ISSN:0003-2654    

DOI:10.1039/AN97398FX001

出版商:RSC    

年代:1973

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97398BX003

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

January, 19731 SUMMARIES OF PAPERS I N THIS ISSUEThe Direct Complexometric Determination of Metals in UnusedLubricating Oils and Additives by Automatic Non-aqueousPotentiometric TitrationMethods are described for the determination of calciuni, barium, zincand magnesium in any combination in additive concentrates and unusedlubricating oils. The samples are dissolved in a mixed aqueous - organicsolvent and titrated directly with 1,2-bis(2-aniinocthoxy)ethanc-NNN”’-tetraacetic acid [ethylene glycol bis( 2-aminoethyl) tetraacetic acid ; lS(;‘l‘A]or 1,2-diaminocyclohexane-NNN’N’-tetraacetic acid (DCTR), the end-pointsbeing indicated by metal ion sensitive electrodes. I3y use of a maskingagent and the appropriate buffer solutions the individual metals in anycombination can be determined following a maximum of three titrations.The results are compared with those obtained by established procedures.The accuracy of the complexometric methods is comparable with that o festablished wet-chemical methods, but the former are up to ten times fastcrand are therefore suitable for blending control.J.N. WILSON and C. 2 . MARCZEWSKIThe British Petroleum Company Limited, T 3 P ICesenrch Centre, Chert ,cy I C o m I ,Sunltury-on-Thames, Middlesex.Analyst, 1973, 98, 42-48.viiThe Direct Determination of Mercury by Atomic-absorptionSpectrophotometry at 184.9 nm by Using a Nitrogen- separatedNitrous Oxide - Acetylene FlameA modified atomic-absorption spectrophotonieter with a nitrogen-purgedoptical path has been used for the direct determination of mercury by atomic-absorption spectrophotometry, by using a nitrogen-separated pre-mixednitrous oxide - acetylene flame and electrodeless discharge lamp source.The high transparency of the fuel-rich flame below 200 nni permits the useof the principal resonance line of mercury at 184.9 nni for its determination.The sensitivity (for 1 per cent. absorption) enhancement obtained by using thisline rather than the 253-7-nm line is fifty-fold; detection limits for rnercury(1)and mercury(I1) of 0.02 p.p.m.and 0.05 p.p.m. are achieved in aqueoussolution.G. F. KIRKBRIGHT, T. S. WEST and P. J. WILSONChemistry Department, Irnpcrial College, London, SW7 2AY.Analyst, 1978, 98, 4!)-52.A Cold Vapour Technique for the Determination of Mercury inGeological Materials Involving its Reduction with Tin(I1)Chloride and Collection on Gold WireA technique for the determination of mercury in geological matcrials isdescribed in which mercury liberated from the sample by tin(ll) chloride iscollected on gold-wire clippings and determined by flameless atomic absorptionafter heating the gold in an easily constructed resistance furnace.Ihringthe heating stage the stream of air used to carry the atomic mercury vapouris diverted through a by-pass. The method gives a five-fold increase insensitivity over reduction with tin(I1) chloride alone and is free from non-atomic-absorption interferences when single-beam detectors are used. Thedetection limit is 0.001 pg of mercury and the coefficient of variation for0.080 pg of mercury is 3-9 per cent.P.C. HEAD and R. A. NICHOLSONInstitute of Geological Sciences, Geochemical Division, 64-78 Gray’s Inn Road,London, WClX 8NG.Analyst, 1973, 98, 63-66January, 19731 THE ANALYST ixCLASSIFIED ADVERTISEMENTST& ra& for classifid advertisewnts i s 369 a lint (or spaceequtvaient of a Line) with an extra charge of lop for theuse of a Box Number. Semi-displayed classifiedadvtrtisemenls arc €4 for single-column inch.Copy for classijied advertisements required not later thanthe r8th OJ the month preccding date of publication whichis on the 16th of each month. Advertisements should beaddressed to J . Arthur Cook, 9 Lluyd Square, London,WCIX 9MA. Tel.: 01-837 6315APPOINTMENT VACANTINSTRUMENTAL METHODS ANALYSTTo be responsible for the administration and operation of instrumentalnicthods of chemical analysis including U.V.emission and X-Raycwission and diffraction techniques.Tlit. successful candidate should hold an H.N.C. or above qualificationi i i cither chemistry or physics and should have previous experienceof this type of work and a knowledge of application to both metallica r i d nonmetallic samples.The Company can offer an attractive salary to the right candidate; i d fringe benefits available include four weeks annual holiday tothose aged 25 or above, contributory superannuation schrmc andtrw life assurance.Applications should he made i n writing giving details of age, qualifica-tions and experience to:John MasonINTERNATIONAL COMBUSTION LTD.Sinfin Lane, Derby DE2 9GJANALYSTrequired byThe EAST AFRICANPESTICIDES CONTROL ORGANISATIONto establish a small analytical laboratory for the veri-fication of pesticide contents of selected technicaland formulated pesticides.The successful candidatewill be a member of EAPCO which meets bi-annuallyto promote the safe and effective use of pesticidesand enforce conditions for the sale and proper use ofefficient and appropriate pesticide-products in EastAfrica.Candidates must have a good Honours degree inChemistry plus at least 4 years post-graduate ex-perience in Pesticide Chemistry. Knowledge of methodanalysis of major pesticides is essential. Salary is ina range of approxlmately €2890- €4050 at present rateof exchange.A Gratuity of 25% of total salary drawnis payable on completion of service.The post descrlbed is partly financed by Britain’sprogramme of a!d to the developing countries ad-ministered by the Overseas Development Admin-istration of the Foreign and Commonwealth Office.Apply toCROWN AGENTSM. Division, 4, Millbank, London,SWlP 3JDfor application form and further particulars statingname, age, brief details of qualifications and ex-perience and quoting reference number M3€/720450/AA D.~~LECTURES & COURSESUNIVERSITY OF SOUTHAMPTONDEPARTMENT OF CHEMISTRYAPPLICATIONS OF I’HO‘TOCHEMlS1’RYThe following self-contained short coursI’s driilirig with sonw appli-cations of photocheniistry arc open to m y applicwt.7th-12th January 1973 13iiviroiiinc.iit;iI arid Aii;ilvtic;il Aslwcts25th-30th March 1953 Polymvr Photochc.rriistry1st-6th April 1973 Iniagc,-forrning i’rocrssrsThe above courses are rcsitleiitial.In addition this followiiig shortcourses, offered as part of an M.Sc. programinc., arc opeii to partici-pants although in these cases accominotfatioii caiiriot I)v arr;uigc.tl.22nd-26th January 1973 Inorganic Photochrrnistry29th January-?rid FebruaryI973 Photochvmical Syri thrsis12th-16th February 195919th-ZSrd February 1973For furthrr details contact5th-9th February 1973 PhotobiologyPhotochemistry of DyrstuflsPhotochfwistry in thc Solid StatcDr. D. Phillips (A),1)epartinc:iit of Ch(-iiiistry,’The Univcmity,Southairiptori SO!) 6NH.BINDINGHave your back numbers of The Analyst boundin the standard binding case.Send the parts and the appropriate index(es)together with a remittance for f2.40 to:W. Heffer & Sons Ltd.Cambridge EnglandBOOKSM ON OG RAPHSREPRINTSorders for all publications ofthe Society (except journals)should be sent direct or througha bookseller to-THE SOCIETY FORANALYTICAL CHEMISTRYBook Department9/ I0 Savile Row,London, WIX IAx THE ANALYST [January, 1973ANALYTICAL CHEMISTRYSECTION MANAGERto join the Process Research Department of our General Chemicals Division at Widnes.Key tasks include thc development and application of analytical tccliiiiqucs to ;L witlc rangeof organic and inorganic compounds and liaison between I<cscarch ant1 I’rotluction units through-out the Division on matters relating to analytical methods.The man appointetl will be directlyrcsponsible to Mr C. Whalley, the current President of the Socicty for Analytic;iI Chemistrya n t l the Analytical Division of the Chemical Society.Candidates must have a sound knowledge of modern analytical procedures a n t 1 particularlyof instrumental methods. They must be able to rccognise the potcntinl ol thc Intwt tlcvclopnicntsi n analytical chemistry; some experience in managing ;t team o l gratluatcs mi(1 assistants iscsscntial.11 competitive starting salary is offered together with all the usual 1)cnclits of a I)rogressivecompany, including generous removal assistance where appropriatc.Applications, quoting reference LA.90, and giving details of ctlucation an(1 car(wr to (la tc,shoultl l)c ;ttltlressed t o :The Divisional Personnel ManagerLAPORTE INDUSTRIES LTDGeneral Chemicals DivisionMoorfield Road, Widnes, Lancs.WA8 OHESENIOR ANALYSTI<oche Products Limited have an opening for a Senior Analyst to be responsiblc for the analyticallaboratory work in their Dalry, Ayrshire factory. This laboratory provides qua1 ity controlinformation on raw materials and final products in synthetic vitamin and drug manufactures a tthis site ; it also offers some analytical service to the plant control antl investigation lahoratories.Applicrtnts must therefore have a t least fivc years’ postgraduate training ant1 experience in thisticltl (i.c.PhL, level) preferably in pharmaceutical chemistry, antl have hat1 experience in control0 1 staff. The person appointed will be required to take charge of a team o f some 16 assistants.ICoche l’roclucts Limited is a member of an international Swiss-bnsctl group of pharmaceuticala n t 1 chemical companies, and the conditions of service arc those to bc cxpcctctl from such anorganisation.Write i n confidence, quoting. rcfcrencc ’r J , for an application forln a n ( l further information, to theI )ircxtor o f Personnel a t :ROCHE PRODUCTS LIMITED15 MANCHESTER SQUARE LONDON W1M 6AJanuary, 19731 THE ANALYST xiBUREAU OF ANALYSEDSAMPLES LTD.NEW SAMPLESChemical StandardsNo. 384 Hycomax I11 Permanent Mag-net AlloyNo. 387 Nimonic 901 Alloy (12% Cr,60/, Mo) Also available asspectroscopic standard.No. 389 High Purity Magnesite stand-ardised for B,03No.390 High Tensile Brass (BS 2874CZ 114)announce the issue of the followingSpectroscopic StandardsNos. 666-670 Nodular Irons contain-ing increments of C, Si,Mn, S , Ni and MgFor further details please write to:-NEWHAM HALL, NEWBY,TS8 9EAor Telephone 0642 37216MI DD LES B ROU G HI TE ESSl D E, EN G LAN D.DECEN NlAL INDEXESTO THE ANALYST1906-1915t .. f2.101916-1925* . . f2.101936-1945* . . €2.101946-1955* . . €51956-1965" . . €6.75Bound in cloth boardst Paper boundObtainable fromThe Society for Analytical ChemistryBook Department9/10 Savile Row,London, WIX IAFSPECIALIST ABSTRACTJOURNALSpublished bySCIENCE AND TECHNOLOGY AGENCYAtomic Absorption and FlameEmission Spectroscopy AbstractsVol.5, 1973, bimonthly S24X-Ray Fluorescence SpectrometryAbstractsVol. 4, 1973, quarterly €24Thi n-Layer Chromatography AbstractsVol. 3, 1973, bimonthly S24Gas Chromatography-MassSpectrometry AbstractsVol. 4, 1973, quarterly $37Nuclear Magnetic ResonanceSpectrometry AbstractsVol. 3, 1973, bimonthly €30Laser-Raman Spectroscopy AbstractsVol. 2, 1973, quarterly €30X-Ray Diffraction AbstractsVol. 1-2, 1973, quarterly €30Neutron Activation Analysis AbstractsVol. 2-3, 1973, quarterly $30Electron Microscopy AbstractsVol. 1, 1973, quarterly S30Liquid Chromatography AbstractsVol. 1, 1973, quarterly €30Electron Spin Resonance SpectroscopyAbstractsVol.1, 1973, quarterly S30Sample copies on request from:SCIENCE AND TECHNOLOGY AGENCY,3 DYERS BUILDINGS, HOLBORN,LONDON, E.C.1, ENGLAND01-405 932xii SUMMARIES OF PAPERS I N THIS ISSUEThe Atomic-absorption Spectrophotometric Determination ofTotal Aluminium in Steel after its Dissolution in a Pressure BombA method is described for the determination of 0.001 to 0.14 per cent.of total aluminium in irons and steels, which does not involve fusion of anoxide residue. The iron or steel is completely dissolved by first subjectingit to conventional treatment with acid in an open beaker and then to attackby acid in a PTFE-lined bomb a t 200 "C. The oxide residue is completelydissolved by attack in the bomb with hydrochloric acid alone or with a mixtureof hydrochloric and hydrofluoric acids, the latter solvent being preferred.Iron(II1) is extracted from the solution of steel, 6 M in hydrochloric acid,with isobutyl methyl ketone and aluminium is then extracted from the aqueousphase buffered a t pH 4-7 with acetylacetone. The concentration of aluminiumin the organic phase is determined by atomic-absorption spectrophotometrywith a nitrous oxide - acetylene flame.Good results were obtained for thedetermination of aluminium in eleven standard irons and steels.J. B. HEADRIDGE and ALAN SOWERBUTTSDepartment of Chemistry, The University, Sheffield, S3 7HF.Analyst, 1973, 98, 57-64.[January, 1973A Study of the Variation with pH of the Solubility and Stabilityof Some Metal Ions at Low Concentrations in Aqueous Solution.Part IAtomic-absorption spectrophotometry has been used to determine traceamounts of metal ions in aqueous solution and to investigate the effect ofpH upon their stability, solubility and adsorption.It was found that for thefifteen metal ions investigated, namely those of aluminium, calcium, cobalt,chromium, copper, iron, magnesium, manganese, molybdenum, nickel, lead,strontium, titanium, vanadium and zinc, a pH of 1.5 or less was necessaryto ensure that all of these metal ions remained in solution a t low levels ofconcentration.A. E. SMITHImperial Chemical Industries Limited, Mond Division, Research and DevelopmentDepartment, Winnington Laboratory, Northwich, Cheshire, CW8 4D J.Analyst, 1973, 98, 65-68.Determination of Oxygen in Semiconductor Materials with aCryogenically Pumped Spark- source Mass SpectrometerA liquid helium cryogenic pump, which is mounted directly on to theion-source chamber of the mass spectrometer, is described.Base pressuresdown to about 8 x 10-10 torr are rapidly obtained within 1 hour of thecommencement of cryogenic pumping and pressures of about torr aremaintained during the analysis. The analytical blank for oxygen is shownto be directly dependent on the residual gas pressure in ths ion-sourcechamber and with liquid helium cryogenic pumping the blank for the deter-mination of oxygen in germanium is not more than 0.02 p.p.m.a. (parts permillion atomic).The quantitative performance of the method has been evaluated byusing germanium and silicon samples that had previously been analysedby infrared spectrophotometry.A mean standard deviation of 19 per cent.was obtained from measurements of oxygen present in silicon covering therange 11 to 35 p.p.m.a. The method has also been applied to the determinationof oxygen and carbon in indium phosphide, and satisfactory agreement a tthe 0-1 p.p.m.a. level has been found with results independently obtainedby gamma-photon activation analysis.J. B. CLEGG, I. G. GALE and E. J. MILLETTMullard Research Laboratories, Redhill, Surrey.Analyst, 1973, 98, 69-74January, 19731 THE ANALYST xiiiANNUAL REPORTSONANALYTICAL ATOMICSPECTROSCOPYVolume 1 , I W lTHIS comprehensive and critical reportof developments in analytical atomicspectroscopy has been compiled frommore than lo00 reports received fromworld-wide correspondents who areinternationally recognised authorities inthe field and who constitute the Editor-ial Board.In addition to surveyingdevelopments throughout the worldpublished in national or internationaljournals, a particular aim has been toinclude less widely accessible reportsfrom local, national and internationalsymposia and conferences concernedwith atomic spectroscopy.Volume 1 covers the year 1971.204 pagesPrice $5000Members of The Chemical Society may buypersonal copies at the special price of S.3-00.Obtainable from-THE SOCIETY FOR ANALYTICALCHEMISTRY,(Book Depart men t) ,9/10 Savile Row, London, W1X 1AFMONOGRAPHSFOR TEACHERSModernAnalyticalMethodsby D.BETTERIDGEand H. E. HALLAMModern Analytical Mefhods is one of TheChemical Society's series of paperbackmonographs which present concise andauthoritative accounts of selected welldefined topics in chemistry for those whoteach the subject at 'A' level and above andfor students of further and higher education.It discusses the principles underlying themost important methods of quantitative andqualitative analysis used today. Samplesfor analysis may arise from diverse sourcesand contain a variety of molecules orelements at various levels of concentration.Thus separation methods, organic reagents,nuclear, electrochemical, spectroscopic andtitrimetric methods are amongst those dealtwith in some detail. Within the bounds ofelementary algebra, equations are developedwhich show how the optimum conditions forthe application of a method may be deducedand conditional constants are usedthroughout.The numerous illustrationssupport the text by clarifying principles or byexemplifying important methods which aredealt with briefly because they do not involvenew principles.234pp 75 diagrams S2.00(CS Members €1.50)ISBN 0 85186 759 6Orders enclosing the appropriateremittance, should be sent to:The Publication Sales Oficer, TheChemical Society, Blackhorse Road,Letchworth, Herts SGS 1HN.For information on other titles in the serieswrite to: The Marketing Officer,The Chemical Society, Burlington House,London W1 V OBNxiv SUMMARIES OF PAPERS IN THIS ISSUEThe Determination of Oxygen and Carbon in Indium Phosphideby High-energy Gamma-photon ActivationHigh-energy gamma-photon activation has been used for the determina-tion of carbon and oxygen in single-crystal indium phosphide a t concentrationsin the range 0.01 to 0.1 p.p.m. m/m and less than 0.05 p.p.m. m/m, respectively.J. S. HISLOP, T. J. WEBBER and D. R. WILLIAMSAnalytical Sciences Division, Atomic Energy Research Establishment, Harwell,Didcot. Berkshire.Analyst, 1973, 98, 75-76.[January, 1973PARTICLE SIZE ANALYSIS1970THE Society for Analytical Chemistry has published i n this book all papers presented atthe Second Particle Size Analysis Conference, held in Bradford in September 1970, andthe full discussions on them.The 35 papers cover all aspects of research i n t o the subject, basically covering the4-year period since the f i r s t conference was held i n Loughborough i n 1966, and includeplenary lectures by the late Professor H. Heywood and by Professor K. Leschonski.The volume i s a companion to “Particle Size Analysis” - the report of the First Con-ference, also published by the Society.Pp. x + 430Price €7975Obtainable from-THE SOCIETY FOR ANALYTICAL CHEMISTRY,(Book Department),9/10 Savile Row, London, WIX IAFMembers of The Chemical Society may buy personal copies at the special price of f6.2

