ISSN:0003-2654    

DOI:10.1039/AN97398FX009

出版商:RSC    

年代:1973

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97398BX011

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

iv THE ANALYST [March, 1973THE ANALYSTEDITORIAL ADVISORY BOARDChairman: H. J. Cluley (Wembley)*L. S. Bark (Salford) W. Kemula (Poland)*G. F. Kirkbright (London)G. W. C. Milner (Harwell)G. H. Morrison (U.S.A.)*G. Nickless (Bristol)*J. M. Ottaway (Glasgow)*G. E. Penketh (Billingham)S. A. Price (Tadworth)D. 1. Rees (London)E. B. Sandell (U.S.A.)A. A. Smales, O.B.E. (Harwell)H. E. Stagg (Manchester)E. Stahl (Germany)A. Walsh (Australia)T. S. West (London)P. Zuman (U.S.A.)R. Belcher (Birmingham)L. J. Bellamy, C.B.E. (Waltham Abbey)L. S. Birks (U.S.A.)E. Bishop (Exeter)E. A. M. F. Dahmen (The NetherlandsjA. C. Docherty (Billingham)D. Dyrssen (Sweden)*W. T. Elwell (Birmingham)*D. C. Garratt (London)*R. Goulden (Sittingbourne)*R. C.Chirnside (Wembley)J. Hoste (Belgium)D. N. Hume (U.S.A.)H. M. N. H. Irving (Leeds)A. G. Jones (Welwyn Garden City)M. T. Kelley (U.S.A.)*A. Townshend (Birmingham)*J. A. Hunter (Edinburgh)* Members of the Board serving on the Executive Committee.NOTICE TO SUBSCRIBERS(other than Members of the Society)Subscriptions for The Analyst, Analytical Abstracts and Proceedings should beThe Chemical Society, Publications Sales Office,Blackhorse Road, Letchworth, Herts.Rates for 1973sent to:(a) The Analyst, Analytical Abstracts, and Proceedings, with indexes . . . . f37.00(b) The Analyst, Analytical Abstracts printed on one side of the paper (withoutindex), and Proceedings . . . . . . . . . . . . . . f38.00(c) The Analyst, Analytical Abstracts printed on one side of the paper (withindex), and Proceedings . .. . . . . . . . . . . . f45.00The Analyst and Analytical Abstracts without Proceedings-(d) The Analyst and Analytical Abstracts, with indexes . . . . . . . . €34.00(e) The Analyst, and Analytical Abstracts printed on one side of the paper (without(f) The Analyst, and Analytical Abstracts printed on one side of the paper (withindex) . . . . . . . . . . . . . . . . . . f35.00index) . . . . . . . . . . . . . . . . . . €42.00(Subscriptions are NOT accepted for The Analyst and/or for Proceedings alonevi SUMMARIES OF PAPERS IN THIS ISSUE [March, 1973Summaries of Papers in this IssueThe Rapid Estimation and Control of Precision byDuplicate DeterminationsStudies on computer-simulated models have provided several new methodsof estimating, studying or controlling analytical precision in real systems.The methods are based upon precision estimators derived from the differencebetween duplicate analyses, and take into account variations in the precisionof a determination with the concentration of the substance being determined.The methods have been checked by applying them to simulated samples ofmany duplicate analyses drawn by Monte Carlo techniques from specifiedpopulations, that is, in effect, from analytical systems with known precisioncharacteristics.Some examples show the application of the methods inpractice.MICHAEL THOMPSON and RICHARD J. HOWARTHApplied Geochemistry Research Group, Imperial College, London, SW7 2BP.Analyst, 1973, 98, 163-160.The Determination of Vitamin E in Food by Colorimetry andGas - Liquid ChromatographyTwo methods for the determination of vitamin E in food are described:a colorimetric procedure for determining total tocopherols, based on theirreaction with iron(II1) chloride and bathophenanthroline (4,7-diphenyl-l,10-phenanthroline) , and a gas - liquid chromatographic method for determiningindividual tocopherols.Techniques for the removal of interfering substancessuch as sterols and tocopherol dimers are given and results obtained byboth methods for the vitamin E contents of some uncooked foods are com-pared. A complete determination can be carried out in 1 day by colorimetryand in 2 days by gas-liquid chromatography.A.A. CHRISTIE, ANNE C. DEAN and BARBARA A. MILLBURNDepartment of Trade and Industry, Laboratory of the Government Chemist, CornwallHouse, Stamford Street, London, SE1 9NQ.Analyst, 1973, 98, 161-167.Chemical Composition of Alleppy Cardamom Oil byGas ChromatographyAn isothermal gas-chromatographic technique used to investigate thechemical composition of cardamom oil is described. A total of twenty-onecomponents were detected by our method. Most of the peaks, representing98.1 per cent. of the oil, have been identified.A. K. S. BARUAH, S. D. BHAGAT and B. K. SAIKIARegional Research Laboratory, Jorhat, Assam, India.Analyst, 1973, 98, 168-171.Fungicide ResiduesFruits by Gas ChromatographyPart 111. The Determination of Residues of Binapacryl in SelectedA simple method for determining residues of binapacryl in selected fruitsis presented. After extraction with a mixture of hexane, diethyl ether anddimethylformamide, binapacryl is separated from interfering co-extractivesby chromatography on a silica gel column and is quantitatively determinedby electron-capture gas - liquid chromatography.A confirmatory chemicaltest for binapacryl is also described.P. B. BAKER and R. A. HOODLESSDepartment of Trade and Industry, Laboratory of the Government Chemist,Cornwall House, Stamford Street, London, SE1 9NQ.Analyst, 1973, 98, 172-175March, 19731 SUMMARIES OF PAPERS IN THIS ISSUEThe Rapid Determination of the Organophosphorus PesticidesDiazinon and Dichlorvos in Blood by Gas ChromatographyA rapid method for the gas-chromatographic determination of diazinonand dichlorvos in blood is described.Protein is precipitated by mixingthe blood with an excess of acetone containing an internal standard and thcsupernatant liquid is injected, directly or after concentration, into the gaschromatograph.Recoveries are satisfactory from 0.1 ml of blood containing 0.1 p.p.m.of pesticide without concentration of the extract. If a 1-ml sample is usedand the extract concentrated, 0.02 p.p.m. can be determined. It is suggestedthat the method should be applicable to a wide range of organophosphoruspesticides.The method is also shown to be effective for determining diazinon intissues and organs, but it is not recommended for this purpose.A.F. MACHIN, M. P. QUICK and D. F. WADDELLBiochemistry Department, Ministry of Agriculture, Fisheries and Food, CentralVeterinary Laboratory, New Haw, Weybridge, Surrey.A thermionic phosphorus detector is used.Analyst, 1973, 98, 170-180.vi iThe Examination of Tetrachlorvinphos and its Formulations forthe Presence of Tetrachlorodibenzo- p-dioxins by a Gas - LiquidChromatographic MethodA gas-chromatographic method for the examination of the insecticidetetrachlorvinphos [Gardona, the Z- or trans-styrene isomer of 2-chloro-1- (2,4,5-trichlorophenyl)vinyldimethyl phosphate] for the presence of tetra-chlorodibenzo-p-dioxins is described.Procedures are described both for the extraction and clesn-up of thesample and for the final analysis to determine tetrachlorodibenzo-p-dioxinsdown to 0.025 p.p.m.or less by gas - liquid chromatography with electron-capture detection.Tetrachlorodibenzo-p-dioxins were not detected in various samples oftetrachlorvinphos and its formulations, either before or after acceleratedstorage for 2 weeks at 55 "C.T. J. N. WEBBER and D. G. BOXShell Research Limited, Woodstock Agricultural Research Centre, Sittingbourne,Kent.Analyst, 1973, 98, 181-189.The Application of the Thin-layer Chromatographic - EnzymeInhibition Technique to Organochlorine InsecticidesThe application of the thin-layer chromatographic - enzymatic techniqueto lindane, Pjf-DDT, methoxychlor, heptachlor, aldrin, dieldrin and endrin hasbeen investigated. By use of this technique, lindane in amounts of 500 ngor more is detected, whereas the other pesticides tested remain undetectedup to 10 pg. Positive results for the tests for organophosphorus and carbamatepesticides with the thin-layer chromatographic - enzymatic technique there-fore require corroboration from other tests to exclude the possibility of thepresence of lindane. Kinetic enzymatic studies have shown that lindane isa non-competitive inhibitor of carboxylesterase, the predominant enzyme ofliver homogenate, which is responsible for the hydrolysis of a chromogenicsubstrate.M. BOGUS2 and T. BORKOWSKIInstitute of Forensic Research, Westerplatte 9, I<rak6w, Poland.Lindane does not inhibit the cholinesterase in liver homogenate.Analyst, 1973, 98, 190-193

