Analytical chemistry


作者: J. G. N. Gaskin,  


期刊: Annual Reports on the Progress of Chemistry  (RSC Available online 1945)
卷期: Volume 42, issue 1  

页码: 247-264




年代: 1945




出版商: RSC


数据来源: RSC



ANALYTICAL CHEMISTRY.1. THE PROTEIN AMINO-ACIDS.RECENT theoretical studies of protein structure, and the increasing interestin protein hydrolysates for clinical use, have within the last few years evokeda wide extension of the analytical methods available for estimating theindividual amino-acids. It is hoped that, by reviewing the most recentwork together with a background of less immediately recent “source”papers, it may be possible to provide a fairly comprehensive summary of thetechniques which have been most effectively used during the last ten years.These techniques are, however, of comparatively little value, unless it isreasonably certain that the amino-acids present after hydrolysis correspondqualitatively and quantitatively to the units originally present.Investig-ations on the unhydrolysed protein have thus 9 considerable significancerelevant to the subsequent analysis of the hydrolysate. In this field, infra-red spectrography has been applied to the estimation in proteins of residueswith distinctive groupings- such as those of arginine and proline.The danger of misconception arising from qualitative changes on hydroly-sis is exemplified by the isolation, from protein hydrolysates, of lanthionine ;this has been shown to be in most cases, and probably in all, a secondaryformation from cysteine residues which are the true protein constituent^.^Effective chemical stability in an amino-acid formed by hydrolysis is not initself evidence that the acid will be liberated unchanged from the corres-ponding radical in the parent protein, since it has been shown * that cystine,serine, and threonine, when combined in proteins, are destroyed by hydrolysisconditions which do not affect them in the free st,ate.Similarly, it is knownthat whole protein is more readily racemised by alkalis than are free amino-acids. For some purposes (e.g., if the hydrolysate is to be analysed by iso-topic, biological, or enzymatic methods which are specific as between opticalisomers) racemization must be regarded as a qualitative transformation.There is evidence that ordinary acid hydrolysis conditions will not normallyinduce serious racemization except after a period several times longer than isrequired for hydrolysis only; the amino-acid most readily racemized ishi~tidine.~ 9 65 Racemization during hydrolysis is, however, reported as asource of error in the enzymatic estimation of glutamic acid.62Quantitative errors may arise by more far-reaching decomposition duringhydrolysis, forming breakdown products which are not of amino-acid nature,A.M. Buswell and R. C. Gore, J . Physical Chem., 1942,46, 575.M. J. Horn, D. €3. Jones, and S. 5. Ringel, J. Biol. Chem., 1941,138, 141.E.g., A. Schoberl, Biochem. Z., 1942, 313, 214.€3. H. Nicolet, L. A. Shinn, and L. J. Saidel, J. Biol. Chem., 1942, 142, 609.A. H. Schein and C. P. Berg; through Dairy Sci. Abs., 1944,5, 197248 ANALYTICAL CHEMISTRY.and are not subsequently determined. indicatethat, as in the case of racemization, there will generally be a reasonablelatitude between the duration required for hydrolysis only, and that requiredfor serious decomposition.The emphasis thus laid on hydrolysis conditions,however, is reflected in a tendency for analysts to prepare a number ofhydrolysates from any one protein, using different agents which are severallymost suitable for each of the different amino-acids expected; e.g., ifcystine is to be estimated, maximum yield with minimum humin formationis achieved by hydrolysis with equal parts of 20% hydrochloric and 90%formic acids. For production of tryptophan, which is unstable to acids,alkalis or trypsin have previously been employed, and, to obtain it in acolourless hydrolysate suitable for colorimetric examination, papain hasbeen used for the hydrolysis of ~ a s e i n .~Actual analysis of the. hydrolysate, especially by techniques of theclassical type depending on solubility differences, is frequently complicatedby inter-effects (e.g., mutual solubility and co-precipitation) between theconstituent amino-acids ; so that a procedure suitable for one amino-acidin a particular hydrolysate may be ineffective for the same acid in anothermixture with different proportions of the same or other amino-acids. It istherefore often desirable, before proceeding to the determination of anysingle acid, to simplify its " background " by a preliminary sorting of thetotal amino-acids into groups.39 The newer methods for this purpose aretherefore now reviewed, though it will be understood that some of thesemay also be applicable, in suitable circumstances, to the estimation ofindividual amino-acids.Among methods utilizing solubility differences, the separation of thecopper salts with different solvents has continued to find wide application,and B.W. Town lo has usefully summarized his series of papers on thistechnique. A critique of the Foreman separation of the dicarboxylic acidswith lime (or baryta) and ethanol has been published.ll Mutual solubilityeffects arising when phosphotungstic acid is used to precipitate the basicamino-acids have been investigated by D. D. van Slyke and others.12 The" partition chromatogram" technique of A. J. P. Martin and R. L. M.Synge 13 must strictly be included among differential solubility methods,though the manipulation is more akin to chromatography.The Martin andSynge column, however, not being in itself adsorptive of the amino-acids,serves only as an inert mechanical support to a solvent. When the amino-acids dissolved in a second immiscible solvent are made to flow along thecolumn, partition occurs between the " mobile " and the " static " liquidA number of studies 59 6,6 R. Borchers and C. P. Berg, J . Biol. Chenb., 1942, 142, 693.7 J. M. R. Beveridge and C. C. Lucas, ibid., 1944, 155, 547.8 E.g., R. J. Block and D. Bolling, Arch. Biochem., 1945, 6, 277.9 M. J. Horn and D. B. Jones, J. BioZ. Chem., 1945,157, 153.10 Biochem. J., 1941, 35, 419.11 K. Bailey, A. C. Chibnall, M. W. Rees, and E. F. Williams, ibid., 1943, 37, 361.12 D.D. van Slyke, A. Hiller, and R. T. Dillon, J . Biol. Chem., 1942,146, 137.l 3 Biochem. J., 1941, 35, 91, 1358; 1943, 37, 79, 86, 313; 1945, 39, 363KELLETT : THE PROTEIN AMINO-ACIDS. 