ISSN:0003-2654    

DOI:10.1039/AN97398BP005

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

Analyst, January, 1973, Vol. 98, $p. 9-18 9 The Analysis of Carbamate and Urea Herbicides by Fluorimetry of Their Dansylated Amine Moieties BY R. W. FREI, J. F. LAWRENCE AND D. S. LEGAY (Trace Analysis Research Centre, Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada) Carbamate and urea herbicides have been analysed by thin-layer chro- matography and fluorimetry of the 1-dimethylaminonaphthalene-5-sulphonyl (dansyl) derivatives of their aniline hydrolysis products. The herbicides were hydrolysed with 1 M sodium hydroxide solution at 80 "C for 40 minutes and then the liberated anilines and amines were extracted into hexane. Aliquots of the hexane solution were spotted on to a chromatographic plate and made to react in situ with a t least a ten-fold excess of dansyl chloride, which was spotted over them.Next, the plate was sprayed until moist with a 20 per cent. solution of triethanolamine in propan-2-01 and the spots were analysed fluorimetrically in situ. The hydrolysis and coupling reaction of the herbicides, fluorescence phenomena and chromatographic properties of the derivatives were investigated. Detection limits of 1 ng per spot taken at a 3: 1 signal to noise ratio and a reproducibility of 3 to 6 per cent. relative standard deviation can be expected and the calibration graphs arc linear up to 300 ng per spot. Natural water samples can be analysed to determine concentrations of herbicides of a few parts per billion (log) with high recoveries and without the necessity of a clean-up step. UREA and N-phenylcarbamate herbicides have been of considerable interest recently because of the advantages of their rapid degradation in the environment.Some of the more important discussions on their mode of action are li~ted.l-~ While the analysis of these compounds by direct gas - liquid chromatographic methods is the procedures require relatively mild gas - liquid chromatographic conditions that are not always practical when carrying out sample analyses. The clean-up problem in gas - liquid chromatography, which often consumes much time and effort, is also an unfavourable factor with this technique. Thin- layer chromatographic methods, which usually involve the formation of coloured derivatives by means of chromogenic spray reagent^,^ have also found wide use in the analysis of these types of corn pound^.^-^^ However, none of these methods is genuinely quantitative at the level of detection (a few nanograms) that is normally required for residue analysis.The combination of thin-layer chromatography with fluorescence offers a considerable advantage as it increases the sensitivity by 10 to 100 times. The determination of Sevin (carbaryl, 1-naphthyl N-methylcarbamate) as its naturally fluorescent hydrolysis product, 1-naphthol, by an in situ fluorimetric thin-layer chromatographic technique13 demonstrated the applicability of such an approach to quantitative pesticide analysis. This work has been extended to include the analysis of non-fluorescent pesticides and fluorigenic spray reagents were developed for the analysis of a number of organophosphate and carbamate insecti- cides.14-16 Although the spray techniques are rapid and simple, they are not selective, and are therefore more susceptible to interferences and give a sensitivity only about ten times greater than for the chromogenic spray procedures.An alternative to these spray methods is fluorigenic labelling, which involves the formation of fluorescent derivatives that are separated by thin-layer chromatography and quantitatively determined by use of fluori- metry in situ. This technique has the advantage that it does not require a spray reaction for the formation of the fluorescent spots, thus avoiding background irregularities caused by uneven spraying. The fluorigenic labelling of non-fluorescent compounds has found a great deal of use in biological and pharmaceutical analyses.Amino-acids, peptides and biogenic amines have @ SAC and the authors.10 FREI, LAWRENCE AND LEGAY: ANALYSIS OF CARBAMATE AND UREA [Analyst, Vol. 98 been fluorimetrically determined for more than a decade as their dansyl (1-dimethylamino- naphthalene-5-sulphonyl) derivatives following separation by thin-layer chromatography or high-voltage electrophoresis. Seiler and Wiechmannl' have recently reviewed progress in this field with a detailed discussion of the reactions of dansyl chloride with amines and phenols. Preliminary work on the analysis of carbamate insecticides by this fluorigenic labelling technique has been carried out by Frei and Lawrence,leJ9 who used dansyl chloride as the labelling reagent.This compound reacts with both amine and phenol hydrolysis products of N-methylcarbamates, resulting in two derivatives suitable for the determination of a few nanograms of carbamate insecticide residues. The reaction procedure has since been investigated20 and results indicate that complete formation of the dansyl derivatives of the carbamates takes place in less than 1 hour. The fluorescence behaviour of these derivatives was examined21 in order to determine the stability of the derivatives with time and to investigate the optimum conditions for their quantitative determination. The thin-layer chromatographic properties of the dansyl derivatives of a number of carbamates have also been studied for both one and two-dimensional separations.22 The application of such a method to the quantitative analysis of N-methylcarbarnates in natural water samples without using clean-up procedures has recently been reported.23 The use of dansyl chloride as a fluorigenic labelling reagent for herbicides in the carbamate and urea classes seems particularly promising and worthwhile because upon hydrolysis these compounds yield amines or anilines that can react with dansyl chloride to form highly fluorescent derivatives.The investigation of the dansyl labelling and quantitative analysis of these compounds is the subject of the present work. EXPERIMENTAL REAGENTS- The common and systematic names of the herbicides used in this work are given in Table I. Stock solutions of these compounds were prepared a t a concentration of 1 mg ml-1 in redistilled analytical-reagent grade dichloromethane. The labelling reagent consisted of a 0.2 per cent.solution of analytical-reagent grade dansyl chloride (1-dimethylaminonaphtha- lene-5-sulphonyl chloride, obtained from Mann Research Laboratories, New York, U.S.A.) in redistilled reagent grade acetone. Spray solutions consisting of 20 per cent. of triethanol- amine in propan-2-01 and 20 per cent. of paraffin oil in toluene were prepared. A 1 M solution of sodium hydroxide was used to hydrolyse the herbicides. Dansyl derivatives of aniline, 3-chloroaniline (obtained from Matheson Coleman and Bell, Norwood, Ohio, U.S.A.) and 3,4-dichloroaniline [obtained from BDH (Chemicals) Ltd., Poole, Dorset] were used as chromatographic standards for derivative identification.All other materials were of analytical-reagent grade quality. TABLE I COMMON AND SYSTEMATIC NAMES OF THE HERBICIDES USED Compound Systematic name Carbamates- IPC (propham) . . .. . . Isopropyl N-phenylcarbamate CIPC (chlorpropham) . . . . Isopropyl N-(3-chloro) phenylcarbamate Swep .. .. .. . . Methyl N-(3,4-dichloro)phenylcarbamate Barban .. .. .. . . 4-Chlorobut-2-yn-1-y1 N-(3-~hlorophenyl)carbamate Linuron . . .. .. . . N-Methyl-N-methoxy-N'- (3,4-dichloro) phcnylurea Diuron . . .. .. . . NN-Dimethyl-N'-(3,4-dichloro)phenylurea Maloran (chlorbromuron) . . N-Methyl-N-methoxy-N'-( 3-chloro-4-bromo)phenylurea Fluometuron . . .. . . NN-Dimethyl-"-( 3-trifluoromethy1)phenylurea Urea- REACTION PROCEDURE- Ten microlitres of a stock solution of the pesticide were placed in a 2-ml centrifuge tube and the solvent was evaporated in a water-bath at 40 "C.To the dry residue, 0.2 ml of sodium hydroxide solution was added and the tube was loosely stoppered and heated in a water-bath at 80 "C for 30 to 40 minutes. The tube was then cooled to room temperature,January, 19731 HERBICIDES BY FLUORIMETRY OF THEIR DANSYLATED AMINE MOIETIES 11 0.2 ml of hexane added and the tube shaken. The phases were permitted to separate and 10 p1 of the organic layer were spotted on to a 20 x 20-cm thin-layer plate at a point 2 cm from the bottom, 4p1 of the dansyl chloride solution being spotted 011 top of the sample spot, The plate was then kept covered with a clean glass sheet in the dark at room temperature for 60 minutes before elution with the chromatographic solvent.CHROMATOGRAPHY- The thin-layer plates were prepared with a Desaga (Heidelberg) thin-layer applicator by applying a slurry consisting of 30 g of silica gel G with calcium sulphate binder (obtained from Machery, Nagel and Co., Duren) and 60 ml of distilled water to the plates at a thickness of 250 pm. The plates were dried at 110 "C for 10 minutes prior to spotting, reaction and elution by the ascending-solvent technique at room temperature in the dark. The eluting solvent mixture was benzene - triethylamine - acetone (75 + 24 + l ) , freshly prepared before use. The plates were developed to a 13-cm solvent front, dried in a stream of cool air, sprayed until moist with one of the spray solutions mentioned above (to enhance and stabilise the fluorescence of the derivatives) and then dried again to remove the propan-2-01 or toluene from the plate.INSTRUMENTAL ANALYSIS- A Zeiss Chromatogram Spectrophotometer PMQ I1 was used in the fluorescence mode for all quantitative measurements. A 365-nm Zeiss filter was used with a mercury lamp as the excitation source and the fluorescence was monitored with an emission slit width of 0.3 mm for all work. Spectra and scanning peaks were recorded on a Honeywell Electronik 194 strip-chart recorder and the peak areas were integrated electronically with an Autolab Vidar 6300 digital integrator. Fluorescence spectra were also recorded in sita with an Aminco-Bowman Spectrophotofluorometer equipped with the thin-layer scanning attach- ment. For the qualitative, visual examination of the developed chromatographic plates, a Camag Universal ultraviolet lamp was used at 350 nm.ANALYSIS OF WATER SAMPLES- Three 500-ml samples of local lake water were spiked to give 10 p.p.b. (parts per 109) each of swep and IPC. The samples and a blank were extracted twice with 50-ml portions of dichloromethane. The combined extracts for each sample were reduced to 1 ml by rotary vacuum evaporation a t room temperature and subsequently transferred to 2-ml centrifuge tubes and evaporated to dryness in a water-bath at 40 "C under a gentle stream of air. To rinse down the sides of the tube and to re-dissolve the residue, 1 or 2 drops of acetone were added followed by 0 6 m l of sodium hydroxide solution. The contents were then treated as described under Reaction procedure.Each sample was spotted on to the thin-layer chromatographic plates in triplicate and compared with standards run on the same plate. Time/minutes Fig. 1. Rates of formation of aniline in 1 M NaOH solution for A, swep; B, linuron; and C, IPC a t 200ng each12 FREI, LAWRENCE AND LEGAY: ANALYSIS OF CARBAMATE AND UREA [AIZUhSt, VOl. 98 RESULTS AND DISCUSSION REACTION CONDITIONS- A 1 M solution of sodium hydroxide was found to be most suitable for the liberation of the anilines from the herbicides. The relative rates of aniline formation of a representative group of herbicides with a 1 M solution of sodium hydroxide can be seen in Fig. 1. These were determined by forming the dansyl derivative of the free anilines in the hydrolysis mixtures and fluorimetrically determining the amount of aniline liberated as a function of time.The hydrolysis is essentially complete in 60 minutes for most of the herbicides studied. A reaction scheme is shown in Fig. 2 for the formation of the derivatives of a typical urea and an N-phenylcarbamate. CA RBAM ATES (Cl PC) 1. NH-C-OCH ‘CH3 20H- ___t UREAS (LINURON) 1. CIL I c1 S02CI NH2 I + (CH3I2CHOH + Cog- CI NH2 I 9 CHsNHOCH3 + COi- CI CI Fig. 2. Dansylation of CIPC and linuron: 1, hydrolysis of the herbicides to give the free chloroaniline; and 2, reaction of the dansyl chloride onsilica gel G layer with the aniline to form the dansyl derivativeJanuary, 19731 HERBICIDES BY FLUORIMETRY OF THEIR DANSYLATED AMINE MOIETIES 13 = A = B I 80 120 Time/minutes Amount of dansyl chloride/pg Fig. 3.Rate of reaction of the free anilines of swep (A) and IPC (B) with a ten-fold excess of dansyl chloride on the thin- layer plates a t room temperature in the dark Fig. 4. Effect of the amount of dansyl chloride on the forma- tion of the aniline derivative of 260 ng of IPC after reaction for 60 minutes on the plate The coupling of the anilines with dansyl chloride takes place more rapidly directly on the thin-layer plates than in solution. In addition, when effecting the reaction in solution it is necessary to reduce the pH of the hydrolysis solution to 9 to 10 before the dansylation reaction will form derivatives in any significant yield. The rate of reaction to form anilines directly on the plate is shown in Fig. 3. About 40 to 50 minutes are required for complete formation of the derivatives; the N-phenylcarbamates form only the aniline derivative. For the N-phenylureas that contain a methoxy substituent on the methylamine moiety of the molecules, only the aniline derivative was found in the chromatogram.The ureas that contain a methylamine or dimethylamine moiety as well as an aniline group (ie., diuron and fluometuron in Table I) form dansyl derivatives with both hydrolysis products. The amount of dansyl chloride spotted over the hydrolysed herbicides was found to influence the results. Fig. 4 shows the effect of varying the dansyl chloride concentration on the yield of dansyl derivative for IPC (200 ng). An excess (about ten-fold) of reagent is required for complete conversion into the dansyl derivative.The dansyl chloride is in solution in acetone, which is an extremely polar liquid and washes the aniline and reagent into the 0 Second - Fig. 6 . Two-dimensional chromato- gram of the dansylation spray reaction with 200ng of hydrolysed swep. The first dimension was eluted with benzene - acetone (96 + 4) and the second with benzene - triethylamine - acetone (75 + 24 + 1). The cross-hatched area is the dansyl spray14 [Analyst, vol. 98 same zone, providing close contact between the two reactants. At the 200ng of herbicide level, the diameter of the spot is not important. However, at low concentrations it is preferable to keep the spots as small as possible for maximum contact between the labelling reagent and aniline. Also, small spots are much more desirable for chromatography because better separations can be achieved.An interesting use of the in situ reaction between the anilines and dansyl chloride is in the elimination, by two-dimensional chromatography, of any interferences that may be encountered. The anilines can be eluted in one direction before dansylation and then sprayed with dansyl chloride solution, as shown in Fig. 5, and eluted in the second dimension after dansylation is complete. To avoid any interference from excess of dansyl chloride, which appears as a broad band at or near the solvent front in the second elution, the plate can be sprayed with dilute base (0.1 M sodium hydroxide solution, for example) before the second elution. The dansyl-OH (1-dimethylaminonaphthalene-5-sulphonic acid) formed does not move in the solvent systems used.As a more polar solvent system is required for elution of the anilines the first dimensional run will remove ‘many interferences that would cause problems in the analysis of the dansyl derivatives in the second elution. In Fig. 5 a two- dimensional separation of a sample of swep is shown. FLUORESCENCE PHENOMENA- The spectra for the dansylated aniline derivatives from IPC and linuron are shown in Fig. 6, while the excitation and emission maxima for the dansylated aniline derivatives of all the herbicides studied by use of the two sprays are given in Table 11. The paraffin oil spray caused a bathochromic shift in the emission maxima of the derivatives as compared with the triethanolamine spray. The spectra of all derivatives are similar for a given spray.This last property is useful if more than one derivative is present as all the derivatives can be scanned under the same instrumental conditions. On the other hand, the fluorescence spectrum is consequently not very useful as a qualitative indication of an aniline. FREI, LAWRENCE AND LEGAY: ANALYSIS OF CARBAMATE AND UREA Excitation Emission 300 400 500 600 700 Wave I en g t h I n rn Fig. 6. Fluorescence spectra recorded on the hminco-Bowman instrument with a 0.2-mm excita- tion slit of the dansylated anilines of IPC and linuron on silica gel G : broken line, IPC; and solid line, linuron The effects of constant irradiation by the ultraviolet excitation light of the Zeiss and Aminco-Bowman instruments on the fluorescence intensities of the aniline derivatives are shown in Fig.7, with use of triethanolamine as the stabilising spray. A greater degree of decomposition is observed with the Zeiss than with the Aminco-Bowman ultraviolet source. This occurrence was also noticed in previous work with N-methylcarbamate derivatives.21 The triethanolamine spray is recommended (Fig. 8) for increased stability of the spots under ultraviolet light and daylight conditions. The results of multiple scanning of the dansylated aniline derivative of IPC are shown in Fig. 8 and they indicate that scanning itself causesJanuary, 19731 HERBICIDES BY FLUORIMETRY OF THEIR DANSYLATED AMINE MOIETIES 15 TABLE I1 FLUORESCENCE MAXIMA OF THE DANSYLATED ANILINE DERIVATIVES OF THE HERBICIDES STUDIED Compound Spray* Barban, CIPC .. .. TEA IPC .. .. .. TEA Swep, linuron, diuron . . TEA Fluometuron . . .. TEA Maloran . . .. .. TEA PO PO PO PO PO Aminco-Bowman equipment$ excitation and emission Zeiss equipment emission? wavelength/nm wavelengthslnm 626 360, 612 632 362, 632 638 366, 636 635 366, 636 626 360,610 636 370, 636 620 360, 606 632 365, 628 610 366, 606 536 366, 636 * TEA = 20 per cent. V/V triethanolamine in propan-2-01; t A 0.3-mm emission slit width and a 365-nm excitation filter were used. $ A 0.2-mm excitation slit width was used. PO = 20 per cent. V / V paraffin oil in toluene. most of the degradation of the spots. However, under normal conditions only one scan per spot is required for a quantitative determination. Storage of the plates covered and in the dark increases the stability of the derivatives by more than 4 weeks with no detrimental effects21 A 15 30 45 Time/m i nu tes Fig.7. Effect of ultraviolet excita- tion light on the fluorescence intensities of the aniline derivatives. Curves A to D were obtained by using the Aminco- Bowman instrument and curves E to H were recorded on the Zeiss instrument. Triethanolamine was used as the spray. Curves A and E,-linuron, diuron, maloran and swep; B and F, CIPC and barban; C and HI fluometuron; and D and G, IPC CHROMATOGRAPHY- A typical thin-layer chromatogram of the dansyl derivatives of a number of herbicides is shown in Fig. 9. The R, values decrease with the increase in number of halogen sub- stituents on the anilines (see also Table I). The aniline derivatives are much more polar than the phenol derivatives of N-methylcarbamates,22 therefore separation of these two classes16 FREI, LAWRENCE AND LEGAY: ANALYSIS OF CARBAMATE AND UREA [AndySt, VOl.98 Number of scans Fig. 8. Influence of scanning on the fluores- cence intensity of the dansylated aniline derivative of IPC; curve A, triethanolamine spray; and B, paraffin oil spray as dansyl derivatives is easy. The dansyl-OH remains at the origin in the solvent systems investigated and hence does not interfere in quantitative analysis. The excess of dansyl chloride that may still be present travels well above the dansylated aniline derivatives and does not interfere. Solvent front 1 1 ; . ? t , 3 4 5 Dimethylamine - derivatives -Start Fig. 9. Thin-layer chromatogram of the dansyl derivatives of five herbi- cides on silica gel G developed with benzene - triethylamine - acetone (76 + meturon; 4, diuron; and 5, linuron.Dimethylamine derivatives are also formed in the reactions with fluo- meturon and diuron 24 + 1 ) ; 1, IPC; 2, CIPC; 3, flue- QUANTITATIVE ANALYSIS- The reproducibility of the method was investigated for linuron determinations and found to be 4.6 per cent. average relative standard deviation with a range of standard deviations of 2.3 to 6-8 per cent., determined on concentrations ranging from 40 to 400ng per spot, with at least four plates for each concentration and a minimum of seven spots per plate (Table 111). The results obtained for linuron can be assumed to be repre- sentative of the herbicides studied because they are structurally similar and the reaction and chromatography stages are the same.January, 19731 HERBICIDES BY FLUORIMETRY OF THEIR DANSYLATED AMINI: MOIETIES 17 Visual detection limits under the ultraviolet lamp were about 5 to long per spot for all compounds studied, while 1 ng per spot or less can be determined with the Zeiss equipment at a 3: 1 signal to noise ratio under optimum conditions. The calibration graphs pass through the origin and are linear up to 200 to 300ng per spot with essentially no change in slope over a 30-minute period when using either spray.TABLE I11 REPRODUCIBILITY STUDIES ON THE DANSYL LABELLING OF LINURON Excitation wavelength 350 nm; emission wavelength 510 nm; emission slit width 0.3 mm Concentration, Relative standard deviation, Average, Signal to noise ng per spot per cent.* per cent.ratio 400 5-3, 4.6, 5.3, 3-8 4.7 240 200 2.3, 4.8, 5.2, 3.2 3.9 200 80 4.5, 6.2, 4.6, 5.3 6.1 05 40 4.1, 6.8, 4.1, 5.1 5-0 60 * Four plates were analysed for each concentration with a minimum of 7 spots per plate. The analysis of local lake water samples spiked with swep and IPC was carried out with little interference from co-extractives. A sample containing only swep at a concentration of 10 p.p.b. gave 102 & 5 per cent. recovery, while samples containing both swep and IPC gave recoveries of 98 In Fig. 10, a thin-layer chromatogram and fluorescence scan of a swep determination are shown. 5 and 84 & 4 per cent., respectively. 0 0 2 t t i I i l Scan- Fig. 10. Chromatogram and fluorescence scan for a 10 p.p.b.swep-containing water sample extract after dansyl chloride labelling ; 1, dansyl-OH and impurity at the origin; 2, dansylated aniline (swep); 3, excess of dansyl chloride; and 4, solvent front and impurity. A Zeiss 365-nm excitation filter was used and the fluorescence monitored at 525 nm with a 0.3-mm slit width. Scanning speed 15 cm min-l; and chart speed 40 cm min-l CONCLUSIONS The dansyl labelling reaction of the anilines resulting from the hydrolysis of the herbicides on thin-layer plates's simple, rapid and reproducible. The method is suitable as an alternative technique to gas - liquid chromatography or as a check method for gas - liquid chromato- graphic results as similar detection limits and precisions can be obtained.24 The quantitative determination of two herbicides (at concentrations of 10 pg 1-l) in natural water samples was successfully carried out without necessitating a clean-up step.18 FREI, LAWRENCE AND LEGAY The labelling reaction with dansyl chloride carried out directly on thin-layer chromato- graphic plates has been found to be applicable to other non-volatile amines, including amino-acids (Lawrence, J.F., and Frei, R. W., unpublished work). This work was supported by funds provided by a Public Health Research Grant (Project No. 602-7-141) of the National Health Grants Program. Additional assistance was provided by the National Research Council of Canada. D. S. LeGay is grateful for the award of a Summer Research Scholarship of the Atlantic Provinces Inter-University Committee on the Sciences (A.P.I.C.S.). 1.2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. REFERENCES Moreland, D. E., A . Rev. PI. Physiol., 1965, 18, 365. Bleidner, W. E., J . Agric. Fd CJzem., 1954, 2, 682. Hilton, H. W., and Nomura, N., Weed.Res., 1964, 4, 216. Sheets, T. J., and Harris, C. I., Residue Rev., 1966, 11, 119. Cohen, I. C., and Wheals, B. B., J . Chromat., 1969, 43, 233. McKone, C. E., Ibid., 1969, 44, 60. Yip, G., J . Ass. Of. Analyt. Chem., 1971, 54, 327. Katz, S. E., and Strusz, R. E., J . Agric. Fd Chem., 1969, 17, 1409. Abbott, D. C., Blake, K. W., Tarrant, K. R., and Thomson, J., J . Chromat., 1967, 30, 130. &ha, F., and Kljajic, R., Ibid., 1969, 40, 304. Skrinde, R. T., J . Wat. Pollut. Control Fed., 1970, 42, 863. Hance, R. J., J . Chromat., 1969, 44, 419. Frei, R. W. , Lawrence, J. F., and Belliveau, P. E. , 2. analyt. Chem. , 1971, 254, 271. Mallet, V., and Frei, R. W., J . Chromat., 1971, 54, 251. Belliveau, P. E., and Frei, R. W., Chromatographia, 1971, 4, 189. Belliveau, P. E., Mallet, V., and Frei, R. W., J . Chromat., 1970, 48, 478. Seiler, N., and Wiechmann, M., in Niederwieser, A., and Paraki, G., Editors, “Progress in Thin-layer Chromatography and Related Methods,” Volume 1, Ann Arbor Science Publishers, Ann Arbor, Mich., 1970, p. 94. Frei, R. W., and Lawrence, J. F., in “I.U.P.A.C. Pesticide Congress, Tel Aviv, Israel, February 1971, Congress Proceedings,” Gordon and Breach, London and New York, 1971, p. 193. Lawrence, J. F., and Frei, R. W., Int. J . Envir. Analyt. Chem., 1972, 1, 317. Frei, R. W., and Lawrence, J. F., J . Chromat., 1971, 61, 174. Lawrence, J. F., and Frei, R. W., Ibid., 1972, 66, 93. Williams, I. H., Residue Rev., 1971, 38, 1. , J , Ibid., 1972, 66, 295. , Ibid., 1972, 67, 87. -- -- Received January 3rd. 1972 Accepted July 24th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800009