ISSN:0003-2654    

DOI:10.1039/AN97398FP025

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

March, 19731 THE ANALYST ixSpeakers:hl. A. Leonard(Queen’s UniversityFOR SALEDepartment of ChemistryADVANCES IN CHEMISTRYSERIESLECTURES & COURSESAPPARATUSSURPLUS FOR SALEHilger Medium Quartz Spectroscope with accessoriesAbel l’lash Point ApparatusPrnsky Martin Flash Point ApparatusRedwood Viscometer No. 1Redwood Viscometer No. 2Isorna tic HeatcrBut yrorefractometrrB.D.H. 1-ovibond TintometerLovibond ComparatorG & T.Shaker for 4 flasksCarnliridge RecorderPlatinum Electrodes for Analysis (unused)B1I:A Jrlly TesterTar Baker-Pectin Strength TesterKek Mill (Laboratory size)Marconi pH metersCarnlxidge pH meterElectric Muffle furnace ( 3 sq. in. cross-section)Stainless Steel Tincture PressBuy now and avoid V.4TTHOMAS McLACHLAN & PARTNERS,4 Hanway Place, London, W1P 9DFAPPOINTMENTS VACANTANALYTICAL INSTRUMENTS LABORATORY.Applicationsare invitrd from persons with a university degree or its equivalent tooperate the day to day functions of the laboratory, and to assist inthe drvclopnient of ‘on-line’ analytical techniques. The laboratoryis well-equipped with a range of instruments and linked to a real-timecomputer system. Experience in mass spectrometric and spectro-scopic methods of analysis is required. Background knowledge ofinstrumentation niaintenance and modification would be an ad-vantage. Applications in writing giving details of experience andnaming two referres to the:-Head of Department of ChemicalIfriginwring and Chemical Technology, Imperial College, Londons w 7 2BX.Belfast).G .Nickless(University of Bristol)A. Townsend(University of Birmingham).I. K. Foreman(Laboratory of the Govt.Chemist)L.S Bark(University of Salford)J. M. Bather(IJ.M.1.S.T.)A Symposium onADVANCES IN RESEARCH INANALYTICAL CHEMISTRY12th and 13th April, 1973The symposium is intended topresent some of the fundamentaland applied analytical chemicalresearch being studied a t present.Short contribution to the dis-cussion papers are invited.IFurther particulars and form of application can be obtained from theRegistrar, U.M.I.S.T., P.O. B ~ x No. 88, Manchester M60 1QD.UNIVERSITY OF BRISTOLDepartment of Inorganic ChemistryM.Sc. COURSE IN ANALYTICAL CHEMISTRY(With Special Reference to Instrumental Methods)OCTOBER 1973-1974Applications are invited from persons who hold or expect to beawarded, an appropriate Honours degree or equivalent qualification.The course offers a complete background to the principles and practiceof instrumental analytical chemistry.The aim is to develop a research approach to analytical chemistry andinvestigation of relevant analytical problems including pollutionstudies will form a major part of the course.Applications and enquiries should be sent to The Registrar, TheUniversity, Senate House, Bristol, as soon as possible.ASSISTANT ANALYSTBAHAMASUp to f4,700 + Gratuity* NO LOCAL INCOME TAX* Free passages* Gratuity 15% of basic salary* 3 year tour* Subsidised Housing* Generous paid leaveThe Ministry of Health requires anAssistant Analyst with a degree inChemistry or Bacteriology and two yearsexperience in a Public Analyst Labora-tory to work under the direction of thePublic Analyst in the area of food, drugsand forensic investigationsApply to: CROWN AGENTS, M.Division, 4, Millbank,London, SW1P 3JD for application form and furtherparticulars stating name, age, brief details ofqualifications and experience and quoting reference number M2X/730161/AADX THE ANALYST [March, 1973APPOINTMENTS VACANT7 I 1 I . 1 rn!yh!cal w IPromotion on merit, to moresenior posts is consideredperiodically, the next being thatof Senior Chemist on the scale so ut h A i ca €3,05643,699 (approximately)Control Board now has vacanciesThe Wheat Industry per annum.Benefits include a 5 dayfor Analytical Chemists in itsQuality Control CerealLaboratory.The work involves thedevelopment of new analyticalmethods and the improvementof existing ones in the field ofwheat, barley, oats and rye andrelated products.Applicants should bequalified at graduate level,preferably in chemistry and/orbiochemistry.They should becapable of carrying outinvestigations independently,initiating new analyticaltechniques and supervisingtechnicians. A knowledge andunderstanding of instrumentaltechniques and continuousflow processes and someexperience in cereal analysiswould be advantageous.scale €2,25242,895(approximately) per annum.Salaries will be on theweek, ample leave privileges, a-holiday savings bonus and travelconcession, a medical benefitscheme, an insurance pensionscheme and a housing loansubsidy scheme.The successfulapplicants will be required tocomply with the Government’simmigration formalities.Travelexpenses will be paid by theBoard.Interviews will be heldat convenient centres in yourcountry of residence.Applications, giving fulldetails of qualifications,experience, nationality, maritalstatus, date of birth and generalhealth condition should be sentas soon as possible to:The ManagerWheat Industry Control BoardP.O. Box 908PRETORIARepublic of South AfricMarch, 19731 THE ANALYST xi c TRACE ELEMENTANALYSISPRO B L E M S?Activation Analysis could be the answer, Thistechnique offers the advantages of high sensitivityand speciflcity, good accuracy and precision, even atsub-p.p.m.concentrations. A bulk analysis isobtained, often non-destructively. The maindisadvantage (the need for a nuclear reactor) can beovercome by making use of the Activation AnalysisService provided by the Universities Research Reactor.If you would like to discuss your particular problem,or would like to receive further details, please contact:Dr. G. R. Gilmore (Activation Analyst),Activation Analysis Service,Universities Research Reactor,Rirley,Nr. Warrington,Lancr.Phone : Warr. 32680,33114BUREAU OF ANALYSEDSAMPLES LTD.announce the issue of the followingNEW SAMPLESChemical StandardsNo.387 Nimonic 901 Alloy (12% Cr,6% Mo) Also available asspectroscopic standard.No. 389 High Purity Magnesite stan-dardised for B,O,No. 390 High Tensile Brass (BS 2874CZ 114)Spectroscopic StandardsNos. 65 1-655 Malleable Irons contain-ing increments of C,Si, Mn, S and PNos. 666-670 Nodular Irons contain-ing increments of C,Si, Mn, S, Ni and MgFor further details please write to:-NEWHAM HALL, NEWBY,MIDDLESBROUGH, TEESSIDE, ENGLAND.TS8 QEAor Telephone 0642 37216SPECIALIST ABSTRACTJOURNALSpublished bySCIENCE AND TECHNOLOGY AGENCYAtomic Absorption and FlameEmission Spectroscopy AbstractsVol. 5, 1973, bimonthly €24X-Ray Fluorescence SpectrometryAbstractsVol. 4, 1973, quarterly €24Thi n-Layer Chromatography AbstractsVol.3, 1973, bimonthly €24Gas 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. 24,1973, quarterly €30Electron Microscopy AbstractsVol. 1, 1973, quarterly €30Liquid Chromatography AbstractsVol. 1, 1973, quarterly €30Electron Spin Resonance SpectroscopyAbstractsVol. 