ISSN:0003-2654    

DOI:10.1039/AN95883FX029

出版商:RSC    

年代:1958

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95883BX031

出版商:RSC    

年代:1958

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95883FP131

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

THE ANALTST xiiiCLASSIFIED ADVERTISEMENTSThe rate fur classijied aduertisements is 5s. a h e (07 Spacequiualent of a ltne), wzth am extra charge of Is. for theuse uf a Box iVumber. Semi-drsplayed classifiedaduertzsements are 60s. per single-column w c h .ANALYTICAL CHEMIST B 1.1'. Ciirinicals Ltd., Oldbnry, have a vacancy for an*Analgtical Rescarch Chemist to take charge of a labora-tory engagcd UII thc invrstigation of analytical problemsassociated with the Plastics Industry with particular referenceto themosrtting resins. Thc work will involve thc iisc ofrniidrrn trchniques, including spectroscopy and gas-c.hrorrrato-graphy together with tht, investigation of new methods ufanalysis. .\.R.I.C. or rquivalent qualification is essential andprrvmus industrial experiencc is dc~sirahle.l'rcfrrrrd age3 - 3 5 yiws. Thi. company offers good conditions of em-plriynent and a generous Pension and Isif(, Insurance schimr..Applications, giving full details, should be made in writingt o the Pcrsonncl Manager, B.I.P. Chrrnicals Lid., Oldbury,Birmin gh ail I.CHELSEA COI.l.EGE 01' SCIENCE AND TECHNOLOGYI)EPAKlMb;ST OF CHEMISTRYANALYTICAL METHODSserif's of post-graduate lectures on 111oi-c rc'ccnt analyticalAtecliniques will bc hrld during thr Antumn 'I'enri unit 7 p.m., cornrnencmg on 7th October, 1938. Theinclude spectroscopic mcthods, so!vmt c,xtraction,squarr wavc polarography, radio-activation, isotop!c dilution,inorganic paper chromatography, silicate analysis, nuclfwiuagnctic rcwnancr arid organic sub-micro analysis.4ltatlrt giving f u l l drtails and an application forni may beohtainccl from the Hcad of thc Chrr11ist1-y D(.partnient, Chi+sea G ~ l l r g ~ of Science and Technology, Manresa KoadLondon, S.\\'.:!.. ~ ~ ~~~SOI,.TH TEES-SIDE HOSI'ITAL MANAGEXESTCOMMITTEE1STAKT HOSPITAL BIOCHEMIST (Basic Grade)PLICATIONS are invited for thc aliovt, post at theCentral Clinical Laboratory Grnrral Hospital, Mlddlrs-brougli. Salary and Conditions of Sprvice will be in accord-aiicc with S.H.S. regulations end applicants should hawas qualllicatioms, an appropnatr science drgrer or A.R.I.C.Applications shoiild be niadr to the Srnirrr Pathologist a tthe abow addres6.HE C.W.S. I'rcsrrvc Group requires a CHEMIST (ageT21, :!(I) ior its Group Central Labordtory a t Middleton,near Manchrster.Higher National Certifiratv. Pi-<.vioiisexpericncc in the Food Induslrv, while, not essential, willhe an advantagr. Salary according to age, academic statusand rxprrience. Contributory Pvnsinn Fiind, cantern,sports club, etc. Forty-on<. hnur, five-day week. Applica-with fiill details of age. education and prrvious es-cc to the C.\\-.S. Limited, Group Manager's Office,ves XVorks, Middlvton, Ncar Manchrster.~~~ ~~ ~~CHELSEA COLLEGE OF SCIENCEDl~PARlllEKT OF CHMnnr?sa Koad, S.\.I course of Lectures and Practical IVork onTHI: CIIEXlISTRY AND MICKOSCOI'Y OFn.ill Ile givrrl Sr.ptcniber, l:I.i?, to Jiily, 1959I,rcturc~iri Chargr: R. G. Minor, F.P.S., F.R.I.C.man, DRUGS ,?AD LVATERThc cours(I is hascd on the Syllabus of thrfor thr 1)iploma of thr Koyal Institrite of Chincludes Analysis and Microscopy of Food, DrugToxicological Analysis.Acts and Rcgulatioris rclatiiig t~Foocl, Unigs and Poisons.. Minor will attcnd on Wdnesday, 17th Scptcml)rr,rticnlars can bc ohtaind on application to the(i i p.ni.. to aclviir students.__ ~ AS i\SSIST..\N?' ANALYST is required for a Br<vt,ryThe position is suitahle for ayoung man, recently qualified, and with soin? food, bcvoragc,Laboi-atory in 1.ondon.CO-OPERATIVE WHOI.ES.\LE SOCIETY LIMITEDH!5 Directors invite applications for the position ofhr main Laboratory being locatid in Manchestrr.Thy appointment involvrs the suprrvision of the wholeIf thv scirntific services of thc organisation and pmhraccsL wide and interesting fickl of rrscarch of consumer goods~ n d Lcvelopriirnt of IirocPssing and niariiifacturr,.In addi-ion to tcxtiks, food inanufacture, crrral and bacteriological.ontn~l arc outstaudingl~~ important. The succrssful candi-late .would tw required to visit thi, Society's various estab-ishriiqnts tlirnughout thc country, assist iii the adniinistration)t,thi. laboratory and prngraninic future rpsearcti policy.The post offers excellent opportunities in technical rcscarchind progress can be aiiticipatvd bccause the prcscnt HPadIf the- Drpartinmt is due to retire within two yrars.('arididatcs must have prrviously held responsible positionsn tf clinical rcsearch and should posscss qualificatimsncluding a g o d Honorirs Drgrei, an11 pr<.feral,ly F.K.I.C.Brarlcll E).Candidates should write in corifidrricc to the Secretary anciIsecritivr Oftict.r, Co-opcrative M'holesale Socicty J,iniitrd,I Balloon Strrrt, Manchrster, 4, mdorsnig envelopes-lcputy Hcnd, Trchriical Research Departriirnt, asking forI fonii of application.rD1: 4 1 3 CTY .HEAD of the Technical Research Dcpartnient,CITY OF BIRMINGHAMPUBLIC HEALTH DHPARTMENTCITY ANALYSTP'1.ICATIONS arc invitid for thc post of City Analystmninicnce In January, 1959. C.andii1atc.s must hulrl tht.scale will depend upon the candidate's rxprnrncr. l'insion~chrnic (including widows arid orphans) : inadical examination.Th- offirer apponited will he requirrd tn dcvotc his a h o l i .time to official duties ,ind the appnuitment will Irr subject i i ,thrcc iiionths' notice vii eithrr sidr.The appointnirnt willhP suhiwt tir tht, ap~iroval o f the, Ministvr of Agricultnn ,Fisheries and Food.Applications, togtthcr with thr nairirs uf thrrc prrsuns towiioni rrfrrenct, inay h e iuadr, shonld stair ag?, clnaliticatmnsand -xpi'm'ncc and sent to thv Zlrdiral Officer of Hcalth,Council Home, Hnniirigharrr. :i, not 1atc.r than loth Scptriiibcr,1958.HEAD OF AN.lI.YTIC.41. KESEXKCH 1.ARORATORYU A I YTICAL CHEMIST rrqulred to take charge ofAmoc<rn Research Laboratory working on organic andinorganic nmti.rials uscd iii uian-riladc fibres industry..Applicant Inust h a w had rxperience in running am1 adrnin-istration of a laboratory and in investigating and devisingnew tt:chriiqriPs.Salary accordha to qualitratioris andff conditions of rniployiiient includ? goodand fivc-day wi,k. Applications shouldications and rxprrience and be addrrs,rd toPrrsonnrl Manager, British Enka Lid.. Aintrw, Liverpool 9.STANDARDS DIVISION ~ ANALYTICAL CHEMISTH E Research Organisation for Sniith 61 Nephew .4ssociatedvacancy in their Standards Divi5ionAnalytical Chemist. This Group ofrr cosmptir and toilet products, aridI preparations and the work will bcmnrvrnrd with t h r r%miinatiort nf voinpvtitivt' prodwts,the preparation of specifications for new products and thcrevision of rxisting oneb, and grnrral rxprrimnilal andanalytical work mithiii tlic fields indicated, both for theKesrarrh Company itself and the ;\.;sociated Companirs.Tkii vacancv is meeting the demands of an eaoandine . I, R c s e ~ ~ r ~ h Organisation and will probide a progressive and+atis,'actory carper for those keeilly interested in this fieldnf chcrrnstry. hlinn,um qualifications art. a St~cond ClassHonours degrer in Chermstry or Graduate Membership ofthe lioyal lnslitutv of Chrniistry. 'The Company's iuainintrrest will he in persons nuder 30 years of age and applicantsshould be i n good health. Writi, giving fnll dctails of agr,. ~~~ ~ ~~~~~~LVO I OVI ROKD Rlodel 13 "Tintorurtm" (for soaps,T>egrtakle oils, fats, waxrs) at LOO each (approxiriiatclytwo-thirds currrnt pnres). Xew and nnused, in originalpacking c a b 6 Cox, 4 i High Street, Edgware, Middlpsrx.CDG 4685 and 3PX8.T W O "OEII1'LIN(;" Model 48 Analytical Balances w'ithKinmiatically arranged arrestnient bearings a t L40 each!appi-nxin,ately two-thirds <mrrmt prircs). KiJw and unused,in original packing cases. Cox, 1 7 High Street, Edgwarv,hliddlrsrx. EDG. 4 i X G and DXSP.iappi-nxinbately in DXS

ISSN:0003-2654    

DOI:10.1039/AN95883BP141

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

AUGUST, 1958 Vol. 83, No. 989 THE ANALYST PROCEEDINGS OF THE SOCIETY FOR ANALYTICAL CHEMISTRY DEATH James Rawson Walmsley. NORTH OF ENGLAND SECTION THE twenty-first Summer Meeting of the Section was held at the Old Swan Hotel, Harrogate, from Friday, June Gth, to Monday, June 9th, 1958. The Chairman of the Section, Mr. A. N. Leather, B.Sc., F.R.I.C., presided over an Ordinary Meeting at 10.15 a.m. on Saturday, June 7th, at which Mr. R. J. Gardner, Publicity Officer of Imperial Chemical Industries Ltd. (Fibres Division) spoke on “Terylene,” illustrating the formation of synthetic fibres and the discovery and uses of Terylene by means of a colour film and model display. On the Saturday evening the party saw “The House by the Lake,” at the Grand Theatre, Leeds, and made a coach tour to Wharfedale, Burnsall and Bolton Abbey on the Sunday afternoon. WE record with regret the ‘eath of hIICROCHEMISTRY GROUP THE fifteenth London Discussion Meeting of the Group was held at 6.30 p.m.on Wednesday, June lSth, 1958, in the restaurant room of “The Feathers,” Tudor Street, London, E.C.4. The Chair was taken by Dr. G. F. Hodsman, BSc., A.1nst.P. A discussion on “The Microdetermination of Elements other than C,H,O,N,S and Cl,Br,I in Organic Compounds” was opened by Alison M, G. Macdonald, B.Sc., M.Sc., Ph.D., A.R.I.C., and R. Belcher, DSc., Ph.D., F.R.I.C., F.1nst.F. BIOLOGICAL METHODS GROUP THE Summer Meeting of the Group was held on Thursday, May 22nd, 1958, when members visited the new Pharmaceutical Research Laboratories of Imperial Chemical Industries Ltd., at Alderley Park, Cheshire. A description of the Research Department by the Division’s External Relations Officer preceded a tour of the laboratories during which an opportunity was given to see and discuss the work of the various sections visited. At the close of the meeting, thanks on behalf of the Group for a most instructive and enjoyable visit were expressed by the Vice-chairman, Dr. J. I. RI. Jones, D.Sc., F.R.I.C. 44 1