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

Analyst, January, 1973, Vol. 98, PP. 19-24 19 The Determination of Malathion and Dichlorvos Residues in Grain REPORT BY THE PANEL ON MALATHION AND DICHLORVOS RESIDUES IN GRAIN OF THE COMMITTEE FOR ANALYTICAL METHODS FOR RESIDUES OF PESTICIDES AND VETERINARY PRODUCTS IN FOODSTUFFS OF THE MINISTRY OF AGRICULTURE, FISHERIES AND FOOD THE Panel was formed in 1969 by the Committee for Analytical Methods for Residues of Pesticides and Veterinary Products in Foodstuffs to study gas - liquid chromatographic methods for the determination of malathion and dichlorvos residues in stored grain. At that time, the Codex Committee on Pesticide Residues had under review a colorimetric method for the determination of malathion residues, which was evaluated by a United Kingdom panel some years ag0.l The Committee for Analytical Methods considered it advisable to establish a gas - liquid chromatographic method that would be more specific and up to date and might effectively replace the colorimetric method.As dichlorvos is being increasingly used in a similar way to malathion on stored grain, it was included in the study. Members of the Committee themselves, or deputed staff from their laboratories, acted as a nucleus of the Panel while representation was invited, and accepted, from the British Agrochemicals Association, the Association of Public Analysts, the Society for Analytical Chemistry and the (then) Pest Infestation Laboratory so as to co-ordinate the work of various interested laboratories in the United Kingdom. The members of the panel are listed in Appendix 111.The terms of reference of the Panel were- “To establish by collaborative study a gas - liquid chromatographic method for residues of malathion and dichlorvos in grain, bearing in mind its application to organophosphorus compounds in other commodities.” The residues limits under consideration for raw grain by the Codex Committee were 8 p.p.m. for malathion and 2 p.p.m. for dichlorvos. Some relevant literature on residue analysis was reviewed by the Panel. Two methods (at that time unpublished) particularly interested the Panel: those of Elgar, Marlow and Mathews2 and Crisp and Tarrant.3 In the method of Elgar et aZ., the powdered grain, moistened with water, is macerated with dichloromethane. The filtered extract is then steam distilled and the distillate extracted and examined by gas - liquid chromatography by using, for preference, a flame-photometric detector (clean-up is not so critical with grain when using this detector).In the method of Crisp and Tarrant, the grain is macerated with three successive portions of methanol and the extract is concentrated. After chromato- graphy on activated charcoal, the eluate is examined by gas - liquid chromatography by use of a phosphorus-sensitive thermionic detector (the initial chromatography serves to remove spurious peaks from the methanol used in extracting the grain). Initial individual investi- gation of these two methods by laboratories showed neither to be completely satisfactory in the hands of workers not already familiar with the methods. The main investigations of the Panel concerned the choice of solvent for extracting malathion and dichlorvos from grain.When spiking grain for this purpose, the Panel con- sidered that, in order to test the method or solvent, after the addition of small amounts of a solution of pesticide to the grain, the flask containing the ground grain and pesticide should be stoppered and allowed to stand for at least 24 hours in a deep-freeze. In this way, the pesticide would be more likely to be absorbed into the grain and therefore correspond more closely to a treated commercial grain than if the sample were analysed immediately after the addition of the pesticide. Ethyl a ~ e t a t e , ~ acetonitrile, dichloromethane - water and acetone were all tested collaboratively as extraction solvents for spiked samples (see Appendix 11) by blending, filtration and gas - liquid chromatography (i.e., without clean-up).However, usually none of these procedures was as effective as methanolic extraction without clean-up (see Table I). Although the recovery of malathion was usually satisfactory, that of dichlorvos 0 SAC; Crown Copyright Reserved.20 PANEL ON MALATHION AND DICHLORVOS RESIDUES IN GRAIN : [Analyst, Vol. 98 TABLE I RECOVERY OF MALATHION AND DICHLORVOS FROM WHEAT WHEN USING SOLVENTS OTHER THAN METHANOL Level of pesticides on Solvent grain, p.p.m. Ethyl acetate . . 0.1 to 0.5 Acetonitrile , . 0.1 to 0-5 Dichloromethane - water, 2 + 1 . . 0-2 to 1-0 Acetone . . . . 0.2 to 1.0 Mean recovery of Recovery of dichlorvos individual determinations relative to methanolic (spiked samples) per cent.extraction (range arid M a e v o s determinations) per cent. mean of individual 90 34 - 71 32 - - 33 to 123, mean 69 83 60 25 to 97, mean 56 - was inadequate in solvents other than methanol. Moisture content affects the extent of recovery of dichlorvos. Comparisons of the extraction of dichlorvos, from a reference sample of wheat treated in a manner similar to that used commercially, by dichloromethane - water, acetone and methanol axe given in Table 11. TABLE I1 COMPARISON OF RECOVERY OF DICHLORVOS FROM TREATED WHEAT BY USING DICHLOROMETHANE - WATER, ACETONE AND METHANOL Laboratory 1 2 3 4 5 6 7 8 9 Mean recovery of pesticide compared with methanolic extraction, per cent. r > Dichloromethane - water Acetone 84 16 65 38 70 30 75 41 79 29 35 39 33 64 57 One determination 97 84) only 14 A Absolute value of methanolic extraction, p.p.m.0.22 0.48 0.19 0.36 0.49 0.82 0.56 0.57 - The actual level of dichlorvos found by methanolic extraction varied with the time of storage before analysis but the ratio of dichloromethane - water or acetone extraction to methanolic extraction would be expected to be independent of the actual level present. Values for this ratio are variable but show clearly that methanol is the best solvent. A collaborative study was undertaken so as to obtain results for the reproducibility of the method with dichlorvos, in which duplicate analyses were made on five 100-g sub-samples on a reference sample of wheat treated in the normal commercial fashion; results are given in Table 111.Again, the actual value varied with the time between exposure and analysis. The standard deviation of individual determinations on a 100-g sub-sample in the range 0.07 to 0.30 pep.". was 0.013 p.p.m. The lowest level of detection, by a single determination, would therefore not be less than 0.02 p.p.m. The recovery of malathion from wheat that was spiked and stored for 24 hours in a closed container is shown in Table IV. Members used their own discretion in choosing the column and detector for the gas- liquid chromatography. A limited study by members showed that the method, which was used successfully on wheat, was also applicable to the determination of residues of these pesticides in barley, sorghum, maize and flour. Very low levels of pesticide (less than 0.06 p.p.m.) may require clean-up.Other organophosphorus pesticides are unlikely to interfere in the determination of dichlorvos during the gas - liquid chromatographic stage. Columns on which the pesticides indicated may interfere in the determination of malathion are: 1.3 per cent. Apiezon L onJanuary, 19731 THE DETERMINATION OF MALATHION AND DICHLORVOS RESIDUES IN GRAIN 21 TABLE I11 DETERMINATIONS OF DICHLORVOS ON TREATED WHEAT Duplicate determinations on 20 g of wheat taken from five 100-g amounts from a common sample Laboratory 1 2 3* 4 5 6 7 8 9 First extract . . Duplicate . . Mean .. .. First extract . . Duplicate , . Mean . . .. First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean .... First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean . . .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. * . .. .. .. .. .. .. Dichlorvos found, p.p.m. r A 0-27 0.28 0.28 0.17 0.14 0.16 0.072 0.072 0.072 0.20 0.21 0.21 0.2 1 0.24 0.23 0.30 0.30 0.30 0.15 0-17 0.16 0.24 0.25 0-25 0.18 0.16 0-17 0.079 0.078 0.079 0.23 0.23 0.23 0.24 0.26 0.25 0-30 0.29 0.30 0.18 0-17 0-18 0.23 0.25 0.24 0.14 0.16 0.15 0-067 0.069 0.068 0.21 0.22 0.22 0.27 0.28 0.28 0-30 0.30 0.30 0.16 0.16 0.16 0.26 0.25 0.26 0.18 0.14 0.16 0.078 0.073 0.076 0.20 0.19 0.20 0.25 0.27 0.26 0.29 0.30 0-30 0.18 0.19 0.19 0.29 0.30 0.28 0.28 0.28 0.37 0.36 0.35 0.29 0.34 0.32 0.32 0-29 0.29 0.29 0.29 0.27 0.28 0.28 0.28 0.28 0.29 0.29 0.29 1 0.24 0.24 0.24 0.14 0.16 0.15 0.069 0.07 1 0.070 0.26 0.22 0.24 0.26 0.27 0.27 0.3 1 0.3 1 0.3 1 0.16 0.19 0.18 0.28 0.3 1 0.30 0.29 0.29 0.29 * Maintained a t room temperature for 1 week before analysis instead of in a deep-freeze.Chromosorb G, formothion (Rm 99), aspon (Rm 103) and paraoxon (Rm 108); 1.3 per cent. butane-1,4-diol succinate on Chromosorb G, schradan (Rm 98), bromophos (Rm 100) and malaoxon (Rm 108); and 20 per cent. DC 200 on Gas-Chrom Q, paraoxon (Rm 94) and fenthion (Rm 107) [Rm represents the position of a peak from the time of injection relative to that of malathion (malathion = loo)]. Malaoxon is not an important metabolite of malathion on grain as the degradation of malathion proceeds by hydrolysis rather than by oxidation.Analytical procedures for dichloroacetaldehyde, a decomposition product of dichlorvos, are so markedly different from those for dichlorvos itself that they could not be included in the work reported here. TABLE I V RECOVERY OF MALATHION FROM SPIKED WHEAT Theore tical Laboratory Recovery, per cent. concentration, p.p.m. 1 87 0.7 76 0.1 2 98 0.1 87 0.5 3 82, 82, 87 0.1 85, 82, 72 0.5 0.1 0.5 The Panel therefore recommends the method given in Appendix I for the determination of malathion and dichlorvos residues in grain.22 PANEL ON MALATHION AND DICHLORVOS RESIDUES IN GRAIN: [Analyst, Vol. 98 Appendix I RECOMMENDED METHOD FOR DETERMINING RESIDUES OF MALATHION AND DICHLORVOS IN GRAIN APPARATUS- Low-speed grinder.High-speed homogeniser. Buchner funnel with a sintered-glass plate-The plate was 6 to 10 cm in internal diameter. Buchner $ask, 250 or 500 ml capacity. Suction pump. Standard flasks. Gas - liquid chromatographic apparatus with detector suitable for determining organophos- phorus compounds. REAGENTS- Methanol-Analytical-reagent grade material, screened for its blank value, was used. A cetone-Analytical-reagent grade. Dichlorvos and malathion-Material of known purity, preferably of analytical-standard grade, was used. METHOD When received, store the samples in a deep-freeze (at -20 "C). EXTRACTION- Grind the undried grain to a powder (care must be taken not to overheat the grain, otherwise losses may occur). Take a 20-g sample for analysis and homogenise it with 40 ml of methanol for 2 minutes.Filter the homogenate through the sintered-glass Buchner funnel by use of a partial vacuum. Transfer the deposit back into the homogeniser and wash it with two 30-ml portions of methanol for 30 s each, refiltering each time. Evaporate the combined filtrates to about 2 ml a t a temperature below 36 "C (otherwise dichlorvos may be lost) and under a partial vacuum. Dilute the concentrate to a suitable volume with acetone, maintaining the same ratio of acetone to methanol in all solutions and in standards so as to avoid inaccurate results. [A dilute solution of dichlorvos in acetone (1 pg ml-1) can be stored for at least 1 week in a refrigerator without any detectable change in its concentration.] Examine the solutions by gas - liquid chromatography.GAS - LIQUID CHROMATOGRAPHY- Most of the work reported above required the use of a phosphorus-sensitive thermionic detector but a flame-photometric detector was also used. The following columns and conditions have been used and found to be satisfactory. (i) Phenyldiethanolamine succinate (3 per cent.) on 100 to 120-mesh Diatomite CQ in a 0.9 m x 6 mm 0.d. glass column operated at 175 "C for dichlorvos and 200 "C for malathion. Retention times were about 3 and 5 minutes, respectively, with a nitrogen gas flow-rate of 30 rnl min-l, a hydrogen flow-rate of 20 ml min-l and an air flow-rate of 350 ml min-l. (ii) Phenyldiethanolamine succinate (10 per cent.) on Gas-Chrom Q in a 1.5 m x 6 mm 0.d. glass column operated at 180 "C. Retention times were 2 minutes for dichlorvos and about 30 minutes for malathion with a nitrogen gas flow-rate of 27 ml min-l, a hydrogen flow-rate of 30 ml min-1 and an air flow-rate of 500 ml min-l.(iii) Phenyldiethanolamine succinate (4 per cent.) on Chromosorb G in a 1.5 m x 6 mm 0.d. glass column operated at 180 "C. The retention time of dichlorvos was 3 minutes with a nitrogen gas flow-rate of 40 ml min-l, a hydrogen flow-rate of 200 ml min-l, an air flow-rate of 32 ml min-l and an oxygen flow-rate of 22 ml min-l. (iv) Yhenyldiethanolamine succinate (10 per cent.) on 100 to 120-mesh Gas-Chrom Q in a 1 m x 3 mm i.d. glass column operated at 140 "C. The retention time for dichlorvos was 2 minutes with a nitrogen gas flow-rate of lOOmlmin-l, a hydrogen flow-rate of 800 ml min-1 and an oxygen flow-rate of 100 ml min-l.With a 4 per cent. phenyldiethanol- amine succinate stationary phase at a temperature of 190 "C, malathion can be conveniently determined.January, 19731 THE DETERMINATION OF MALATHION AND DICHLORVOS RESIDUES IN GRAIN 23 ( v ) Butane-1,4-diol succinate (1.3 per cent.) on acid-washed silanised 100 to 120-mesh Chromosorb G in a 1.5 m x 3 mm i.d. glass column operated at 175 "C for dichlorvos and 220 "C for malathion. (vi) Butane-l,4-diol succinate (1.3 per cent.) on Chromosorb G in a 1.5 m x 6 mm 0.d. glass column operated at 200 "C. The retention time for malathion was 4 minutes with a nitrogen gas flow-rate of 28 ml min-1, a hydrogen flow-rate of 30 ml min-l and an air flow-rate of 500 ml min-1.(vii) Butane-1,4-diol succinate (1.3 per cent.) on silanised 60 to 80-mesh Chromosorb G in a 1.2 m x 4 mm i.d. stainless-steel column operated a t 170 "C for dichlorvos. The retention time for dichlorvos was 1.2 minutes with a nitrogen gas flow-rate of 70 ml min-l, a hydrogen flow-rate of 28 ml min-1 and an air flow-rate of 340 ml min-l. (viii) Diethyleneglycol succinate (6.4 per cent.) on 100 to 120-mesh Aeropak 30 in a 0-4 m x 3 mm 0.d. glass column operated at 135 "C for dichlorvos and 215 "C for malathion. Retention times were 1.7 minutes and 2.1 minutes, respectively, with a nitrogen gas flow-rate of about 15mlmin-1, a hydrogen flow-rate of 15mlmin-l and an air flow-rate of about 170 ml min-l. ( i x ) Diethyleneglycol succinate (5 per cent.) on 100 to 120-mesh Chromosorb W in a 1.6 m x 4 mm id.glass column operated at 160 "C for dichlorvos and 230 "C for malathion. Retention times were 6.3 and 9.5 minutes, respectively, with a nitrogen gas flow-rate of 30 ml min-l, a hydrogen flow-rate of 15 to 25 ml min-1 and an air flow-rate of 400 ml min-l. (x) OV-1 (2 per cent.) on 100 to 120-mesh Diatomite CQ in a 0.9 m x 6 mm 0.d. glass column operated at 120 "C for dichlorvos and 150 "C for malathion. Retention times were 3 and 4 minutes, respectively, with a nitrogen gas flow-rate of 30mlmin-1, a hydrogen flow-rate of 20mlmin-l and an air flow-rate of 350mlmin-l. ( x i ) GE XE-60 (2 per cent.) on 100 to 120-mesh Gas-Chrom Q in a 0.9 m x 6 mm 0.d. glass column operated at 120 "C for dichlorvos and 180 "C for malathion.The nitrogen gas flow-rate was 30 ml min-1, the hydrogen flow-rate 110 ml min-l and the oxygen flow-rate 30 ml min-l. (xii) GE XE-60 (3 per cent.) on 100 to 120-mesh Gas-Chrom Q in a 1.5 m x 4 mm i.d. glass column operated at 140 "C for dichlorvos and 195 "C for malathion. The nitrogen gas flow-rate was 30mlmin-1, the hydrogen flow-rate 15 to 25ml min-l and the air flow-rate 400 ml min-l. (xiii) GE XE-60 (2.5 per cent.) with 0.25 per cent. Epikote 1001 on 80 to 100-mesh Gas-Chrom Q in a 1.5 m x 6 mm 0.d. glass column operated a t 178 "C for dichlorvos and 200 "C for malathion. Retention times were 0.6 minutes and 3 minutes, respectively, with a nitrogen gas flow-rate of 25mlmin-1, a hydrogen flow-rate of 24mlmin-l and an air flow-rate of 400 ml min-l.(xiv) GE XE-60 (2 per cent.) and 0-2 per cent. Epikote 1001 on 100 to 120-mesh Aero- pak 30 in a 1.5 m x 3 mm 0.d. glass column at 135 "C for dichlorvos and 215 "C for malathion. Retention times were 1.4 minutes for dichlorvos and 2.1 minutes for malathion, with a nitrogen gas flow-rate of 15 ml min-l, a hydrogen flow-rate of 15 ml min-l and an air flow-rate of 170 ml min-l. (xv) DC 200 (5 per cent.) on 70 to SO-mesh Aeropak 30 in a 1.5 m x 3 mm 0.d. stainless- steel column at 165 "C for dichlorvos and 230 "C for malathion. The nitrogen gas flow-rate was 25 ml min-l, the hydrogen flow-rate 15 ml min-l and the air flow-rate 200 ml min-l. The retention time for dichlorvos was 2.8 minutes. (mi) SE-30 (5 per cent.) on 70 to 80-mesh Aeropak 30 in a 1.5 m x 3 mm 0.d.stainless- steel column at 115 "C for dichlorvos and 157 "C for malathion. Retention times were 2 minutes and 12 minutes, respectively, with a nitrogen gas flow-rate of 20 ml min-l, a hydrogen flow-rate of 18 mlmin-l and an air flow-rate of 220mlmin-l. (xvii) SE-30 (5 per cent.) on 100 to 120-mesh Embacel in a 1.2 m x 6 mm 0.d. stainless- steel column at 150 "C for dichlorvos and 190 "C for malathion. Retention times were 2-3 minutes and 3.8 minutes, respectively, with a nitrogen gas flow-rate of 60 ml min-l, a hydrogen flow-rate of 28 ml min-l and an air flow-rate of 400 ml min-1. (xviii) QF-1 (2-5 per cent.) and 0.25 per cent. Epikote 1001 on 100 to 120-mesh Celite in a 1.5 m x 4 mm i.d. glass column operating at 150 "C for dichlorvos and 190 "C for malathion.Retention times were 3-0 minutes and 9.5 minutes, respectively, with a nitrogen24 PANEL ON MALATHION AND DICHLORVOS RESIDUES IN GRAIN gas flow-rate of 30 ml min-1, a hydrogen flow-rate of 15 to 26 ml min-l and an air flow-rate of 400 ml min-l. (xix) E 301 (2.5 per cent.) and 0.25 per cent. Epikote 1001 on 100 to 120-mesh Gas- Chrom Q in a 1.5 m x 6 mrn 0.d. stainless-steel column operating at 150 "C for dichlorvos and 190 "C for malathion. Retention times were 3.3 minutes and 5.2 minutes, respectively, with a nitrogen gas flow-rate of 60 ml min-l. It is suggested that one of the succinate ester types of stationary phase be used unless another suitable column is readily available. Recorder signals for the pesticides should normally be 40 per cent. or more of full-scale deflection.Appendix I1 METHOD OF FORTIFICATION OF SAMPLES OF GRAIN FOR RECOVERY EXPERIMENTS Circulate 20 g of ground grain in a 250-ml ground glass necked flask (Quickfit and Quartz) by using a wrist-action shaker. Then add slowly the required amount of pesticide in 1 ml of acetone at a rate of about one drop every 2 s and mix for 6 minutes by further shaking. Remove the solvent by using an intermittent, light stream of air. Stopper the flask and store it in a deep-freeze overnight to allow penetration into the grain. This sample should undergo extraction on the following day. Appendix I11 MEMBERSHIP OF THE PANEL The membership of the Panel was: Mr. W. Cassidy (Chairman), Mr. S. Bailey, Mr. P. M. Brown (from October, 1970), Mr. G. J. Dickes (from March, 1970), Mr. L. Donegan, Mr. J. W. Edmunds, Mr. F. B. Fishwick, Mr. D. C. Holmes, Mr. A. Holder (from October, 1970), Dr. D. F. Horler, Mr. F. R. Johnson (from October, 1970), Mr. A. F. Machin (until March, 1970), Mr. B. L. Mathews, Mr. M. P. Quick (from March, 1970), Mr. K. Rawlings (until October, 1970), Mr. R. C. Spalding (until June, 1970) and Dr. N. A. Smart (Secretary). REFERENCES 1. 2. 3. 4. Report by the Malathion Panel, Analyst, 1960, 85, 915. Elgar, K. E., Marlow, R. G., and Mathews, B. L., Ibid., 1970, 95, 875. Crisp, S., and Tarrant, K. R., Ibid., 1971, 96, 310. Watts, R. R., and Storherr, R. W., J . Ass. OH. A g r i ~ . Chem., 1965, 48, 1158. Received July 20th, 1972 Accepted August 24th, 1972 COMMITTEE FOR ANALYTICAL METHODS FOR RESIDUES OF PESTICIDES AND VETERINARY PRODUCTS IN FOODSTUFFS (DR. N. A. SMART, SECRETARY), MINISTRY OF AGRICULTURE, FISHERIES AND FOOD, PLANT PATHOLOGY LABORATORY, HATCHING GREEN, HARPENDEN , HERTFORDSHIRE.

