biol. Zentr. .Boll. Chim.-farm. .Boll. SOC. ital. Biol. sperim. .Bot. Gaz. .Brit. Abs. .Brit. Dental J . .Brit, J . Exp. Path. .Brit. J . Ophthalmol. .Brit. J . Pharmacol. .Brit. J . Radiol. .Brit. J . Urol. .Brit. Med. J . .Bull. Acad. polonaise .Bull. Acad. roy. Belg. .Bull. Acad. Sci. Roumaine ..Bull. A d . Sci. U.R.S.S. .Bull. Amer. Ceram. SOC. .Bull. Amer. Phys. SOC. .Bull. analyt. ,Bull. Biol. Med. exp. l7.R.S.SBull. Chem. SOC. Japan .Bull. China. SOC. Romdne .FULL TITLE.Annales de Physique.Annual Reports on the Progress of Chemistry.Annual Review of Biochemistry.Annals of Science.Annales du Secteur d’Analyse physicochimique, InstitutAnnales de la, Soci6te scientifique de Bruxelles.Liebigs Annalen der Chemie.Deutsche Apotheker-Zeitung.Archives of Biochemistry.Archiv fur das Eisenhuttenwesen.Archiv fur experimentelle Pathologie und Pharma-Archiv fur klinische Chirurgie.Archiv fur Mikrobiologie.Archiv der Pharmazie.Archives des Sciences biologiques (U.R.S.S.).Archives des Sciences physiques et naturelles.Archives de Zoologie experimentale et generale.Arkiv for Kemi, Mineralogi och Geologi.Atti (Rendiconti) della Reale Accademia Nazionaledei Lincei.Classe di scienze fisiche, matematichee naturali, Roma.Atti della, Reale Accademia della Scienze di Torino.de Chimie gBn6rale (U.R.S.S.).kologie.Australian Journal of Experimental Biology andAvhandlinger utpitt av det Norske Videnskaps-AkademiMedicine.i Oslo, MatemLtisk-naturvidenskapelig Kfasse.Berichte der deutschen chemischen Gesellschaft.Berichte der deutschen botanischen Gesellschaft.Berichte der deutschen keramischen Gesellschaft.The Biochemical Journal.Biochemical Society Symposia.Biochemische Zeitschrift.Biochimica.Biochimica e t Biophysica Acta.Biokhimiya.Biological Reviews.Biologisches Zentralblatt.Bolletino Chimico - farmaceutico.Bolletino della Societh italiana di Biologia sperirnentalla.Botanical Gazette.British Abstracts.British Dental Journal.British Journal of Experimental Pathology.British Journal of Ophthalomology.British Journal of Pharmacology and Chemotherapy.British Journal of Radiology.British Journal of Urology.The British Medical Journal.Bulletin internationale de l’Acad6mie polonaise desSciences e t des Lettres.Bulletin de 1’AcadBmie royale de Belgique.Classe desSciences.Bulletin de la Section ScientSque de l’Acad6mieRoumaine.Bulletin de 1’Academie des Sciences de l’U.R.S.8.Bulletin of the American Ceramic Society.Bulletin of the American Physical Society.Bulletin Analytique.Bulletin de Biologie et MBdicine experimentale deBulletin of the Chemical Society of Japan.Bulletinul de Chimie pura si aplicata a1 Societatii1’U.R.S.S.Romhe de ChimiePRINCIPAL REFERENCES USED M CHEMICAL SOCIETY PUBLICATIONS. 