1, 1973, quarterly €30Sample copies on request from:SCIENCE AND TECHNOLOGY AGENCY,3 DYERS BUILDINGS, HOLBORN,LONDON, E.C.1, ENGLAND01 -405 932xii SUMMARIES OF PAPERS IN THIS ISSUEThe Analysis of Crops to Determine Neutral Conjugates of anN-Hydroxymethyl Derivative of Monocrotophos InsecticideFollowing the application to plants of the organophosphorus insecticidemonocrotophos (AZODRIN; dimethyl cis-1-methyl-2-methylcarbamoylvinylphosphate) , an N-hydroxymethyl derivative can sometimes be detected asa breakdown product.This product is capable of conjugation with plantsugars, and the present paper describes the development of an analyticalmethod for the determination of this conjugate.In the laboratory, the conjugate can be converted into a complex mixtureof enzyme inhibitors by reaction with hydrochloric acid. However, if thereaction is carried out in the presence of butane-2-thiol, the reaction can becontrolled, and there is quantitative conversion into an s-butyl thioether.This derivative can be determined down to a level of 0.03 p.p.m.by a cholin-esterase inhibition technique, and added specificity can be obtained bycarrying out the inhibition reaction directly on a thin-layer plate afterchromatography.The verification of the procedure by using radioisotope techniques isdescribed.[March, 1973K. I. BEYNON, K. E. ELGAR, B. L. MATHEWS and A. N. WRIGHTShell Research Ltd., Woodstock Agricultural Research Centre, Sittingbourne, Kent.Analyst, 1973, 98, 194-201.Applications of Sensitised Reactions. SpectrophotometricDetermination of Tin in Steel with Catechol Violet andCetyltrimethylammonium BromideModification of the visible spectrum of the catechol violet - tin(1V)complex by the addition of surfactants and basic dyes has been studied inan attempt to improve the catechol violet procedure for determining tin.On adding cetyltrimethylammonium bromide, the cetyltrimethylammoniumsalt of the catechol violet - tin(1V) complex anion is probably formed incolloidal solution and dispersed by the excess of the surfactant.The Brilliantgreen cation reacts with the complex anion in a similar manner and the saltformed can be dispersed with poly(viny1 alcohol). In the latter instance,however, the bathochromic shift in the main absorption band of the complexis not associated with increased sensitivity.The spectrophotometric method of determining tin with catechol violetand cetyltrimethylammonium bromide has been modified and applied to thedetermination of tin in steel.Preliminary separation of tin is achieved byextracting tin(1V) iodide into toluene. Results obtained with a wide rangeof British Chemical Standard steels indicate a high precision for the recom-mended procedure. Lactic acid is used to mask molybdenum, chromium,vanadium, nickel and cobalt, which interfere a t the levels that are commonlyfound in steels. The recommended procedure is not applicable to steels withcopper to tin ratios greater than 20: 1 owing to precipitation of copper(1)iodide at the extraction stage.A. ASHTON, A. G. FOGG and D. THORBURN BURNSDepartment of Chemistry, Loughborough University of Technology, Loughborough,Leicestershire, LEll 3TU.Analyst, 1973, 98, 202-208March, 19731 THE ANALYST xiiiDECENNIAL INDEXESTO THE ANALYST1906-19151... €2101916-1925* . . €2.101936-1945* . . €2.101946-1955* . . €51956-1965” . . €675* Bound in cloth boardsf Paper boundObtainable fromThe Society for Analytical ChemistryBook Department9/10 Savile Row,London, WIX IAFBOOKSM ON OG RA PH SREPRINTSorders for all publications ofthe Society (except journals)should be sent direct or througha bookseller to-THE SOCIETY FORANALYTICAL CHEMISTRYBook Department9/10 Savile Row,London, WIX IAFPARTICLE SIZE ANALYSISI970THE Society for Analytical Chemistry has published in this book all papers presented atthe Second Particle Size Analysis Conference, held i n Bradford in September 1970, andthe full discussions on them.The 35 papers cover all aspects of research into the subject, basically covering the4-year period since the f i r s t conference was held i n Loughborough in 1966, and includeplenary lectures by the late Professor H.Heywood and by Professor K. Leschonski.The volume is a companion to “Particle Size Analysis” - the report of the First Con-ference, also published by the Society.Pp. x + 430Price €7.75Obtainable 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 €6.2xiv SUMMARIES OF PAPERS IN THIS ISSUEA Study of the Variation with pH of the Solubility and Stabilityof Some Metal Ions at Low Concentrations in Aqueous Solution.Part I1Atomic-absorption spectrophotometry was used to dcterminc trace levclsof metal ions in aqueous solution in order to invcstigatc the effect of pHupon their stability, solubility and ease of adsorption during storage.It wasfound that of the thirteen metal ions investigated, namely those of gold,barium, bismuth, cadmium, indium, lithium, palladium, platinum, rhodium,ruthenium, antimony, tin and thallium, with the exception of lithium, noneis stable over the pH range from 1 to 11, and that to ensure the stability ofsolutions containing these ions it is necessary to acidify them to pH 1 withhydrochloric acid immediately after sampling.A. E. SMITHImperial Chemical Industries Limited, Mond Division, liesearch ant1 1 )cvclopmentDepartment, IVinnington Laboratory, Northwich, (:hc.sliirc,, C\VX 41 ) J .LMarch, 1973Analyst, 1973, 98, 209-212.A Comparison of a Wet Pressure Digestion Method withOther Commonly Used Wet and Dry-ashing MethodsA wet-digestion method for the determination of metals and pliosphorusin biological material that involves the use of pressure is described andcomparisons are made with the standard dry and wet-ashing mcthods andthe sulphuric acid - hydrogen peroxide method.Big sage ( A rtemisia tvidczztata)was chosen as the test material. The pressure method has tlic advantage ofbeing able to digest many samples at the same time (to the extent that thecations sought and phosphorus can be extracted into dilute nitric acid) withvery little equipment, small volumes of acid and with a low digestion tem-perature (that of hot tap water), with the precision of more laborious standardmethods.WILLIAM J.ADRIANColorado Division of Wildlife, Fort Collins, Colorado, U.S. A.Analyst, 1973, 98, 213-216.Continuous Determination of Ammonia in Condensed Steamand High-purity Boiler Feed-water by Using a PotentiometricAmmonia ProbeThe Electronic Instruments Ltd. 8002-6 Ammonia Probe lias beeninvestigated for its use in the continuous determination of ammonia incondensed steam and boiler feed-water. The technique is simple and ofadequate precision and accuracy for use in the power industry. The standarddeviations of single measurements made on 0.1 and 0.44 p g nil-1 ammoniasolutions not more than 12 hours after standardisation were 0.008 and0.04 pg ml-l of ammonia, respectively.D. MIDGLEY and K. TORRANCECentral Electricity Research Laboratories, Kelvin Avenue, Lcathcrhcad, Surrey.Analyst, 1973, 98, 217-222.Improvements to an Automatic Counter for RadioactiveDeposits on PlanchetsA method for linking a counter - printer system to an automatic samplecarrier holding twenty-four planchets is described. Serial counting of thesamples can be indefinitely replicated on a basis of either prc-determinedcounts or pre-determined time, the results being printed out on to a paperstrip.C. P. LLOYD-JONES and E. J. SKERRETTUniversity of Bristol, Department of Agriculture and Horticulture, Iiescarch Station,Long Ashton, Bristol.Analyst, 1973, 98, 223-226