ISSN:0003-2654    

DOI:10.1039/AN9739800019

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

Analyst, January, 1973, Vol. 98, @. 25-33 25 Effect of Isoelectric Focusing on the Amino-acid Composition of Proteins BY S. JACOBS (National Institute for Medical Research, Ridgeway, Mill Hill, London, N . W. 7) The normal isoelectric focusing method has been used to separate various proteins into fractions. The fractions and the parent proteins have been hydrolysed and the amino-acid composition of each has been determined by ion-exchange chromatography. Evidence was obtained that shows that the parent protein is modified by the normal isoelectric focusing procedure; the sulphur-containing amino-acids cystine and methionine were shown to be present partly in the form of cysteic acid and methionine sulphoxides, in a greater proportion than with hydrolysates of proteins isolated by other separation procedures.Prolonged treatment of the protein by the isoelcctric focusing procedure adversely affects other amino-acid residues in the molccule also. An improved method has been devised so as to protect the protein isolated by the isoelectric focusing procedure and to prevent the oxidation or modification of the protein, which would otherwise occur during the normal isolation procedure. THE theoretical principles of isoelectric focusing were demonstrated by Svenssonl in 1961 , and Vesterberg and Svensson2 developed a series of synthetic ampholytes suitable for produc- ing a natural equilibrated pH gradient. A sucrose gradient was used to prevent convective disturbances .3 The technique of isoelectric focusing has been used by several workers to isolate different proteins that displayed heter~geneity~--~ when examined by this method.The ion-exchange resin chromatography of the hydrolysates of proteins, isolated by the normal isoelectric focusing procedure, has been performed in this laboratory. In each instance, the amounts of cysteic acid and methionine sulphoxides present in the hydrolysates of proteins isolated by isoelectric focusing exceeded those in proteins isolated by other separation procedures. The amino-acid compositions of component fractions of the proteins thyrotrophin, or-glycoprotein and bovine ribonuclease have been determined, and the results showed that the protein was modified during the normal isoelectric focusing procedure. The latter procedure has therefore been changed in order to prevent such alterations in the amino-acid composition of the isolated protein. EXPERIMENTAL MATERIALS- The ion-exchange resins that were used to examine the hydrolysates of proteins separated by the isoelectric focusing procedures were supplied by Permutit Company Ltd.and Beckman Instruments Ltd. The bovine ribonuclease A, Grade V (chromatographically pure), was supplied by Miles Seravac Ltd., Maidenhead, Berkshire. The ampholine ampholytes used for producing the natural pH gradients in the sucrose gradient columns for isoelectric focusing were supplied by LKB Produkter AB. REAGENTS- were prepared with conductivity water as previously described.10 The properties of these resins were given in a previous paper.9 All reagents were of analytical-reagent grade unless otherwise stated, and all solutions Indanetrione hydrate, normal laboratory grade. Indanetrione hydrate stock solution.Indanetrione hydrate reagent. Methyl Cellosohe (2-methoxyethanol) , technical grade. Sodium acetate trihydrate. Sodium acetate bufler solution, 4.0 M, +H 5.5. The above six reagents were prepared as described previou~ly.~ @ SAC and the author.26 JACOBS: EFFECT OF ISOELECTRIC FOCUSING ON THE [Anahst, vol. 98 Sodium citrate buffer solution, 0.2 M, p H 3.25-This was produced in bulk amounts of 20 litres for use with the Beckman amino-acid analyser. Each batch of 20 litres of buffer solution contained 160g of sodium hydroxide, 420g of citric acid, about 214ml of concen- trated hydrochloric acid, 1.0 g of Brij 35, 200 ml of 2,2'-thiodiethanol (thiodiglycol) and 2 ml of octanoic acid, The octanoic acid was of laboratory-reagent grade. Sodium citrate buffer solution, 0.2 M, @H 4.25-This buffer solution was produced in bulk amounts of 10 litres for use with the Beckman amino-acid analyser. Each batch of 10 litres of buffer solution contained 80 g of sodium hydroxide, 210 g of citric acid, about 47 ml of concentrated hydrochloric acid, 100 ml of 2,2'-thiodiethanol, 1.0 g of Brij 35 and 1.0 ml of octanoic acid.Sodium citrate b u f e r solzttion, 0.35 M, j5H 5.28-Each batch of 20 litres of this buffer solution contained 280 g of sodium hydroxide, 491 g of citric acid, approximately 135 ml of concentrated hydrochloric acid, 2 g of Brij 35 and 2 ml of octanoic acid, but contained no 2,2'-thiodiethanol because no sulphur-containing amino-acids were determined during the elution of amino-acids on the column used to elute basic amino-acids.11 APPARATUS- The isoelectric focusing was performed on the LKB standard equipment with the 8100 elect rof ocusing column. Some comparative isoelectric focusing experiments were performed on the preparative density gradient electrophoresis apparatus supplied by Instrumentation Specialities Co.Inc. (ISCO), Lincoln, Nebraska, USA. The amino-acid analyses of the protein hydrolysates were performed on a Beckman Amino-Acid Analyser, Model 120C, by using the two-column method previously described12 with ion-exchange resins of improved q ~ a l i t y . ~ PROCEDURE- The isoelectric focusing was performed on 10 to 12-mg amounts of the ribonucIease A by using the standard LKB or the ISCO apparatus.The LKB 8100 electrofocusing column was maintained at 5 "C. The sucrose gradient was adjusted so as to have a concentration that varied from 50 to 5 per cent. of sucrose, and the solution surrounding the cathode con- tained 50 per cent. of sucrose and 1 per cent. of tetraethylenemethylenediamine. The ampholine ampholytes had a pH in the range 7 to 10. The total volume of the solution containing the sucrose gradient was 110 ml, and the concentration of the ampholine in this gradient was 1 per cent. A 10-ml volume of aqueous solution containing the antioxidant was introduced carefully so as to form a layer above the sucrose gradient. The 10ml of 10 per cent. solution of ascorbic acid contained no ampholine, whereas the 10ml of either 10 or 5 per cent.2,2'-thiodiethanol as antioxidant contained 0.2 ml of 40 per cent. ampholine solution. A volume of 2 to 3 ml of 0.5 per cent. orthophosphoric acid was added to the solution surrounding the anode. The contents of the isoelectric focusing columns were eluted and monitored as fractions by measuring the absorbance at a wavelength of 280 nm in a Unicam SP500 spectrophoto- meter. The fractions containing the individual separated proteins were pooled and chromato- graphed on a column of Amberlite IRC 50 CG Type I1 200 to 400-mesh ion-exchange resin of diameter 6 mm and height approximately 20 mm. The column was equilibrated against 0.01 M phosphate buffer at pH 6.97. The sugar was removed by washing the column with 3 ml of water.The protein and most of the ampholine were eluted with 1.0 M sodium chloride solution. The fractions collected from the IRC 50 column were monitored again for protein content by measurement of the absorbance at 280 nm and the appropriate fractions containing protein were pooled. This eluate of pooled fractions was added to a Sephadex G-25 column of height 20 cm and diameter 2.0 cm, which had previously been equilibrated against 0.01 M ammonium hydrogen carbonate solution. The protein was eluted with a solution of 0.01 M ammonium hydrogen carbonate and concentrated by freeze-drying for 16 hours. The proteins were hydrolysed in 6~ hydrochloric acid at 105 "C for 24 hours, after first removing air from the hydrolysis mixture by passing a rapid stream of nitrogen through it for 1 hour.13 The amino-acid compositions of the hydrolysates of the proteins were determined by means of an improved system of analysis described previously.9 Ribonuclease A was used as the test material.January, 19731 AMINO-ACID COMPOSITION OF PROTEINS TABLE I AMINO-ACID COMPOSITION OF BOVINE RIBONUCLEASE A, BATCH UK3, The experiments were carried out in the LKB 8100 electrofocusing column BEFORE AND AFTER ISOELECTRIC FOCUSING Percentage of total residues f A t 27 Amino compound Methionine sulphoxides Threonine .. .. Serine .. .. Glutamic acid . . Proline . . .. Glycine . . . . Alanine . . . . Cystine . . .. Valine .. .. Methionine . . .. Isoleucine . . .. Leucine . . .. Tyrosine . . .. Phenylalanine . . Lysine . . .. Histidine .. .. Arginine . . .. Oxidised cystine . . Oxidised methionine Total masslpg . . Isoelectric point . . Cysteic acid . . .. Aspartic acid . . .. .. .. . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. Original protein 0.17 0.56 13.00 7.45 9-99 11.31 2.55 1.43 6.69 5-7 1 5.45 3-27 1-20 1.87 7.30 3.41 10.10 3.40 4-79 2.9 14.6 1459 - Protein treated once Peak I Peak I1 Peak I11 2.54 0.68 1.70 0.90 0.96 1.00 13.37 12.97 13.10 7.69 7.42 7.56 9.95 9-60 10.13 11-86 11-19 11.64 2.29 2.77 2.55 1.56 1.42 1.69 6-71 6.36 6-6 1 2.91 5.43 3.81 6-16 6.29 6-19 2.43 2.96 2.13 1-62 1.60 1-53 1.86 1.92 1.91 6.79 7.42 6-6 1 3.32 3.43 3.4 1 9.80 9-22 10.03 3-65 3-32 3.67 4.72 5-06 4.75 P 46.7 11.1 30.9 27.0 24.5 32-0 1772 402 1664 8-95 9.10 9.40 Protein treated twice, peak I1 1.00 1.43 13.17 7.43 9.84 11.62 2.58 1.41 6-88 5-05 6.21 1.76 1.62 1.93 6.83 3-45 10.07 3-06 4.48 16.5 44.8 558 9.10 Peak 11 in presence of ascorbic acid, treated once 0.17 0.39 12.96 7-02 9.37 11.38 2.47 1.33 6-30 5.9 1 6.12 3.36 1-61 1.81 7.28 3-46 9.73 3.58 5.09 2.8 10.3 6984 9.10 RESULTS The isoelectric focusing of a commercially available chromatographically pure sample of bovine ribonuclease A is illustrated in Table I.The amino-acid composition of the original sample, together with those of the separated fractions indicated by peaks I, I1 and I11 in Fig. 1, are shown. The protein contained in peak I1 was isolated by the normal LKB I "H0 Peak II PH 7.0 W c 2 L w 2 Fraction number (fraction volume, 1.0 ml) Fig. 1. Fractions of ribonuclease A isoelectrically focused in the absence of ascorbic acid.(LKB equipment)28 JACOBS: EFFECT OF ISOELECTRIC FOCUSING ON THE [Analyst, Vol. 98 procedure and freed from sucrose and ampholine, and a portion of the product was treated again in the isoelectric focusing column by the normal procedure with LKB equipment (Fig: 2). The amino-acid composition of the twice-treated protein is given in Table I. The original protein was isoelectrically focused in the presence of 10 ml of 10 per cent. ascorbic acid solution and only two fractions resulted (Fig. 3), viz., a main peak that corresponded to peak I1 and a very small peak that corresponded to peak I11 of the three peaks that resulted from the original protein sample. The amino-acid composition of the main peak obtained in the presence of ascorbic acid is shown in the final column in Table I.0.6 PH PH 10.0 7.0 0 Fraction number (fraction volume, 1.0 ml) Fig. 2. Fractions of bovine ribonuclease A obtained when the main component (peak 11, Fig. 1) is treated a second time by isoelectric focusing (LKB equipment) In Table I1 are shown the results of the analyses of the main peak (11) obtained by isoelectric focusing of bovine ribonuclease A in the presence of ascorbic acid (Fig. 3) or 2,2’-thiodiethanol as antioxidant (Fig. 4). The amino-acid composition of the original protein is also given in Table I1 for reference purposes. 2 .o 1.5 Q, t m -e 1.c a n a 0-E 0.I PH 10.0 Peak II ,u 10 20 30 40 50 60 3 Fraction number (fraction volume, 1.25 ml) Fig. 3. Fractions of ribonuclease A separated in the presence of ascorbic acid (LKB equipment)January, 19731 AMINO-ACID COMPOSITION OF PROTEINS TABLE I1 ANALYSES OF BOVINE RIBONUCLEASE A (PEAK 11) IN THE PRESENCE AND ABSENCE OF ANTIOXIDANT The LKB apparatus was used, and the results relate to batch UK3, except those in the final column, which relate to batch UK6 29 Amino compound Methionine sulphoxides Threonine .. .. Serine .. .. Glutamic acid . . Proline . . .. Glycine . . .. Alanine . . .. Cystine . . .. Valine .. .. Methionine . . .. Isoleucine . . .. Leucine . . .. Phenylalanine . . Histidine . . .. Oxidised cystine . . Oxidised methionine Cysteic acid . . .. Aspartic acid .. Tyrosine . . . . Lysine . . .. Arginine . . .. .. .. .. .. .. .. .. .. .. . . .. .. .. .. .. .. .. . . . . .. . . Original protein 0.17 0.56 13.00 7.45 9-99 11.31 2-55 ' 1-43 6.69 5.7 1 5.45 3.27 1.20 1.87 7.30 3.41 10.10 3.40 4-79 2.9 14.6 Percentage of total residues A 1 In presence of I n absence of antioxidant antioxidant A I -I - 6 per cent.10 per cent. Treated Treated Ascorbic 2,2'-thiodi- 2,2'-thiodi- once twice acid ethanol ethanol 0.68 1.00 0.17 0.61 0-69 0.96 1.43 0.39 0.37 1.93 12.97 13.17 12.96 12.96 13.39 7-42 7.43 7.62 7.50 7.66 9-60 9-84 9-37 9-67 9.27 11.19 11.62 11-38 11.35 11.59 2.77 2.58 2.47 2.48 2.44 1.42 1.41 1.33 1.37 1-35 6-36 6.58 6.36 6.43 6.66 6.43 5.05 5.9 1 6.86 6.24 6.29 6-2 1 6-12 6.20 6.60 2-96 1-76 3.36 3.29 1-54 1.60 1.52 1.61 1-63 1.85 1-92 1.93 1.81 1.82 1.87 7.42 6-83 7.28 6.94 7-21 3-43 3.45 3.46 3-37 3.38 9.22 10.07 9.73 10.03 9-36 3.32 3.66 3.58 3.80 3.69 5.06 4.48 6-09 4.7 1 4.59 11.1 16-5 2.8 9.9 11-6 24-5 44.8 10.3 10.0 85.7 The results of the analyses of two batches of bovine ribonuclease A and the fractions resulting from them after isoelectric focusing on both the LKB and ISCO equipment are shown in Table I11 (see Figs.1 and 5). leak I I Peak Ill & 0*0' Ib i0 30 40 50 60 70 80 Fraction number (fraction volume, 1-1 ml) Fractions of bovine ribonuclease separated in the presence of 6 per cent. 2,2'-thiodiethanol solution (LKB equipment) Fig. 4. D30 O.OL 1'0 ;o 3b 40 El0 6L ;o sto do 1;o JACOBS: EFFECT OF ISOELECTRIC FOCUSING ON THE TABLE I11 ISOELECTRIC FOCUSING OF BATCHES OF BOVINE RIBONUCLEASE A [AnaZyst, Vol. 98 110 Amino compound Methionine sulphoxides Cysteic acid . . . . 