485Abbreviated Title.Bull. Exp. Biol. M d . .Bull. Hlth. Org. .Bull. Hyg.Bull. Imp. Inst. .Bull. Inst. Min. Met. .Bull. Inst.Phys. Chem. Res.,Bull. Johns Hopkins Hosp. .Bull. SOC. chim. .Bull. SOC. chim. Belg. .Bull. SOC. chim., Belgrade .Bull. SOC. Chim. biol. .Bull. SOC. sci. Bretagne .Bur. Stand. J . Res. .TokyoCanad.Chem. .Canad. J . Res. .C a d . Med. Assoc. J..Cellulosechem. .Cereal Chem. .Chem. Abs.Chem. Analyst .Chem. and Ind. .Chem. Ber.Chem. B’abr. .Chem. Obzor .Chem. Reviews .Chem. Zentr. .ChemieChim. e l’I&.Chim. et Ind. .ChimiaChinese J . PhysicsCold Spring Harbor Symp.Coll. Czech. Chem. Comm. .Coll. Trav. chim. Tchdcosl. .Compt. rend. .Compt. rend. Acad. Sci.Compt. rend. SOC. Biol. .Compt. rend. Trav. Lab.Contr. Boyce Thompson Inst.Current Sci. .Dansk Tidsskr. Farm. .Deut. med. Woch. .DieChemie .Discuss.Faraday SOC. .coiEoi& J . , U.S.S.R. .U.R.S.S.CarlsbergE. African Med. J , .Edin. Med. J. .Elektrotech. 2. .Eng. Min. J . .Ergebn. Enzymforsch. .Ergebn. exakt. Naturwiss. .Ergebn. Physiol.Ergebn. Vitamin- u. Homnon-Experientia . forsch. .FULL TITLE.Bulletin of Experimental and Biological Medicine.Bulletin of the Health Organisation of the League ofBulletin of Hygiene.Bulletin of the Imperial Institute, London.Bulletin of the Institution of Mining and Metallurgy.Bulletin of the Institute of Physical and ChemicalBulletin of the Johns Hopkins Hospital.Bulletin de la Soci6t6 chimique de France.Bulletin de la Soci6t6 chimique de Belgique.Bulletin de la Soci6t6 chimique, Belgrade.Bulletin de la Soci6t6 de Chimie biologique.Bulletin de la Soci6t6 scientifique de Bretagne.Bureau of Standards Journal of Research (now J .Res.Canadian Chemistry and Process Industries.Canadian Journal of Research.Canadian Medical Association Journal.Cellulosechemie.Cereal Chemistry.Chemical Abstracts.Chemist Analyst.Chemistry and Industry.Chemische Berichte (superseded Ber.) .Die Chemische Fabrik.Chemicky Obzor.Chemical Reviews.Chemisches Zentralblatt.Chemie.La Chimica e 1’Industria.Chimie et Industrie.Chimia.Chinese Journal of Physics.Cold Spring Harbor Symposium on QuantitativeCollection of Czechoslovak Chemical Communications.Collection de travaux chimiques de TchBcoslovaquie.Colloid Journal, U.S.S.R.Comptes rendus hebdomadaires des SBances deComptes rendus de l’Acad6mie des Sciences deComptes rendus hebdomadaires de S6ances de laComptes rendus des Travaux du Laboratoire Carls-Contributions from the Boyce Thompson Institute.Current Science.Dansk Tidsskrift for Farmaci.Deutsche medizinische Wochenschrift.Die Chemie.Discussions of the Faraday Society (first published1947 ; before that Faraday Society discussions werepublished as part of Trans.Faraday SOC.) .East African Medical Journal.Edinburgh Medical Journal.Elektrotechnische Zeitschrift.Engineering and Mining Journal.Ergebnisse der Enzymforschung.Ergebnisse der exakte Naturwissenschaften.Ergebnisse der Physiologie.Ergebnisse de: Vitamin- and Hormonforschung.Experientia.Nations.Research, Tokyo.Nut. Bur.Stand.).Biology.l’Acsd6mie des Sciences.U.R.S.S.Soci6th de Biologie.berg486 PRINCIPAL REFERENCES USED IN CHEMICAL SOCIETY PUBLICATIONS.Abbreviated Title,Fed. Proc.Finska Kem. M&d.GazzettaGeneesk. Tijdschr. iederl.:Geol. Mag.Helv. Chim. Acta .Helv. Phys. Acta. .Helv. Physiol. Pharmacol. ActaInd. Chem. Chem. Manuf. .Ind. chim. belg. .Ind. Eng. Chem.Ind. Eng. Chem. Anal. .Indian J . Med. Res. .Indian J . Physics .Ing.