ISSN:0003-2654    

DOI:10.1039/AN97398BP031

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

MARCH, 1973 THE ANALYST Vol. 98, No. I164 The Rapid Estimation and Control of Precision by Duplicate Determinations BY MICHAEL THOMPSON AND RICHARD J. HOWARTH (Applied Geochemistry Research Group, Imperial College, London, SW7 2BP) Studies on computer-simulated models have provided several new methods of estimating, studying or controlling analytical precision in real systems. The methods are based upon precision estimators derived from the difference between duplicate analyses, and take into account variations in the precision of a determination with the concentration of the substance being determined. The methods have been checked by applying them to simulated samples of many duplicate analyses drawn by Monte Carlo techniques from specified populations, that is, in effect, from analytical systems with known precision characteristics.Some examples show the application of the methods in practice. IT is rare to encounter analytical laboratories in which precision is regularly measured and controlled. Usually the precision of a method is established during the development stage by the replicate analysis of a few samples judged to be typical of the material that is to be analysed. This is a necessary stage in development, but does not account for any addi- tional sources of variation that occur in day-to-day work, such as those due to small modi- fications in technique, which all analysts evolve, the varying skill of different analysts, an unexpected change in the nature of the samples or some unobserved operational or instru- mental factor. This situation is tolerated at one extreme, when the precision of a method is considerably better than that Iequired for the application, and at the other extreme, when it is not recognised that bad analytical results are stemming from failure to achieve the necessary precision.In applied geochemical surveys very large numbers of samples of stream sediment, soil, rock, etc., are analysed at a high rate to determine elements of interest in studies of mineral exploration or agricultural trace element problems. To ensure the economic viability of such an approach the analytical methods are reduced to the bare essentials by reducing the materials and labour required to a minimum. The variability of the analytical results produced is therefore usually the highest that can be accepted for the application.In this situation the measurement and control of precision is of great importance because any additional source of variability is liable to render the analytical results meaningless. METHODS OF ESTIMATING PRECISION- Methods that have been used to estimate the precision within a batch of analytical results suffer from certain drawbacks. The most straightforward consists in the replicate analysis of a few selected samples and the subsequent calculation of the standard deviation. Unless random sampling is used the samples selected may not be representative of the batch. Indeed, there is a risk that the replicates may be especially chosen because they are not representative, i.e., if they are selected from the extremes of the concentration range to demonstrate the variability in precision over the range.If this method is used the analyst becomes involved in time-consuming calculations. The “statistical series” method has been much used in mineral exploration work.1 This method is based on the analysis of a series of mixtures of two bulk samples of material selected so that their matrices are similar to the samples analysed in the batch, and the concentration of the element sought varies over the whole range found in the samples of the batch. The precision is obtained in terms of a single value for standard deviation. The statistical basis of this method has been criticised on the grounds that no allowance is made for variation in the absolute or relative precision over the concentration range.2 Again, the bulk samples chosen for the “statistical series” may not represent the samples that were analysed in the batch, and the analyst is involved in carrying out calculations.There is the additional risk that the mixing of the two bulk samples may not be completely effective. @ SAC and the authors. 153154 THOMPSON AND HOWARTH: RAPID ESTIMATION AND [Analyst, VOl. 98 Analysts have always used duplicate analyses to gauge the reproducibility of their results, but comparatively rarely use the methods that are available to make these duplicate results quantitative in terms of precision. These methods again assume that the standard deviation is invariable, and therefore only apply to samples that fall within a very limited concentration range.In one method use is made of the formula s = dCd2/2n, where s is the estimate of standard deviation, d is the difference between the duplicates and n is the number of pairs of determination^.^ In another method the difference between the duplicates is regarded as the range of two values, and use is made of the range as an estimator for standard deviation by multiplying the range by a f a ~ t o r . ~ ~ ~ However, the range of two values has a peculiar sampling frequency distribution and single values are apt to be misleading. In the present work we describe studies carried out by computer simulation techniques concerning the sampling frequency distribution of duplicate analyses in analytical systems when the standard deviation varies with concentration. Simple graphical and computational methods for estimating precision have been evolved, tested and applied to actual cases arising in geochemical practice.DEFINITIONS- Definitions of precision and detection limit vary from laboratory to laboratory. The methods of estimation described in this paper can be adapted to suit definitions other than ours. By precision we mean twice the coefficient of variation expressed as a percentage, or P, = 200 alp., where p is the mean and a the standard deviation of a normal (gaussian) distribution N(p,a). By detection limit we mean the concentration at which P, = 100, or p = 2a. SAMPLING DISTRIBUTION OF PRECISION ESTIMATORS BASED ON DUPLICATE MEASUREMENTS- If a pair of duplicate samples, x1 and x2, are taken from a parent population N(p,ap), the sampling distribution of the difference between these values (x, - x,) is also normal, N(O,aa), and it can be shown that a d = 2/Tap.The distribution of the absolute difference between duplicate values lxl - x21 is, in effect, the positive half of N(O,(Td) (see Fig. 1). The value of up can then be obtained from a population of duplicate values in three different ways: by cal.culation of ad/2/3;-by obtaining the mean value of Ixl - x21, or d (it can be showns that d = 0.798 a d , or 1.128 up) ; and by obtaining the median value of Ixl - xzI, or Md. From tables of the area under the normal curve we find that Md = 0.675 a d , or 0.955 up. From each of these methods one can obtain an estimate (Sp) of ap based on a sample of n duplicate pairs. For large values of n the sampling variance can be derived analytically' and var S, is 0.50 ai/n, 0.58 ui/n and 1.35 aJn for the three methods, respectively.Hence, the use of the median (Md) is less precise than the use of the other two