ISSN:0003-2654    

DOI:10.1039/AN9588300441

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

442 BENTLEY AND BURGAN : POLYNUCLEAR HYDROCARBOBS IN [Vol. 83 Polynuclear Hydrocarbons in Tobacco and Tobacco Smoke Part I. 3 : 4 - Benzopyrene BY H. R. BENTLEY AND J. G. BURGAN (The Imperial Tobacco Comeany Ltd., Research Department, Raleigh Road, Bristol, 3) A method is described for determining the concentration of 3 : 4-benzo- pyrene in tobacco and in tobacco-smoke condensate by using the fluorescence spectrum of the hydrocarbon. IT has been suspected for many years, largely by analogy with coal tar, that the water- insoluble “tar” forming the condensable fraction of tobacco smoke would be found to contain polynuclear aromatic hydrocarbons. An essential difference, however, between normal tobacco smoke and coal tar is that, whereas the latter is the by-product of a carbonisation process occurring in the absence of air, tobacco smoke contains the products of combustion of leaf constituents and of distillation of volatile substances in the presence of air.Considerable amounts of leaf constituents are also carried over into the smoke by a process of steam-distillation and entrainment, and, since these substances are removed rapidly from the burning zone, they are to a large extent chemically unaltered. Polynuclear aromatic hydrocarbons are thought to be formed during pyrolysis of many substances in a restricted supply of air between about 750” and 1600” C by a process involving first a breakdown into methylene radicles and hydr0gen.l The methylene radicles dimerise t o ethylene, which breaks down further to hydrogen and “nascent acetylene.” This partly decomposes to carbon, hydrogen and methane and partly polymerises to polynuclear aromatic hydrocarbons with further loss of hydrogen. A mechanism of this sort may explain the formation of carcinogenic tars during the carbonisation in the absence of air of the organic materials that were tested by K e n n a ~ a y ~ ? ~ and Kennaway and Samp~on.~ These carbonisa- tion conditions are, however, not representative of those in the burning zone of a cigarette, and normal tobacco smoke is, a priori, unlikely to contain appreciable amounts of polynuclear aromatic hydrocarbons.This may not be so in tars produced by heating tobacco in closed ~ t i l l s , ~ ~ 6 ~ 7 ~ 8 ~ ~ which may be expected to resemble carbonisation tars more closely, and this essential difference was noted by some of the earlier workerslOJ1 and also in more recent times.12 Because of its important biological effects, and also because of the comparative ease with which it can be recognised in mixtures, attention has been directed first to 3:4-benzo- pyrene.However, many workers who analysed tobacco tar by methods applicable to coal tar failed to detect the h y d r o c a r b o n . l l ~ ~ ~ J ~ ~ ~ ~ An early report by Roffos that tobacco tar contained 3 : 4-benzopyrene arose by a confusion of nomenclature. The hydrocarbon for which spectroscopic evidence was obtained by Roffo was, in fact, 1 : 2-benzopyrene, which he believed t o be the highly carcinogenic isomer. It is now evident that one of the reasons for the earlier failures to detect 3 : 4-benzopyrene in tobacco tar was that the order of magnitude of the concentration present was not appre- ciated.It has recently become clear that the smoke from cigarettes smoked under conditions simulating the human habit does in fact contain small amounts of 3 : 4-benzopyrene. There is, however, little agreement among workers in this field as to the amounts present. For example, Cooper and Lindseyx6 give a figure of about 1 pg per 100 cigarettes smoked, equivalent to 0.2 p.p.m. by weight of condensable material; Wynderl’ gives the concentration as 2 p.p.m. by weight of condensable matter; Alvord and Cardonls found a range of 8 to 18 pg per 100 cigarettes smoked, equivalent to 1.6 to 3.6 p.p.m. by weight of condensable matter; Latarjet, Cuzin, Hubert-Habart, Muel and Royerl9 found 1.2 pg per 100 cigarettes smoked, equivalent to 0.2 p.p.m.by weight of condensable matter: and Bonnet and Neukomm20 found 2-2 pg per 100 cigarettes smoked, equivalent to about 0.4 p.p.m. by weight of condensable matter. Differences in smoking technique are probably responsible for some of these variations. In the work now reported, cigarettes have been smoked under conditions that are thought to resemble most closely those of the human habit.2IAugust, 19581 TOBACCO AND TOBACCO SMOKE. PART I 443 In our experience, which we believe to be general, it has not yet been possible to isolate pure polynuclear hydrocarbons from cigarette-smoke condensate. Even after extensive chromatographic separation, the 3 : 4-benzopyrene-containing fractions contain a large excess of extraneous material, which contributes an intense background absorption in the 300 to 400-mp region and an intense fluorescence a t a somewhat longer wavelength.The typical appearance of the absorption spectrum of a purified 3 : 4-benzopyrene-containing fraction is shown in Fig. 1. In this, the absorption peaks a t 365 and 385 mp, which are characteristic of the hydrocarbon, can be seen as small inflexions against a generalised background absorp- tion. A method for determining substances by the heights of absorption peaks under these conditions is that of Morton and Stubbs,22 which has been applied to the determination of vitamin A in fish oils and of anthracene in petroleum. The small and indeterminate peaks obtained from purified cigarette-smoke fractions, however, makeit impossible to use this method for 3 : 4-benzopyrene.For example, a requirement of the method of Morton and Stubbs is that linear irrelevant absorption must be assumed to be present over the region of the peak used in the determination. This assumption cannot be made for certain fractions of tobacco tar. As will be shown later, the analytical method finally adopted depends on the use of fractions that show the characteristic banded fluorescence spectrum of 3 : 4-benzopyrene, and these fractions invariably also show small inflexions in the absorption spectrum charac- teristic of 3 : 4-benzopyrene. On the other hand, fractions closely adjacent to these on the chromatogram have been found to show apparently typical 3 : 4-benzopyrene inflexions without displaying the characteristic fluorescence bands of the hydrocarbon.The possibility that the absence of typical fluorescence in these fractions might be due to quenching was excluded by the addition of small amounts of pure 3 : 4-benzopyrene; the characteristic bands then appeared with the correct intensity. It cannot, therefore, be assumed that the back- ground absorption is linear for the purpose of measuring peak heights. I I 350 400 Wavelength, mp Fig. 1. Absorption spectrum of purified 3: 4- The values reported hitherto in the literature for the 3 : 4-benzopyrene content of tobacco smoke have all been found by the absorption method. Because they show considerable discrepancies, it was thought that it would be useful to develop, for comparison, an analytical method based on an entirely different principle.The proposed method, based on fluorescence, has therefore been devised. The application of the method to some problems of current interest is shown by the results in Table I. For mixed cigarettes representative of those that have a large sale in the United Kingdom, a large number of replicate determinations permits the reproducibility of the method to be assessed. For the twenty-five results listed in the first section of Table I, the mean maximum 3 : 4-benzopyrene content per 500 g of cigarettes is 4.9 pg, with a range 1.5 to 8.0 pg. This concentration is equivalent to about 0.2 p.p.m. by weight of condensable matter and agrees with the results of Cooper and Lindsey,16 Waller15 and Latarjet, Cuzin, Hubert-Habart, Muel and Royer.19 The concentrations found by other workers, which are considerably higher than this, are therefore not typical of the cigarettes on sale in the United Kingdom smoked under our conditions.benzopyrene-containing fraction444 BEiVTLEY AND BURGAN : POLYNCCLEAR HYDROCARBONS IN [Vol. 83 The results listed in Table I also show that there is no difference in the 3 : 4-benzopyrene content of the smoke from cigarettes containing only American or Rhodesian tobacco, although the chemical composition of these two types of leaf differs appreciably. Also, there is no more 3 : 4-benzopyrene in the smoke from cigarettes made entirely of cut tobacco stems, which have a very high cellulose and lignin content and are practically free from alkaloids.TABLE I DETERMINATION OF 3 : 4-BENZOPYRENE IN TOBACCO SMOKE, LEAF AND STEM Source Naterial analysed Mixed cigarettes typical of current United Tobacco . . production American cigarettes . . . . .. Rhodesian cigarettes . . . . . . . . Cigarettes made from tobacco stems Smoke . . Tobacco . . Smoke .. Stem . . * . Maximum 3 : 4-benzopyrene content per 500 g of 6.0 3.0 8.0 1.5 5.0 2.0 4.0 1.5 3.0 6.0 8.0 7.0 5.5 3.0 4.0 2.7 7.0 3.0 5.0 3.0 6.0 7.0 7.0 8.0 7.0 2.0 2.75 5.0 5.0 2.5 3.0 2.0 source, pg 3.0 3.5 6.0 2.5 2.0 3.0 2.5 5.0 4.0 4.0 5.0 Not 0.2 found The figures for unburnt tobacco and tobacco stem show that after normal curing and manufacture these materials, as might be expected, have become contaminated with 3 : 4- benzopyrene by contact with atmospheric dust and soot.The low figure for stem as compared with lamina is presumably due to the much lower surface area - weight ratio of the former. XETHOD REAGESTS- Light petroleum-Light petroleum, boiling range 40" to 60" C, free from aromatic hydro- carbons, is percolated through chromatographic alumina, distilled and stored over sodium wire. Benzene-Benzene (crystallisable) is washed three times with concentrated analytical- reagent grade sulphuric acid, once with water, twice with 2 N sodium hydroxide solution and then again with water until the washings are neutral. I t is then dried over anhydrous sodium sulphate, distilled repeatedly until the residue (50 ml from 2 litres) does not fluoresce and stored over sodium wire. Diefhyl ether-Anaesthetic ether B.P.is dried over sodium wire. Acetone-Laboratory-reagent grade acetone is used without further