-chim. .Inorg. Synth. .Inst. int. Chim. Solvay .Indib. .Iowa State Coll. J . Sci. .J . .J . Agric. Chem. SOC. Japan.J . Agric. Res. .J . Agric. Sci. .J . Amer. Ceramic SOC. .J . Amer. Chem. SOC. .J . Amer. Med. Assoc. .J . Amer. Oil Chem. SOC.J . Amer. Pharm. Assoc.J .Appl. Chem., U.S.S.R.J . Appl. Physics, U.S.S.R.J . Assoc. 08. Agric. Chem.J . Bact. .J . Biochem., Japan .J . Biol. Chem. ..T. Biol. Chem. Sci. Proc. .J . Cell. Comp. Physiol. .J . Chem. Educ. .J . Chem. Phys. .J . Chem. SOC. Japan .J . Chim. phys. .J . Chinese Chem. SOC. .J . Clin. Invest. .J . Coun. Sci. Ind. Res.,J . Econ. Entomol. .J . Electrochem. Assoc. JapanJ . Electrochem. SOC. .Australia>. Endocrinol. .J . Exp. Biol. .J . Exp. Med. .J . Exp. 2001. .J . Franklin Inst.J. Gen. Chem., U.S.S.R.J . Gen. Physiol. .J. Geol. .J . Hyg. .J . Immunol. .FULL TITLE.Federation Proceedings.Finska Kemistamfundets Meddelanden (SuomenGazzetta chimica italiana.Kemistiseuran Tiedonantoja).Geneeskundig Tijdschrift voor Nederlandsch-Indie.Geological Magazine.Helvetica Chimica Acta.Helvetica Physica Acta.Helvetica Physiologica e t Pharmacologica Acta.Industrial Chemist and Chemical Manufacturer.Industrie chimique belge.Industrial and Engineering Chemistry.Industrial and Engineering Chemistry : AnalyticalIndian Journal of Medical Research.Indian Journal of Physics.Inghieur-chimiste.Inorganic Syntheses.Institut international de Chimie Solvay.Iowa State College Journal of Science.Journal of the Chemical Society.Journal of the Agricultural Chemical Society of Japan.Journal of Agricultural Research.Journal of Agricultural Science.Journal of the American Ceramic Society.Journal of the American Chemical Society.Journal of the American Medical Association.Journal of the American Oil Chemists' Society.Journal of the American Pharmaceutical Association.Journal of Applied Chemistry, U.S.S.R.Journal of Applied Physics, U.S.S.R.Journal of the Association of Official AgriculturalJournal of Bacteriology.Journal of Biochemistry, Japan.Journal of Biological Chemistry.Scientific Proceedings of the American Society ofJournal of Cellular and Comparative Physiology.Journal of Chemical Education.Journal of Chemical Physics.Journal of the Chemical Society of Japan.Journal de Chimie physique.Journal of the Chinese Chemical Society.Journal of Clinical Investigation.Journal of the Council for Scientific and IndustrialJournal of Economic Entomology.Journal of the Electrochemical Association of Japan.Journal of the Electrochemical Society (commencedpublication 1948, 93 ; volume numbering the same asTrans.Electrochem. Soc . ) .Journal of Endocrinology.Journal of Experimental Biology.Journal of Experimental Medicine.Journal of Experimental Zoology.Journal of the Franklin Institute.Journal of General Chemistry, U.S.S.R. (formerlyJournal of General Physiology.Journal of Geology.Journal of Hygiene.The Jourial of Immunology.Edition (now Analyt. Chem.).Conseil deChimie.Chemists.Biological Chemists (bound with J . Biol. Chem.).Research, Australia.chemical part of J. Russ. Phys. Chem. Soc.)PRINCIPAL REFERENCES USED IN CHEMICAL SOCIETY PUBLICATIONS. 