methods, although it will be influenced to a lesser extent by gross errors and can be found graphically. The fortuitous close approximation of Md and ap means that up can be estimated from the median without any calculation. Fig. 1. Position of the mean ( d ) and median (Md) of the half-normal curve relative to its standard deviationpd. Md = 0.675 Ud; d = 0.798 UdMarch, 19731 CONTROL OF PRECISION BY DUPLICATE DETERMINATIONS 155 The foregoing arguments are based on many pairs of duplicate values selected from a single normal population.In practical analytical terms this would correspond to values drawn at random in pairs from any analytical results on the same sample, which would have no advantage over simply determining the standard deviation on a set of replicates. The information that is required in practice is the precision of the method over the whole con- centration range of the samples, including any variation caused by matrix variations in the samples, This information can be obtained from duplicate results on a large number of samples. In statistical terms it is equivalent to drawing two values at random from each of R different populations N(pi,ai), i = 1,R. Even if all the means were equal, there would still be variations in 01 from sample to sample because of variations in the matrix of the substance being determined.(In the analysis of stream sediments, soils, etc., this variation is likely to be considerable.) However, it does not invalidate the conclusions drawn previously from consideration of statistics on a single sample, because the measured analytical variance will simply include variance due to variation of the sample matrix. VARIATION OF PRECISION WITH CONCENTRATION- In any analytical system the precision of determination will vary over the concentration range of the samples. This fact must be taken into account if the concentration range is wide. In a system in which the substance determined occurs in the same matrix in all the samples, the standard deviation of measurement (uC) will increase with concentration, usually as a linear function.* This can be expressed as where a, is the standard deviation at zero concentration, c is the true concentration (equivalent to p above) and k is a constant for the system.In terms of precision this relationship becomes where k, = 200 k. The detection limit, obtained by substituting P, = 100 per cent., is made quantitative as In almost all analytical methods kp is much less than 100, so that the detection limit becomes c = 20,. Thus, the precision falls steadily from 100 per cent. at the detection limit to an asymptotic value of k, at high concentrations. This model of precision variation has been used in all the computer simulations when samples with a range of concentrations have been considered, and has been confirmed in the actual analytical systems to which we have applied the method.TABLE I .. * . (1) .. - (2) .. * * (3) uc = ou + kc .. .. .. P, = 200 ~ O / C + kp .. .. c = 2a,J(l - kp/100) .. .. PRECISION ESTIMATES BASED ON STANDARD DEVIATIONS OF 500 VALUES OF 400 (XI - X,)/(X, + X 2 ) I N A SIMULATED SYSTEM N(lO,a) U 0.01 0.1 1.0 2.6 6.0 10.0 20.0 Pc = 200 o / p Estimated precision 0.200 0.202 2.00 2-04 20.0 20.2 60.0 63.2 loot 116 200 262 400 622 t Detection limit. A DIRECT PRECISION ESTIMATOR- The standard deviation estimators previously referred to can be converted into direct precision estimators by dividing by an estimate of the concentration. For duplicate determina- tions this estimate would be the mean value (x, + x2)/2, so the precision estimator is 400 (x, - x,)/(x, + x,). The deviation of the distribution of this function from the normal curve is not great when the precision is less than 100, Le., above the detection limit.This can be seen in Table I where the precision of the parent population is compared with estimates based on values of 400 (x, - x,)/(xl + x,) selected a t random. * Deviations from this rule can occur if measurements are made beyond the normal analytical range, e . g . , on non-linear parts of calibration graphs.156 THOMPSON AND HOWARTH: RAPID ESTIMATION AND [ATZdySt, VOl. 98 This estimator requires more calculation to give precision by graphical plotting, but can be of use when direct precision values are required and the duplicate data are handled by computer, as in some larger projects. SIMULATION TECHNIQUES- In order to obtain a general impression of the basic form of the frequency distributions Ixl - x21 and 21x, - x2!/(x1 + x,), a large-sample Monte Carlo method was used.Values of c were taken at fixed intervals over the concentration range of interest and used to generate pairs of values selected at random from populations of the form N(c,ac), where uc is defined by substituting appropriate values of k and a. in equation (1). Fig. 2 shows a typical form of the frequency distribution of the relative difference 21x1 - x,l/(xl + x,) with concentration. Log RD ‘3 4--1 Fig. 2. Isometric view of the frequency distribution of relative difference (RD), 2 I x1 - x2 I /(xl + x 2 ) , as a function of concentration c (p.p.m.) for a simulated analytical system (uC = 0.01 + 0.04~).The vertical axis corresponds to relative frequency In a typical set of analytical samples, however, the concentrations will not be uniformly distributed but will be less frequent towards the extremes of the range. In geochemical samples the distribution of trace-element concentrations often tends towards the log-normal [where y = log (c) is normally distributed], and such a model has been used in producing a simulated population of duplicate values for testing the estimation methods. A number of values (R) were picked a t random from a suitable population F ( y ) = N(p,u), and were transformed as ci = lOyi, i = 1,R to create a set of parent concentrations (ci). A pair of values (x1,x2) were then generated from the R populations N(ci,uci), i = 1,R, to give a set of R duplicates very similar to those which would be obtained from an actual set of samples, with the exception that there would be no gross errors, as would occasionally be encountered in practice.These duplicate values were then used to test the various methods of deriving the concentration - precision relation- ship, which was, of course, pre-determined. RESULTS OBTAINED FROM SIMULATED VALUES- Computational method-This method is based on the standard deviation estimator .Said% Sets of duplicate values were simulated as described above, and sorted into increasing orderMarch, 19731 CONTROL OF PRECISION BY DUPLICATE DETERMINATIONS 157 of concentration, as estimated by the value of (x, + x,)/2. These values were then taken in successive groups of twelve and within each group the mean value of (x, - x2)/2 and the corresponding value of Sd/z/Fwere calculated.The relationship between the estimated means and the estimated standard deviations was then found by linear regression. The results obtained corresponded closely with the original generating function, as shown in Table 11. In practice, this method would be most appropriate in circumstances when the analytical data are processed by use of a computer, i.e., when long runs of data are produced automatically at a high rate. TABLE I1 THE ACTUAL AND ESTIMATED RELATIONSHIP BETWEEN c AND oc I N TWO SIMULATED SYSTEMS System 1 System 2 Actual - x t e d Actual -Estimated 1.00 0.92 2.50 2.03 2 0~0100 0.0095 0.100 0.121 A more simple method requires the analysis of the regression of Ix, - x21 on (x, + x z ) / 2 .The regression coefficients are divided by 1.1284 to give the relationship between oc and c directly. However, this method would be more sensitive to the effects of gross errors than would the median technique. Its use is illustrated in Fig. 3, where it was used to derive the relationship shown. 