249phase; and the total effect approximates to that of a large number of two-solvent separations, in the same way that the equivalent of a large series ofsuccessive distillations is effected in the operation of a fractional distillationcolumn. The amino-acids are thus separated into isolable bands, which aremade visible by an indicator of appropriate pH range, incorporated in thecolumn. A specially suitable indicator, not readily leached out, has beendescribed.l* Martin and Synge found the acetyl derivatives of the amino-acids most convenient in this application, but the copper salts of the un-acetylated acids have also been used; the separated bands are then directlyvisible by their blue colour, and are quantitatively assessed by iodimetrictitration of their copper content.15 The partition chromatogram principleis raised to a higher power in the " two-dimensional chromatogram,') 16, 23in which the solid support is cellulose (filter-paper), and the static liquidphase is the moisture inherent in it.After capillary irrigation with oneimmiscible mobile-phase solvent, the paper sheet is rotated in its ownplane through QO", and irrigated with a different mobile phase. By thistwofold treatment, 22 amino-acids in only 400 pg. of wool protein hydrolysatewere qualitatively separated into distinct spots, which were made visible incharacteristic colour by spraying the paper with ninhydrin solution andheating.The co-ordinates of location of each spot, being determined bytwo different constants (vix., partition coefficients in two different water-solvent systems) of the amino-acid concerned, are characteristic of eachamino-acid .Applications of true chromatography have been somewhat limited. J. L.Wachtel and H. G. Cassidy l7 found that charcoal as an adsorbent tended todecompose the amino-acids, both by aminolysis and more fundamentally.Bleaching earth has been more successfully used for selective adsorption ofthe diamino-acids ; l8 and glutamic and aspartic acids were isolated on acid-washed alumina, and separated from one another by fractional elution withsuitably buffered acidic solutions.l 9 Ion-exchange resins of the Amberlitetype have been similarly applied to separation of the basic acids by R. J.Block,20 and this variant seems so far the most likely to be useful inpractice.21An electrolytic separation of the basic acids was effected by A. A.Albanese,22 who claimed that this method, owing to the minimum of rnanipu-lation involved, avoided many risks of nitrogen loss (e.g., by adsorption onprecipitates) which other methods incur. It. I,. M. Synge 23 has electrolyseda mixture containing monoamino-acids, a diamino-acid, aspartic acid, andethanolamine, placed in a trough hollowed in a block of silica jelly stiffened'' H. F. Liddell and H. K. Rydon, Biochem. J . , 1944, 38, 68.l 5 T.Wieland and H. Fremerey, Ber., 1944, 77, 234.l6 R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J., 1944, 38, 224.l7 J. Amer. Chem. SOC., 1943, 65, 665. l6 F. Turba, Ber., 1941, 74, 1829.l9 F. Turba and M. Richter, ibid., 1942, 75, 340.2o PTOC. SOC. Exp. Biol. Med., 1942, 51, 252; through Chem. Abs., 1943, 37, 899.21 Cf. R. K. Cannan, J. Biol. Chem., 1944,152, 401.22 Ibid., 1940, 134, 467. 23 Biochem. J., 1945, 39, 358, 363250 ANALYTICAL CHEMISTRY.with paper pulp and buffered at pH6. The migration of ions towardselectrodes a t the ends of the block is traced by pressing a sheet of paper onto the block and “ developing ” by spraying with ninhydrin and heating;when ionophoresis through the block has proceeded sufficiently far, the printsof separate bands of ions can be recognised on the sheet.The strong baseethanolamine moves rapidly towards the cathode, the diamino-acid lysinemore slowly, and the monoamino-acids most slowly ; the dicarboxylic acid,aspartic, migrates towards the anode. The basic acid ornithine also movestowards the cathode rapidly enough to separate it from the monoamino-acids. With the guidance of the paper “print,” segments of the blockcontaining the separated components can be cut out for further treatment.Specific methods for the estimation of individual amino-acids may bereviewed under five heads : (A) solubility methods, (B) colorimetric andother chemical reactions, (C) microbiological, (D) enzymatic, and (E) iso-topic dilution.(A) Solubility Methods.Advances have been made in the search for precipitants of higherspecificity. M.Bergmann Z4, 25 observed that the introduction of organicmolecules into the co-ordinated envelopes of certain chromium diamminesincreased their specificity as amino-acid precipitants. Thus, althoughReinecke’s salt (ammonium tetrathiocyanatodiamminochromate) precipitatesboth proline and hydroxyproline, yet only proline is precipitated if the twoammonia molecules are replaced by two of aniline ; whereas the trioxalato-chromate complex is specific for glycine. Salts of various amino-acids with‘‘ dioxanilic ” and ‘‘ dioxpyridic ” acids, in which the co-ordinated envelopescontain respectively pyridine and aniline with oxalate residues, have sub-sequently been described.26 The use of naphthalene- p-sulphonic acid andflavianic acid as precipitants has prompted an extensive examination ofother sulphonic acids of possibly higher specificity, chiefly derived fromanthraquinone, diphenylamine, and a~obenzene.~’ Both these types ofprecipitant have been applied in the new technique of the “ solubilityproduct method.” In this procedure, no attempt is made a t quantitativeprecipitation of the complex (amino-acid with precipitant) in an absolutesense ; the concentration of the amino-acid ion in a solution is calculated fromthe extent to which a weighed addition of the complex dissolves, first againstthe common-ion effect of the amino-acid originally present, and then whenthis effect is increased by the addition of a weighed amount of pure amino-acid.If the solubility product [amino-acid ion] [precipitant ion] has not24 J . Biol. Chem., 1935, 110, 471.26 M. Bergmann and S. W. Fox, ibid., 109, 317.26 M. Bergmann, ibid., 1938, 122, 569.27 W. H. Stein, G. Stamm, C - Y . Chou, and M. Bergmann, ibid., 1942,143,121. Someadvantages in the use of flavianic (dinitronaphtholsulphonic) acid to precipitate arginineas the monoflavianate instead of the more usual diilavimate are noted by Beveridge andLucas (ref. 39)KELLETT : THE PROTEIN AMINO-ACIDS. 251identical values a t the two different concentrations involved, a factor isintroduced into the calculation to correct for this.28* 299 30(B) Colorimetric and other Chemical Reactions.Only a selection can be given from numerous papers describingestimations based on chemical, including colorimetric, reactions. Many ofthese papers, however, have full bibliographies of earlier and alternativemethods.Clycine and alanine have been determined by their reaction with nin-hydrin, yielding respectively formaldehyde and a~etaldehyde.~~ Theformer aldehyde is subsequently estimated colorimetrically with chromo-tropic (1 : 8-dihydroxynaphthalene-3 : 6-disulphonic) acid, and the latterwith p-hydroxydiphenyl.Valine and leucine, after aminolysis with nitrous acid, have been deter-mined by oxidation of their isopropyl groups to acetone with chromic acidunder pressure .32Xerine and threonine (and other p-hydroxy-a-amino-acids if present) areconverted into aldehydes by treatment with periodic acid.The form-aldehyde produced from serine can be estimated with dimedon 33 or colori-metrically with chromotropic acid.34 The production of acetaldehyde fromthreonine is much influenced by conditions, and is hardly to be relied uponas quantitative ; when formaldehyde is simultaneously formed, the acet-aldehyde can be simply isolated by volatilization in a stream of air.35 Aserious drawback in these methods is the danger of secondary reactionsbetween the aldehydes and unchanged amino-acid~.