487Abbreviated Title.I d . Eng. J . Chem..J . I d . Hyg. .J . Indian Chem. SOC. .J . Indian Inst. Sci. .J . Infect. Dis. .J . Inst. Brew. .J . Inst. Elect. Eng. .J. Inst. Metals ..J , Inst. Petrol. .J . Int. SOC. Leath. Chem.J . Iron Steel Inst. .J . Lab. Clin. Med. .J . Marine Biol. Assoc.J . Marine Res. .J . Nutrit.J . Oil Colour Chem. Assoc.J . Org. Chem. .J . Pediat. .J . Pharm. Belg. ,J . Pharm. Chim.J . Pharm. Pharmacol. .J . Pharm. SOC. Japan .J . Pharmacol. .J . Phys. Chem. .J . Phys. Chem., U.S.S.R. .J . Phys. Colloid Chem. .J . Phys. Radium .J . Phys., U.S.S.R. .J . Physiol.J . Polymer Sci. .J.pr. Chem. .J . Proc. Austral. Chem. Inst.J . Proc. Roy. Soc. N.S.W.J . Res. Nut. Bur. Stand...J . Roy, Inst. Chem. .J . Roy. Microscop.SOC. .J . Rubber Res. .J . Russ. Phys. Chem. SOC. .J . S. African Chem. Inst. .J . Sci. Ind. Res., India .J . Sci. Instr. .J . SOC. Chem. Ind. .J . SOC. Chem. Ind., JapanJ . SOC. Dyers and Cot. .J . Text. Inst. .Kgl. Danske Videnskab.Selsk.Kgl. fysiogr. Sallsk. LundForh.Kgl. Norsie Vidensk. ' Selsi.Porh. .Kgl. Norske VGensk. ' Selsk:Skrifter .Kolloid-Beih. .Kolloid Z. .Lancet ..FULL TITLE.Journal of Industrial and Engineering Chemistry (nowJournal of Industrial Hygiene and Toxicology.Quarterly Journal of the Indian Chemical Society.Journal of the Indian Institute of Science.Journal of Infectious Diseases.Journal of the Institute of Brewing.Journal of the Institution of Electrical Engineers.Journal of the Institute of Metals.Journal of the Institute of Petroleum.Journal of the International Society of Leather TradesJournal of the Iron and Steel Institute.Journal of Laboratory and Clinical Medicine.Journal of the Marine Biological Association ofJournal of Marine Research.Journal of Nutrition.Journal of the Oil and Colour Chemists' Association.The Journal of Organic Chemistry.Journal of Pediatrics.Journal de Pharmacie de Belgique.Journal de Pharmacie et de Chimie.Journal of Pharmacy and Pharmacology.Journal of the Pharmaceutical Society of Japan.Journal of Pharmacology and Experimental Thera-The Journal of Physical Chemistry.Journal of Physical Chemistry, U.S.S.R.Journal of Physical and Colloidal Chemistry (formerlyJournal de Physique et le Radium.Journal of Physics, U.S.S.R.Journal of Physiology.Journal of Polymer Science.Journal fur praktische Chemie.Journal and Proceedings of the Australian ChemicalInstitute.Journal and Proceedings of the Royal Society of NewSouth Wales.Journal of Research of the National Bureau of Standards(formerly Bur.Stand. J . Res.).Journal of the Royal Institute of Chemistry.Journal of the Royal Microscopical Society.Journal of Rubber Research.Journal of the Russian Physical and Chemical Society(now obsolete: cf. J . Gen. Chem., U.S.S.R.).Journal of the South African Chemical Institute.Journal of Scientific and Industrial Research, India.Journal of Scientific Instruments.Journal of the Society of Chemical Industry.Journal of the Society of Chemical Industry, Japan.Journal of the Society of Dyers and Colourists.Journal of the Textile Institute.Kongelige Danske Videnskabernes Selskab, Mathe-matisk-fysiske Meddelelser.Kongliga fysiografiska Siillskapets i Lund Forhandlinger.Kongelige Norske Videnskaber Selskabs