0.8 '1 Fig. 3. Values of]lxl xz.I versus (xl + x,)/2 per cent., obtained by spectrographic determinations of potassmm, also showing the medians (- - -) and functional relationship (-) So = 0.001 + 0-103c, estimated by linear regression Graphical method-In this method a graph of the mean estimator (x, + x 2 ) / 2 ueYsus the standard deviation estimator Ixl - x21 is plotted on log - log graph paper* for each (x1,xa) pair. The points are divided into successive concentration ranges, each containing 10 to 20 points, and the median value of Ixl - x21 in each range is marked.A curve fitted (by eye) through the medians would be taken as showing the relationship between c and crc. Fig. 4 shows such a graph with the medians drawn in; the line representing the generating relationship between c and uc is shown for comparison purposes. * Linear graphs can be used if the concentration range is small.158 THOMPSON AND HOWARTH: RAPID ESTIMATION AND [Ana&St, VOl. 98 The speed and success of this method renders it particularly suitable for routine work when, as is normally the practice, the analyst writes down and checks the results. Another advantage of this method in practice stems from the fact that the median value is only slightly affected by extreme values such as would be caused by a gross error... .. b . - 0 * t - . . . - . * - b . SOME PRACTICAL EXAMPLES- In Fig. 5, the application of the graphical method given above to a rapid atomic-absorp- tion method for determining zinc in stream sediments is illustrated, in a project in which every tenth sample is determined in duplicate. This chart shows a feature not encountered in the computer simulations, owing to the limited resolution of the digital read-out of the instrument, viz., that the observed differences between duplicates consist mainly of small multiples of the last readable figure. This does not affect the median if differences of zero are plotted as half of the last readable figure. This artifact does not appear in Fig.3, in which the results derived from the deter- mination of potassium by use of a direct-reading spectrograph are plotted. In this instance the resolution of the read-out is much lower than the detection limit for the method. CONTROL OF PRECISION- When large numbers of duplicate pairs are available it is easy to obtain the relationship between uc and c over the concentration range. In batches containing less than 200 samples, when only 10 per cent. of the samples are analysed in duplicate, a good estimate of this relationship cannot be made from the relatively few points scattered over the concentration range. However, it is much easier to ascertain whether the points conform to some previously decided standard of precision, either selected by some external requirement, or by previous experience with the method.This conformity can be determined by drawing various per- centiles calculated from the arbitrary precision standard on the chart before plotting the points. The method is illustrated in Fig. 6, which shows a set of data which have better precision than the arbitrary standard of P, = 20 per cent., as delineated by the 90th percentile value as a function of concentration. The combination most useful for precision control is that of the 90th and 99th percentiles, which enables the analyst to establish immediately whether his set of data is conforming to the arbitrary standard, and also whether any points are present that probably belong to a different population from the remainder (Le., gross errors).March, 19731 CONTROL OF PRECISION BY DUPLICATE DETERMINATIONS - 1: .I I 1 1 1 1 1 1 1 I I l l l l l l l I I 1 1 1 1 1 1 1 I I l l l U 159 00 Fig. 5. Values of Ixl - xz I vetws (xl .+ x z ) / 2 p.p.m., obtained for atomic-absorption determinations of zinc, also showing the medians (- - -) and graphically estimated functional relationship (-) S , = 3.0 + 0.033~ CONCLUSION An assumption inherent in this approach has been that the characteristics of the system are invariant within the time period of the observations. Thus, the method would be invalid if applied to sets of duplicate pairs in which significant systematic bias was present between the corresponding results. However, random variations on a time scale that was short in relation to the sampling interval would merely contribute to the over-all variability.10 ( X , +X2)/2 Fig. 6. Values of Ixl - x21 versus (xl + x,)/2 parts per loD, obtained by atomic-absorption deter- minations of mercury, also showing the 90th and 99th percentiles as a function of concentration for Pc = 20 per cent.160 THOMPSON AND HOWARTH For example, if all the duplicate determinations were made within a batch of analyses the method would give an estimate only of the within-batch precision rather than of the over-all characteristics of the analytical system. If, however, some samples were re-analysed in a subsequent batch, perhaps after re-setting the instrument, by introduction of a new batch of reagent, or by a different analyst, then some systematic difference between the correspond- ing results might be detectable (by a significance test) superimposed upon the random vari- ations.In such an instance the methods of estimation outlined would not give a reliable value for precision. Finally, if the same procedure were used on many successive batches a valid estimate of the over-all variance would be obtained, consisting of the sum of the within-batch variances and the variance due to any systematic differences between batches. The project of which this work forms a part is supported by a grant from the Natural Environment Research Council for an investigation under the direction of Professor J. S. Webb into the applicability of statistical techniques to the interpretation of regional geo- chemical data. Computer time has been provided by the Imperial College Computer Centre. The authors thank the referees and Dr. P. J. Brown of the Department of Mathematics, Imperial College, for their helpful comments on the statistics. 1. 2. 3. 4. 5. 6. 7 . REFERENCES Craven, C. A. U., Trans. Instn Min. Metall., 1953-4, 63, 651. Stern, J. E., “A Statistical Problem in Geochemical Prospecting,” M.Sc. Thesis, University of Youden, W. J., “Statistical Methods for Chemists,” Chapman and Hall, London, 1951, p. 16. Dean, R. B., and Dixon, W. J., Analyt. Chew .,,‘1951, 23, 636. Eckschlager, K., translated by Chalmers, R. A., Elandt, R. C., Technometrics, 1961, 3, 551. Kendall, M. G., and Stuart, A., “The Advanced Theory of Statistics,” Volume 1 , Griffin, London, Received September llth, 1972 Accepted November 27th, 1972 London, 1959. Errors, Measurements and Results in Chemical Analysis,” Van Nostrand Reinhold Co. Ltd., London, 1969, p. 89. 1969, pp. 235 to 243.