purification, Alumina-Woelm alumina, neutral grade, activity 1, is used. 3 : 4-Benzopyrene solzttion-A solution of the commercial material in light petroleum is percolated through alumina (Woelm neutral grade), and the principal fluorescent band is eluted with 40 per cent. v/v of benzene in light petroleum. The hydrocarbon is recovered from the eluate by evaporation and then recrystallised from light petroleum and dried in vucuo. The product has log(e382mp) = 4.46 (literature 4.47). PROCEDURE FOR RECORDING FLUORESCENCE SPECTRA- Fluorescence spectra are obtained with a Hilger medium-quartz spectrograph and photographed. In the examination of chromatographic fractions for the presence of 3 : 4- benzopyrene, Ilford HPS plates (5 inches x 4 inches) are used with an exposure time of 1 to 4 minutes.In the determination of 3:4-benzopyrene, Ilford HP3 plates (5 inches x 4 inches) are used with an exposure time of 2 to 12 minutes. The optical system used is show~n in Fig. 2.August, 19581 TOBACCO AND TOBACCO SMOKE. PART I 445 The source of exciting radiation, A, is a “black-glass” 125-watt mercury-arc lamp used in conjunction with a polished Wood’s glass filter, C. This transmits a group of lines in the 365-mp region, which excites the visible fluorescence of many polycyclic hydrocarbons. By means of a large glass condensing lens, B, and a concave mirror, F, arranged as shown, the exciting radiation is focused on the sample cell, E. This is a I-cm quartz cell, with lid, turned slightly off axis so as to deflect as much stray mercury light as possible from the spectrograph slit.To reduce the amount of stray mercury light still further, a matt-black shield, D, is placed in front of the cell, ” G Silt B C F A = 125-watt “black-glass” mercury-arc lamp (G.E.C. type MBW/U) B = Condensing lens C = Wood’s glass filter (Chance 0x1, 2 mm thick) D = Matt-black shield E = Quartz sample cell F = Concave mirror G = Lens of short focal length Fig. 2. Optical system used for recording fluorescence spectra The fluorescent light emitted from the cell passes through an aperture in the centre of the concave mirror and is focused on the spectrograph slit by means of a lens of short focal length, G.The whole of the optical system is conveniently mounted on the optical bench of a spectrograph. PROCEDURE FOR DETERMINING 3 : 4-BENZOPYREIiE IN CIGARETTE SMOKE- The cigarettes used in this work are conditioned a t 60 per cent. relative humidity and 70” F before smoking, and are smoked under the standard conditions described elsewhere.= The smoke is collected by electrostatic precipitation, use being made of the automatic smoking machine described elsewhere.21 The precipitated smoke solids (about 25 g) from 500 g of cigarettes are extracted from the autosmoker glass tubes with a mixture of equal volumes of diethyl ether and 2 N hydrochloric acid. The combined ether extracts are separated and washed, successively, with four 100-ml portions of 2 N hydrochloric acid, 100 ml of water, four 100-ml portions of 2 N sodium hydroxide and three 100-ml portions of water.The solution of the neutral fraction of smoke condensate in ether is then dried over anhydrous sodium sulphate, filtered, and evaporated on a steam-bath. The residue is recovered by evaporation three times from successive small volumes of light petroleum; it is then dissolved in 50 ml of light petroleum and transferred to a column of 115 g of alumina in a glass tube of 28 mm diameter, protected from direct sunlight. The chromatogram is developed with 500 ml of light petroleum and then, successively, with 250-ml portions of 10, 20 and 30 per cent. v/v benzene - light petroleum mixtures and finally with sufficient 100-ml portions of 40 per cent. v/v benzene - light petroleum mixture to elute all the 3:4-benzopyrene from the column.Each fraction is evaporated to dryness, care being taken to remove all traces of benzene, and re-dissolved in 5 ml of light petroleum. The fluorescence spectrum of each fraction is recorded, and those fractions containing 3 : 4-benzopyrene are combined. The combined fractions are concentrated to small volume and transferred to a column of 10 g of alumina in a glass tube of 16 mm diameter, protected from direct sunlight. The chromatogram is developed with 50 ml of light petroleum, successive 25-ml portions of 10, 20 and 30 per cent. v/v benzene-light petroleum mixtures and finally sufficient 25-ml portions of 40 per cent. v/v benzene - light petroleum mixture to elute all the 3 : 4-benzopyrene.The fractions are evaporated to dryness, care again being taken to remove all benzene, re- dissolved in 5 ml of light petroleum and examined for the presence of 3 : 4-benzopyrene as before. Fractions containing 3 : 4-benzopyrene are combined and evaporated on a steam-bath.446 BENTLEY AND BURGAN : POLYNUCLEAR HYDROCARBONS IN [Vol. 83 This volume depends on the amount of 3 : 4-benzopyrene present, but the concentration of 3 : 4-benzopyrene in it must be such that the following requirements are met- (i) The bands in the fluorescence spectrum a t 403, 408 and 427 m p are visible. (ii) The fluorescence due to the maximum amounts of 3:4-benzopyrene to be subse- quently added to aliquots for the purpose of determination must not be appreciably quenched in the solution.When necessary, this requirement is checked by means of a microphotometer, use being made of the strong 403-mp band. The concentration of 3 : 4-benzopyrene in this solution is determined by the comparison on a single photographic plate of the fluorescence spectra of the unknown solution, of standard solutions of 3:4-benzopyrene in light petroleuin and of aliquots of the unknown solution containing the same added concentrations of 3 : 4-benzopyrene as the standard solutions. For a photographic plate in the normal exposure ranges, the plate density is directly proportional to log(1ight intensity). With the low light intensities involved in this work, however, the plates are very much underexpcsed and the increase in plate density with increasing light intensity, and hence with increasing concentration of 3 : 4-benzopyrene, is found to be nearly linear for solutions of the pure hydrocarbon in light petroleum.Hence, by visual comparison of the spectra of the unknown solution and the solutions containing added 3 : 4-benzopyrene, it is possible to determine the level of added 3 : 4-benzopyrene a t which the concentration of the hydrocarbon in the unltnown solution has been doubled, and hence what this concentration is. With the proposed procedure, the final recovery of pure 3 : 4-benzopyrene added to solutions of smoke condensate in diethyl ether before the initial extraction with hydrochloric acid is 85 to 90 per cent., which provides a correction factor for determinations on normal smoke condensates. The residue is dissolved in a suitable known volume of light petroleum.PROCEDURE FOR DETERMINING 3 : 4-BENZOPYRENE I N LEAF AND CIGARETTE TOBACCO- The leaf is stemmed and cut before extraction. Manufactured cigarette tobacco is extracted without further preparation. Cut leaf or cigarette tobacco (200 g) is extracted with acetone for 4 hours in a Soxhlet extractor. The extract is evaporated under reduced pressure on a steam-bath, and the residue is hydrolysed by boiling under reflux for 2 hours with 100 ml of 10 per cent. w/v ethanolic potassium hydroxide. The resulting: solution is concentrated under reduced pressure on a steam-bath, diluted with water and repeatedly extracted with diethyl ether. The combined ether extracts are washed, successively, with three 50-ml portions of 2 N hydrochloric acid, 50 ml of water, three 501-ml portions of 2 N sodium hydroxide and three 50-ml portions of water; they are then dried with anhydrous sodium sulphate and evaporated under reduced pressure on a steam-bath. The residue is recovered by evaporation three times from successive small volumes of light petroleum, dissolved in 50 ml of light petroleum and transferred to a column of 115 6; of alumina, exactly as for the analysis of smoke condensate. With leaf extracts it is usually unnecessary to re-chromatograph the combined 3 : 4-benzopyrene-containing fractions.The 3 : 4-benzopyrene content of the com- bined fractions can then frequently be determined by direct comparison on a photographic plate of the fluorescence spectrum of the unknown solution with the spectra of standard solutions of the hydrocarbon; otherwise the determination is carried out by the method described for smoke condensate.For pure 3 : 4-benzopyrene added to tobacco before extrac- tion and hydrolysis of the extract, the recovery from the final chromatogram fractions is 75 per cent., which provides a correction factor for determinations on normal tobaccos. We thank the Directors of The Imperial Tobacco Company (of Great Britain and Ireland) Ltd. for permission to publish this paper. REFERE~CES 1. 2. 3. 4. 6, R~~~~ F 2 m r t ~ n n m i . A daaa,,*, r r-..... inn- 4 1 1 - 7 - Falk, H. L , and Steiaer, P. E., Cancer Res., 1952, 12, 30. Kennaway, E. L., Brzt. Med. J., 1925, 2 , 1. -, Bzochem. J . , 1930, 24, 497. Kennaway, E. L., and Sampson, B., J . Path. Btzct., 1928, 31, 609.447 August , 19581 TOBACCO AND TOBACCO SMOKE. PART I 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Sugiura, K., Amer. J . Cancer, 1940, 48, 41. Flory, C. M., Cancer Res., 1941, 1, 262. Campbell, J. M., Brit. J . Exp. Path., 1939, 20, 122. Schurch, O., and Winterstein, A., 2. Krebsforsch., 1935, 42, 76. Wynder, E. L., Graham, E. A., and Croninger, A. B., Cancer Res., 1953, 13, 855. Cooper, E. A., Lamb, F. W. M., Sanders, E., and Hirst, E. L., J . Hyg., 1932, 32, 293. Wynder, E. L., Graham, E. A., and Croninger, A. B., Cancer Res., 1953, 13, 855. Waller, R. E., Brit. J . Cancer, 1952, 6, 8. Cooper, R. L., and Lindsey, A. J., Ibid., 1955, 9, 304. Wynder, E. L., Brit. Med. J . , 1957, i, 1. Alvord, E. T., and Cardon, S. Z., Brit. J . Cancer, 1956, 10, 498. Latarjet, R., Cuzin, J., Hubert-Habert, M., Muel, B., and Royer, R., Bull. Cancer, 1956, 43, 180. Bonnet, J., and Neukomm, S., Helv. Chim. Acta, 1956, 39, 1724. Sharman, C. F., and Iles, W. G., J . Appl. Chem., 1957, 7, 384. Morton, R. A., and Stubbs, A. L., Analyst, 1946, 71, 348. Received September 24th, 1957