Forhandlinger.Kongelige Norske Videnskaber Selskabs Skrifter.Kolloid-Beihefte.Kolloid Zeitschrift.Lancet.Ind.Eng. Claem.).Chemists.the U.K.peutics.J . Phys. Chem.)488 PRINCIPAL REFERENCES USED IN CHEMICAL SOCIETY PUBLICATIONS.Abbreviated Title.Makromol. Chem. .Mem. Coll. Aaric. Kyoto .Mem. Coll. Sci. Kyoto . .Mem. Inst. Chem. Ukrain.Mem. Manchester Lit. Phil.Mem. R. Accard. Ital. .Acad. Sci.SOC.Metal Ind. .Metallforsch. .Metallw..Mikrochem. .Mikrochem. Mikrochim. ActaMikrochim. Acta .Min. Mag.Monatsh. .Nach. Ges. Wiss. GdttingenNaturwiss:.Nederl. Tzjds. Natuurk.Neues Jahrb. Min. .New England J . Med. .New Phytol.Nord. med. Tidssir. 1Nuovo Cim.Nutrit. Abs. Rev.Oesterr. Chem.-Ztg. .Org. Synth.Paint Oil Chem. Rev. .PfEiig. Arch. ges. Physiol.Pharm. Acta Helv. ,Pharm. J . .Pharm. Weekblad‘Pharm. Zentralh.Pharm. Ztg. .Pharmazie .Phil. Mag.Phil. Trans. .Physica .Phys. Review .Physikd. 2. .Physikal. Z. SovietunionPhysiol. Reviews .Plant Physiol. .Pogg. Ann.Post Grad. Med. >.Poultry Sci. .Proc. Acad. Nat. Sci. .Proc. Arner. Acad. Arts Sci.Proc. C a d . Phil. Soc..Proc. Chem. SOC..Proc. Qeol. Assoc.Proc. Imp.Acad. TokioProc. Indian Acad. Sci. .Proc. Indiana Acad. Sci. .FULL TITLE.Die Makromolekulare Chemie.Memoirs of the College of Agriculture, Kyoto ImperialMemoirs of the College of Science, Kyoto ImperialMemoirs of the Institute of Chemistry, UkrainianMemoirs and Proceedings of the Manchester Literary andMemorie della Reale Accademia d’Italia. Classe diMetal Industry.Metallforschung .Metallwirtschaft, Metallwissenschaft, Metalltechnik.Mikrochemie.Mikrochemie vereinigt mit Mikrochimica Acta.Mikrochimica Acta.Mineralogical Magazine and Journal of the MineralogicalMonatshefte fur Chemie und verwandte Teile andererNachrichten von der Gesellschaft der WissenschaftenNaturwissenschaften.Nederlandsch Tij dschrift voor Natuurkunde.Neues Jahrbuch fur Mineralogie, Kristallographie andPetrographie.New England Journal of Medicins.New Phytologist.Nordisk medicinisk Tidsskrift.Nuovo Cimento.Nutrition Abstracts and Reviews.Oesterreichische Chemiker-Zeitung.Organic Syntheses.Paint, Oil and Chemical Review.Pflugers Archiv fur die gesamte Physiologie desPharmaceutica Acta Helvetiae.Pharmaceutical Journal.Pharmaceutisch Weekblad.Pharmazeutische Zentralhalle f.Deutschland.Pharmazeutische Zeitung.Pharmazie.University.University.Academy of Sciences.Philosophical Society.Scienze fisiche, matematiche e naturali.Society.Wissenschaften.zu Gottingen.Menschen und der Tiere.Philosophical Magazine (The London, Edinburgh andPhilosophical Transactions of the Royal Society.Dublin).Physic;.Physical Review.PhysikaIische Zeitschrift.Physikalische Zeitschrift der Sowjetunion.Physiological Reviews.Plant Physiology.Poggendorfs Annalen (now Annalen).Post Graduate Medical Journal.Poultry Science.Proceedings of the Academy of Natural Sciences ofProceedings of the American Academy of Arts andProceedings of the Cambridge Philosophical Society.Proceedings of the Chemical Society (ceased in 1914).Proceedings of the Geologists’ Association.Proceedings of the Imperial Academy, Tokyo.Proceedings of the Indian Academy