~~ An unpublishedmodification giving quantitative results is, however, mentioned by R. L. M.S ~ g e . ~ ~Papers have been published on the elaboration of the Sullivancolorimetric reaction with sodium P-naphthaquinone-4-sulphonate 3' andthe modification of the Vassel method using p-aminodimethylaniline.3*Estimation of cystine from the sulphur content of the mercaptide precipi-tated by cuprous oxide may be unreliable, since methionine has been foundin such a precipitate even after rigorous purification.39Cystine.28 M.Bergmann and W. H. Stein, J . Biol. Chem., 1939,128, 217; 129, 609.29 H. R. Ing and M. Bergmann, ibirE., p. 603.30 S. Moore and W. H. Stein, ibid., 1943,150, 113.31 B. Alexander and A. M. Seligmann, ibid., 1945,159,9 ; 160,51.32 J. Roche and M. Mo~~rgue, Coinpt. rend. Xoc. biol., 1943,137, 766; through Chem.35 B. H. Nicolet and L. A. Shim, J . Biol. Chem., 1941,139, 687.34 M. J. Boyd and M. L. Logan, i b d . , 1942,148,279.35 A. J.P. Martin and R. L. M. Synge, Biochem. J . , 1941,35, 294.s 6 A. Neuberger, ibid., 1944,38, 309.3 7 F. A. Csonka, H. Lichtenstein, and C. A. Denton, J . Biol. Chem., 1944,156, 571 ;38 D. K. Mechan, ibid., 1943,151, 643.39 J. M. R. Beveridge and C. C. Lucas, Bwchem. J., 1944, 38, 88.Abs., 1945, 39, 3313.R. J. Evans, ibid., p. 373252 ANALYTICAL CHEMISTRY.Methionine. Interference by cystine when methionine is determinedby the McCarthy-Sullivan colour reaction with nitroprusside has beenin~estigated.~~ A chemical determination has been described by E. F.Beach and D. M. T e a g ~ e , ~ l based on demethylation with hydriodic acid,forming homocysteine. Under the experimental conditions, homocysteinepasses into a thiolactone ring which does not ’form a cuprous mercaptide;cystine can therefore be removed in this form, after which the thiolactonering is opened with sodium hydroxide, and further treatment with cuprousoxide then precipitates only the homocysteine equivalent to the originalmethionine.Estimation of methionine by means of its periodide has beendescribed by T. F. Lavine; 42 this compound is stable to thiosulphate,which can therefore be used to remove excess of iodine before decomposingthe periodide with acid and titrating the iodine thus liberated. Otheramino-acids which form periodides are allowed for by a blank in which themethionine present is oxidised with potassium iodate to its sulphoxide,which does not form a periodide.Phenylalanine can be estimated by a method 43 based, like the earlierKapeller-Adler method, on the presence of the nitratable benzene ring.Nitration yields the dinitro-compound, which is reduced to the diamine,and this gives a red colour with sodium @-naphthaquinone-4-sulphonate.Other nitratable rings present (tyrosine and tryptophan) are eliminated bypermanganate oxidation.Tyrosine is still most conveniently estimated by hlillon’s reaction, forwhich satisfactory conditions have been worked out by J .W. H. L ~ g g . ~ ~Tryptophan estimation by colorimetric means has been reviewed byM. X. Sullivan and W. C. Hess 45 (cf. Horn and Jones 9).Glutamic acid on heating under pressure a t pH 3-4 undergoes ring-closure to pyrrolidonecarboxylic acid, and a van Slyke determination oftotal amino-nitrogen before and after ring closure has been proposed as ameasure of the glutamic acid present.46 An alternative chemical treatmentof glutamic acid is its oxidation with chloramine-.r to p-cyanopropionic acidand subsequent hydrolysis to succinic acid, which is then estimated by itsoxygen absorption in the presence of succin~xidase.~~A rginine estimation by the Weber-Sakaguchi colour reaction witha-naphthol and a hypohalite solution has been critically discussed by A.A.Albanese and J. E. F r a n k s t ~ n . ~ ~Improved conditions for the Koessler-Hanke colour reactionwith diazotized sulphanilic acid have been described by H. T. MacPhers~n.~~Histidine.W. White and F. C. Koch, J . Biol. Chem., 1945,158, 535.4 1 Ibid., 1942, 142, 277.4 2 Ibid., 1943, 151, 281.43 W.C. Hess and M . X. Sullivan, Arch. Biochem., 1944, 5, 165.4 4 Biochem. J., 1937, 31, 1422; 1938, 32, 775.4 5 J . Biol. Chem., 1944, 155, 441.4:i H. S . Olcott, $bid., 153, 71.4 7 P. P. Cohen, Biochem. J., 1939, 33, 551.4 8 J . Biol. Chem., 1945, 159, 185. 4 9 Riochem. J . , 1942, 38, 59KELLETT : THE PROTEIN AMINO-AO'CDS. 253(C) Microbiological Methods.Microbiological assays for most of the commoner amino-acids have nowbeen described, utilizing LactobaciZEus spp. 50 or Leuconostoc mesenteroides. 51Quantitative assessment may be either by a turbidimetric measure of thegrowth of the organism, or by titration of the acid liberated in its metabolism.Using Streptococcus fcecalis, a uniform routine method allowing of a completeanalysis for histidine, arginine, lysine, leucine, isoleucine, valine, methionine,threonine, tryptophan, and phenylalanine on 1.5 g.of sample has beendescribed.52 Considerable evidence is now available as to the specificityof this type of estimation. Complete specificity as between optical isomersis by no means invariable, since d- and Z-aspartic acids are equally wellutilized by L. DeEbruckii, though this organism responds to serine only inthe Z-form.53 d-Leucine and 1-glutamic acid s5 can a t least partly replacethe natural isomers. There is also evidence that amino-acids may be oftenpartly replaced by the corresponding deaminated (a-hydroxy- or a-keto-)compounds.54, 55 These observations may perhaps be correlated with theconclusion that d-glutamic acid is not utilized as such by the organism;comparison of its irregular " delayed " response curve with the regular curvegiven by glutamine suggests that the acid is utilized only after conversioninto the amine, which may be to some extent formed also from the deaminatedor optically isomeric c0mpounds.5~ As regards glutamic acid, this resultmight perhaps be expected, since the unit is probably present in proteins asglutamine more often than as glutamic acid; but it does not appear that thesame explanation will apply to aspartic acid, which is not readily replaced incultures by asparagine.56Attention has been directed to allowances for error due to the presenceof undefined growth-stimulants either in the basal medium 57 or in thehydrolysate under examination.58 There would also appear to be aomedanger of the test organism varying in its response to certain amino-acids,since it has been reported 59 that L.arabinosus in the course of culture canreadily develop a strain capable of entirely dispensing with histidine or anyobvious source of histidine such as indole or anthranilic acid. A