ISSN:0003-2654    

DOI:10.1039/AN9588300442

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

August, 19581 TOBACCO AND TOBACCO SMOKE. PART I 447 Determination of DDT and Chlorobenzilate Occurring Together in Spray Deposits BY E. A. BAKER AND E. JOHN SKERRETT (Research Station, Long Ashton, Bristol) Mixtures of DDT and chlorobenzilate from spray deposits on plant surfaces are separated on alumina columns, and the two constituents are determined by procedures based on the colours developed from their nitration products. CHLOROBENZILATE (ethyl 4 : 4’-dichlorobenzilate, I) is a non-phytocidal spray material developed for the control of mite infestations of plants. It has been used successfully for controlling the citrus-bud mite and may prove to be of value against other species, such as the big-bud mite of blackcurrants. For this latter purpose it would be convenient to use DDT [l : 1 : 1-trichloro-2 : 2-di(9-chlorophenyl)ethane, 111 in conjunction with the acaricide for the simultaneous control of the currant-leaf midge.In order to assess the distribution and the levels of the deposits, it is necessary to have available methods for the determination of DDT and chlorobenzilate when used together in such sprays. Several methods have been described for the determination of DDT1s2J and chlorobenz- ilate4J occurring singly in spray deposits on plants, but Harris4 has been the only worker to consider mixtures of the two. He separated chlorobenzilate from DDT by treatment with ethanolic potassium hydroxide solution. This reagent hydrolysed the former to the potassium salt of the acid I11 and dehydrohalogenated the DDT to DDX (IV).OH H OH KO2 NO, I I cc1, After dilution with water, the DDX was removed with light petroleum, and, after The acid was then nitrated to acidification, the acid I11 was extracted with diethyl ether.448 BAKER AND SKERRETT: DETERMINATION OF DDT AND [Vol. 83 a tetranitro compound (V), which was determined colorimetrically. The method did not measure the DDT present, but our preliminary experiments showed that the DDX removed in the light petroleum washes could have been used to determine the original level of DDT. However, chromatographic methods of separation were investigated in order to reduce both time and apparatus requirements. It was thought that such a separation was feasible, as chlorobenzilate, with its tertiary carbinol hydroxyl group, might be strongly adsorbed on a hydrophilic column through which DDT would pass.EXPERIMENTAL Initial experiments with silica and alumina columns indicated a quantitative separation of chlorobenzilate from DDT in carbon tetrachloride solution, the final traces of DDT being eluted from the column with small portions of carbon tetrachloride. Unfortunately, in later experiments with plant extracts, silica columns failed to retain materials that interfered with the colorimetric determination of DDT, and the use of this chromatographic medium had to be abandoned. Incomplete recoveries of chlorobenzilate from alumina columns in early stages of the work were attributed to the formation of the insoluble sodium salt of the acid, 111, with traces of alkali in the alumina. This trouble was overcome by carefully neutralising, washing, drying and conditioning the alumina before use.6 During the preliminary experiments, it was found to be desirable to modify slightly the established methods for the individual toxicants.In Harris's method4 for the deter- mination of chlorobenzilate, the nitration mixture is slowly warmed over a period of 30 minutes to 85" C and then heated in a steam-bath for 1 hour. It was found that the initial heating period could be omitted and the flask containing the reaction mixture placed directly into boiling water. However, under these conditions a minimum heating period of 1 hour was necessary. Saturated sodium sulphate solution was used to dilute the reaction mixture ; this gave sharper and much more rapid separations during the ether extractions. Difficulties were caused by the growth of Fusarium spp.in the sodium sulphate solution, which was prevented by bubbling sulphur dioxide through the solution for a short time. This development of mould was rather surprising, although a few analogous instances'$* have been reported. 2-Ethoxyethanol (ethyl Celllosolve) was preferred to benezene as solvent for the nitration product, which has been showns to be a tetranitrobenzophenone derivative (V). Methanolic potassium hydroxide solution was preferred for the colour development, as it gave less trouble from carbonate precipitation than did the ethanolic solution. The maximum colour developed after 30 minutes and was stable for a further 2 hours. Determination of DDT was based on the nitration method of Schechter, Soloway, Hayes and Haller.' When solutions of plant extract were evaporated, it was found to be desirable to add a little oxalic acid to prevent loss of insecticide. (The stearic acid used by Harris for a similar purpose in the determination of chlorobenzilate was found to cause interference in the final colour development.) The time of nitration was reduced to 10 minutes,l0 and the cooled acid mixture was diluted with saturated sodium sulphate solution.2-Ethoxyethanol was again used to dissolve the nitration product, the final blue colour being developed with 5 per cent. ethanolic potassium hydroxide solution and measured at 390 mp with a Unicam SP600 spectrophotometer. In both procedures it was found that traces of rubber, silicone and Apiezon stopcock lubricants interfered with the final dissolution of the nitro compounds.However, a stiff paste of bentonite and analytical-reagent grade glycerol proved to be satisfactory, and the addition of a glass bead appeared to be a further aid to the dissolution of residues. METHOD APPARATUS- All-glass apparatus is used throughout. Chromatographic columns are prepared in tubes, as shown in Fig. 1. The small glass projections are of use when the tubes are positioned in the Bolton extractors during the extraction stage.August, 19581 CHLOROBENZILATE OCCURRING TOGETHER I N SPRAY DEPOSITS 449 I I REAGENTS- Use a Widmer column with a proportionating head for all fractionations. Diethyl ether-Purify technical grade ether by fractionation over sodium.Methanol-Purify B.P. grade methanol by fractionation after the addition of sodium. Ethanol-Purify technical grade absolute ethanol by fractionation after the addition of sodium. 2-Ethoxyethanol-Purify technical grade ethyl Cellosolve by distill- ation after the addition of sodium. Carbon tetrachloride-Purify the analytical-reagent grade material by fractionation. Nitration mixture-Cautiously add an equal volume of analytical- reagent grade concentrated sulphuric acid to well stirred fuming nitric acid, also of analytical-reagent grade. Stearic acid solution-Twice recrystallise stearic acid from ethanol, and prepare a 0.5 per cent. solution in light petroleum (boiling range 40" to 60" C). Oxalic acid solution-Prepare a 0.5 per cent. solution of analytical- reagent grade oxalic acid in acetone.- i m m im - Fig. 1. Modified Sodzum sulphate solution, saturated-Saturate distilled water with chromatographic tube sodium sulphate, and pass sulphur dioxide through the solution for a few minutes. Methanolic potassium hydroxide solution, 5 per cent.-Freshly prepare this solution in the cold, and filter before use. Ethanolic totassium hydroxide solution, 5 t e r cent.-Freshly prepare this solution in the cold, and filter before use. Alumina-Stir 500 g of H-grade alumina with 300 ml of 0.2 per cent. v/v hydrochloric acid for 2 hours. Allow the mixture to settle, and then decant the supernatant liquid. Repeat this procedure with a further 300 ml of 0.2 per cent. hydrochloric acid. Wash the alumina twice with 300-ml portions of distilled water, stir it with 300 ml of distilled water and then with 300ml of 0.3 per cent.v/v acetic acid for 1 hour. Filter the alumina on a Buchner funnel, and dry at 120" C. Break up any lumps, spread in a thin layer on a silica tray and heat a t 600" C for 3 hours. Allow to cool from approximately 200" C in a closed container, and condition the alumina to Brockmann activity IIP by placing a beaker containing 15 ml of water in the container for 3 days. Stopcock lubricant-Add sufficient bentonite to analytical-reagent grade glycerol to form a viscous paste after being stirred. PROCEDURE FOR SEPARATING THE MIXTURE- Extract 1 g of the plant material for 15 minutes in a Soxhlet extractor with 25 ml of carbon tetrachloride. Lightly plug the bottom of a chromatographic tube with cotton-wool that has been extracted with hot ethanol, add 5 g of alumina and tap the column to ensure that the packing is uniform. Use another small plug of cotton-wool to prevent displacement of the top of the column.Pour the cooled plant extract on to the column, and wash with five 5-ml portions of carbon tetrachloride. Collect the eluate, and use it for the determination of DDT. Place the column in a Bolton extractor, and extract for 2 hours with 35 ml of hot ethanol. Use this extract for the determination of chlorobenzilate. PROCEDURE FOR DETERMINING DDT- Add 2 ml of oxalic acid solution and a glass bead to the eluate, and evaporate to dryness at approximately 55" C and a pressure of 20 cm uf mercury. To the cooled residue add 2 ml of nitration mixture, and carefully rotate the flask to wet any particles of solid.Im- merse the flask in a boiling-water bath for 10 minutes, occasionally swirling the contents. Cool the flask in an ice - water mixture, and add 75 ml of saturated sodium sulphate solution. Transfer the contents of the flask to a separating funnel with 70 ml of diethyl ether. Shake, discard the aqueous layer, and wash the ethereal layer successively with 25 and 1Oml of 5 per cent. potassium hydroxide solution and 15 ml of saturated sodium sulphate solution.450 BAKER AND SKERRETT [Vol. 83 Run the ethereal solution through a 15-mm layer of anhydrous sodium sulphate into a 100-ml round-bottomed flask containing a glass bead of diameter 5 mm. Evaporate the solution to dryness in a water bath at 55" C, and add 0.5 ml of 2-ethoxyethanol.Rotate the flask so that the bead assists the dissolution of the residue, cool, and then add 10.0 ml of 5 per cent. ethanolic potassium hydroxide solution. After 5 minutes, measure the colour at 390 mp with a Unicam SP600 spectrophotometer, and interpolate the result on a curve prepared from standard mixtures of plant extracts and :DDT (m.p. 107.5" C). PROCEDURE FOR DETERMINING CHLOROBENZILATE- Add 2ml of stearic acid solution to the extract obtained from the separation, and evaporate the mixture to dryness at approximately 55' C and a pressure of 20 cm of mercury. To the cooled residue add 5 ml of nitration mixture, and immerse the flask in a boiling-water bath for 1 hour. Cool the flask in an ice - water mixture, and add 75 ml of saturated sodium sulphate solution.Transfer the contents of the flask to a separating funnel with 70ml of diethyl ether, and shake. Discard the aqueous layer, and wash the ethereal layer successively with 25 and 10 ml of 5 per cent. potassium hydroxide solution and 15 ml of saturated sodium sulphate solution. Run the ethereal solution through a 15-mm layer of anhydrous sodium sulphate into a 100-ml round-bottomed flask containing a gla!js bead of diameter 5 mm. Evaporate the solution to dryness at 55" C, and add 0.5 ml of 2-ethoxyethanol. After swirling the bead round the flask to assist dissolution, add 10.0 ml of 5 per cent. methanolic potassium hydroxide solution. After 30 minutes, measure the colour developed at 538 mp with a Unicam SP600 spectrophotometer, and determine the chlorobenzilate by using a curve prepared from standard mixtures of plant extracts and chlorobenzilate (m.p.37" to 38.5" C). RESULTS Mixtures prepared by adding known amounts of DDT and chlorobenzilate to extracts of 1 g of untreated leaves were analysed by the proposed method. The results shown in Table I indicate good recoveries. TABLE I RECOVERIES OF DDT AND CHLOROBENZILATE FROM SYNTHETIC MIXTURES Amount of Amount of Amount of Amount of DDT added, chlorobenzilate added, DDT found, chlorobenzilate found, Crg r g r g Crg 10 10 10.5, 10.0 10.0, 9.8 20 20 21.0, 20.5 19.9. 19.7 50 50 49.7, 49.5 49.7, 49.5 75 75 73.5, 72.5 72.5, 72.0 100 100 98.0. 97.0 98.0, 95.0 200 200 193, 192 197, 196 We thank Professor H. G. H. Kearns and Dr. J. T. Martin for their interest in the work, and Messrs. Geigy Ltd. for the gift of a sample of chlorobenzilate. REFERENCES 1. 2. 3. 4. 6 . 6. 7. 8. 9. 10. Schechter, M. S., Soloway, S. B., Hayes, R. A., and Haller, H. L., Ind. Eng. Chsm., Anal. Ed., Butterfield, D. E., Parkin, E. A., and Gale, M. M., J . Soc. Chew. Ind., 1949, 68, 310. Amsden, R. C., and Walbridge, D. J., J . Agric. Food Chem., 1954, 2, 1323. Harris, H. J., Ibid., 1956, 3, 939. Blinn, R. C., Gunther, F. A., and Kilbezen, M. J., Ibid., 1954,2, 1080. Brockmann, H., and Schodder, H., Ber., 1941, 74, 73. Starkey, R. L., and Waksman, S. A., J . Bact., 1943, 45, 509. Pulst, C., Jahr. wiss. Botan., 1902, 37, 205. Skerrett, E. J., and Baker, E. A., Ann. Rep. Agric. Hort. Res. Sta., Bristol, 1958, 130. Martin, J. T., and Batt, R. F., Ibid., 1953, 121. 1945, 17, 704. Received January loth, 196.8