of Science.Proceedings of the Indiana Academy of Science.Philadelphia.SciencesPRINCIPAL REFERENCESAbbreviated Title.Proc.K. Ned. Akad. Wet. .Proc. London Math.Soc. .Proc. Mayo Clin. .Proc. Nat. Acad. Sci. .Proc. Nat. Inst. Sci. India .Proc. Phys. SOC.Proc. Roy. Irish Acad..Proc. Roy. SOC. .Proc. Roy. SOC. Edinburgh .Proc. Roy. SOC. Medicine ,Proc. SOC. Exp. Biol. N.Y. .Publ. Amer. Assoc. Adv. Sci.Publ. Hlth Rep., Wash. .Quart. J . Exp. Physiol. .Quart. J . Ceol. SOC. .Quart. J . Pharm.Quart. Reviews .Radiology .Rec. Trav. chim. .Rep. Brit. Assoc. .Research .Rev. B r a d . Chim. .Rev. Fac. Sci. Istanbul .Rev. Immunol. .Rev. I d . min. .Rev. Mod. Physics .Rev. sci. .Rev. Sci. Instr. .Ric. sci. .Roczn. Chem. ,X. African J . Sci. .Schweiz. med. Woch. .Sci. J . Roy. Coll. Sci. .Sci. Papers Inst. Phys. Chem.Res., TokyoSci. Proc. R. Dublin Xoc. .Sci.Progress .Sci. Rep. Tohoku .Sitzungsber. Akad . Wiss.WienSitzungsber. Ges. Bef ord.Naturwiss. MarburgSkand. Arch. Physiol. .Smithson. Misc. Coll. .SOC. Xci. Fenn. Phys. Math.Spectrochim. Acta .Sth. Med. J .Siiddeut. A p o t h h g .Suomen Kem. .Svensk Kem. Tidskr. .Symp. SOC. Exp. Biol..Tidsskr. Kjemi .Trans. Amer. SOC. Metals .Trans. Electrochem. SOC. .Trans. Faraday SOC. .Trans, Illinoia Acad. Sci. .USED IN CHEMXCAL SOCIETY PUBLICATIONS. 489FULL TITLE.Proceedings of the Koninklyke Nederlandsche AkademieProceedings of the London Mathematical Society.Proceedings of the Staff Meetings of the Mayo Clinic.Proceedings of the National Academy of Sciences.Proceedings of the National Institute of Science ofProceedings of the Physical Society of London.Proceedings of the Royal Irish Academy.Proceedings of the Royal Society.Proceedings of the Royal Society of Edinburgh.Proceedings of the Royal Society of Medicine.Proceedings of the Society for Experimental Biology andPublications of the American Association for thePublic Health Reports, Washington.Quarterly Journal of Experimental Physiology.Quarterly Journal of the Geological Society of London.Quarterly Journal of Pharmacy and Pharmacology.Quarterly Reviews of the Chemical Society.Radiology.Recueil des Travaux chimiques des Pays-Bas et de laReports of the British Association for the AdvancementResearch.Revista Brasileira de Chimica.Revue de la Facult4 des Science de l’Universit6 d’Istan-Revue d’Immunologie.Revue de I’Industrie minerale.Reviews of Modern Physics.Revue scientifique, Paris.Review of Scientific Instruments.Ricerca scientifica.Roczniki Chemji.South African Journal of Science.Schweizerische medizinische Wochenschrift.The Scientific Journal of the Royal College of Science.Scientific Papers of the Institute of Physical andScientific Proceedings of the Royal Dublin Society.Science Progress.Science Reports, Tohoku Imperial University.Sitzungsberichte der Akademie der Wissenschaften inSitzungsberichte der Gesellschaft zur BeforderungSkandinavisches Archiv fur Physiologie.Smithsonian Miscellaneous Collection.Societas Scientiarum Fennica, Cormnentationes Physico-Spec trochimica Ac ta.Southern Medical Journal.Suddeutsche Apotheker-Zeitung.Suomen Kemistilehti (formerly also known