morehelpful kind of variation is exhibited by Neurosporn crassa, which normallycan be cultured in a medium containing only inorganic salts and a sourceof organic carbon, with the addition of biotin; by ultra-violet irradiation or'O E.g., S. Shankman, M. S. Dunn, and L. B. Rubin, J . Biol. Che-m., 1943,150, 305,477; 151,511." E.g., M. S. Dunn, M. N. Camien, S. Shankman, and L. B.Rockland, ibid., 1946,159, 653.5 2 J. L. Stokes, M. Gunness, M. Dwyer, and M. C. Caswell, ibid., 160, 35.53 J. L. Stokes and M. Gunness, ibid., 157, 561.5 4 D. M. Hegsted, ibid., p. 741.5 5 L. R. Hac, E. E. Snell, and R. J. Williams, ibid., 159, 273.56 L. R. Hac and E. E. Snell, ibid., p. 291.'' E. J. chu and R. J. Williams, ibid., 1944, 155, 9.5 8 E. C. Wood, Nature, 1945, 155, 632.59 L. D. Wright and H. R. Skeggs, J . Biol. Chem., 1945,159, 615254 ANALYTIUAL OHEMISTRY.other means, however, mutant strains, having specific requirements for asingle additional orgavic compound, can be generated. The productionof a strain to the growth of which lysine is essential, and the bio-assayof lysine by the use of this organism, have been described by A.H.Doermann.60(D) Enzymatic Methods.A promising development from the bio-assay is the use of enzymepreparations isolated from the micro-organisms. From cultures of variouscoliform bacteria, decarboxylase preparations have been obtained which arespecific respectively to lysine, tyrosine, glutamic acid, histidine, and orni-thine; and mixtures containing these acids have been analysed by mano-metric estimation of the carbon dioxide liberated from each by its appro-priate enzyme.61, 62 Arginine decarboxylase has also been obtained,though not as yet separated from the lysine enzyme.63 The enzymes aresomewhat sensitive to inhibition by inorganic salts and various organiccompounds, e.g., hydroxylamine. They appear to be entirely specific asbetween optical isomers ; this is consistent with the reason suggested under(C) for non-specificity in certain microbiological assays, since the productionof a substance such as glutamine from any one of several precursors is ofcourse not possible by a decarboxylase alone.It is remarkable, however,that the enzymes do appear to some extent effective against hydroxy-derivatives of their normal substrates, whether substituted in the aliphaticchain (hydroxylysine, p-hydroxyglutamic acid) or benzene ring (dihydroxy-phenylalanine) ; but the converse is not true, since tyrosine decarboxylasedoes not act on phenylalanine.(E) Isotopic Dilution.H. H. Ussing’s proposal to estimate the quantity of any oneamino-acid from its effect in diluting a measured addition of the same acidin which one of the elements is present as an isotope, has been applied byG.L. Foster and his co-workers G5* 661 67 to aspartic and glutamic acids,leucine, glycine, lysine, arginine, phenylalanine, and tyrosine. Deuteriumwas the label element chosen by Ussing, but the American workers havepreferred 15N, determining the proportion of this isotope in the pure amino-acid as added, and again as subsequently re-isolated, by the mass spectro-graph. The isohopic preparation as added being necessarily synthetic andtherefore racemic, while the acids in the hydrolysate (except glycine) areoptically active, a somewhat laborious series of crystallizations is required,to ensure that the re-isolated acid consists only of the naturally-occurringstereoisomer ; this course is, however, found more practicable than either*O J .Biol. Chem., 160, 95.81 E. F. Gale and H. M. R. Epps, Biochem. J., 1944, 38, 232, 242.62 E. F. Gale, ibid., 1945, 39, 42, 46.64 Nature, 1939, 144, 977.d B D. Rittenburg and G. L:Foster, ibid., 1940,133, 737.67 D. Shernin, ibid., 1945, 199,1439.6s E. S. Taylor and E. F. Gale, ibid., p. 52.6 5 G. L. Foster, J. Biol. Chem., 1945, 159, 431GASKM : ORGANIC REAGENTS IN INORGANIC ANALYSIS. 255of the alternatives, uiz., quantitative racemization of the acid in the hydroly-sate, or resolution of the racemic isotope preparation before addition. Amarked advantage of the method is that isotope concentration is a verysharp criterion of the purity of the isolated amino-acid, since 5% con-tamination by another amino-acid causes a difference of 5% in the isotopeconcentration, but can cause only a much smaller change in properties suchas nitrogen content or optical rotation which are common to the contaminantand the compound being isolated. E.G . K.2. ORGANIC REAGENTS IN INORGANIC ANALYSIS.As the title of this section indicates, attention has been devoted to thequantitative use of organic reagents in inorganic analysis, and no attempthas been made to cover the whole field of their application in analyticalpractice. This section of the subject appears to have been dominated bythe modern ability to obtain mechanical measurement of colour, either theabsorption or the transmission; hence many more reagents are described asgiving suitable colour reactions than as being suitable for gravimetric work.This insistence on colour has been carried to some lengths, for methods aredescribed where coloured precipitates are actually maintained in a dispersedstate to enable a colour measurement to be made.At other times colourstandards are used, prepared from a different substance.The devotion to colour in its turn has been governed by the desirabilityfor speed in the examination of large numbers of similar samples and also bythe necessity for the determination of minute amounts of an element wherethe colour reaction is the most sensitive test. Fortunately a number ofcomparisons have been made between methods already established and thenewer colour methods, and on the whole it may be said that the latter are notinferior.Although it is the sincere hope of the Reporter that he has not omittedany important contribution in the last year or two to this section of analysis,it should be nevertheless pointed out that the scope of organic reagents,even in inorganic analysis, is very wide and difEcult to arrange in anythinglike a systematic survey.It would have been preferable to have made thevarious headings refer to the reagents, but for reference purposes it has beenconsidered that headings under the names of the elements determined aremore satisfactory and this order has been followed. Lead, copper, and ironhave easily attracted the most attention, followed by bismuth, cobalt,tungsten, boron, beryllium, nickel, zinc, and cadmium, and these elementshave been reviewed under separate headings.Then follow two groups ofelements, the first being those less frequently encountered, and the second amiscellaneous group which have not attracted much attention.Lead.-Mention of lead almost inevitably draws attention to “ dithi-zone,’’ and considerable work has been done with this reagent. In a com-prehensive study P. A. Clifford 1 has enumerated the metals reacting withJ. Assoc. 