0.5 Aspartic acid Threonine .. Serine . . Glutamic acid Proline . . Glycine . . Alanine . . Cystine . . Valine . . Methionine . . Isoleucine . . Leucine .. Tyrosine . . Phen ylalanine Lysine . . Histidine . . Arginine . . Oxidised cystine - .. .. .. .. .. . . . . . . . . .. .. .. .. .. .. .. .. Percentage of total residues A Batch UK3 in A r 7 LKB apparatus /-=-, ISCO apparatus Peak I1 Peak I1 ,-*-. without with ascorbic ascorbic Original acid acid . . 0.17 0.68 0.17 . . 0.56 0.96 0.39 . . 13.00 12.97 12.96 . . 7.45 7-42 7-62 . . 9-99 9.60 9.37 . . 11-31 11.19 11.38 . . 2-55 2.77 2.47 . . 1-43 1.42 1-33 . . 6-69 6.36 6.36 . . 6-71 6.43 6-91 . . 5.45 6.29 6.12 . . 3.27 2.96 3.36 . . 1.20 1.60 1.61 . . 1-87 1.92 1.81 . . 3-41 3.43 3-40 . . 10.10 9.22 9-73 . . 3.40 3-32 3.68 .. 4-79 6.06 6.09 . . 2.9 11.1 2.8 . . 7-30 7-43 7-28 Without ascorbic acid 2-93 0-28 13.16 7.70 9-96 11.52 2.78 1-48 6-69 2.84 6-90 3.46 1.42 1.97 6.98 3.38 9.91 3.39 4.18 50.7 With * ascorbic acid 0.43 0.84 13.28 7.65 9-83 11.36 2-69 1.40 6.43 6.58 6-70 3-18 1.31 1.86 7.20 3-34 10.06 3.46 4-61 6.9 Batch UK6 in ISCO apparatus* W l t h o u t h ascorbic ascorbic Original 0.63 0.63 13.15 7-47 9.41 11-28 2-03 1.37 6.39 6.63 6.98 3-29 1.69 1.93 7.42 3-42 10.23 3-65 4.48 10.1 acid 1.29 0.78 13.63 7.68 9.79 11.72 2.37 1.39 6-70 3.85 6-34 2.80 1.66 1-77 7-03 3.34 9.78 3.66 4.44 24.9 acid 1.40 0.13 13-49 7.62 9.64 11.81 2.55 1.40 6.77 3-86 6-52 3.63 1-81 1.78 6.87 3-29 9.72 3.62 4.30 27.7 * Column maintained a t 4 "C for the ISCO apparatus. The amino-acid compositions and isoelectric points of fractions of a glycoprotein and thyrotrophin isolated by the normal isoelectric focusing procedure with the LKB equipment are given in Table IV.1.5 Peak I I Fig. 5. Fractions of bovine ribonuclease A separated by the ISCO apparatus in the presence of ascorbic acidJanuary, 19731 AMINO-ACID COMPOSITION OF PROTEINS TABLE IV AMINO-ACID COMPOSITION OF PROTEINS AFTER NORMAL ISOELECTRIC FOCUSING I N THE LKB APPARATUS Percentage of total residues 31 Amino compound Cysteic acid . . .. Aspartic acid . . 0 . Methionine sulphoxides Threonine . . ,. Serine . . .. .. Glutamic acid . . .. Proline . . .. .. Glycine . . .. .. Alanine . . .. * . Cystine . . .. .. Valine . . . . .. Methionine . . .. Isoleucine . . * . Leucine * . .. Tyrosine .... Phenylalanine . . .. Lysine . . .. .. Arginine .. .. Glucosamine . . .. Galactosamine . . . . Histidine . . .. Oxidised cystine . . Isoelectric point . . .. .. .. .. .. .. .. .. .. .. .. .. . . . . .. .. .. .. .. .. .. .. .. a-Glycoprotein*/pmol - A B 0.017 0.016 Trace Trace 0.147 0.185 0.098 0.127 0.092 0.121 0.176 0.231 0.065 0.082 0.089 0.117 0.099 0-130 0.007 0.02 1 0.084 0-112 0.014 0.016 0.053 0.064 0.124 0.141 0.037 0.049 0.066 0.075 0.035 0.043 0.112 0.143 0.040 0.057 0.042 0.058 54.8 28.0 5-1 5.5 - - ceThyrotrophin/pmol 0.1831 0,0441 0.662 0.879 0.600 0-705 0.696 0-432 0.710 0,295 0.667 0-268 0.344 0,379 0.587 0.366 0.418 0.180 0.852 0.268 0.321 23.8 8.25-8.30 * Focused in 6 M urea solution after neuramidase treatment. The relative positions or p l values of bovine ribonuclease A fractions after separation by isoelectric focusing under different conditions are shown in Table V.TABLE V RELATIVE POSITIONS OF FRACTIONS FROM BOVINE RIBONUCLEASE A AFTER SEPARATION BY ISOELECTRIC FOCUSING Bovine ribonuclease system Fractions Isoelectric point Peak I 9.4 Peak I1 9-25 Peak I11 8.95 Without ascorbic acid . . . . .. .. With ascorbic acid . . . . . . Peak I1 9.15-9.20 Peak I11 8.95-9.00 Peak I 9-10 Peak I1 9.0 Peak I11 8.8 With 5 per cent. 2,2’-thiodiethanol . . . . Peak 11, treated twice without ascorbic acid Peak I Peak I1 9.1 5-9.2 0 9.06-9.10 Peak I - Peak I1 9.26 Peak I11 - \Vith 10 per cent. 2,2’-thiodiethanol . . . . DISCUSSION The heterogeneity of proteins found by other workersG8 after the treatment of the proteins by the normal isoelectric focusing procedure has been confirmed in the present study.When the solution in the column of sucrose density gradient containing the frac- tionated components of a protein has reached the stage of equilibrium, and is eluted directly and monitored for absorbance at 280 nm, the elution pattern indicates the individual com- ponents, In addition, the portions of solution finally eluted show absorbance values that32 [Analyst, vol. 98 increase steadily (see Figs. 1 to 5 ) to a maximum. These highly absorbing portions correspond to the layer of sucrose at the top of the sucrose gradient. It is common for a greyish film to be formed on the top of the sucrose gradient at the interface between the sucrose solution and the aqueous solution containing the phosphoric acid used to surround the anode.The high absorbance value at 280nm and the presence of trace amounts of insoluble material indicate the possibility of the presence of denatured protein at the interface between the sucrose column and the aqueous solution floating above the column. The analyses of the component fractions of proteins isolated by isoelectric focusing for amino-acids show that cysteic acid and methionine sulphoxides occur to an extent greater than in proteins isolated by chromatography, electrophoresis or ultrafiltration. The hydrolysates of cc-glycoprotein, thyrotrophin and bovine ribonuclease A have been examined in the present work by the improved system of automatic analyses for amino-acids described earlier.9 In a preliminary communication,14 it was shown that appreciable amounts of cystine and methionine were present in their oxidised states in the hydrolysates.Gordon and Louis15 examined an a, acute phase globulin of rats by the normal isoelectric focusing procedure and obtained two fractions. They reported that the isoelectric focusing led to alterations in at least some of the protein molecules. Bovine thyrotrophins have been separated by Fawcett, Dedman and Morrisls by using the normal isoelectric focusing procedure with the LKB 8100 apparatus. They reported the presence of four components, each with a different isoelectric point, and they gave the biological activities of three com- ponents. In a previous cornmunication,l4 the degree of oxidation of a thyrotrophin of iso- electric point 8-65 to 8-70 was reported as 13 per cent., but in the thyrotrophin of isoelectric point 8.25 to 8.30 reported in the present paper, the degree of oxidation of cystine was 23.8 per cent.It is possible that the electrolytic oxidation of a protein during isoelectric focusing may produce fractions with apparently specific isoelectric points. Each fraction recognised on the elution pattern after monitoring the eluate for absorbance a t 280nm may consist of a mixture of protein molecules, each of different charge, as monomers, dimers or polymers whose total charges may differ so little from each other that they cannot be resolved in the pH gradient. The experimental results obtained from the analyses of the bovine ribonuclease A frac- tions for amino-acids show that when the major fraction (peak 11) was isoelectrically focused a second time, the amount of the arginine residues in the protein thus isolated decreased from 5.06 to 4.48 per cent.(compared with 5-09 per cent. of arginine in peak I1 when the isoelectric focusing was performed in the presence of ascorbic acid as antioxidant). This degradation of arginine in the fraction in peak I1 (see Fig. 1) when submitted twice to iso- electric focusing might occur as the result of reduction of the original amino-acid during the electrochemical procedure. Arginine is known to be converted into ornithine by any one of four well known reaction^,^^,^^ and alkaline solutions may cause the conversion of arginine into ornithine. The system of chromatography used to examine the hydrolysates of proteins in this study would not resolve ornithine, if it was present, from lysine, and in the analysis of peak I1 obtained from bovine ribonuclease A subjected twice to isoelectric focusing (see Table I) the decrease in arginine was accompanied by an increase in the apparent amount of lysine (cf., results for lysine in Table I).It is possible that the enlarged peak that is normally occupied by lysine contained a small amount of ornithine produced by the electrolytic decomposition of a portion of the arginine residues in the bovine ribonuclease A molecules. The oxidation of cystine and methionine, which occurs when isoelectric focusing is used in the normal procedure described by the original authors,lP2 can be controlled by the use of a suitable antioxidant such as ascorbic acid.The bovine ribonuclease A used in these experiments was fractionated into three components when it was submitted to isoelectric focusing in the presence of 10 ml of either 5 per cent. (Fig. 4) or 10 per cent. thiodiethanol solution on the LKB column. Only two fractions of the protein were obtained when the ribonuclease A was examined in the presence of ascorbic acid (Fig. 3) with the LKB apparatus. The isolation of bovine ribonuclease A in the ISCO isoelectric focusing equipment from a commercial supply of the protein produced only two components, whereas three fractions were separated by the LKB equipment. The pH gradient used with the ISCO apparatus was produced by an ampholine mixture designed to yield a pH range of 8 to 10, extended by means of arginine, triethanolamine and diaminoethane to a pH of approximately 11.5.JACOBS: EFFECT OF ISOELECTRIC FOCUSING ON THEJanuary, 19731 AMINO-ACID COMPOSITION OF PROTEINS 33 The normal isoelectric focusing procedure adversely affected the tyrosine residues in the bovine ribonuclease A sample. The differences in the results for the percentage tyrosine residue values when the main component (peak 11) of bovine ribonuclease A was isolated in the presence of ascorbic acid and also after treatment once or twice by the normal isoelectric focusing procedure, are indicated in Table I. This effect on the percentage tyrosine residue value is shown also in Table 111, which includes results obtained by using both the LKB and ISCO isoelectric focusing equipment.The earlier observation^^^^^^ on the oxidation of the sulphur-containing amino-acids of a-glycoprotein and thyrotrophin have been supported by similar observations on the corre- sponding amino-acids of the commercial samples of bovine ribonuclease A, which was selected as a test material because of its relatively high concentration of sulphur-containing amino- acids. Provided that a suitable antioxidant is used to protect the sucrose density gradient, the protein can be isolated with minimum modification during the isoelectric focusing procedure, which takes approximately 16 hours. The author is indebted to Mr. R. A. Faulkes for skilled technical assistance, and thanks Dr. J. Fawcett and Dr. D. Leaback for the use of the LKB and ISCO isoelectric focusing columns, respectively. Loan of the ISCO apparatus from Shandon-Southern Instruments Ltd. is gratefully acknowledged. The author also acknowledges gifts of samples of ct-glyco- protein, for which he thanks Dr. A. H. Gordon, and of cc-thyrotrophin, for which he thanks Dr. Fawcett. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. REFERENCES Svensson, H., Acta Chem. Scand., 1961, 15, 326. Vesterberg, O., and Svensson, H., Ibid., 1966, 20, 820. Kolin, A., J . Chem. Phys., 1954, 22, 1628. Vesterberg, O., Biochim. Biophys. Acta, 1968, 168, 218. Quast, R., and Vesterberg, O., Acta Chern. Scand., 1968, 22, 1499. Lewis, U. J., Cheever, E. V., and Hopkins, W. C., Biochiwt. Biophys. Acta, 1970, 214, 4987. Norton, I. L., Pfunderer, P.. Stringer, C. D., and Hartman, F. C., Biochemistry, 1970, 9, 4952. Kaplan, L. J., and Foster, J. F., Ibid., 1971, 10, 630. Jacobs, S., Analyst, 1970, 95, 370. -, Chem. G. Ind., 1955, 944. -, Lab. Pract., 1963, 12, 557. -, Protides Biol. Fluids, 1964, 11, 463. -, Meth. Biochem. Analysis, 1966, 14, 177. -, Protides Biol. Fluids, 1971, 18, 499. Gordon, A. H., and Louis, L. N., Biochem. J., 1969, 113, 481. Fawcett, J. S., Dedman, M. L., and Morris, C. J . O., FEBS Lett., 1969, 3, 250. Schultze, E., and Winterstein, E., 2. physiol. Chem., 1898-1899, 26, 1. Greenstein, J. P,, and Winitz, M., Editors, “Chemistry of the Amino Acids,” Volume 3, John Wiley Received January 26212, 1972 Accepted JuZy loth, 1972 & Sons, New York, 1961, p. 2477.