as ActaSvensk Kemisk Tidskrift.Symposia of the Society of Experimental Biology.Tidsskrift for Kjemi og Bergvesen.Transactions of the American Society of Metals.Transactions of the Electrochemical Society.Transactions of the Faraday Society.Tramactions of the Illinois Academy of Science.van Wetenschappen.India.Medicine, New York.Advancement of Science.Belgique.of Science.bul .Chemical Research, Tokyo.Wien.der gesamten Naturwissenschaften zu Marburg.Mathematicae.Chemica Fennica)490 PRINCIPAL REFERENCES USED IN CHElKICAL SOCIETY PUBLICATIONS.Abbreviated Title.Tram.Inst. Chem. Eng. .Trans. N.Y. Acad. Sci. .Trans. Roy. SOC. Canada ,Tram. Roy.SOC. Trop. Med.Tram. State Inst. Appl. Chem.Tram. Wisconsin Acad. Sci.Ukrain. Biochem. J . .Ukrain. Chem. J . .U.X. Bur. Min. .Usp. Chim. .W i d . Ann. .2. anal. Chem. .2. angew. Chem. .2. angew. Min. .2. angew. Phys. .2. anorg. Chem. .2. Biol. .Z. Elektrochem. .HY9.U.S.S.R.2. Elektrotech. .2. ges. exp. Med. .Z. Krist. .2. Metallk.2. Naturforsch. .Z. Physik .Z. physikal. Chem. .Z. physikal. chem. UnterrichtZ. physiol. Chem. .Z. tech. Physik .Z. Unters. Lebensm. .2. Vitaminforsch. .2. Wirts. Zuckerind. .2. wiss. Phot. *Zauod. Lab. .Zentr. Bakt. .Zentr. Min.. .FULL TITLE.Transactions of the Institution of Chemical Engineers.Transactions of the New York Academy of Sciences.Transactions of the Royal Society of Canada.Transactions of the Royal Society of Tropical MedicineTransactions of the State Institute of Applied Chemistry,Transactions of the Wisconsin Academy of Sciences,Ukrainian Biochemical Journal.Ukrainian Chemical Journal.United States Bureau of Mines (Bulletins, TechnicalUspechi Chimii.Wiedemanns Annalen der Physik.Zeitschrift fur analytische Chemie.Zeitschrift fur angewandte Chemie (now Angew. Chem.) .Zeitschrift fur angewandte Mineralogie.Zeitschrift fur angewandte Physik.Zeitschrift fur anorganische und allgemeine Chemie.Zeitschrift fur Biologie.Zeitschrift fur Elektrochemie (und angewandte physi-kalische Chemie).Zeitschrift fur Elektrotechnik.Zeitschrift fur die gesammte experimentelle Medizinzugleich Fortsetzung der Zeitschrift fur experimentellePathologie und Therapie.Zeitschrift fur Kristallographie.Zeitschrift fur Metallkunde.Zeitschrift fur Naturforschung.Zeitschrift fur Physik.Zeitschrift fur physikalische Chemie, Stochiometrie undZeitschrift fur den physikalischen and chemischenHoppe-Seylers Zeitschrift fur physiologische Chemie.Zeitschrift fur technischen Physik.Zeitschrift fur Untersuchung der Lebensmittel.Zeitschrift fur Vitaminforschung.Zeitschrift der Wirtschaftsgruppe Zuckerindustrio(Verein der Deutschen Zucker Industrie).Zeitschrift fur wissenschaftliche Photographie, Photo-physik und Photochemie.Zavodskaja Laboratorija.Zentrrtlblatt fur Bakteriologie, Parasitenkunde undInfektionskrankheiten.Zentralblatt fur Mineralogie, Geologie and Palliontologie.and Hygiene.U.S.S.R.Arts and Letters.Papers and Reports of Investigation).Verwandtschaftslehre.Unterricht YliINTED IN GREAT BHITdJN BYRTCFIARD CLAY AND COMPANY, IITI).,BUNQAY, SWFOLH

ISSN:0365-6217    

DOI:10.1039/AR9504700483

出版商:RSC    

年代:1950

数据来源: RSC