ISSN:0003-2654    

DOI:10.1039/AN9588300447

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

August, 19581 BATH 451 The Ultra-violet Spectrophotometric Determination of Sugars and Uronic Acids BY I. H. BATH (Chemistry De$artment, National Institute for Research in Dairying, Shinfceld, Reading, Berks.) A simple ultra-violet spectrophotometric method is described for the determination of microgram amounts of aldo- and keto-hexoses, pentoses and uronic acids in a single pure solution. The sugars and uronic acids are heated in 90 per cent. sulphuric acid solution, and the optical densities are measured a t the appropriate wavelength. The characteristics of the absorp- tion spectra of the reaction products are given. The method is particularly suited to the determination by one procedure of each of a series of carbohydrates that has been separated by paper chromatography. Interference by the usual chromatographic solvents, with the exception of n-butyl alcohol, is negligible.Experiments in which known mixtures of sugars were separated on paper chromatograms, eluted from the paper and analysed by the method show satisfactory recoveries. THE reaction of sulphuric acid with carbohydrates has been extensively studied in recent years. Holzman, MacAllister and Niemannl observed the spectral characteristics of some monosaccharides in 79 per cent. w/w sulphuric acid during their study of the carbazole reaction. who showed the possibility of making use of the ultra-violet absorption spectra of sulphuric acid solutions of the saccharides for analytical purposes. The nature of the reaction between sulphuric acid and carbohydrates has been further investigated by Love4 and more extensively by Rice and Fishbein516 and Rice.' The object of the work described in this paper was to formulate a rapid method that is suitable for the determination of several different sugars and uronic acids, after each has been obtained in a single pure solution.Previously, a combination of several methods was required to determine a series of aldo- and keto-hexoses, pentoses and uronic acids. The use of concentrated sulphuric acid alone, as a convenient reagent, has been successfully studied, and a suitable procedure has been developed. METHOD APPARATUS- The reaction is carried out in hard-glass test-tubes, 150 mm x 15 mm, lightly closed with a small test-tube, 50 mm x 12.5 mm, in the mouth of each to act as condensers during the heating and to exclude dust particles.Twenty-four tubes are accommodated in a carrier to allow easy and rapid change from the boiling-water bath to cold water. The absorption spectra and optical densities of the solutions were measured in 1-cm cells with a Unicam SP500 spectrophotometer. REAGENT- Sdphwic acid, 98 per cent.-Analytical-reagent grade. PROCEDURE- Add 6-ml portions of 98 per cent. sulphuric acid from a burette to the test-tubes, and thoroughly chill in an ice - water bath. Place 1-ml layers of the aqueous carbohydrate solutions, containing up to 100 pg, on the acid from a pipette, and then thoroughly mix by stirring with a glass rod while the tubes remain in the cooling bath. Heat the resulting solutions containing 90 per cent. w/w of sulphuric acid by immersing the tubes for exactly 5 minutes (30 minutes for glucuronolactone) in a bath of rapidly boiling water, and then cool to room temperature in cold water. Include a reagent blank containing 1 ml of water instead of carbohydrate solution with each set of tubes.Measure the optical densities of the solutions at the appropriate wavelength of maximum absorption for the sugar or uronic acid, namely, arabinose and ribose at 287 mp, glucurono- lactone at 295 mp, galacturonic acid at 301 mp, xylose at 316 mp and fructose, galactose, glucose, mannose and sucrose at 322 mp. Further carbohydrates were studied by Ikawa and Niemann,2452 BATH : THE ULTRA-VIOLET SPECTROPHOTOMETRIC [Vol. 83 EXPERIMENTAL SPECTRA OF REACTION PRODUCTS IN 90 PER CENT.SULPHURIC ACID- The hexoses, fructose, galactose, glucose and mannose, and the disaccharide sucrose, exhibit an absorption maximum at 322 mp and another less intense peak at 257 mp. Xylose is similar, although the peak of maximum absorption is at 316 mp. Ribose and arabinose have maxima at 287 and 316mp, the former being of greater intensity for ribose, but of almost equal intensity for arabinose. The uronic acids, under similar conditions, have only a single absorption peak; glucuronolactone at 295 mp and galacturonic acid at 301 mp. The ultra-violet absorption curves for these carbohydrates are shown in Figs. 1 and 2, and it can be seen that the optical density at the wavelength of maximum absorption varies for equal concentrations of the different carbohydrates.Wavelmgth, mp Fig. 1. Absorption spectra of the reaction products formed after heating with sulphuric acid for 5 minutes a t 100°C. 0, 100 pg of galacturonic acid per ml; A, 50 pg of fructose per ml; A, 50pg of ga.lactose per ml; a, 1OOpg of ribose per ml; +, 50pg of xylose per ml TIME OF HEATING- The determination of the optimum time of heating was made with solutions of sugars and uronic acids in 90 per cent. w/w sulphuric acid (see Fig. 3). The sugars used in the investigations were either the analytical-reagent grades or the laboratory-reagent grades (obtained from the British Drug Houses Ltd. and L. Light & Co. Ltd.), dried in vacw over silica gel. Maximum absorption occurred after heating for 2 to 5 minutes and it decreased slowly with further heating, except with glucuronolactone, when maximum absorption occurred after heating for 30 minutes and thereafter remained practically constant.As any slight variation in the period of heating arou:nd 5 minutes has a negligible effect on the measured optical density, 5 minutes were taken as the optimum time of heating for all the carbohydrates studied except glucuronolactone. For this, a time of heating of 30 minutes is required for maximum sensitivity of the method, but a shorter period can be used, as the curve of optical density against lactone concentration is linear after heating for only 5 minutes. It was also observed that fructose exhibited the same maximum optical-density value if kept for 30 minutes at 20" C as that found ;after heating for 5 minutes at 100" C, which provides an alternative procedure for this sugar if preferred.The similar behaviour of fructose in 79 per cent. sulphuric acid has been noted by Ikawa and Niemann.2August, 19581 DETERMINATION OF SUGARS AND URONIC ACIDS Wavelength, m p Absorption spectra of the reaction products formed after heating with sulphuric acid for 5 minutes at 100°C. 0, 50pg of sucrose per ml; A, IOOpg of mannose per ml; 0, 100 pg of glucuronolactone per ml; 0, 50 pg of glucose per ml; x , 100 pg of arabinose per ml Fig. 2. ".4 I I /--- n-. 0 4 I IP 0 2 4 6 8 1 0 Time of heating, minutes Fig. 3. Change in optical den- sity with time of heating the carbo- hydrate solutions with sulphuric acid a t 100' C. 0, 100 pg of galacturonic acid per ml; A, 50pg of fructose per ml; 0, 1OOpg of glucuronolactone per ml; 0, 60pg of glucose per ml; , 50pg of galactose per ml: ., 50pg d ribose per ml; +, 50 pg of of xylose per ml; x , 100 pg of arabinose per ml 453 Concentration of carbohydrate, yg per rnl Fig.4. Change in optical density with concentration of carbohydrate. Solutions were heated with sulphuric acid in accordance with the proposed procedure. 0, galacturonic acid; A, mannose; a, fructose; 0, glucose; A, galactose; , ribose; a, glucuronolactone; +, xylose; X, arabinose454 BATH : THE ULTRA-VIOLET SPECTROPHOTOMETRIC [Vol. 83 The change in optical density with concentration obeys Beer's law for most sugars, and the use of a standard calibration graph for each sugar (see Fig. 4) overcomes any slight deviation from linearity. CHANGE IN OPTICAL DENSITY WITH CONCENTRATION OF SUGARS- STABILITY OF REACTION PRODUCTS- The optical density at the wavelength of maximum absorption remains unaltered if the test-tubes are left standing for 3 hours at room temperature in the light, and no significant change in optical density was observed with fructose, galactose, glucose, galacturonic acid, ribose and xylose over a period of 24 hours.In practice, the optical density is measured shortly after the solution attains room temperature. ACCURACY OF THE METHOD- The procedure described has been applied in quadruplicate, at each of five different concentrations, to each of the sugars and uronic acids. The results for glucose, which are typical, are shown in Table I. The variance is homogeneous through the range of 20 to 100 pg (9 = 1.50, 4 degrees of freedom).The coefficient of variation at 20 pg was 1.9 per cent. and at 100 pg only 0.35 per cent. The corresponding coefficients of variation a t 20 pg and 100 pg, respectively, of arabinose were 1.3 per cent. and 0.45 per cent., fructose, 2.4 per cent. and 0-52 per cent., sucrose, 3.4 per cent. and 0.77 per cent. and xylose, 1.1 per cent. and 0.27 per cent. TABLE I ACCURACY OF THE DETERMINATION OF GLUCOSE BY THE METHOD Glucose Mean Range of Standard concentration, optical density clptical densities deviation r g Per ml 0.0051 I 20 0.379 0.372 to 0.389 40 0.750 0.746 to 0.753 60 1.111 1.105 to 1.114 80 1.454 1.445 to 1.459 100 1.764 1.755 to 1.770 This replication and the fact that the standard curves can be readily reproduced show that the method can be used to give reliable determinations of the sugar content of pure solutions.APPLICATIONS OF THE METHOD The method has its primary use in the determination of microgram amounts of mono- and disaccharides eluted from chromatograms after separation with normal solvent mixtures, e g . , as in my experience with the analysis of plant carbohydrate extracts and hydrolysates. A low and almost constant value is found, owing to the elution of chromogenic material from chromatography paper, but this does not interfere with the determination, provided a portion of each chromatogram free from sugar spots is eluted and determined, and the blank value so obtained is subtracted from that of the carbohydrates. TABLE I1 THE RECOVERY OF CARBOHYDRATES SEPARATED BY PAPER CHROMATOGRAPHY The solvent system used was ethyl acetate - acetic acid -water (3 + 1 + 3) AND DETERMINED BY THE METHOD Sugar mixture I P Amount Amount Re- applied, found, covery, r g Pg % Galacturonic acid 62.4 63.4 101.6 Galactose ..Glucose . . .. Fructose . . . . Arabinose . . 62.3 61.4 98.6 Xylose . . .. Ribose .. . . 62.4 62.4 100.0 - - - - - - - - - - - - Sugar mixture I1 Sugar mixture I11 Amount Amount Re- applied, found, covery, r g llg % - - - 62.5 60.8 97.3 62.4 62.2 99.7 - - - - - - - - - 60.0 59.0 98.3 Amount Amount Re- applied, found, covery, PLP rg % - - - 62.4 63.0 101.0 - - - - - - 62.6 64.3 102.7 60.0 61.2 102.0August, 19581 DETERMINATION OF SUGARS AND URONIC ACIDS 455 Any traces of ethyl acetate, acetic acid, pyridine or benzene from developing solvents, which may remain on the chromatogram and be eluted with the sugar, have been found not to interfere with the determination, but the use of solvents containing n-butyl alcohol leads to erroneous results. In experiments to determine the recovery of sugars from a known mixture separated by paper chromatography and subsequently analysed by the method, the recovery ranged from 97.3 to 102.7 per cent, The results in Table I1 show that the accuracy is satisfactory.DISCUSSION OF RESULTS The method provides an accurate and rapid means of determining aldo- and keto- hexoses, pentoses and uronic acids. The reaction conditions are similar for all thesugars and uronic acids, but the optical densities are measured at different wavelengths in the ultra- violet spectrum.As the only reagent required is sulphuric acid, the frequent preparation of unstable colour-forming reagents as, for example, is found to be necessary for anthrone,* orcinoP and o-aminodiphenyl,lO is avoided. The chromogenic compounds formed are unusually stable and possess definite absorption peaks, and the standard curves, which obey Beer’s law within the range of concentrations normally encountered, can be readily reproduced, and only one curve need be prepared for each carbohydrate. Experiments have shown that the reproducibility of readings and the recovery of a mixture of carbohydrates separated by paper chromatography and subsequently determined by the method are satisfactory. The method can be used for the determination, by one simple procedure once they have been separated, of each carbohydrate present in plant hydrolysates, whereas hitherto a combination of methods has been necessary. I thank the Agricultural Research Council for the award of a Research Studentship, during the tenure of which this study was carried out, and Dr. M. J. Head for his interest and advice and Miss R. L. Rutherford for technical assistance. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. REFERENCES Holzman, G., MacAllister, R. V., and Niemann, C., J . Biol. Chem., 1947, 171, 27. Ikawa, M., and Niemann, C., Ibid., 1949, 180, 923. -- , Arch. Biochem. Biophys., 1951, 31, 62. Lo&, R. M., Biochem. J., 1953, 55, 126. Rice, F. -4. H., and Fishbein, L., J . Amer. Chem. SOL, 1956, 78, 1005. -- , Ibid., 1956, 78, 3731. Rice: F. A. H., Ibid., 1956, 78, 6167. Yemm, E. W., and Willis, A. J., Biochem. J., 1954, 57, 508. Bruckner, J., Ibid., 1955, 60, 200. Timell, T . E., Glaudemans, C. P. J., and Currie, A. L., Anal. Clzem., 1956, 28, 1916. Received February 17th, 1968