08. Agric. Chem., 1943, 26, 26256 ANALYTICAL CHEMISTRY.dithizone and has listed certain of the important properties of the metalliccomplexes. He has shown that interference from the complexes of bismuthand tin in the final photometric measurements can be detected, and hasindicated a possible method whereby bismuth and lead may be simultaneouslydetermined. At the same time, a procedure for the separation of smallquantities of lead from a possible interfering element, thallium, was outlined.Applying the method to the examination of urine, Clifford showed that theresults were quite comparable with those of other established methodsrequiring much larger quantities of the original material ; the possibleerrors in the method were examined and eliminated.It has been pointed out that in micro work the bismuth interferencemay be eliminated by extraction of the bismuth complex a t pH 2, and thattitanium and aluminium can interfere with the photometric measurements.This extraction of bismuth may be lengthy, and to avoid it, K.Bambachand R. E. Burkey recommend that the normal acid (‘ stripping ” be re-placed by an aqueous (‘ stripping ” a t pH 3.4, whereby, unless excessivebismuth is present, the lead is completely separated. Using dithizone, thelead in numerous toilet preparations has been successfully determined *and the method has proved of value in the examination of soil, plants, andfood.The blue-violet insoluble precipitate formed with lead salts and carminicacid in acid solution has been proposed as a sensitive test for detecting afew pg. of lead. E. A. Leibmann has examined the reactions of diphenyl-carbazide with lead, and V. I. Kuznetsov 8 proposes testing for quadrivalentlead with paper impregnated with anthraquinone- 1 -azo-4-dimethylamine.Electrolytically deposited lead dioxide may be dissolved in an acetic solutionof tetramethyldiaminodiphenylmethane and the resulting blue colour usedfor the determination of the lead.gCopper.-Numerous reagents have been discussed in recent literaturefor use in the determination of copper.Substituted amides of dithio-carbonic acid l o can be used satisfactorily for its detection, and rapidity inthe quantitative determination of the metal in aluminium alloys can beobtained by the colorimetric method using diethyldithiocarbarnate.ll9 12, l3Extraction of a copper solution with chloroform containing 8-hydroxy-quinoline will, at the appropriate pH, remove the copper-oxine complexJ. Schultz and M. A. Goldberg, Ind. Eng. Chem. Anal., 1945, 15, 155.Ibid., 1942, 14, 904F. H.Buckwalter, Proc. Sci. Sect. Toilet Goods Assoc., 1943-44, No. 1, 22.0. Braadlie and H. Bargh, Tidskr. Kjemi Berg., 1942,2,88 ; Chem. Zentr., 1943.11,J. V. Dubsky, Chem. Listy, 1940, 34, 91; Chern. Zentr., 1941, I, 1576.J . Appl. Chem. U.S.S.R., 1943, 16, 238.M. Fields, New Zealand J . Sci. Tech., 1942, 23B, 224.345.’ Lab. Prakst. U.S.S.R., 1941, 16, No. 10-11, 23.lo E. Geiger and H. G. Muller, Helv. Chim. Acta, 1943, 26, 996.l1 F. W. Haywood, Analyst, 1943, 68, 206.l2 S. Bertoldi, Alluminio, 1943, 12, 37 ; Chem. Zentr., 1944, I, 452.l3 R. F. Partridge, Ind. Eng. Chem. Anal., 1946, 17, 422GASKIN : ORGANIC REAGENTS IN INORGANIC ANALYSIS. 257and the absorption maximum of this extract has been measured.14 H.T.Liem l5 has suggested that Quinosol or Superol can, with advantage, replace8-hydroxyquinoline in the determination of copper, aluminium, and zinc.Attention has been paid to the separation of copper from zinc and cadmium ;apart from interference by silver and gold, 8-hydroxyquinolinecarboxylicacid is a suitable reagent for this purpose.16 With copper and cadmiumonly present, the quinaldic acid separation, originally proposed by P. Rayand M. K. B0se,17 has been shown to be reasonably, l9 Ob-jections to the method have not been supported by spectrographic examin-ation 2o of the separated metals, the copper containing only 0~05-0~07~0 ofcadmium, and the cadmium only 0.3% of copper.Two methods 21, 22 are available for using isonitroso-3-phenylpyrazolone,interference from cadmium being prevented by addition of ammoniumtartrate, which addition also prevents co-precipitation of a number ofelements.Highly coloured cuprous complexes are formed with o-phenanthroline and its derivative^,^^ and the first named can be used forthe spectrophotometric determination in the vbsence of interfering elements.=Salicylaldoxime 25 is used for copper in steel, and in nickel-plating baths 26the dithizone method is useful. If the dithizonates are extracted a t pH 3,the transmittance of the copper dithizonate may be measured spectrophoto-metrically, provided interference from silver, mercury, bismuth, and stannoustin is eliminated with acid potassium iodide. Gold, platinum, and palla-dium, which might interfere, are fortunately rarely en~ountered.~~ Com-plexes formed between metal thiocyanates, including copper, and a-naphthylamines are quantitatively precipitated,28 and the copper thiocyanate-pyridine complex can be extracted with cblor~form.~~ The o-toluidinecomplex is regarded as the best for a colorimetric determination.Dithio-oxamide (rubeanic acid) forms a precipitate with a copper solution, but theprecipitate can be dispersed with gum arabic and colorimetric measurementsobtained, provided interference due to the colour of the reagent be elimin-ated.3O Curves relating copper concentration and colour have been obtained14 T. Moelier, Ind. Eng. Chem. Anal., 1943, 15, 346.15 Pharm. Tjdschr. Nederl. Indie, 1942, 19, 13.16 J.R. Gilbreath and H. M. Haendler, Ind. Eng. Chem. Anal., 1942,14, 866.1 7 Z . anal. Chem., 1933, 95, 400.19 C. E. Pritchard and R. C. Chirnsido, ibid., p. 244.2o A. K. Majumdar, J . Indian Chem. SOC., 1944, 21, 24.21 V. Hovorka and J. Vorisek, Chem. Listy, 1942,36, 73; Chem. Zentr., 1942,11, 573.22 V. Hovorka and J. Vorisek, Chem. Listy, 1943, 37, 5; Chem. Zentr., 1943, I, 1700.23 M. L. Moss. M. G. Mellon, and G. F. Smith, I d . Eng. Chem. Anal., 1942,14, 931.2' M. L. Moss and M. G. Mellon, ibid., 1943,15, 116.2 6 M. Jean, Bull. SOC. chim., 1943, 10, 201.26 B. B. Knapp, Proc. Amer. Electroplaters SOC., 1944, June, 109.27 G. H. Bendix and D. Grabenstetter, Ind. Eng. Chem. Anal., 1943, 15, 649.2 * F. B. Ubeda and R. Alloza, Anal. Fis.Quim., 1941, 37, 350.29 L. A. Gulyaeva and E. S. Itkina, J . Appl.,Chem. U.S.S.R., 1944,17, 252.30 A. Ringbom and F. Sundman, Finska Kem. Medd., 1942, 51, 42; Chem. Zentr.,l8 A. K. Majumdar, Analyst, 1943, 88, 242.1943, I, 761.REP.-VOL. XLTI. 258 ANALYTICAL CHEMISTRY.for this reagent.31 R. J. Sherman 32 has described a solution of 5-bromo-2-aminobenzoic acid in sodium hydroxide which gives good quantitativeprecipitation with copper of a complex which can be washed, dried, andweighed.I7ron.