ISSN:0003-2654    

DOI:10.1039/AN9739800025

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

34 Amzlyst, January, 1973, Vol. 98, @@. 34-39 The Polarographic Determination of Tripolyphosphate Ions and of Tripolyphosphate and Nitrilotriacetic Acid in Admixture BY F. AL-SULIMANY AND ALAN TOWNSHEND (Department of Chemistry, University of Birmingham, P.O. Box 363, Birmingham, B16 2TT) Tripolyphosphate ions in amounts from 2 to 20pmol are determined by measurements of the decrease in the height of the cadmium polarographic wave by using a trimetaphosphate buffer at pH 7.0. Up to 6 mol per cent. of orthophosphate or pyrophosphate and 50 per cent. of trimetaphosphate or tetrametaphosphate ions do not interfere. Tripolyphosphate and nitrilo- triacetic acid in admixture can be determined by measuring the reduced height of the cadmium wave and the cadmium - nitrilotriacetic acid complex wave.THE present paper describes attempts to develop a rapid polarographic method for the determination of tripolyphosphate ions in the presence of other polyphosphates. It also describes attempts to determine tripolyphosphate and nitrilotriacetic acid in admixture. One of the most important chemical properties of polyphosphate ions is their ability to form complexes with metal ions, but the polarographic behaviour of these complexes has received surprisingly little attention. Existing polarographic methods for the determination of polyphosphates do not involve complex formation. Cahn and Kolthoff precipitated as little as 9mg of pyrophosphate ions with cadmium, and determined the cadmium content of the precipitate polarographically ; at pH 3.6, orthophosphate did not interfere.Tripoly- phosphate (350 mg) has been precipitated with tris(ethy1enediamine)cobalt dichloride solution and the excess of I precipitant determined polarographically.2 In the present study, the effect of polyphosphate ions on the d.c. polarographic behaviour of cadmium ions has been investigated. DETERMINATION OF TRIPOLYPHOSPHATE BY DECREASE OF CADMIUM WAVE HEIGHT- Initial experiments showed that at pH 7, tripolyphosphate at a concentration of M decreased the height of the polarographic wave of the cadmium ion although no precipitate was formed. The half-wave potential of the wave was unchanged, and no wave appeared at more negative potentials. It was assumed that a stable, soluble complex was formed and that the reduction of the cadmium from the complex occurred at potentials beyond the base electrolyte discharge potential. The metaphosphates examined (trimetaphosphate and tetrametaphosphate) had no effect on the cadmium wave, but pyrophosphate ions at a concentration of 1 0 - 3 ~ gave a precipitate with an equal concentration of cadmium ions.The height of the cadmium wave decreased with increasing tripolyphosphate concen- tration, but in the ammonium acetate buffer solution used initially the decrease was rather small (Fig. 1). This phenomenon was ascribed to competitive complexing of cadmium by ammonia and acetate ions3 In a non-complexing solution (2 x M sodium perchlorate at pH 7.0), a much greater decrease was obtained (Fig. 1). The metaphosphates again had no effect a t the 2 x M concentration level but this concentration of pyrophosphate also precipitated cadmium.Pyrophosphate ion concentrations of greater than 10 mol per cent. of that of tripolyphosphate also gave increased reduction of the height of the wave given by cadmium in the presence of tripolyphosphate (see Table I), although in this instance no precipitate was formed unless a large amount of pyrophosphate was present. Moreover, although the metaphosphates alone did not affect the height of the cadmium wave, they reduced the amount by which tripolyphosphate ions decreased the cadmium wave height (Table I). The metaphosphate effects are relatively small and level out at higher metaphos- phate concentrations. @ SAC and the authors.AL-SULIMANY AND TOWNSHEND 35 F Tripolyphosphate ion concentration/M x Fig.1. Effect of tripolyphosphate ions on the wave height of 1.0 x M cadmium in: 1, 2 per cent. ammonium acetate solution; 2, 2-0 x 10-4 M sodium perchlorate solution; and 3, 4.0 x lo-' M sodium trimetaphosphate solution The above findings suggested the use of a metaphosphate buffer solution to eliminate the interference by metaphosphates by ensuring the presence of a metaphosphate concen- tration that is sufficient to give a constant enhancement to the cadmium wave height. As trimetaphosphate had the greater effect, it was selected for further investigation as a buffer The use of a solution of trimetaphosphate at a concentration of 4 x 10-2 M and at pH 7.0. enabled a concentration of 4 x 10" M of tripolyphosphate to be determined without inter- ference from a less than loA3 M concentration of trimetaphosphate, but the solution became turbid before the polarogram had been recorded completely.A reduction in the concentration of trimetaphosphate (to 4 x M) eliminated this problem. In the latter solution, tripoly- phosphate ions had an increasingly large effect on the cadmium wave height as the pH increased to 7.0. At a pH of more than 7.5 cadmium again gave a precipitate with trimetaphosphate, so that pH 7-0 was selected as the optimum for the determination of tripolyphosphate. TABLE I EFFECT OF POLYPHOSPHATES ON CADMIUM WAVE HEIGHT IN THE PRESENCE oI; TRIPOLYPHOSPHATE AND 2 x 10-4 M SODIUM PERCHLORATE SOLUTION AT p~ 7.0 Tripolyphosphate concentration/M x 10-4 r 1 2.5 5.0 10.0 A - I \ - Pyro- Pyro- Tetrameta- Trimeta- phosphate phosphate phosphate phosphate concen- concen- concen- concen- trationl Wave tration/ Wave trationi Wave tration/ Wave M x 10-6 height/cm M x height/cm M x heightlcm M x heightlcm 0 6.5 0 5.7 0 5-7 0 3.9 2 6.6 6 5.6 1 6.2 1 4.3 4 6.1 8 6.2 10 6.2 2 4.6 6 6.7 10 4.7 100 6.2 5 4.6 The maximum concentrations of polyphosphate in the final solution that could be tolerated in the determination of various amounts of tripolyphosphate under the recom- mended conditions are summarised in Table 11.Orthophosphate and pyrophosphate ions cause serious interference if present in amounts greater than 6 mol per cent. of the tripoly- phosphate concentration.36 AL-SULIMANY AND TOWNSHEND : POLAROGRAPHIC DETERMINATION [Analyst, Vol. 98 M could be tolerated in the determination of a concentration of 4 x low4 M of tripolyphosphate.Nitrilotriacetic acid, which has been proposed as an alternative detergent to tripolyphosphate, decreased the cadmium wave both in the presence and absence of tripolyphosphate ions. This effect is described in more detail below. A nitrilotriacetic acid concentration of about 5 x 1 0 - 5 ~ is the maximum that can be tolerated in the determination of a concentration of 4 x 10-4 M of tripolyphosphate without correcting for the nitrilotriacetic acid concentration as described below. Chloride and sulphate ions at concentrations of M and less than 5 x Higher sulphate concentrations decreased the cadmium wave height. It was also established that calcium and magnesium in concentrations of less than M, respectively, had no effect.TABLE I1 APPROXIMATE MAXIMUM TOLERABLE CONCENTRATIONS OF POLYPHOSPHATES I N THE DETERMINATION OF TRIPOLYPHOSPHATE Maximum toleration/M x A Tripolyphosphate r 7 concentration/ Pyro- Trimeta- Tetrameta- Ortho- M x 10-4 phosphate phosphate phosphate phosphate 2.0 0.7 5 7 - 4.0 2 60 50 3 8.0 3 60 60 - A typical calibration graph is given in Fig. 1, line 3. The standard deviation for six determinations of tripolyphosphate concentration between 2.85 x and 8.1 x low4 M was 0-2 x M. Although a number of interferences could not be eliminated, the method proved to be satisfactory for the rapid analysis of solid tripolyphosphate samples. The method was tested on polyphosphate samples previously analysed by an ion-exchange - molybdenum-blue procedure and the results are summarised in Table 111.All were within one standard deviation of the results obtained by the reference method. TABLE I11 ANALYSIS OF POLYPHOSPHATE SAMPLES Sample content, per cent. of pyrophosphate* tripolyphosphate* 0.7 64.1 0.7 53-2 0.8 62.2 1.2 62.2 1.4 62.6 1-0 62.4 0.0 41.9 A f \ Tripolyphosphate found, per cent. 64.8 63.6 63.6 63.6 63.6 64.2 41-9 1.3 per cent. of tripolyphosphate is approximately equivalent to 0.1 cm of wave height. * Calculated as phosphorus pentoxide. DETERMINATION OF TRIPOLYPHOSPHATE AND NITRILOTRIACETIC ACID IN ADMIXTURE- Nitrilotriacetic acid forms a complex with cadmium that shifts the half-wave potential from -0.58 V for free cadmium ions to -1.10 V. In the presence of an excess of cadmium, two waves are observed, E+ = -0.58 and -1.10 V (see Fig.2). The increase in height of the wave at -1.10 V with increasing nitrilotriacetic acid concentration has been used by a number of workers4-' as the basis for the determination of small amounts of nitrilotriacetic acid. When tripolyphosphate ions are also present the wave at -0.58 V is further depressed. The total wave height is also reduced somewhat, but unexpectedly the height of the cadmium - nitrilotriacetic acid wave is increased (Fig. 2). The heights of both waves obtained for various nitrilotriacetic acid and tripolyphosphate concentrations are given in Table IV. The results in Table IV show that any given combination of nitrilotriacetic acid and tripolyphosphate concentrations below certain limits gives rise to a unique pair of wave heights.A graph of the first wave height against the second wave height can be plottedJanuary, 19731 OF TRIPOLYPHOSPHATE IONS AND NITRILOTRIACETIC ACID (C1 (dl (4 ( f 1 Fig. 2. Effect of tripolyphosphate ions on the wave of 1.0 x M cadmium in the presence of 5.0 x M ; (d), 6.0 x lo-"; (e), 8.0 x 1 0 - 4 ~ ; and (A, 1.0 x M. All waves start a t -0.4V. Sensitivity = 7 pA full-scale deflection, when using a ,drop-life timer M nitrilotriacetic acid: (a) 0.0 M ; ( b ) , 2.0 x lo-' M ; (c), 4.0 x 37 and the points of equal nitrilotriacetic acid concentration and of equal tripolyphospliate concentration joined by tie lines, as illustrated in Fig. 3. Such a graph can be used as a calibration graph for the determination of nitrilotriacetic acid and tripolyphosphate in admixture in any combination of concentrations up to 5 x M of each component.TABLE IV EFFECT OF NITRILOTRIACETIC ACID AND TRIPOLYPHOSPHATE ON CADMIUM WAVE HEIGHTS Wave heigh t/cm Tripol yphosphate Nitrilotriacetic acid r-----h-----7 concentration/M x concentration/M x Wave 1 Wave 2 0.0 2.0 4.0 6.0 8.0 10.0 0.0 2.5 6.0 0.0 2.6 6.0 0.0 2.6 5.0 0.0 2.5 5.0 0.0 2.5 5.0 0.0 2.5 5.0 9.0 0.0 6.9 0.4 4.6 2.4 6.8 0.0 4.3 1.1 2.2 3.3 4.5 2.9 1.1 3.1 1.4 0.45 1.8 0*56 0.4 0.0 1.7 4.0 0.0 2.3 4.5 0.0 2-86 4.1 0.8 0.0 0.6 2.5 0.6 3.9 EXPERIMENTAL APPARATUS- A standard calomel reference electrode was used. REAGENTS- Wilson (Mfg) Ltd. ; other chemicals were of analytical-reagent grade. Polarograms were obtained on a Polariter PO4 polarograph fitted with a drop-life timer.Polyphosphate solutions were prepared from sodium salts provided by Albright and38 AL-SULIMANY AND TOWNSHEND : POLAROGRAPHIC DETERMINATION [Andyst, Vol. 98 PROCEDURE FOR THE DETERMINATION OF TRIPOLYPHOSPHATE I N A SOLID SODIUM POLY- PHOSPHATE SAMPLE- Dissolve 0.2 g of the sodium polyphosphate sample (containing less than 5 mol per cent. of orthophosphate or pyrophosphate and less than 50 per cent. of trimetaphosphate or tetra- metaphosphate) in 50 ml of distilled water. Transfer by pipette 0.8 ml of this solution into the polarographic cell, followed by exactly 2.0 ml of cadmium nitrate solution (1.0 x M), 10.0 ml of trimetaphosphate solution (8.0 x lo4 M, pH 7-0), and 5.2 ml of water. Adjust the solution to pH 7.0 (by using a pH meter) with 1.0 x M perchloric acid, noting the volume of acid added, and make the volume up to exactly 20 ml with water.After passing nitrogen through the solution for 2 minutes, record the cadmium polarographic wave, starting at -0.40 V v e i s m S.C.E. and measure the wave height. Determine the tripolyphosphate con- centration from a calibration graph obtained by applying the above procedure to standard tripolyphosphate samples. PROCEDURE FOR THE DETERMINATION OF TRIPOLYPHOSPHATE IN AQUEOUS SOLUTIONS- 4 x lo-' mol of tripolyphosphate. Use the above procedure to analyse 6.0 ml or less of solution containing not less than Height of second wave/cm 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Fig. 3. Relation between height of first and second cadmium waves ( E , = -0.66 and - 1.08 V) for various amounts of tripolyphosphate and nitrilo- triacetic acid.Solid lines : concentrations of nitrilotri- acetic acid are M x and broken lines: concen- trations of tripolyphosphate are M xJanuary, 19731 OF TRIPOLYPHOSPHATE IONS AND NITRILOTRIACETIC ACID 39 PROCEDURE FOR THE DETERMINATION OF NITRILOTRIACETIC ACID AND TRIPOLYPHOSPHATE IN Dissolve 0-5g of the solid mixture (containing less than 25 per cent. of nitrilotriacetic acid) in 50 ml of water. Transfer into the polarographic cell, by pipette, 0.8 ml of this solution and follow the procedure given above for tripolyphosphate. Measure the height of both polarographic waves and obtain the nitrilotriacetic acid and tripolyphosphate concentrations from a calibration chart similar to Fig.3, prepared from the results obtained with suitable standard mixtures subjected to the procedure. As for tripolyphosphate alone, aqueous solutions can be analysed by taking a sample of 6.0 ml or less, containing at least mol each of nitrilotriacetic acid and tripolyphosphate, the exact limits depending on the relative concentrations (Le., smaller amounts of nitrilo- triacetic acid can be determined when the tripolyphosphate concentration is relatively high). The authors thank Professor R. Belcher for his interest and encouragement. They also thank Mr. I. L. Jones and Mr. S. Greenfield, Albright and Wilson (Mfg) Ltd., Oldbury, for their advice and for the provision of polyphosphate samples. F. Al-Sulimany thanks the University of Birmingham for the provision of research facilities and the University of Riyadh, Saudi Arabia, for financial support. REFERENCES ADMIXTURE- 1. 2. 3. 4. 5. 6. 7. Cahn, G., and Kolthoff, I. M., Ind. Engng Chem., Analyt. Edn, 1942, 14, 886. Golubchik, E. M., Zav. Lab., 1969, 35, 926; Analyt. Abstr., 1970, 19, 2193. Andress, K. R., and Nachtrab, R., 2. anorg. allg. Chem., 1961, 311, 22. Asplund, J., and Wanninen, E., Analyt. Lett.. 1971, 4, 267. Daniel, R. L., and LeBlanc, R. B., Analyt. Chem., 1959, 31, 1221. LeBlanc, R. B., Ibid., 1959, 31, 1840. Farrow, R. N. P., and Hill, A. G., Analyst, 1965, 90, 241. Received January loth, 1972 Accepted September 21st, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800034