ISSN:0003-2654    

DOI:10.1039/AN9588300451

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

August, 19581 DETERMINATION OF SUGARS AND URONIC ACIDS 455 Applications of Gas - Liquid Chromatography The Examination of Solvents from Plastic Adhesives BY J. HASLAM AND A. R. JEFFS (Imperial Chemical Industries Ltd., Plastics Division, Welwyn Garden City, Herts.) Details of the methods that have been used in the examination of mixed solvents from plastic adhesives, spray laquers, etc. , are given. The isolation of the solvent, its gas - liquid chromatographic separation on polar and non-polar columns and the infra-red and chemical tests on the separated products are described. IN the analytical examination of adhesives, plastic paints, etc., containing various mixed solvents, it is often necessary to express opinions on the composition of the solvent mixture that has been used.Within the past 2 years we have examined a large number of materials, for example, adhesives for bonding plastics to plastics, plastics to metal and plastics to glass,456 HASLAM AXD JEFFS: APPLICATIONS OF [Vol. 83 as well as plastic spraying lacquers, polymer coating solutions and inks suitable for printing on plastic materials. We have found gas - liquid chromatography to be of great value in the examination of these preparations and the purpose of this paper is to give details of the methods that we have found to be most useful. It is first necessary to isolate the solvent mixture in a clean condition from the sample under test. The following method has given excellent results in the analysis of a varied range of preparations. METHOD OF ISOLATING THE SOLVENT MIXTURE FROM THE COMPOSITION UNDER TEST- It is a modification of the vacuum-depolymerisation apparatus originally used by Haslam and S0ppet.l Approxi- mately 6 g of the composition are introduced into the bottom of tube A.This tube is then placed in a solid carbon dioxide - methanol bath at -80” C and the open end, B, is then sealed in a flame. The open end, C, is now attached to a vacuum-pump and the apparatus is evacuated, tube A being kept at -80” C; with the Viicuum pump still running the apparatus is then sealed at the constriction, D, by means of a hand torch. An H-tube is constructed having the dimensions shown in Fig. 1. D ,I 0 rnni Fig. 1. Modified vacuum-depolymerisation Tube A is now removed from the solid carbon dioxide - methanol bath and is replaced by tube E.As tube A gradually attains room temperature, the solvent tends to be volatilised from A to E. This transfer is assisted by warming tube A in a heated water bath or even in a heated oil-bath if the solvent is not readily volatilised. I t is often useful to allow this recovery process to proceed overnight. The dimensions of tube A are made purposely large, as some compositions tend to froth during this recovery process. When the material in A is observed to be “dry,” the seal of the apparatus is broken at D and tube E is cut off below the connecting tube. The mixed solvent in a clean condition is now ready for the preliminary gas - liquid chromatographic test. It has been reported that some workers introduce the composition directly on to an asbestos pad at the top of a gas - liquid chromatographic column and allow the solvent to evaporate in the gas stream.Apart from the difficulty of introducing such viscous samples by syringe, our experience is that the drops of the composition “harden” on the outside and trap some solvent on the inside. The result is that the solvent dries out slowly and gives a trace that is not a chromatogram. Alternatively, if a high-temperature vaporiser is used, there is a tendency with some preparations to get a chromatogram of the solvent 9lus depoly- merisation products. We believe that our met hod, although more time-consuming, is to be preferred. PRELIMINARY GAS - LIQUID CHROMATOGRAPHIC TEST-- of the mixed solvent. u / -’ apparatus The purpose of this test is to obtain preliminary information about the general complexityAugust, 19581 GAS - LIQUID CHROMATOGRAPHY 457 The gas - liquid chromatographic test is carried out on 1 drop of the isolated solvent; the column is 6 feet long, of $-inch nominal bore and packed with 30 per cent.w/w of dinonyl phthalate on Celite 545. The Celite 545 is graded by elutriation in the manner described by James and Martin.2 The temperature of the column is maintained at 100' C. Fig. 2. Gas -liquid chromatograms on a 6-foot column of 30 per cent. w/w of (a) Light petroleum, boiling range below (c) Light petroleum, boiling dinonyl phthalate on Celite 545 a t 60" C. 40' C. range 60" to 80' C . (b) Light petroleum, boiling range 40" t o 60' C. (d) Light petroleum, boiling range 80" to 100' C Sample Fig.3. Gas -liquid chromatograms on a 6-foot column of 30 per cent. wjw of dinonyl phthalate on Celite 545 a t 100°C. (a) Light petroleum, boiling range 60" to 80°C. (b) Light petroleum, boiling range 80' to 100" C. (c) Light petroleum, boiling range 100" to 120' C. (d) Light petroleum, boiling range above 120' C The exit pressure is adjusted to 150 mm of mercury and, with a rate of flow of 2.0 litres of nitrogen per hour, the pressure drop along the length of the column is approximately 450 mm of mercury. A katharometer, at room temperature, is used as sensing mechanism.458 HASLAM AND JEFFS : APPLICATIONS OF [Vol. 83 Visual examination of the chromatogranl will quickly indicate the general boiling range of the solvent mixture. If components are present that are rapidly eluted from the column at 100" C, it is desirable to repeat this preliminary separation, but at a column temperature of 50" C.This test is particularly valuable for ascertaining the presence or absence of petroleum fractions and solvent naphtha. These solvents show characteristic patterns, examples of which are given in Figs. 2, 3 and 4. Fig. 2 shows chromatograms of the lower boiling petroleum fractions at a column temperature of 50" C. Figs. 3 and 4 (a) show chromatograms of higher boiling petroleum fractions and Fig. 4 (b) the chromatogram of a typical solvent naphtha carried out at a column temperature of 100" C. If the very high-boiling petroleum fractions are encountered as solvents, e.g., petroleum distillate, boiling range 190" to 275" C, and kerosine, boiling range 210" to 250" C, some difficulty wiU be experienced.I t can be seen from Fig. 4 (a) that, at a column temperature of 100" C, the chromatogram of white spirit takes 100 minutes to complete. The chromato- gram of kerosine, for instance, looks similar to that of white spirit, but the former contains many higher boiling constituents that may not be eluted from the column at 100" C. In any case of doubt, the column temperature is raised to 130" C (the maximum permissible with this stationary phase) and the flow rate is greatly increased to facilitate the removal of such constituents. min. min. (b) Fig. 4. If the preliminary test indicates that solvents are present that boil at temperatures of the order of 140" C or less, it is desirable t o proceed to the gas - liquid chromatographic test on polar and non-polar columns.In our experience in this type of work, solvents boiling above 140°C will normally, at this stage, have been identified as particular high-boiling petroleum fractions. An occasional instance of a composition containing fl-butyl lactate, b.p. 188" C, was encountered. GAS - LIQUID CHROMATOGRAPHIC TESTS ON POLAR AND NON-POLAR COLUMNS- The purpose of this test is to separate, as far as possible, the individual components of a mixed solvent on columns of quite different character, i.e., on (a) a non-polar column containing paraffin wax as stationary phase, and (b) a polar column containing tritolyl phosphate as stationary phase. The idea underlying this test was first put forward by James and Martin? Gas - liquid chromatograms on a 6-foot column of 30 per cent.w/w of dinonyl phthalate on Celite 545 at 100" C. (a) White spirit. (b) Solvent naphthaAugust, 19581 GAS - LIQUID CHROMATOGRAPHY 459 Further, in the course of this test the corrected relative retention times of the individual components, i.e., relative to pure benzene, are determined on both columns. The figures are used in the identification of the separated constituents. The paraffin wax column is 12 feet long, of $-inch nominal bore and packed with 33.3 per cent. w/w of paraffin wax (congealing point 54" C) on 52 to 60-mesh Johns Manville Silocel C22 firebrick; the column is heated by means of a steam jacket at 100" C. The exit pressure of the column is adjusted to 150 mm of mercury and the inlet pressure controlled so as to give a flow rate of 2.0 litres of nitrogen per hour through the column.The tritolyl phosphate column is similar to the paraffin wax column, except that 33.3 per cent. w/w of tritolyl phosphate is substituted for the paraffin wax. This column is enclosed in the same steam jacket as the paraffin wax column. A portion of the sample is first diluted with about one-fifth of its volume of pure benzene and four chromatograms are run, i.e., with and without benzene on each of the columns. Visual examination of the four chromatograms will indicate whether or not benzene is present in the mixed solvent under examination. The corrected relative retention times are now calculated for the individual components separated on each column.The method of calcu- lation is illustrated by the example given in Fig. 5 , which represents the chromatogram on the tritolyl phosphate column of a mixed solvent containing ethanol, ethyl acetate, rt-butyl alcohol, n-butyl acetate and added benzene. Comparison of the results obtained on the two columns with the calibration charts shown in Table I will then indicate the composition of most mixed solvents. This Table has been prepared by the examination of known mixtures containing added benzene. Certain solvents, on a given column, were found to have cor- rected relative retention times similar to that of benzene. Such solvents were examined on their own, pure benzene being run immediately before and after the test substance; the average value for benzene was then used in computing the value for the test substance.It was noted that the alcohols did not give very reproducible results on the paraffin wax column and the values given are the average of several results. Calculation of corrected relative retention times- (24'7-2.1) = 0.52 (32'7-2.1) - 0.71 (107.4-2.1) Peak A (45.5-2.1) Peak (45.5-2.1) - Peak c (45.5 - 2.1) = 2*43 (123.8-2.1) - 2.80 (45.5 - 2.1) - PeakiD Solvent mixture sample identified as- A = Ethanol B = Ethyl acetate C = n-Butyl alcohol D = n-Butyl acetate Fig. 5. Specimen gas - liquid chromatogram of a mixed solvent on a 12-foot column of 33.3 per cent. wjw of tritolyl phosphate on Silocel C22 for determining the corrected relative retention times Apart from this anomaly, we find in practice that the reproducibility of the test is of the order of +0.02 to 0.03 units up to a corrected relative retention time of 1.0, f0.04 to 0.08 units for 1.0 to 3.5 and k0.1 to 0.15 units for above 3.5.It may be necessary, therefore, that, in order to obtain two peaks in a chromatogram from a mixture of two substances, their corrected relative retention times should differ by as much as 0.08 units up to a corrected relative retention time of 1 and 0.15 to 0.25 units for a corrected relative retention time above 1. It will be realised from these figures that difficulties may arise because of the overlap of peaks, and for this and other reasons we find it invaluable when there is any doubt to carry out separations on columns with isolation of the separated components and infra-red examination of the separated products.For example, in our experience a mixture of methyl acetate and acetone gives a single peak on both columns, but such a mixture would not460 HASLAM AND JEFFS: APPLICATIONS OF [Vol. 83 deceive an infra-red spectroscopist. A corresponding instance would be that of a mixture of m-xylene and 9-xylene. TABLE I CALCULATED RELATIVE RETENTION TIMES FOR VARIOUS SOLVENTS ON 12-FOOT COLUMNS O F 33.3 PER CENT. OF PARAFFIN WAX ON SILOCEL c22 AND 33.3 PER CENT. OF TRITOLYL PHOSPHATE ON SILOCEL C22 Solvent Parafin wax column- Acetaldehyde . . .. Methyl formate . . .. Methanol * . .. Ethanol . . .. * . Acetone . . .. .. Methyl acetate . . .. isoPropyl alcohol . . Ethyl formate . . .. Allyl alcohol . . .. Diethyl ether .. . . Methylene dichloride . . tert.-Butyl alcohol . . n-Pentane . . .. n-Propyl alcohol . . Ethyl methyl ketone . . Ethylidene dichloride . . sec.-Butyl alcohol . . Diisopropyl ether . . Methyl propionate . . isoPropyl acetate . . Chloroform . . .. Tetrahydrofuran . . Ethylene dichloride . . Methyl n-propyl ketone n-Propyl acetate . . Ethyl propionate . . Carbon tetrachloride . . Methyl n-butyrate . . Dioxan . . .. .. Propylene dichloride . . isoPropyl propionate . . isoButyl methyl ketone Trichloroethylene . . isoOctane * . .. n-Heptane . . * . isoButyl acetate * . Methylcyclohexane . . n-Butyl acetate .. Toluene . . .. .. n-Octane .. .. Ethylbenzene . . .. Di-n-butyl ether .. p-Xylene . . .. .. o-Xylene .. .. Vinyl acetate . . .. Ethyl acetate . . .. isoButyl alcohol * .n-Butyl alcohol .. cycloHexane . . .. m-Xylene . . .. Bo i 1 in g - point, "C 20.2 31.5 64.6 78.3 56.0 57.1 82.4 54.2 97.1 34.6 40.1 82.5 36.1 77.0 97.2 80.0 77.2 57.3 99.5 67.5 79.9 108.1 88.9 61.2 65.0 83.5 102.3 118.0 101.6 99.1 76.7 102.3 101.4 80.8 96.4 111.3 116.8 86.7 99.2 98.4 117.2 100.8 126.2 110.8 125.6 136.2 142.4 138.4 139.3 144.0 Calculated relative retention time 0.09 0.11 0.13 0.17 0.18 0.23 0.23 0.23 0.25 0.26 0.27 0.28 0.29 0.35 0.36 0.40 0.43 0.44 0.50 0.52 0.54 0.55 0.58 0.60 0.62 0.72 0.81 0.83 0.93 0.94 1.06 1.09 1.12 1.14 1.17 1-25 1.28 1.35 1-31 1.40 1.51 1-91 2.13 2.32 3.10 4.6 4.8 5.2 5.3 6.1 Solvent Trifolyl phosphate column- n-Pentane .. .. Diethyl ether . . .. Acetaldehyde . . .. Methyl formate . . .. Diisopropyl ether . .Methanol .. .. isoOctane * . .. Ethyl formate . . .. Methyl acetate . . .. n-Heptane .. .. cycZoHexane . . .. Acetone . . .. .. Methylene dichloride . . Ethanol . . .. .. tert.-Butyl alcohol . . Ethylidene dichloride . . isoPropy1 alcohol . . Methylcyclohexane . . Carbon tetrachloride . . Methyl propionate . . isoPropy1 acetate . . %-Octane .. . . Tetrahydrofuran . . Ethyl methyl ketone . . Chloroform . . .. sec.-Butyl alcohol . . n-Propyl alcohol . . Ethyl propionate . . Trichloroethylene . . n-Propyl acetate . . Ethylene dichloride . . Methyl n-butyrate . . ZsoPropyl propionate . . Methyl n-propyl ketone Propylene dichloride . . Dioxan .. .. I . Toluene . . .. . . isoButyl methyl ketone Vinyl acetate . . .. Ethyl acetate . . .. Allyl alcohol . . .. isoButyl alcohol ..isoButyl acetate . . n-Butyl alcohol . . .. Di-n-butyl ether .. n-Butyl acetate .. Ethylbenzene . . . . p-Xylene . . .. .. m-Xylene . . .. o-Xylene .. .. Boiling- point, "C 36.1 34.6 20.2 31.5 67.5 64.6 99.2 54.2 57.1 98.4 80.8 56.0 40.1 78.3 82.5 57.3 82.4 77.0 100.8 77.2 76.7 79.9 88.9 126.6 65.0 80.0 61.2 99.5 97.2 99.1 86.7 97.1 101.6 83.5 102.3 111.3 102.3 108.1 96.4 117.2 101.4 110.8 116.8 11 8.0 142.4 126.2 136.2 138.4 139.3 144.0 Calculated relative retention time 0.11 0.17 0.19 0.22 0.29 0.36 0.37 0.38 0.40 0.42 0.45 0.46 0.47 0.52 0.53 0.57 0.58 0.59 0.66 0.70 0.76 0.77 0.79 0.85 0.86 0.89 0.99 1.14 1.15 1.24, 1.26 1.26 1.35 1.37 1.46 1.48 1.55 1.85 1.76 1-93 1.95 2.10 2.12 2.41 2.41 2.78 4.1 4.2 4.4 5.3 CHROMATOGRAPHIC SEPARATION AND INFRA-RED :EXAMINATION OF SEPARATED PRODUCTS- The great advantage of this method is that, idthough the column may have to be loaded with a comparatively large amount of sample, and hence there may be a considerable loss in column efficiency, once the products have been.separated their infra-red identification is usually unequivocal.August, 19581 GAS - LIQUID CHROMATOGRAPHY 461 The principle of this method, which involves the trapping of the separated products after passage through the column, has been described previously? In the interval, the trapping system has been modified and reduced in size. Its dimensions are shown in Fig. 6. Nichrome wire covered with Electrical leads to variable transformer Fig. 6. Modified trapping apparatus The choice of column, load and conditions of separation in this test are largely governed by the boiling range of the mixture as indicated by the preliminary chromatogram. The column temperature is adjusted so that complete separation between the peaks, with a suitable load, is indicated by the record.I t must be borne in mind, however, that it is usually necessary to separate at least 0.05 ml of a particular constituent in order to obtain a satis- factory infra-red spectrum, although work is being carried out to extend the range of the tests so that much smaller amounts of substance can be dealt with. With very volatile constituents it may be necessary to separate rather more than this amount. We have pointed out previously4 that a single peak in a chromatogram does not always indicate the presence of a single substance.Recently, a spray lacquer was examined; this contained 16 per cent. of poly(viny1 chloride - vinyl acetate) copolymer, 9 per cent. of dioctyl phthalate plasticiser and 75 per cent. of mixed solvent. The preliminary chromatogram of the mixed solvent showed four quite separate peaks. The constituents corresponding to these four peaks were condensed in separate cold traps. Infra-red examination indicated that trap 1 contained a mixture of methyl acetate and acetone (a mixture difficult to resolve on many columns). Trap 2 contained tetrahydrofuran and traps 3 and 4 contained benzene and toluene, respec- tively. The mixture in fact contained five components, although only four were indicated in the chromatogram. I t is often desirable to ascertain the approximate composition of the solvent mixture and this is carried out by the method described below.DETERMINATION OF THE APPROXIMATE COMPOSITION OF SOLVENT MIXTURES- From the chromatograms obtained as described it is usually possible to estimate the approximate composition of an unknown solvent mixture. One or two synthetic mixtures similar to the estimated composition of the sample are examined under the precise conditions of test as with the unknown mixture. The composition of this latter mixture is then deduced by inspection of the chromatograms. With experience, the accuracy is normally quite adequate for most work of this type. If at any time greater accuracy is required, a calibration for each component with an internal marker is necessary when nitrogen is used as the carrier gas in conjunction with a katharometer detector. Finally, on occasion, use may be made of chemical tests on separated products.462 CROSSLEY AND THOMAS: THE SEPARATION OF SOME [Vol.83 In addition to the infra-red identification, it is possible to prepare chemically suitable derivatives of the isolated products. Moreover,, certain tests may be made directly on the separated products (diluted with carrier gas) as they leave the gas - liquid chromatographic column. For example, the presence of ketones may be readily proved by bubbling the exit gases through a trap containing 2 : 4-dinitrophenylhydrazine reagent .6 This reagent is prepared by dissolving 1 g of 2 : 4-dinitr0pheny:lhydrazine in 15 ml of concentrated sulphuric acid and diluting to 400 ml with water. The solution is set aside overnight in a refrigerator, after which it is filtered from the excess of reagent. The filtrate is then diluted to 500ml with water, 0.05 to 0.1 pl of acetone will produce a visible turbidity with 0.5 ml of this reagent. Even smaller amounts of acetone, i.e., of the order of 0.01 pl, may be detected by extraction of the reaction product with 1 ml of spectroscopically pure cyclohexane; the extract is examined spectrophotometrically against a corresponding extract of the reagent. Then again, we have shown that chemical tests may be applied in order to decide whether, for example, formaldehyde is produced from methanol under specific conditions of chromatographic test. For this test the exit gases were passed through phenylhydrazine reagent, after which the principle of Schryver’s test was applied in a very sensitive test for formaldehyde. CHEMICAL TESTS ON SEPARATED PRODUCTS- It seems to us that there are many other possibilities for this form of test. We thank Mr. H. A. Willis for his valuable assistance in the infra-red work and Mr. M. Green for his general assistance in the development of these methods. REFERENCES 1. 2. 3. 4. 6. Haslam, J., and Soppet, W. W., Analyst, 1950, 75, 63. James, A. T., and Martin, A. J. P., Biochem. J , 1952, 50, 679. -,- , Analyst, 1952, 77, 915. Haslam, J., and Jeffs, A. R., J . Apfil. Chern., 1957, 7 , 24. Perkin - Elmer Instrument News, 1957, 8, No. 4. Received Febvuary 25th, 1958