--o-Phenanthro1ine7 ax’-dipyridyl, and sulphosalicylic acid are allsatisfactory reagents for the colorimetric determination of iron. J. P.Mehlig and H. R. Hulett 33 recommend spectrophotometric measurement ofthe colour produced by ferrous ion with either o-phenanthroline or its nitro-derivative.Factors affecting the development of the colour34 are theorder in which the reagents are added, the speed of the addition, the presenceof phosphate, the temperature, and the time allowed for development of thecolour ; the addition of a buffer, sodium citrate, a t a temperature exceeding20” is advantageous. As little as 0-05 pg. of iron can be detected with thisreagent or aa’-di~yridyI.~~ Certain derivatives of o-phenanthroline are notpreferred in place of the parent sub~tance.~3The production of coloured ferrous complexes with either aa’-dipyridylor aa’a’’-tripyridyl may be accomplished by using a wide range of reducingagents.36 K. Buch 37 considers hydrazine sulphate good for this purposeand recommends the addition of an ammonium acetate buffer when formingthe colour.Sulphosalicylic acid was proposed by E. I. Nikitina,38 and H.Pfeiffer 39 records its satisfactory use.Of the five reagents, sulphosalicylic acid , salicylic acid, thiocyanate,aa‘-dipyridyl and o-phenanthroline, the last is regarded as the most satis-factory and reliable,40 a finding which is confirmed by a comparative study 41of this reagent and the A.O.A.C. thiocyanate method. The thiocyanateappears slightly less reliable, chiefly because of the instability of the reagentand the difficulty of reproducing the colour-concentration curve. Despitethe technical difficulties of the production and storage of the reagent, thetitanium chloride titration is reliable and easy to use, and results obtainedby it agree well with the colorimetric figures.The colour of iron withthioglycollic acid is also regarded as more stable than the thiocyanate co10ur,42although the latter is more reliable after a definite time interval.An iron colour used in the analysis of minerals and brasses, sensitiveeven in the presence of fluorides, phosphates, tartrates, citrates, and oxalates,is obtained with ferric iron and disodium 1 : 2-dihydroxybenzene-3 : 5-31 E. J. Center and R. M. Macintosh, Ind. Eng. Chem. Anal., 1945,17, 239.a2 J . SOC. Chem. Ind., 1942, 61, 164.83 Ind. Eng. Chem. Anal., 1942‘, 14, 869.34 S. L. Bandemer and P. J. Schaible, ibid., 1944, 16, 317.36 H. Borei, Biochem. Z . , 1943, 314, 359.36 M. L. Moss and M. G. Mellon, I d .Eng. Chem. Anal., 1942, 14, 862.57 Finska Kern. Medd., 1942, 51, 22; Chem. Zentr., 1943, I, 700.* 8 Zavod. Lab., 1940, 9, 629; Khim. Referat. Zhur., 1941, 4, No. 1, 84.a, 2. anal. Chem., 1943, 126, B. Bencze, Mezogazdasagi Kututaeok, 16, 61 ; Chem. Zentr., 1943, 11, 548.E. J. Benne and A. J. Snyder, J . Assoc. Off. Agric. Chena., 1944, 27, 526.H. van Dam, Ing. china., 1942, 26, 131 ; Chem. Zentr., 1943, I, 2323GASKIN : ORGANIC REAGENTS IN INORGANIC ANALYSIS. 259disulphonate.43 A linear relationship between concentration and colourreading between 0 and 10 pg. has been demonstrated with nitroso-R-salt.44A rapid method depends on the brown colour formed in slightly acid ironsolution with dimethylglyoxime and hydrazine hydr~chloride,~~ and ironcan be removed from a solution, before nickel and cobalt determinations,with hexamethylenetetramine and triethan~lamine.~~Bismuth.-The grouping NR*CS*S*C( SH):N found in thiodiazoledithioland phenyldithiodiazolonethiol is responsible for the formation of colouredprecipitates with the metals of the hydrogen sulphide If theappropriate bismuth complex is peptised with gum acacia, accurate figuresfor bismuth are given by colour rnea~urements.~~~ 49 When phenyldithio-diazolone is used there is considerable interference from other metals, 50and the bismuth complex is best formed from the potassium salt of thereagent in the presence of gum acacia.If the gum is omitted, the precipitatecan be weighed as such. M. Kuras 51 has also noted the reactions of thesulphide-group metals with mercaptoazoles, with particular reference tobismuth.The capacity of these compounds to form metallic salts is thiazole>imidazole > oxazole .s2As already mentioned, T. MoellerI4 has determined the optimum pHrange for the extraction of various metals with chloroform containingoxine, and H. G. Haynes 53 describes complete separation of the bismuthcomplex at pH 52-54 by using oxine alone.I n the absence ofzinc, manganese, and antimony, phenylarsonic acid can be used a t pH 5.1-5.3, and separation from a number of metals is possible if potassium cyanideis added.54 Similarly, the same reagent can be used if the solution isbuffered, preferably with ammonium acetate.55 a-Picoline methiodide i i asuitable reagent for detecting bismuth.56 Small amounts (0.0008-0-0018 yo)in copper have been determined with dithi~one,~' a reagent whose value forthis purpose has already been mentioned under " lead.''BeryZZium.--p-Nitrobenzeneazoresorcinol was first proposed as a spottest for beryllium by A.S. Komarovskii and N. S. P o l u e k t ~ v , ~ ~ but it hasbeen suggested not to be reliable for less than O-8y0 of beryllium.59Substituted arsonic acids have proved of value.43 J. IT. Yo0 and A. L. Jones, I n d . Eng. Chem. A w l . , 1944, 16, 111.4 4 C. P. Sideris, H. Y. Young, and H. H. Q . Chun, ibid., p. 276.4 5 P. van Stein, Clxmist-Analyst, 1945, 34, 15.4 7 A. K. Majumdar, J. I n d i a n Chem. SOC., 1942, 19, 396.48 Idem, Sci. and Cult., 1942, 7, 458.4 O Idem, J .I n d i a n Chem. Xoc., 1944, 21, 240.5 1 Chem. Obzor., 1941, 16, 17; Chem. Zentr., 1941, 11, 84.62 M. Kuras, Chem. Obzor., 1942, 17, 41; Ghem. Zentr., 1944, I, 39.53 AnuZyst, 1945, 70, 129.64 A. K. Majumdar, J. I n d i a n Chem. SOC., 1944, 21, No. 4, 119.6s Idem, ibid., p. 157.66 K. Whelan and F. J. Welcher, J . Chem. Educ., 1943, 20, 246.s7 Y. Yao, I n d . Eng. Chem. Anal., 1945, 1'7, 114.s8 Mikrochem., 1934, 14, 315.4 6 A Kundert, ibid., p. 8.6o Idem, ibid., p. 347.F. Kulcsar, Eng. Min. J . , 1943, 144, No. 12, 103.REP.-VOL. XLII I 260 ANALYTICAL CHEMISTRY.W. Stross and G. H. Osborn 60 subsequently elaborated a photometric method,using the same reagent and, despite the presence of alumina, were able tocorrelate colour measurement (photometrically) with beryllium content overthe range 0.005-18% Be.The colour comparison could not be madevisually. The same authors further extended the use of the reagent to theanalysis of minerals,c1 and F. Kulcsar 62 uses it to detect beryllium in copperbase alloys.It has been found possible to determine 0.005~0 of beryllium by measuringthe fluorescence of the complex formed between the BeO, ion and morin(1 : 2 : 3 : 5-tetrahydroflavonol), aluminium causing no interferen~e.~~ Thesame author separates chromatographically beryllium, aluminium, andmagnesium with quinalizarin.Boron.