ISSN:0003-2654    

DOI:10.1039/AN9739800153

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

Analyst, March, 1973, Vol. 98, pp. 161-167 161 The Determination of Vitamin E in Food by Colorimetry and Gas - Liquid Chromatography BY A. A. CHRISTIE, ANNE C. DEAN AND BARBARA A. MILLBURN (De9artment of Trade and Industry, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SE1 9NQ) Two methods for the determination of vitamin E in food are described: a colorimetric procedure for determining total tocopherols, based on their reaction with iron(II1) chloride and bathophenanthroline (4,7-diphenyl-l,10- phenanthroline) , and a gas - liquid chromatographic method for determining individual tocopherols. Techniques for the removal of interfering substances such as sterols and tocopherol dimers are given and results obtained by both methods for the vitamin E contents of some uncooked foods are com- pared.A complete determination can be carried out in 1 day by colorimetry and in 2 days by gas - liquid chromatography. VITAMIN E consists of eight naturally occurring tocopherols, all of which have different biological activities and are readily oxidised, especially under alkaline conditions in the presence of light. The group of compounds comprises four tocols, usually designated a-, /I-, y- and 8-, and four analogous tocotrienols. Of these compounds, a-tocopherol (5,7,8-trimethyl- tocol) is the form most often found in foods, although in some oils, such as corn oil, the ,f3- or y-form (5,s- or 7,s-dimethyltocol) predominates. The biological activities of the members of this group of compounds depend to some extent on the criteria used in their evaluation, but, in general, a-tocopherol is by far the most potent, the other tocopherols and tocotrienols having activities from 1 to 50 per cent.of that of a-tocopherol.1 Methods of analysis that would be of general applicability to a wide range of foods were required and several possible techniques were considered. Direct spectrophotometry is of little value for application to food on account of the low intensity of absorption of tocopherols in ultraviolet light. Spectrofluorimetry provides a very sensitive means of detecting tocopherols but rigorous purification of solvents is essential, otherwise high background values are obtained that off set the advantages of the technique. In a method developed by the Analytical Methods Com- mittee of the Society for Analytical ChemistryJ2 the individual tocopherols are separated by two-dimensional paper chromatography, but the procedure is not well adapted to the routine determination of vitamin E in food.Thin-layer chromatography is widely used in studies of vitamin E metabolism and is of great value in obtaining a rapid assessment of the forms of the vitamin present in food; a recent collaborative study has shown, however, that quanti- tative recoveries are The colorimetric method based on the reduction of iron(II1) ions by the tocopherols and reaction of the iron(I1) with bathophenanthroline (4,7-diphenyl- 1,lO-phenanthroline) to form the red iron(I1) complex4 is sensitive and reliable, provided that interfering substances are removed and care is taken to prevent oxidation of the vitamin.The main disadvantages are that individual tocopherols cannot be distinguished and that they do not give the same absorbances on either a mass or a molar basis. Gas - liquid chromatography offers the greatest possibilities for the separation and determination of individual tocopherols in a single process with a sensitivity of about 0.01 pg. However, as with most other methods, /I- and y-tocopherols are not completely separated and appear as a single peak on the chromatogram. After preliminary trials, the colorimetric and gas - liquid chromatographic methods showed the most promise and were selected for further examination with a range of foods. EXPERIMENTAL SAMPLE PREPARATION- and sampled immediately. products were sampled directly.Egg and milk samples were freeze-dried. Peas, liver and baked beans were macerated Other Lettuce was deep-frozen and crumbled while frozen. 0 SAC: Crown Copyright Reserved.162 CHRISTIE et al. : DETERMINATION OF VITAMIN E I N FOOD [Analyst, VOl. 98 REAGENTS- All reagents should be of recognised analytical quality. 4,7-DiPhenyl-l ,lO-phenanthroline (bathophenanthroline) reagent-Dissolve 0.05 g in 50 ml of absolute ethanol. Store the solution in a dark bottle in a refrigerator and renew it monthly. Iron(II1) chloride reagent-Dissolve 50 mg of FeCl,.GH,O in 100 ml of absolute ethanol. Dilute 5 ml of this solution to 20 ml with absolute ethanol. Store both solutions in dark bottles in a refrigerator. Renew the solutions monthly. n-Hexane-Spectrosol grade.Digitonin, €32.-Material supplied by Koch-Light Laboratories Ltd. was suitable, Diethyl ether, peroxide free. Cyclohexane, M3.C.-Hopkin and Williams. Ethanol, absolute. L-Ascorbic acid. a-D-Tocopherol standard solutions-Prepare a stock solution containing 1 mg of a-D-toco- pherol per millilitre of absolute ethanol. Store it in a refrigerator and renew it at 3-monthly intervals. Prepare a dilute standard solution containing 10 pg of a-D-tocopherol per millilitre of absolute ethanol. Renew the solution at weekly intervals. Aluminium oxide, neutral-M. Woelm, Eschwege, Germany. De-activate the material by adding 10 ml of water to 100 g of aluminium oxide, shaking the mixture well and leaving it in a closed container overnight. Silica gel for dry column chromatography-M.Woelm. Iron(II1) chloride - 2,2'-bipyridyl spray reagent-A solution containing 0.2 per cent. of iron(II1) chloride and 0.5 per cent. of 2,2'-bipyridyl in ethanol. Orthophosphoric acid solution-Dilute 1 ml of orthophosphoric acid (sp. gr. 1-75) to 150 ml with absolute ethanol. REAGENTS AND APPARATUS FOR GAS - LIQUID CHROMATOGRAPHY- Silylating agent-Bis(trimethylsily1)acetamide from Phase Separations Ltd. Pye Gas Chromatograph 104, Model 64-Fitted with a flame-ionisation detector and 2.7 m x 4 mm glass columns packed with 3 per cent. methylsilicone OV-1 on Gas-Chrom Q. The column temperature was 225 to 240 "C and the carrier gas was nitrogen a t a flow-rate of 40 to 60 ml min-1. The recorder chart speed was 5 mm min-1. Internal standard: preparation of a-tocopheryl propiortate-Dissolve 1 SO g of a-tocopherol in a mixture of 5 ml of dry acetone and 1-25 ml of dry pyridine. Add a mixture of 0.28 ml of propionyl chloride and 5 ml of dry acetone dropwise with stirring.Leave the mixture at room temperature overnight in a fume cupboard, then pour it into a separating funnel containing a mixture of 50ml of diethyl ether and 30ml of water. Shake the mixture, separate the ethereal layer and wash it with two 30-ml volumes of 2 N hydrochloric acid followed by two 30-ml volumes of 0.5 N potassium hydroxide solution and finally with water until the solution is neutral. Evaporate the ethereal solution and dissolve the residue con- taining the a-tocopheryl propionate in 2 ml of cyclohexane. Purify the ester by passing the solution down a column containing 100 g of alumina de-activated with 10 per cent.of water and elute it with a 2 per cent. solution of diethyl ether in cyclohexane. Collect 10-ml fractions of the eluate and examine them by thin-layer chromatography for the presence of a-tocopherol. Combine the pure fractions of a-tocopheryl propionate that are eluted from the column first and concentrate the combined fractions to a yellow oil. Weigh the oil and re-dissolve it in cyclohexane to give a solution 1 ml of which is equivalent to 1 mg of a-tocopheryl propionate. Dilute this stock solution with cyclohexane so that 1 ml of the diluted solution is equivalent to 100 pg of a-tocopheryl propionate. Check the concentration of the solution as described below by determining colorimetrically the a-tocopherol that is produced by refluxing the solution with 60 per cent.m/V potassium hydroxide solution for 5 minutes. Store the standard solution in a refrigerator and make a fresh dilution monthly. The stock solution is stable for at least 1 year if it is stored at deep-freeze temperatures (about -15 "C).March, 19731 BY COLORIMETRY AND GAS - LIQUID CHROMATOGRAPHY COLORIMETRIC PROCEDURE 163 SAPONIFICATION AND EXTRACTION- Weigh accurately an amount of food containing 100 to 200 pg of tocopherol (preferably 2 to 5 g, but see below) into a saponification flask. Add 500 mg of ascorbic acid and 10 ml of ethanol and boil the mixture under reflux with a stream of oxygen-free nitrogen passing through the liquid. Add 5 ml of 60 per cent.m/V aqueous potassium hydroxide solution