ISSN:0003-2654    

DOI:10.1039/AN9588300455

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

462 CROSSLEY AND THOMAS: THE SEPARATION OF SOME [Vol. 83 The Separation of some Coal-tar Food Colours by Paper Electrophoresis BY J. CROSSLEY AND J. D. R. THOMAS* (Department of Chemistry and Biology, South-East Essex Technical College, Dagenham, Essex) The behaviour during electrophoresis on paper of some coal-tar food colours in different electrolytes is described and discussed. Results show that, in suitable electrolytes, it is possible to separate individual colours from mixtures by paper electrophoresis. LEDERER~ gives an excellent account of the basic principles and applications of paper electro- phoresis, and includes a brief survey of published work on the electrophoresis of dye-stuffs. However, the only work published on the separation of food colours by paper electrophoresis is that of Mori and Kimura2 and Mori,3 who have examined the behaviour of colours under different conditions of electrolyte, filter-paper, current and so on, and William~,~ who describes the electrophoretic isolation at pH 12 of dye-stuffs from biscuits, jams and cream confectionery.The chromatographic isolation of food colours has received greater attention, although it is only 6 years since Tilden5 remarked that, of the many applications of paper partition- chromatography, relatively few dealt with the separation and isolation of dye materials. Many papers have since appeared that describe methods for the chromatographic separation of coal-tar food colours. Some workers have used the column te~hnique,~?' but many others have dealt with separations on paper.8 to l4 Fujiil') summarises the chromatographic behaviour of no less than ninety-five artificial coal-tar dyes, and Verma and Dasll have determined the RF values, in thirteen eluting agents covering a wide pH range from acid to alkaline conditions, of forty-five dyes commonly used in foodstuffs.The identification of colouring matter in specific foodstuffs has been dealt with by some workers, and the range of foodstuffs examined includes fruit squash,8 ~aviare,~ wines, syrups and so on12 and jelly ~rysta1s.l~ * Present address : Newport and Monmouthshire College of Technology, Newport, Monmouthshire.August, 19581 COAL-TAR FOOD COLOURS BY PAPER ELECTROPHORESIS 463 Of the twenty-six food colours examined by Mori and Kimuraa and Mona by paper electrophoresis, only three are among the permitted16 coal-tar food colours, namely, amaranth, tartrazine and indigo carmine.We now describe an extension of the work to an examination of the behaviour of erythrosine BS, tartrazine, indigo carmine, ponceau MX, ponceau 4R and ponceau 3R. METHOD APPARATUS- ELECTROLYTE SOLUTIONS- An E.E.L. (Evans Electroselenium Ltd.) electrophoresis apparatus was used. Acetic acid, N. B u f e r solution, PH 4.0-Add 6.0 ml of 0.1 N sodium hydroxide to 750 ml of 0.1 M Buffer solution, pH B.O--Add 85.5 ml of 0.1 N sodium hydroxide to 750 ml of 0.1 M Buffer solution, PH 8.0-Add 702 ml of 0.1 N sodium hydroxide to 750 ml of 0.1 M Sodium tetraborate solution, 1 per cent. w l v . Ammonium hydroxide solution, 0.1 N. PREPARATION OF DYE SOLUTIONS- Prepare aqueous solutions of erythrosine BS, tartrazine, indigo carmine, ponceau MX, ponceau 4R and ponceau 3R, so that 50 ml of each solution contain 10 mg of the dye.When a mixture is to be used, prepare a solution containing 10 mg of each constituent per 50 ml. PROCEDURE- Fill the four compartments of the electrophoresis bath to the same level with the relevant electrolyte solution. Cut a Whatman No. 1 filter-paper strip (5 cm wide) to suitable length (34 cm) . Draw a faint pencil line across the filter-paper strip, 5 cm from one end. Thoroughly moisten the strip with the electrolyte solution in the bath, place it across the “bridge,” and apply evenly at the pencil line 0.02 ml of the relevant dye solution, which corresponds to 4 pg of each dye. When there is more than one strip in the bath, take care that all the samples are at the same end.Place the glass cover on the bath and connect to the power unit. Place the polarity switch in the position indicated by the position of the sample, i.e., if the pencil mark is at end A of the bath, place switch in position A also and vice versa. This makes the sample end the cathode compartment. (The dyes in each instance migrate towards the anode, although some of those examined by Mori and Kimuraz migrate towards the cathode, e g . , Bismarck brown and rhodamine 6G.) Allow electrophoresis to take place by switching on the power unit and adjusting the current to a suitable value. After a suitable time interval, end the experiment by switching off the power unit and disconnecting the electrophoresis bath.Remove the strips and dry them in an oven at 110” C for 5 minutes, the strips being suspended loopwise from a glass rod by means of “bulldog” clips at either end. potassium hydrogen phthalate, and dilute to 1.5 litres. potassium dihydrogen orthophosphate, and dilute to 1.5 litres. potassium dihydrogen orthophosphate, and dilute to 1.5 litres. The electropherogram is then ready for examination. RESULTS The dyes were subjected to paper electrophoresis singly and also as constituents of Table I shows the distances Migration distances mixtures, the observed migration distances being reproducible. migrated by the dyes, both singly and as constituents of mixtures. of less than 2 mm are shown as zero. DISCUSSIOS OF RESULTS Table I shows a number of peculiarities and trends that call for comment.Migration distances in a given time for the dyes in the nearly neutral electrolytes, i.e., buffer solutions pH 6.0 and 8.0, are relatively small when compared with the much greater distances observed under more acid or alkaline conditions. This would suggest that separa- tions can be carried out more effectively under distinctly acid or alkaline conditions. This464 CROSSLEY AND THOMAS: THE: SEPARATION OF SOME [Vol. 83 is found not to be so, as the advantage gained by greater migration distances is lost because of the considerable and often rapid tailing and fading that occurs under these extreme condi- tions. It is worthy of note, however, that erythrosine BS, tartrazine and indigo carmine have been separated in 15 minutes with N acetic acid as electrolyte, the respective migration distances being 0, 16 and 7 mm at a current density of 0.6 mA per strip, tailing and fading being only slight.TABLE I MIGRATION DISTANCES OF DYES hligration distance of- Current - c 1 density, erythro- tartra- Electrolyte mA Time, sine BS, zine, per5cm hours mm mm Acetic acid, N 0.6 2 0 130* 1.7 2 Buffer solution, Buffer solution, 1.7 2 0 23 Buffer solution, 2.0 2 0 15 Single dyes- - - pH 4.0 1.7 1.75 0 74* pH 8.0 pH 8.0 Sodium tetraborate solution, 1 per cent. 2.0 1.5 9 103t Ammonium hydroxide, 0.1 N 1.7 2 0 83* Dye mixtures (each horizontal line corresponds to a mi;vture)- Acetic acid, N 0.6 0.25 0 16 0.6 1.5 0 90* Buffer solution, pH 4.0 1.7 1.75 0 70* Buffer solution, pH 6.0 1.7 2 0 24 1.7 2 0 23 Buffer solution, pH 8.0 2.0 2 0 18 2.0 2 0 18 Sodium tetraborate solution, 1 per cent.2.0 1-5 - - 2-0 1.5 10 85 t 0.1 N 1.7 2 0 83* Ammonium hydroxide, indigo carmine, mm 52t 35s 9 8 - 38* 101 7 40 t 32t 10 8 7 7 - -: 9: ponceau 4 R mm - 37* - 26 30 100 - - - - - 27 - 30 76 80 - ponceau 3R. mm - 20* - 0 0 11 - - - - - 0 - 0 9 10 - * Tailing and fading. 7 ’Tailing. $ Fading. 11 Two bands, but the band at 5 Slight tailing and fading. mm is faint. In experiments in which electrophoresis was carried out for different time intervals, the other conditions being kept constant, an approximately linear relationship between migration distance and time was observed (com:pare Moris), e.g., for tartrazine and indigo carmine in N acetic acid for 15 and 90 minutes, the respective migration distances were 16 and 90 mm for tartrazine and 7 and 40 mm for indigo carmine.It is often observed that, when dyes are present in a mixture, they exert a “dragging” effect on one another. This is particularly marked with tartrazine and ponceau 4R in 1 per cent. sodium tetraborate solution, the migration distance of tartrazine being reduced from 103 mm for the single dye to 85 mm for the dye present in a mixture; corresponding figures for ponceau 4R are 100 and 80mm. The four red dyes, erythrosine BS, ponceau MX, ponceau 4R and ponceau 3R, were chosen for this investigation because of their similarity in colour and, in so far as the ponceaus are concerned, their similarity in structure. As .is to be expected, and as the results show, their separation by paper electrophoresis is more difficult than for the other dyes.Ponceau 4R, however, presents little difficulty, as it migrates readily in every instance. Except in 1 per cent. sodium tetraborate solution, erythrosine BS, ponceau MX and ponceau 3R didAugust, 19581 COAL-TAR FOOD COLOURS BY PAPER ELECTROPHORESIS 465 not separate, although separation would be possible in N acetic acid, except for the considerable tailing and fading that occurs in this electrolyte. When the six dyes are present in one mixture, separation into four distinct bands takes place in 1 per cent. sodium tetraborate solution, a fifth band of indigo carmine being completely faded. Two dyes that cannot be separated in this electrolyte are erythrosine BS and ponceau 3R.The difficulty in separating ponceau MX and ponceau 3R in many electrolytes can probably be attributed to the similarity in structure (ponceau 3R has one extra methyl group). The observations of Anderson and Martin,14 who examined the effect of substituents on the R, values when dye-stuffs were chromatographically extracted with isobutyl alcohol saturated with 2 N hydrochloric acid, are of interest in this connection. They report that the R, value of a mono-azo dye-stuff is not influenced by substituent groups such as methyl, but is increased by a decrease in the number of sulphonic acid groups. These trends cannot hold for paper electrophoresis, as it is possible to separate ponceau MX and ponceau 3R in both iV acetic acid and 1 per cent. sodium tetraborate solution.Further, ponceau MX, a mixture of dyes with methyl groups in different positions, has been separated into two bands in 1 per cent. sodium tetraborate solution. Also, the effect of the sulphonic acid group appears to be the reverse of that for chromatographic separation, i.e., an increase in the number of sulphonic acid groups increases the migration distance, as illustrated by ponceau 4R (three sulphonic acid groups) and ponceau MX and ponceau 3R (two sulphonic acid groups). CONCLUSIOM With a suitable choice of electrolytes it is possible to separate food-colouring materials by paper electrophoresis. Distinct separations of dyes from mixtures are possible, as long as there is a difference of 3 to 4 mm between the migration distance of each dye. This difference must of necessity be greater when tailing occurs.Two to three hours are usually sufficient to isolate the components into distinct zones, which can be cut out, the dyes eluted with distilled water and absorption spectra recorded. With suitable colour filters, which are available from the manufacturers, the E.E.L. scanner can also be used for quantitative evaluation. We thank Solmedia Ltd. for a gift of ponceau MX, ponceau 4R and ponceau 3R. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. REFERENCES Lederer, M., “Introduction to Paper Electrophoresis and Related Methods,” Elsevier Publishing Co., Amsterdam, 1955. Mori, I., and Kimura, M., J . Pharm. SOC., Jafian, 1954, 74, 179. Mori, I., Ibid., 1954, 74, 181. TVilliams. T. F.. Analvst, 1957. 82, 211. Tilden, D.“H., J. Ass: 03. Agric. Chem., 1952,35, 423. McKeown, G. G., Ibid., 1954, 37, 527. Graichen, C., Sclar, R. N., Ethelstein, N., and Freeman, K. A., Ibid., 1955, 38, 792. Mitra, S. N., and Chatterji, R. K., J . Inst. Chem., India, 1955, 27, 169. Panopoulos, G., and MBgaldoikonomos, J., Chim. Anal., 1954, 36, 68. Fujii, S., Bull. Nut. Hyg. Lab., Tokyo, 1955, 73, 335. Verma. M. R.. and Ram Ti Das. T. Sci. I n d . Res.. C. India. 1956. 15. 186. Deshusses, J.,.and DesbaGmes, g, Mitt. Lebensmitt. ‘Hyg., Bern, 1956, 47, 15. Netta, I., An%. Falsif, 1957, 50, 166. Anderson, J. R. A., and Martin, E. C., Anal. Chim. Acta, 1953, 8, 530. “The Colouring Matter in Food Regulations, 1957,” H.M. Stationery Office, London, 1957. Received March 19th, 1968

ISSN:0003-2654    

DOI:10.1039/AN9588300462

出版商:RSC    

年代:1958

数据来源: RSC