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

40 Analyst, January, 1973, Vol. 98, $$. 4041 Studies on the Analytical Chemistry of Hafnium and Zirconium Part III.* Spectrofluorimetric Determination of Hafnium in the Presence of Zirconium by Using Quercetin BY TH. KOUIMTZIS AND ALAN TOWNSHEND (Department of Chemistry, University of Birmingham, P.O. Box 363, Birmingham, B16 2TT) Hafnium a t concentrations of 0-2 per cent. m/m and above in hafnium - zirconium mixtures can be determined spectrofluorimetrically via its complex with quercetin in 55 per cent. perchloric acid. There is no interference from zirconium. RECENTLY, a method was published1 for the determination of hafnium in the presence of zirconium, based on the observation that in 57.5 per cent. perchloric acid, hafnium forms a fluorescent complex with quercetin whereas zirconium forms a non-fluorescent complex.The fluorescence was measured on a simple filter fluorimeter, and the lower limit of 1 per cent. of hafnium in zirconium was imposed by the inability of the instrument to distinguish smaller amounts of hafnium above the fluorescence emission of quercetin itself. It was felt that the lower limit could be significantly reduced by using a spectrofluorimeter because the accurate control of excitation and emission wavelengths might give improved discrimina- tion between the fluorescence of the hafnium complex and that of quercetin, and the superior electronic specification of the instrument should give increased sensitivity to the hafnium chelate. This paper briefly describes the confirmation of these suggestions.EXPERIMENTAL The chemicals and reagent solutions used were as described previous1y.l APPARATUS- A Baird Atomic SF lOOE spectrofluorimeter was used, with exit and entrance slits set at 8 pm. The scale used (coarse gain = 30) was adjusted to give a reading of 100 with 1.0 x M quinine in 1 M sulphuric acid, when using excitation and emission wavelengths of 350 and 450 nm, respectively. Further slits were used to direct the excitation and emission beams so that they passed through only a minimal length of solution in the 1 x 1-cm cell. DETERMINATION OF HAFNIUM AT CONCENTRATIONS OF 0.2 PER CENT. m/m AND ABOVE IN Prepare a perchloric acid solution of hafnium plus zirconium as described previous1y.l To an accurately measured aliquot, containing not more than 6 x lo-' mol of zirconium plus hafnium, add exactly 1.5 ml of 10-3 M quercetin and exactly 3-2 ml of 72 per cent.perchloric acid. Make the solution up to 10 i 1 with 60 per cent. perchloric acid. Measure the fluores- cence intensity by using excitation and emission wavelengths of 396 and 498 nm, respectively. Read the hafnium concentration from a calibration graph of fluorescence intensity versus hafnium concentration, prepared from the results of hafnium standards that have been taken through this procedure. RESULTS AND DISCUSSION A reinvestigation of the fluorescence of the hafnium - quercetin complex in 57 per cent. perchloric acid showed that the fluorescence intensity was greatest when the excitation wavelength was 396 nm and the emission was measured at 498 nm. At these wavelengths, the hafnium fluorescence intensity was three times greater, relative to that of the uncomplexed quercetin, than in the simple fluorimetric procedure.In the 57 per cent. acid, zirconium up to a concentration of 4 x l o - 5 ~ had no effect on the fluorescence, which is the same ZIRCONIUM- * For details of Part I1 of this series, see reference list, p. 41. @ SAC and the authors.KOUIMTZIS AND TOWNSHEND 41 level as in the previous procedure. Reduction of the acid concentration to 55 per cent., however, gave a tolerance of 6 x 1 0 - 5 ~ zirconium, but further reductions in the acidity gave reduced tolerances. Hence the spectrofluorimetric determination in 56 per cent. acid allows an approximately five-fold reduction in the level of hafnium that can be determined in the presence of zirconium, i.e., from 1 per cent.when using the fluorimetric procedure to 0.2 per cent. when using the present method. Fluorescence intensities measured by the spectrofluorimetric procedure are recorded in Table I. Further increases in the zirconium concentration gave high results for smaller amounts of hafnium and low results for the larger amounts of hafnium given in Table I. TABLE I EFFECT OF ZIRCONIUM ON THE FLUORESCENCE INTENSITY OF THE HAFNIUM - QUERCETIN COMPLEX IN 55 PER CENT. PERCHLORIC ACID Hafnium concentration/rn x lo-' .. . . 0.0 2.0 3.0 5-0 10.0 15.0 20.0 25.0 Intensity (no zirconium present) . . .. . . 58 62 64 68 83 93 106 118 7 Intensity (zirconium concentration 6.0 x 10-5 M) 60 63 64 70 79 93 102 116 Morin gives a more sensitive response to hafnium, but zirconium also gives a slight fluorescence with the reagent in strongly acidic so1utions.l It is possible that a spectro- fluorimetric study of this reagent would permit even greater discrimination between zirconium and hafnium. The authors thank Professor R. Belcher for his interest, and Th. Kouimtzis thanks the Greek Ministry of National Economy for the award of a research scholarship. REFERENCE 1. Brookes, A., and Townshend, A., Analyst, 1970, 95, 781. Reference 1 is to Part I1 of this series. Received February 26th, 1972 Accepted September 18th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800040

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

42 Analyst, January, 1973, Vol. 98, pp. 4248 The Direct Complexometric Determination of Metals in Unused Lubricating Oils and Additives by Automatic Non-aqueous Potentiometric Titration* BY J. Tu’. WILSON AND C. 2. MARCZEU’SKI (The British Petroleum Company Limited, BP Research Centre, Chertsey Road, Sunbury-on-Thames, Middlesex) Methods are described for the determination of calcium, barium, zinc and magnesium in any combination in additive concentrates and unused lubricating oils. The samples are dissolved in a mixed aqueous - organic solvent and titrated directly with 1,Z-bis (2-aminoethoxy)ethane-NNN’N’- tetraacetic acid [ethylene glycol bis(2-aminoethy1)tetraacetic acid ; EGTA] or 1 , 2-diaminocyclohexane-NNN’N’-tetraacetic acid (DCTA) , thc end-points being indicated by metal ion sensitive electrodes.By use of a masking agent and the appropriate buffer solutions the individual metals in any combination can be determined following a maximum of three titrations. The results are compared with those obtained by established procedures. The accuracy of the complexometric methods is comparable with that of established wet-chemical methods, but the former are up to ten times faster and are therefore suitable for blending control. THE blending of organometallic additives with automotive lubricants to give a closely specified product has presented a problem in process control analysis that has persisted for many years. The required control is best effected by the determination of the metals, usually calcium, barium and zinc, that are present, sometimes singly, but more often in combination.Magnesium may also be present, and an increase in the use of this metal seems likely to take place, All of these metals can readily be determined by spectroscopic methods. However, it may be uneconomical or impractical to install and maintain the necessary equipment at small or remotely situated blending centres with a limited output in both range and amount. Blending control at such centres frequently depends upon the determination of sulphated ash, which gives an indication of the total metal content. Because such a method is time consuming and can give misleading results, there remains the need for a procedure that is rapid, selective, simple and inexpensive. The use of EDTA and similar complexing agents for this purpose has recently received considerable attention, and several titrimetric procedures have been propo~ed.l-~ Of these, the non-aqueous procedure’ is non-selective, while those involving extraction with acid2s3 create problems of emulsion formation and incomplete recovery of the metals.More recently, very simple, direct methods have been used to determine calcium and ~ i n c . ~ - ~ However, these methods are less satisfactory for samples that contain barium, and are limited to the determination of total metals and zinc if more than two metals are present. This paper describes a direct potentiometric method that is capable of determining any combination of the metals calcium, barium, zinc and magnesium in either additive concen- trates or unused lubricating oils.A wide range of oils and additives has been analysed to determine calcium, barium and zinc and a limited number of determinations of magnesium have also been carried out. The results compare well with those obtained by use of estab- lished wet-chemical procedures or atomic-absorption spectroscopy. Duplicate analyses to determine calcium, barium and zinc can be made in 21, hours, and in proportionately less time for fewer metals. Only a little additional analysis time is required if magnesium is also to be determined. OUTLINE OF METHOD- The oil or additive is dissolved in a mixed aqueous - organic solvent, buffered to pH 7 to 8 and titrated potentiometrically with EGTA [ethylene glycol bis(2-aminoethy1)tetraacetic acid; 1,2-bis(2-aminoethoxy)ethane-NNN’N’-tetraacetic acid]. This titration enables the EXPERIMENTAL * Presented a t the Third SAC Conference, Durham, July 12th to 16th.1971. Q SAC and the authors.WILSON AND MARCZEWSKI 43 total metal content to be calculated. A second inflection is observed if magnesium is present. The titration is repeated after the addition of sulphate ions to mask barium, which can be determined by difference. Zinc is determined separately at pH 4 to 496 by titration with DCTA (1,2-diaminocyclohexane-NNN~W-tetraacetic acid). Calcium is determined either directly or from the difference between appropriate titrations, depending upon the other metals present. Dialkyldithiophosphate ions interfere in the determination and, if present, are removed from the analytical solutions by an anion-exchange procedure before titration.APPARATUS- A block diagram of the apparatus is shown in Fig. 1. The solution to be titrated is contained in a 250-ml tall-form beaker and stirred on a magnetic stirrer. The titrant is added from an automatic incremental dispenser at the rate of 0.05 ml per 10 s (Fison or Hook and Tucker dispensers are suitable). The calomel reference electrode has a modified salt bridge comprising a mixture of 2-methylpropan-2-01 and water (9 + 1) saturated with potassium chloride. Two indicating electrodes are required: when zinc is to be determined with DCTA an electrode of stout silver wire (99.9 per cent. silver) is used whereas a J-type mercury-pool electrode, as described by Reilley, Schmid and Lam~on,~ is used for all other determinations.The electrodes are connected to a millivoltmeter and recorder (100 mV full scale), fitted with a source of backing- off potential and a sensitivity adjustment. The glass column used in the anion-exchange process consists of a 1 cm diameter glass tube of about 25ml capacity, fitted with a straight burette tap with a PTFE key and a 1.6 mm diameter jet. A reservoir of 250 ml capacity is fitted to the top of the tube (Fig. 2). I Stirrer I Chart recorder H Millivoltmeter Fig. 1. Block diagram of apparatus for the determination of additive metals REAGENTS- Fig. 2. Anion- exchange column All reagents should be of analytical-reagent grade unless otherwise specified. Methanol. 2-Methylpropan-2-oI, laboratory-reagent grade. Chlorobenxene, laboratory-reagent grade.Nitric acid, 4.5 per cent. V/V. Acetic acid, 10 per cent. V/V. Sulphuric acid, 0.02 N. Amberlyst A-29 anion-exchange resin, Rohm and Haas, U.S. A .-This material is obtainable in the U.K. from BDH (Chemicals) Limited. Ethanolamine, 8.8 per cent. V/V-Dilute 8.8 ml of laboratory-reagent grade ethanolamine to 100 ml with a mixture of 2-methylpropan-2-01 and chlorobenzene (1 + 4). Prepare a fresh solution weekly.4.4 [Analyst, Vol. 98 Dithizone (diphenyzthiocarbaxone) solution, 0.02 per cent. m/V-Dissolve 0.02 g of dithizone in 100ml of chlorobenzene. Store in darkness. Standard calcium solution, 0-001 M-Dry calcium carbonate (CaCO,) in an oven for 2 hours at 120 "C and cool it in a desiccator. Dissolve 0-1001 g in the minimum amount of warm 2 N nitric acid and dilute the solution to 1 litre with water. Standard zinc solution, 0.001 M-Treat granular zinc with 2 N hydrochloric acid, decant off the acid and wash the zinc thoroughly with water, methanol and finally diethyl ether.Dry the granules under vacuum, dissolve 0.1308 g in the minimum amount of warm 2 N nitric acid and dilute the solution to 2 litres with water. EGTA solution, about 0.001 M-Dissolve 0.38 g of laboratory-reagent grade EGTA (free acid) in 20 ml of warm 0.2 N sodium hydroxide solution, add 80 ml of water and dilute to 1 litre with methanol. Determine the pH with a pH 6 to 8 indicator paper (the pH should be within the range 6.5 to 7.5). If necessary, adjust the pH by the dropwise addition of 0.2 N sodium hydroxide solution. Standardise the solution against the standard calcium solution by the procedure described under Standardisation of titrants.DCTA solution, about 0.001 M-Dissolve 0.35 g of laboratory-reagent grade DCTA (free acid), dilute to 1 litre and check the pH, exactly as described for the preparation of EGTA solution. Standardise the solution against the standard zinc solution by the procedure described under Standardisation of titrants. CONDITIONS OF ANALYSIS- Complexing agents-Because of the relatively low stability constant of the EGTA - magnesium complex, it is possible to obtain the second inflection that distinguishes this metal from calcium, barium and zinc (Fig. 3, B). EGTA cannot be used to determine zinc in the presence of the other metals; for this purpose it is necessary to use DCTA, which forms an unusually stable zinc complex, and therefore enables this metal to be titrated in the presence of the others under relatively acidic conditions.WILSON AND MARCZEWSKI : DIRECT COMPLEXOMETRIC DETERMINATION Fig. 3. Typical titration curves : A, total metals; and B, calcium and magnesium, showing second inflection Electrodes-The mercury-pool electrode is used extensively to indicate metal-ion con- centration and gives reproducible end-points for all of the titrations with EGTA. It is not, however, satisfactory for the titration of zinc with DCTA, for which the recommended silver electrode gives excellent results. The salt bridge of the calomel reference electrode is modified to control the amount of water entering the titration medium.January, 19731 OF METALS I N UNUSED LUBRICATING OILS AND ADDITIVES 45 Titration medium-A solvent mixture of 20ml of chlorobenzene, 40 ml of water and 130 ml of 2-methylpropan-2-01 was adopted after extensive experimentation.It was designed to give homogeneous solutions of up to 1 g of oil, yielding sharp, reproducible end-points. PREPARATION OF ANALYTICAL SOLUTIONS- Weigh accurately into a 100-ml calibrated flask sufficient oil or additive to provide a maximum of 0-2 mmol of total metals, and ideally 0.02 to 0.07 mmol of each individual metal. Add about 50ml of chlorobenzene, swirling the flask to dissolve the sample, and make up to the mark with chlorobenzene. In the absence of dialkyldithiophosphates-For each determination required (see Table I), transfer by pipette duplicate 5-ml aliquots of the chlorobenzene solution into 250-ml tall-form beakers.To each aliquot add 15 ml of chlorobenzene, 130 ml of 2-methylpropan-2-01 and 40ml of water, mix thoroughly, and complete the analysis by the appropriate procedure described under Method I or Method 11. In the presence of dialkyldithiophosfihate-Prepare a 20-ml bed of Amberlyst A-29 anion- exchange resin in the chloride form (as supplied) as follows. Place a small pad of glass-wool in the glass column (see under Apparatus), locating it immediately above the tap. Transfer an aqueous slurry containing 9 g of resin to the column with water, opening the tap sufficiently to allow a gentle flow so that the resin settles into an evenly distributed bed free from channels and air bubbles.During this and all subsequent operations do not allow the liquid level to fall below the top of the resin bed. Depending on the nature of the eluting agent, the maximum flow-rate through an exchange column to this specification will be up to 20 ml min-l. Wash the bed successively with 100 ml of methanol and 100 ml of chlorobenzene, applying each solvent in several portions and a t the maximum flow-rate. Close the tap as the liquid level reaches the top of the bed after the final addition of chlorobenzene. Transfer to the column, by pipette, a 5-ml aliquot of the sample solution. Open the tap so that the sample enters the column over a period of 20 to 30 s and collect the effluent in a 250-ml tall-form beaker containing 90 ml of 2-methylpropan-2-01, Wash the column consecutively with 5 ml of chlorobenzene and, in 10-ml portions, 40 ml of 2-methylpropan-2-01 and 50ml of water, continuing to collect the effluent as before.Elute the column at the maximum flow-rate, but close the tap before adding each different solvent as the liquid level reaches the top of the resin bed. Mix the total effluent thoroughly and complete the analysis by the appropriate procedure described under Method I or Method I1 (Note 1). Prepare the column for re-use by repeating the washing cycle with 100 ml of methanol and 100 ml of chlorobenzene. Process a duplicate 5-ml aliquot of the sample solution and prepare similar duplicate solutions for each determination required (see Table I). METHOD I- Determination of total metals and magnesium-To the prepared solution add, by use of a pipette, 1 ml of 4.5 per cent.nitric acid solution and 1 ml of 8.8 per cent. ethanolamine solution. Stir the mixture on the magnetic stirrer. The pH should be 7 to 8. Introduce the calomel and mercury electrodes into the solution and titrate with 0.001 M EGTA solution while stirring rapidly. Record the volume of titrant added. Carry out a blank determination by using 5 ml of chlorobenzene in place of the 5-ml aliquot of sample solution, including the ion-exchange procedure when appropriate. Determination of barium in the presence of zinc and calcium-Repeat the determination of total metals as described above, with the addition of 1.0 ml of 0.02 N sulphuric acid to the sample solution following the addition of nitric acid and ethanolamine (Note 2).Again, carry out a blank determination, including 1.0 ml of 0-02 N sulphuric acid in the reagents used. NOTES- solvent environments. to be constant at 10 ml. 2-methylpropan-2-01 and 40ml of water, the column retaining the additional 10ml of water. it is necessary to record only the first end-point. 1. Unlike gel-type resins, macroreticular resins do not swell or shrink appreciably in different The solvent retention volume of the spccified resin bed can be considered The analytical solution thus contains 20 ml of chlorobenzene, 140 ml of 2. If magnesium is present a second inflection may be observed. For the barium determination46 WILSON AND MARCZEWSKI : DIRECT COMPLEXOMETRIC DETERMINATION [ A n a & f , Vol. 98 METHOD II- Determination of zinc in the +resence of calcium and barium-To the prepared solution add, by use of a pipette, 1 ml of 10 per cent.acetic acid solution and 1 ml of the 0.02 per cent. dithizone solution. Stir the solution on the magnetic stirrer. The pH should be about 4.5. Introduce the silver and calomel electrodes into the solution and titrate with O-OOl M DCTA solution while stirring rapidly. Record the volume of titrant added. Carry out a blank determination by using 5 ml of chlorobenzene in place of the aliquot of sample, including the ion-exchange procedure when appropriate. STANDARDISATION OF TITRANTS- Standardise the EGTA solution against 5-ml aliquots of the standard calcium solution by using Method I, and the DCTA solution against 5-ml aliquots of the standard zinc solution by Method 11.Dilute each aliquot with 35 ml of water, 140 ml of 2-methylpropan-2-01 and 20 ml of chlorobenzene and add the appropriate buffer solution. Then determine the reagent blanks . CALCULATION OF RESULTS- of two or more of the additive metals. Table I shows the determinations that are necessary in order to analyse any combination TABLE I DETERMINATIONS REQUIRED TO ANALYSE VARIOUS METAL COMBINATIONS Metals present Determine Calcium ; barium Calcium; zinc Total metals: zinc Barium; zinc Total metals; zinc Calcium; barium ; zinc Total metals ; barium Total metals; barium; zinc The additional presence of magnesium does not affect the required determinations. The calculation of results will vary with the metals present, as follows. Calcium and barium present- Calcium, per cent.m/m Barium, per cent. m/m Calcium and zinc present- .. .. .. - (V3 - b2) x 80.16 x MI W - [(V, - bJ - (V3 - b,)] x 274.72 x M I W .. - - Calcium, per cent. m/m Zinc, per cent. m/m Barium and zinc present- Barium, per cent. m/m (V4 - b3) x 130.76 x M , W .. .. . . - IS [(Vl - b,)+-- $(V4 - b3)] x 274.72 x M , W - - Zinc, per cent. m/m, see equation (4). Calcium, barium and zinc present- Calcium, per cent. m/m = MI M2 [(V3 - b,) -- (V4 - b 3 ) ] x 80.16 x MI W .. . . .. .. .. .. Barium, per cent. mlm., see equation (2); zinc, per cent. m/m, see equation (4).January, 19731 OF METALS IN UNUSED LUBRICATING OILS AND ADDITIVES 47 Magnesium present-The magnesium content is indicated by the second end-point in the titration of total metals, irrespective of the other metals present.(V, - VJ x 48.64 x M i W Magnesium, per cent. m/m = and V , and V , are the volumes of EGTA taken to reach the first and second end-points, respectively, in the titration of total metals; V , is the volume of EGTA used in the barium titration; V , is the volume of DCTA used in the zinc titration; b,, b, and b, are respectively the total metals, barium and zinc contents blanks; M , and M , are the molarities of the EGTA and DCTA solutions, respectively; and W is the mass of the sample in grams. RESULTS ADDITIVE CONCENTRATES- The results of the analysis of several additive concentrates containing one or two metals are shown in Table 11. All the determinations were carried out by using Method I, zinc being determined in sample numbers 8 and 9 by masking the barium with sulphate. The alternative wet-chemical procedures were I.P.110 (for barium), I.P. 111 (for calcium), polarography with the Shandon Southern Instruments A 1660 cathode-ray polarograph (for zinc) and A.S.T.M. D 811 (for magnesium). TABLE I1 DETERMINATION OF METALS IN ADDITIVE CONCENTRATES Sample No. 1 2 3 4 5 6 7 8 9 10 11 Metal sought Calcium Calcium Barium Magnesium Zinc Calcium Barium Calcium Barium Barium Zinc Barium Zinc Barium Magnesium Calcium Magnesium Metal content, per cent. mlm, found by- established EGTA titration wet-chemical (Method I) methods * r A > 11.5 11.4, 11.7 11-5 11.5, 11.6 3-46 3.43, 3.64 7-60 7-60, 7-40 7.09 7-05, 7.76 5.54 6.64, 6.63 3-78 3.76, 3.76 0.48 0.48, 0.61 6.70 6.96, 7-00 1.40 1.33, 1-38 0.04 0.30, 0.32 7-60 7-89 7.60 7-30 12.7 23-0, 13.1 13.4 13.2, 13.2 11.6 11.6 11.5 12.0 UNUSED LUBRICATING OILS- Eight samples of unused lubricating oils, providing a comprehensive range of additive formulations, were analysed to determine calcium, barium and zinc.All contained zinc dialkyldithiophosphate and were subjected to the ion-exchange procedure. For comparison purposes they were analysed by the wet-chemical methods and also by atomic-absorption spectroscopy. The results are given in Table 111. A limited statistical programme, which took into account results from several blends, indicated that for all three metals duplicate determinations should not differ from one another by more than 5 per cent. of the mean value (95 per cent. probability). The reproducibility has not been determined.DISCUSSION The determination of metals in lubricating oils by the complexometric methods described is appreciably more rapid than by the classical procedures. Atomic-absorption spectroscopy is a faster technique when a series of similar blends are to be analysed, although not when48 WILSON AND MARCZEWSKI TABLE I11 DETERMINATION OF METALS I N UNUSED LUBRICATING OILS Metal, per cent. mlm Sample No. 1 2 3 4 5 6 7 8 7 Atomic- absorp- tion spectro- scopy 0.023 0-023 0.056 0.056 0.095 0.095 0.12 0.12 0.19 0.20 0.54 0.55 1.07 1.12 2-23 2-28 7 Calcium Estab- lished wet- chemical methods 0.023 0.023 0.055 0.056 0.094 0.091 0.12 0.12 0.19 0.19 0.54 0.54 1.18 1-18 2.23 2.23 A EGTA titration 0.022 0.022 0.052 0.053 0.096 0-093 0.092 0.10 0.18 0.18 0.51 0.50 1-16 1-18 2.32 2.36 7 Atomic- absorp- tion spectro- scopy 0.12 0.13 0.041 0-041 0.092 0.093 0.97 0.95 0.18 0.18 0.36 0.36 0.18 0.19 0.62 0.63 Barium Estab- lished wet- chemical methods 0.13 0.13 0.043 0.041 0.10 0.10 1.01 1.00 0.21 0.2 1 0.39 0-38 0.18 0.19 0.63 0.66 A 1 EGTA titration 0.13 0.14 0.040 0.036 0.088 0.088 1.11 1.11 0.18 0.18 0.39 0.38 0.19 0.18 0.63 0.61 r Atomic- absorp- tion spectro- scopy 0.030 0-030 0.13 0.13 0.070 0.072 0.063 0.062 0-15 0-14 0.22 0.22 0.091 0.093 0.42 0.38 1 Zinc Estab- lished wet- chcmical methods 0.028 0.028 0.12 0.12 0.068 0.069 A- 1 DCTA titration 0.028 0.027 0.12 0.12 0.069 0.069 0.059 0.059 0.060 0.058 0.14 0.13 0-13 0.13 0.22 0.21 0.22 0-21 0.092 0.091 0.090 0,087 0.43 0.44 0.42 0.43 the numbers of samples are small.The complexometric apparatus is simpler and easier to maintain than an atomic-absorption spectrophotometer and the precisions of the three methods are comparable. Two samples gave poor results. Sample number 9 of the additive concentrates (Table 11) in which the barium to zinc ratio was 20:1, gave a very low value for zinc by Method I. A similarly low result was obtained when the zinc determination was repeated by using Method 11. Hooks and Noar5 have noted a depression of the zinc results in the presence of much larger amounts of barium, although not to the same extent. In sample number 4 of the lubricating oils (Table 111) the barium to calcium ratio was about 10 : 1. In comparison with the values obtained by the other methods used, the value for barium was high while that for calcium was low. Co-precipitation of calcium with barium in the presence of sulphate ions could, in part, explain this phenomenon. Neither of these anomalous results seriously interferes with the suitability of the method for most control purposes. Permission to publish this paper has been given by the British Petroleum Company Limited. 1. 2. 3. 4. 5 . 6. 7. REFERENCES Gerhardt, P. B., and Hartmann, E. R., Analyt. Chem., 1957, 29, 1223. Fisher, W., J . Inst. Petrol., 1962, 48, 290. A.S.T.M. Standards, Part 17, Appendix IV, Proposed Method, American Society for Testing Materials, Philadelphia, Pa., 1964. Crump, G. B., Analyst, 1969, 94, 465. Hooks, R. W., and Noar, J. W., Ibid., 1969, 94, 473. Crump, G. B., and Noar, J. W., J. Inst. Petrol., 1970, 56, 180. Reilley, C. N., Schmid, R. W., and Lamson, D. W., Analyt. Chem., 1958, 30, 953. Received July 31st, 1972 Accepted August 24th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800042