-The quinalizarin method for the determination of boron in steelshas been developed and found to give good res~Its,6~ accurate determinationshaving been obtained over the range 0~0005-0~003~0 of boron.g5 It ispossible to apply the method to corrosion-resistant steels containing 0.001-0.005 % of the element, intensities being measured photoelectrically.66 Arelated method 67 for the analysis of plant ashes uses alizarin-S, and in plantand fertiliser ashes 0.1 pg.of boron can be detected with certainty if chromo-trope-2B is the reagent.68. A delicate test for boron, in the absence offluorides, can be made with Solway purple (C.I. 1073) in concentratedsulphuric acid together with a very dilute solution of l-amino-4-hydroxy-anthraquinone in the same a ~ i d . 6 ~ Colorimetric determination, usingpentamethylquercetin, has been proposed, the necessary colour standardsbeing potassium chromate solutions.70Tungsten.-Cost and scarcity of the reagent cinchonine have beenfa6tors encouraging the search for an alternative, and rhodamine-B 'l andP-naphthaquinoline 72 are recommended.F. W. Box 73 considers thatneither cinchonine nor rhodamine-B will effect complete precipitation oftungstic oxide, and in the common methods cinchonine is slightly inferiorwhere amounts between 0.019 and 0.2 g. of tungsten are to be determined.J. H. Yoe and A. L. Jones, as a result of work continued over a number of6o J . SOC. Chem. Ind., 1944, 63, 249,61 Metallurgia, 1944, 30, No. 175, 3.62 Chemist-Analyst, 1945, 34, 28, 29, 39.63 G. Venturello, Ric. Sci., 1942, 13, 726; Chem. Zentr., 1943, I, 2519.64 G. A. Rudolph and L. c'. Flickinger, Steel, 1943, 112, No.14, 114, 131-139, 149.6 6 L. C. Flickinger, Proc. Conf. Natl. Open Hearth Cmm. Iron Steel Div. Amer.6 6 S. Weinberg,.K. L. Proctor, and 0. Milner, Ind. Eng. Chem. Anal., 1945,17, 419.67 D. Dickinson, Analyst, 1943, 68, 106.6 8 A. Stettbacher, Mitt. Lebensm. Hyg., 1943, 34, No. 1-2, 90.69 F. A. Radley, Analyst, 1944, 69, 47.' 0 K. Neelakantam and S. Rangaswami, Proc. Indian Acad. Sci., 1943, 18A, 171.7 1 J. T. Oats, Eng. Min. J . , 1943,144, No. 4, 72,7 2 B. A. Platunov and N. M. Kirillova, Uchenye ZapGki Leningrad Gosudarst Univ.,73 Analyst, 1944, 69, 272.Inst. Mining Met. Eng., 1944, 27, 197.Ser. Kkim. Nauk., 1940, No. 5, (54), 269; Khim. Referat. Zhur., 1941, 4, No. 4, 73GASKIT : ORUANIC REAGENTS IN INORaANIU ANALYSIS. 261years by many analysts, have shown that in the hands of a skilledanalyst anti- 1 : 5-di- (p-methoxyphenyl) -5 - hydroxyamino- 3- oximino- 1 -pen-tene gives good gravimetric results for tungsten in ores and alloy^.^^^ 75Toluene-3 : 4-dithiol produces a bluish-green complex with a few pg.oftungsten, and green complexes with molybdenum and rhenium. The colourof the latter can be suppressed by stannous chloride, and tungsten has beenthus satisfactorily determined in steel. 76Nickel.-In addition to its normal use, dimethylglyoxime can be usedfor colorimetric determinations of nickel in a number of ways, of which asolution of the complex in pyridine is suitable for minute am0~nt.s.'~ Treat-ment of the nickel solution with bromine,781 79 or bromine andbefore the addition of ammoniacal dimethylglyoxime also produces satis-factory colours.Aniline molybdate forms an insoluble double salt withnickel salts,81 a useful reaction, and the insoluble compound of nickel anddiguanide sulphate can be dissolved in a known sulphuric acid and theexcess acid titrated.82Coba2t.-For some time now, or-nitroso- p-naphthol has been the acceptedreagent for this metal, but recently the p-nitroso-a-naphthol has beendescribed as being more sensitive, and a detailed method, bot!h for the pre-paration of the and for its use, has been described.83 The insolublecobalt complex is dissolved in benzene to provide a solution suitable for acolorimetric method. 84 By destroying other metal complexes with nitricacid, a rapid photometric determination of cobalt in steels is possible usingnitro~o-R-salt,~~ a reagent which has also found application in the analysisof sintered metal carbides.g6 Cobalt in parts per million can be detected andmeasured by the colour formed with tripyridyl (2 : 6-di-2-pyridylpyridinehydrochloride), provided copper, nickel, and iron be first separated andcyanide and dichromate be absent.87Zinc and Cadmium.-Sodium anthranilate,*8 in the presence of a smallamount of mineral acid, or anthranilic acid, is a suitable reagent for thedetermination of these metals.Precipitation is effective at 100°,89 and thesolution of anthranilic acid recommended is 0.5 mol. in a litre of N-sodiumi4 Virginia J . Sci., 1943, 3, 301.75 I n d .Eng. Chem. Anal., 1944, 16, 45.7 7 E. Passamaneck, I n d . Eng. Chem. Anal., 1945, 17, 257.H. Seaman, ibid., 1944, 16, 354.79 A. M. Mitchell and M. G. Mellon, ibid., 1945, 17, 380.G. R. Makepeace and C. H. Craft, ibid., 1944,16, 375.81 E. Pozzi-Escot, Anal. Quint. Labs. Invest. Cient. Ind., Peru, Oct. 1943, 9.8 2 A. K. Majumdar, J . Indian Chem. SOC., 1943, 20, 289.83 W. Jung, C. E. Cardini, and M. Fuksman, Anal. Asoc. Q u h . Argentina, 1943, 31,84 C. E. Cardini, W. Jung, and M. Fuksman, ibid., p. 191.8 G H. E. Cox, Analyst, 1944, 69, 235.s7 M. L. Moss and M. G. Mellon, I n d . Eng. Chem. Anal., 1943, 15, 74.8 8 H. Funk, 2. anal. Chem., 1942,123, 241 ; Chem. Zentr., 1942, I, 2805.7 ° C. C. Miller, Analyst, 1944, 69, 109.122.F.W. Haywood and A. A. R. Wood, J . SOC. Chem. Ind., 1943, 62, 37.P. Wenger, Helv. Chim. Acta, 1942, 25, 1499262 AXALYTIUAL CYHEMISTRY.hydroxide.g0 In a buffered acetic acid solution zinc can be separated frommixtures containing magnesium and aluminium, or from solution containhgeither of these metals, with 8-hydro~yquinaldine.~~ Small quantities ofzinc are identified with d i t h i ~ o n e . ~ ~ As in the determination of copper,8-hydroxyquinoline can be replaced by Quinosol and Superol. l 5 For cadmiumalone, F. Feigl and L. I. Mirandag3 have employed the red compoundproduced when a cadmium salt is mixed with the complex formed by aa'-dipyridyl and ferrous sulphate. This red compound is suitable for gravi-metric work and can be weighed as such; alternatively, the cadmium iseventually determined with mercaptobenzothiazole.Gallium, Germanium, Hafnium, Indium, Niobium, Osmium, Selenium,Tantalum, Titanium, Thorium, Uranium, and Zirconium.-Substitutedarsonic compounds, already mentioned for bismuth, frequently appear asreagents for some of the above elements.Thus, in a comprehensive reviewof the possible reagents for thorium, P. Wenger and R. Duckett 94 recommendphenylarsonic acid and record that quadrivalent, cerium and zirconiumbehave similarly. The p-hydroxy-derivative of this reagent has been criticallyreviewed as a reagent for zirconium.g5 The precipitates are difficult to collectin certain conditions of acidity ; nevertheless, under the correct conditions,a large number of metals are separated by one precipitation, thoriumrequiring two.The determination is possible in the presence of titaniumonly if the amount of that element is small, and tungsten and tin both tendto co-precipitate. 5-Chlorobromamine acid has also been proposed foruse with z i r c ~ n i u m . ~ ~ If a mixture of o-aminophenylarsonic acid andsalicylaldehyde is added to an acetic acid solution of a selenium salt an intenseyellow colour is produced sensitive to one part of selenium in 2,000,000.The colour is formed by all elements precipitated by arsonic acid, e.g.,titanium and thorium, and only o-aminoarsonic acids and o-hydroxyaldehydescan produce the rea~tion.