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

Analyst, January, 1973, Vol. 98, $9. 49-52 49 The Direct Determination of Mercury by Atomic-absorption Spectrophotometry at 184.9 nm by Using a Nitrogen-separated Nitrous Oxide - Acetylene Flame BY G. F. KIRKBRIGHT, T. S. WEST AND P. J. WILSON (Chemistry Department, Imperial College, London, SW7 2A Y) A modified atomic-absorption spectrophotometer with a nitrogen-purged optical path has been used for the direct determination of mercury by atomic- absorption spectrophotometry, by using a nitrogen-separated premixed nitrous oxide - acetylene flame and electrodeless discharge lamp source. The high transparency of the fuel-rich flame below 200 nm permits the use of the principal resonance line of mercury at 184-9 nm for its determination. The sensitivity (for 1 per cent. absorption) enhancement obtained by using this line rather than the 253.7-nm line is fifty-fold; detection limits for mercury(1) and mercury(I1) of 0.02 p.p.m. and 0.05 p.p.ni.are achieved in aqueous solution. To date the direct determination of mercury by flame atomic-absorption spectroscopy has been undertaken by using the mercury 253.7-nm line that corresponds to the spin forbidden transition 6s2 lS,- 6p3PI0. The sensitivities achieved at this line have varied from 2.0 to 10.0 p.p.m.1s2 The principal resonance line of mercury corresponding to the allowed transi- tion 6s2 lS, - 6p1P1 lies at 184.9 nm, and the high background absorption from most flames, and from the atmosphere at this wavelength, prevents the use of this line for the deter- mination of mercury.It has been widely predicted, however, that the sensitivity attainable with atomic-absorption spectrophotometry at this line should be as much as fifty times greater than that a t 253.7 nm. Non-flame devices, which are usually purged with a non- absorbing inert gas, can be used for the determination of mercury at the 184.9-nm line, when a vacuum monochromator is also used. L’vov and Khartsyzov3 were able to operate with the 184.9-nm line in this way by using a graphite furnace atomiser and a vacuum monochromator, and achieved a sensitivity thirty times greater than that achieved at the 253.7-nm line. Recent work in this lab~ratory*-~ has demonstrated the remarkable transparency of the separated nitrous oxide - acetylene flame, and its use to determine the elements sulphur and iodine, whose principal resonance lines occur below 200 nm in the vacuum ultraviolet region of the spectrum.The transparency of this flame at short wavelengths is due to the lack of absorbing oxygen species in the interconal zone of the flame and the separation of the oxidising outer mantle by nitrogen shielding. This flame was used for the determination of sulphur a t 180.7 nm and iodine at 183.0 nm; it was expected that it might be used with a commercial atomic-absorption spectrophotometer, as modified by the use of a nitrogen- purged monochromator so as to permit detection of radiation below 200nm for the deter- mination of mercury at 184.9 nm. A microwave-excited mercury electrodeless discharge lamp was included as the source. APPARATUS- A Perkin-Elmer, Model 290B, atomic-absorption spectrophotometer was used.The apparatus was modified as previously described,6 with provision of a nitrogen-purged optical path between the source and flame, and between the flame and the photomultiplier detector via the grating monochromator. An R166 solar blind photomultiplier (Hamamatsu T.V., Japan) was fitted instead of the IP 28a (RCA Ltd.) instrument previously used; although this tube gave rise to a reduced signal intensity, the signal to background and signal to noise ratios observed at the 184.9-nm line were greatly improved. The mercury electrodeless discharge lamp source was made from silica tubing (i.d. 8 mm, wall thickness 1 mm) so as to form a bulb 240mm in length containing about 5mg of elemental mercury. EXPERIMENTAL @ SAC and the authors.50 KIRKBRIGHT 81 d.: DETERMINATION OF MERCURY BY ATOMIC-ABSORPTION [Ana&St, VOl. 98 RESULTS AND DISCUSSION The spectrophotometer used in this work does not have direct wavelength calibration. The location of the mercury 184.9-nm line was therefore found by calibration with sources that produced the mercury 253.7, cadmium 228.8, iodine 206.1, arsenic 193-7 and iodine 183.0-nm lines: the signal to background ratio at 184.9 nm obtained by using a spectral slit width of 20 nm was about 200: 1 with the mercury electrodeless discharge lamp source used. A4s expected, the signal intensity from the source was attenuated by the flame background absorption. When the optical path was purged and the nitrogen-shielding gas to the flame switched on without a flame burning, 100 per cent.transmission was easily set with a 20-nm slit width; the unshielded nitrous oxide - acetylene flame then produced 45 per cent. trans- mission compared with this value, whereas when separated by nitrogen shielding the trans- mission through the flame rose to 80 per cent. These values must be compared with those obtained through a 5-cm path length air - acetylene flame at 184.9 nm with nitrogen purging of the optical path. Fig. 1 shows the effect of the acetylene flow-rate on the flame background absorption in the nitrous oxide - acetylene flame. The numerical values shown are those of the spectrophotometer fuel control scale. Maximum transmission was obtained at values corresponding to a slightly fuel-rich flame that exhibited a red interconal zone about 40 mm in height; all absorption values were made in a region 3 to 6 mm above the primary reaction zone, As the mercury electrodeless discharge lamp produced very intense radiation at 184-9 nm, high sensitivity was obtained even at an operating power of only 10 W from the microwave generator, although above 26 W the absorption signal did begin to drop slowly, probably because of self-absorption broadening of the 184.9-nm line.c; loot- al 0 L- n r' E E l- .- cn .- C 20 6l I I I I 1 fuel flow-rate (instrument settings) Fig. 1. Effect of variation in acetylene flow-rate on flame background absorption a t 184.9 nm. Fuel flow decreases as instrument settings increase 13.6 13.7 13.8 13.9 14 SENSITIVITY, DETECTION LIMITS AND INTERFERENCES- With the optimum fuel flow-rate, spectral slit width and source operating power condi- tions established, the sensitivity values for 1 per cent.absorption shown in Table I were obtained for mercury(I1) in aqueous solution by using the 253.7 and 184.9-nm lines, and TABLE I SENSITIVITY AND DETECTION LIMITS FOR THE ATOMIC-ABSORPTION SPECTROPHOTOMETRY OF MERCURY Sensitivity (1 per cent. absorption), Linelnm Analyte Flame p.p.m. 253.7 HgINO, Air - C2H2 1.0 HgINO, Separated N20 - C,H, 2.5 Hg11(N0,)2 Air - CsHa 3.0 184.9 HgINO, Air - C,H, 0-05 HgINO, Separated N,O - C,H, 0.05 HgIVO,) 2 Air - C,H, 0.1 0.1 HgII(NO,)z Separated NaO - C,H, Detection limit, p.p.m. 2.0 3.0 5-0 0.1 0.02 0.3 0.05January, 19731 61 6-cm path length air - acetylene and separated nitrous oxide - acetylene flames.Fig. 2 shows the calibration graphs obtained for mercury(I1) and mercury(1) by using the 184-9-nm line in the concentration range between 1 and 50 p.p.m. in the separated nitrous oxide - acetylene flame. For comparison, the calibration graph obtained for mercury(1) in the same flame at 253.7 nm is also shown. The detection limits, defined as the concentration in parts per million of mercury in aqueous solution that produces a signal equal to twice the standard deviation in the background recorded near the limit of detection, are also shown in Table I. As shown in Fig. 2, the introduction of mercury as mercury(1) rather than as mercury(I1) produces an enhancement in absorption similar to that previously 0bserved.l The effect has been attributed to the disproportionation of the Hg,2+ ion to produce free elemental mercury : Hg22+ -+ Hg2+ + Hgo and provides a partial pressure of mercury vapour very low in the flame.No significant chemi- cal or physical interference effect has been observed in the absorbance signal produced by a 10 p.p.m. mercury(I1) solution in the presence of fifty-fold mass excesses of copper, cobalt, aluminium, antimony, chromium, sodium, nickel, magnesium, zinc, tin, iron, chloride, nitrate, phosphate, sulphate and fluoride. SPECTROPHOTOMETRY WITH A NITROUS OXIDE - ACETYLENE FLAME I 0 Mercury concentration, p.p.m. Fig. 2. Calibration graph for mercury: A, mercury as Hg(1) by use of a separated nitrous oxide - acetylene flame a t 184.9 nm; B, mercury as Hg(1I) by use of a separated nitrous oxide - acetylene flame at 184.9 nm; and C, mercury as Hg(1) by use of an air - acetylene flame at 253.7 nm CONCLUSION The results of the studies described in this paper suggest that the separated nitrous oxide - acetylene flame is an efficient atomising medium for the direct determination of mercury at its 184.9-nm resonance line.The sensitivity obtained for mercury(I1) is approxi- mately fifty times greater than that of the best results obtained by using conventional atomic-absorption spectrophotometry, and the instrumentation used with an air - acetylene flame for the determination of mercury at the 253.7-nm line. For relatively high concentra- tions of mercury, however, use of either flame at 253.7 nm may still possibly be the most convenient if sample dilution is to be avoided.Although with extremely intense electrodeless discharge lamp sources other flames, such as those of air - acetylene and nitrogen - hydrogen- entrained air, may provide sensitivity for 1 per cent. absorption at 184.9 nm similar to that obtained in the separated nitrous oxide - acetylene flame, by avoidance of the relatively large sample dilution effect by the flame gases that occurs in the separated nitrous oxide - acetylene52 KIRKBRIGHT, WEST AND WILSON flame, this result is achieved only at the expense of a lower flame transmission. The high flame background absorption then results in severe degradation of the signal to noise ratio obtainable, i.e. , poorer detection limits. The enhancement of the mercury absorbance at 253.7 nm when mercury(1) is nebulised rather than mercury(I1) , due to disproportionation of the Hg,2+ ion,l is also observed when the 184.9-nm line is used.The ratio of the 1 per cent. absorption sensitivity for mercury(1) observed at 184.9 nm to that at 253.7 nm in both nitrous oxide - acetylene and air - acetylene flames (50 : 1 and 20 : 1, respectively) corresponds approximately to the value of 30:l expected from calculation of the ratio of the peak absorption coefficients (K0184.9/K0253.,) by. using the classical equation for KO,’ which involves the different wavelengths, Doppler half-wdths and oscillator strengths of the lines. A similar enhancement in 1 per cent. absorption sensitivity was also observed for mercury(I1) in the air - acetylene flame at 184.9 nm compared with 253.7 nm. With the minimum sample volume requirement of 2 ml with our apparatus, the absolute limits of detection attainable for mercury(1) and mercury(I1) become 40 and 100 ng, respectively; the improvement in sensitivity obtained should enable the pre-concentration of mercury by solvent extraction, which is at present required in many analytical procedures when the final determination is made at 253.7 nm in an air - acetylene flame, to be avoided. We are indebted to Beckman Instruments Inc. for the provision of a research grant to one of us (P.J.W.) in support of this work. REFERENCES 1 . 2. 3. 4. 5. 6. 7 . Hingle, D. N., Kirkbright, G. F., and West, T. S., Analyst, 1967, 92, 759. “Perkin-Elmer Methods Handbook,” Perkin-Elmer Corporation, Nomalk, Connecticut, U.S.A. L’vov, B. V., and Khartsyzov, A. D., Zh. Prikl. Spektrosk., 1969, 11, 413. Kirkbright, G. F., and Ranson, L., Amzlyt. Chem., 1971, 43, 1238. Kirkbright, G. F., and Marshall, M., Ibzd., 1972, 44, 1288. Kirkbright, G. F., West, T. S., and Wilson, P. J., Atomic Absorption Newsletter, 1972, 11 ( 3 ) , 53. Walsh, A., Spectrochim. Actu, 1955, 7 , 108. Received July 17th, 1972 Accepted August 31st, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800049

出版商:RSC    

年代:1973

数据来源: RSC