~' The same author 98 has examined the colourreactions of thorium, uranium, and other metals with four complex reagentscontaining arsenic. The cupferron test is suitable for the micro-determin-ation of ~ranium.~9After the separation by distillation and chloroform extraction of thechlorides of osmium and germanium, two organic reagents are available forthe determination of the elements.Osmium has been shown to react readilywith thiourea to form red complexes suitable for colorimetric measurements,as little as 2-5 p.p.m. having been found in a meteorite,l and quinineD. Gunev, Khim. Indust., 1942, 20, 170; Chem. Zentr., 1942, 11, 1157.91 L. L. Morritt and J. K. Walker, Ind. Eng. Chem. Anal., 1944,16, 387.s2 R. Vanossi, Anal. SOC. Cient. Argentina, 1942, 134, 73.93 Anal. Assoc. Quim. Brazil, 1943, 2, 131.s 5 A. Claasen, Rec. Trav.chim., 1942, 61, 299; Chem. Zentr., 1942, I , 3124.s 6 J. H. Yoe and L. G. Overholser, Ind. Eng. Chem. Anal., 1943,15, 73.S 7 V. I. Kuznetsov, J . Gen. Chem. U.S.S.R., 1944, 14, 897.98 Compt. rend. Acud. Sci. U.R.S.S., 1941, 31, 898 (in English).99 P. M. Isakov, J. Appl. Chem. U.S.S.R., 1943,16, 326.E. E. Sandell, Ind. Eng. Chem. Anal., 1944, 16, 342.O4 Helv. Chim. Acta, 1942, 25, 1110QASKM : ORQANIU REAGENTS IN INORGANIC ANALYSIS. 263tannate will precipitate germanium present aa tetrachloride in chloroformor carbon tetra~hloride.~ It has also been found that germanium is com-pletely precipitated with 5 : 6-benzoquinoline as a complex oxalate whichcan be ignited to the oxide and ~ e i g h e d . ~F. Feigl and P. E. Barbosa 5 describe an unusual reagent prepared bymixing an aqueous solution of ethylenediamine with a large excess of silverchromate.The ionic equilibrium in a saturated solution of the resultingcomplex can be disturbed by hydrogen ions, by metal ions forming complexeswith ethylenediamine, and by acid and acid salts, with only trace solubility,which are strong absorbents for the diamine. This last reaction is shownby the appropriate compounds of molybdenum, t'ungsten, tantalum, andniobium, by amorphous silica and zeolites, but not by crystalline silica.Niobium in weak acid solution, and tantalum in sulphuric acid, producecolours with pyrogallol suitable for the determination of the elements.6Gallium has been separated from aluminium with 5 : 7-dibromo-8-hydroxy-quinoline, since aluminium does not form a complex with the reagent.'The precipitate can be ignited with oxalic acid and weighed as the oxide.T. Moeller * has described the use of 8-hydroxyquinoline for indium andmentions the interfering elements, whilst 1 -phenyl-3-methylpyrazoline hasshown promise with hafni~m.~An existing method for the determination of titanium with oxine hasbeen re-examined, and reasons given for errors in the results.lO The separ-ation by this reagent of the metal from aluminium is possible at pH 5-6-6.5in oxalic acid solution, but not in malonic acid, The detection of titaniumwith sodium 1 : 8-dihydroxynaphthalene-3 : 6-disulphonate under prescribedconditions is regarded as more sensitive than the hydrogen peroxide orthymol test.llCalcium, Magnesium, Mercury, Phosphorus, Potassium, Titanium, andVanadium.-Recent work on organic reagents for the above elements hasnot been extensive, but some of the methods described are of interest.Thepolarographic method combined with an organic reagent has proved valuablein the determination of both calcium and magnesium. Small amounts ofcalcium are precipitated with a known amount of picrolonic acid, and theexcess acid is measured polarographically in the filtrate from the calcium. l2High results in the amperometric titration were found to be due to absorp-R. Vanossi, Anal. Asoc. Quim. Argentina, 1944, 32, 164.Idem, Anal. Asoc. Cient. Argentina, 1945, 139, 29.H. H. Willard and C. W. Zuehlke, Ind. Eng. Chem. Anal., 1944,16, 322.Minist.Agr. Dept. Nac. Producao Min. Lab. Producao Min. (Brazil), 1942, Bol. No.M. S. Platonov and N. F. Krivoshlykov, Trudy Vsesoyuz Konf. Anal.Khirn., 1943,E. Gastingsr, 2. anal. Chem., 1944, 126, 373.E. L. Wallace and A. R. Armstrong, Virginia J. Xci., 1943, 3, 292.5, 72.2, 359.a Ind. Eng. Chern. Anal., 1943, 15, 270.lo A. Claasen and J. Visser, Rec. Tvav. chim., 1941, 80, 715.l1 R. Vanossi, Anal. Asoc. Quim. Argentina, 1944, 32, 5 .l2 G. Cohn and I. M. Kolthoff, J. BWZ. Chem., 1943,147,705264 ANALYTICAL CHEMISTRY.tion of the picrolonic acid by the filter paper, for which sintered-glass crucibleswere successfully substituted. l3 It is possible to measure the concentrationof oxine with the polarograph, and such measurements made before and afterthe addition of a magnesium salt give good figures for magnesium in suchmaterials as tap water and plant ash.14 Magnesium in water has also beendetermined by making use of the magnesium lake formed with Titan-yellow,the amount of which lake, suitably dispersed, can be measured spectro-photometrically.Vanadium has been detected in steel by the yellow colour formed withthe reagent obtained by adding phosphoric acid to an alcoholic solution ofbenzidine until the precipitate redissolves,16 and in rocks by treating achloroform solution containing the vanadium with oxine followed by sodiumazide.17 The oxine produces a reddish colour which changes to green withthe azide. The precipitation of vanadium by cupferron in various con-centrations of sulphuric acid is possible, but the method does not separatetitanium and aluminium, and in the presence of aluminium, both vanadiumand aluminium are precipitated by the addition of a neutral electrolyte.l*A. Steigmann has devised a specific test for mercuric chloride usingspeciaIIy prepared membranes treated with dithizone,lg and quantitativeprecipitation is obtained with the metal thiocyanate and a-naphthylamine.26A variation of the normal cobaltinitrite method for potassium involvesthe determination of the nitrogen in the cobaltinitrite precipitate withphenoldisulphonic acid,Z0 and a reagent suitable for both detection anddetermination of the metal is 4 : 6-dinitroben~ofuroxan.~~J. G. N. G.J. G. N. GASKIN.E. G. KELLETT.1s G. Cohn and I. M. Kolthoff, J. BioZ. Chem., 1943, 148, 711.12 K. G. Stone and N. H. Furman, Ind. Eng. Chem. Anal., 1944,16,596.15 E. E. Ludwig and C. R. Johnson, ibid., 1942,14, E. Trepka-Bloch, 2. anal. Chem., 1943,125, 276.1' R. Vanossi, Anal. Asoc. Cient. Argentina, 1943, 135, 97.18 M. G. Raeder andT. Aakre, Kgl. Norske Videnskab. Selskab. Porh., 1942, 16,19 J . SOC. Chena. Ind., 1943,82,43.20 E. M. Emmert, Proc. Amer. SOC. Hort. Sci., 1944, 45, 311.21 H. Rathburg and A. Scheurer, Die Chemie, 1943, 56, 123; Chem. Zentr., 1943, II,75 (in English) ; Chem. Zentr., 1943,II, 2184.152


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