and reflux the solution under nitrogen for a further 15 minutes. If it should be necessary to use an amount of sample greater than 5 g, use a volume of potassium hydroxide solution in millilitres equivalent to the mass in grams of the sample and a volume of absolute ethanol equivalent to twice that of the potassium hydroxide solution; also, increase the amount of ascorbic acid to 1 g for amounts of sample above 20 g. Wash the condenser and nitrogen inlet with 20 ml of ethanol and cool the flask under running water. Transfer the solution quanti- tatively into a separating funnel and extract the unsaponifiable matter by shaking the solution for 2 minutes with 150 ml of peroxide-free diethyl ether. Wash the ethereal layer with four 50-ml volumes of distilled water and reduce it to a small volume on a rotary evaporator.Add about 5 ml of absolute ethanol and evaporate the mixture so as t o remove trace amounts of water. Transfer the solution into a small flask with dry diethyl ether and evaporate it so as to remove trace amounts of ethanol. Add cyclohexane and re-evaporate the mixture so as to remove trace amounts of diethyl ether. Dissolve the residue in the minimum volume of cyclohexane (1 to 2 ml). COLUMN CHROMATOGRAPHY- Pour 15 g of de-activated alumina into a chromatographic column (25 x 1 cm, fitted with a sintered glass disc) containing about 35 ml of cyclohexane, tapping the column during the process so as to remove air bubbles. Add anhydrous sodium sulphate to a depth of about 1 cm.Transfer the residue after saponification and extraction to the top of the column with a few millilitres of cyclohexane and elute the column with 50 ml of cyclohexane at a flow-rate of 1 to 2 ml min-l. Discard the eluate. Replace the eluting solvent with a 7 per cent. solution of dry diethyl ether in cyclohexane and collect 80 ml of eluate when determining a-tocopherol or 150 ml when determining a-, y- and 6-tocopherols. Evaporate this fraction to dryness and dissolve it in absolute ethanol in a calibrated flask so as to give a final volume of 25 ml. Use a 3-ml aliquot of this solution to test for the presence of tocopherol dimers, and if they are present, remove them by dry column chromatography as described below before proceeding with the colour reaction.In the absence of dimers, use a 5-ml aliquot directly for the colour reaction. DETECTION OF TOCOPHEROL DIMERS- Concentrate 3 ml of the ethanolic solution of the tocopherols by evaporation almost to dryness and spot 20 p1 of the concentrated solution on to a thin-layer chromatographic plate of silica gel G. Develop the plate immediately with chloroform and spray it with iron(II1) chloride - bipyridyl solution. Tocopherols and dimers appear as red spots with R, values of 0.3 to 0.5 and 0.7 to 0.8, respectively. If dimers are present, remove them from a 20-ml aliquot of the ethanolic solution by dry column chromatography before proceeding with the colour reaction. REMOVAL OF TOCOPHEROL DIMERS BY DRY COLUMN CHROMATOGRAPHY- Evaporate a 20-ml aliquot of the ethanolic solution to dryness and re-dissolve the residue in 5 ml of cyclohexane.Add 0.3 g of silica gel and remove the solvent by evaporation in a stream of nitrogen. Transfer the sample, adsorbed on the silica gel, on to a glass column (30 cm long x 1.5 cm i.d.) fitted with a sintered disc of porosity 1 and containing 25 g of silica gel.6 Elute the column with chloroform by adding the solvent dropwise so that the solvent front steadily descends the column without allowing excess of solvent to collect at the top of the column. Collect the dimers in the first 15ml of eluate and the tocopherols in the next 75 ml. Evaporate the chloroform from the tocopherol fraction, re-dissolve the residue in 25 ml of ethanol and proceed with the colour reaction on a 5-ml aliquot of the ethanolic solution.164 CHRISTIE etf al.: DETERMINATION OF VITAMIN E I N FOOD [AndyStf, VOl.98 COLOUR REACTION- To prepare the standards, transfer 0, 1, 2, 3, 4 and 5 ml of a 10 pg ml-l a-tocopherol solution by pipette into 10-ml calibrated flasks and dilute the volume to 5 ml with absolute ethanol. Add 1 ml of bathophenanthroline solution to standards and samples, stopper the flasks and mix the solutions well. Carry out the next two stages of the determination by covering the flasks with aluminium foil and working in subdued light. Add 1 ml of iron(II1) chloride solution and mix thoroughly. Exactly 2 minutes later, add 1 ml of phosphoric acid solution and mix again. The flasks can now be brought into the light. Dilute the solu- tions to 10 ml with absolute ethanol, mix and read the absorption of the solutions at 534 nm against absolute ethanol.From the set of standards, determine the concentration of tocopherol in the sample solutions as micrograms per 10ml. Calculation- micrograms per 10ml x dilution factor mass of sample (g) Tocopherollpg g-l = The dilution factor is 5 when dimers are absent and - 25 when dimers are present. 20 GAS - LIQUID CHROMATOGRAPHIC PROCEDURE In this procedure, the saponification, extraction and column-chromatographic stages are the same as those for the colorimetric method. An aliquot of the ethanolic solution containing the purified sample is further treated to remove sterols in two stages before determination of the vitamin by gas - liquid chromatography. REMOVAL OF STEROLS- Transfer a known amount of a-tocopheryl propionate standard solution, equivalent to 100 to 150 pg of tocopherol, by pipette, into a centrifuge tube and remove the solvent by gentle evaporation in a stream of nitrogen.To the residue, add a 10-ml aliquot of the ethanolic solution of the purified sample, mix thoroughly, dilute the mixture with water so that it contains 72 per cent. V/V of ethanol, allow it to stand at about -15 "C for 1 hour and centrifuge. Decant the clear liquid into a solution prepared by dissolving 150 mg of digitonin in 5 ml of 72 per cent. V/V ethanol. Wash the residue in the centrifuge tube with a few millilitres of 72 per cent. V/V ethanol at about 0 "C, centrifuge to remove the solid and add the washings to the digitonin solution. Allow the digitonides to precipitate by leaving the solution at about -15 "C, preferably overnight.Filter the liquid into a separating funnel, extract the solution with two 20-ml volumes of carbon tetrachloride and remove the solvent by evaporation in a small evaporating flask. Dissolve the residue in 0.5 ml of n-hexane. REMOVAL OF TRACE AMOUNTS OF STEROLS ON A CELITE - DIGITONIN COLUMNB- Dissolve 300 mg of digitonin in 5 ml of water with warming and add the solution to 10 g of Celite that has been dried a t 110 "C overnight. Grind these constituents well together in a mortar and pack 3 g of the mixture into a 12 x 1-cm glass column fitted with a sintered- glass disc of porosity 2, by using n-hexane. The solid, which is used only once, keeps for 2 weeks in a screw-topped container.Transfer the sample on to the column and elute it with n-hexane at the rate of 0.5 ml min-1. Collect 15 ml of eluate and evaporate it just to dryness. PREPARATION OF TRIMETHYLSILYL ETHERS- Add 0.2 ml of cyclohexane, containing 50 pg of squalene as a marker, to dissolve the residue in the sample tube, followed by 0.2 ml of bis(trimethylsily1)acetamide. Mix well and leave the mixture at room temperature for 15 minutes. Evaporate off the solvent in a slow stream of nitrogen while warming the mixture. Add 0 6 m l of cyclohexane to the residue and again evaporate the solution just to dryness. Dissolve the residue in 0.5 ml of cyclohexane and inject 1 to 2 pl of the solution into the gas chromatograph.March, 19731 BY COLORIMETRY AND GAS - LIQUID CHROMATOGRAPHY Cahdation- micrograms of internal standard as tocopherol x area of tocopherol peak x 2.5 Tocopherol/pg g1 = area of internal standard peak x mass of sample (g) 165 RESULTS The results obtained for the vitamin E content of several uncooked foods by colorimetry and gas-liquid chromatography are given in Table I.For the recovery studies, 1OOpg of a-tocopherol were added to the samples before saponification. TABLE I COMPARISON OF RESULTS OF COLORIMETRIC AND GAS - LIQUID CHROMATOGRAPHIC DETERMINATIONS OF VITAMIN E I N FOOD Egg (dried) . . Margarine . , Olive oil . . .. Corn oil . . . . Peas (frozen) . . Baked beans (tinned) Lettuce . . ,. Liver (uncooked) Butter . . .. Milk (freeze-dried) Vitamin E/pg g-l I A > Gas - liquid chromatography 1 Colorimetry E- p- + y- S- Total ..94 96 0 0 96 . . 99 76 24 0 100 96 74 24 0 98 .. 108 104 0 0 104 . . 520* 119 395 0 514 .. 8* 0 6 0 6 0 5 0 5 .. 10* 3 7 0 10 .. 12* 5 7 0 12 .. 6 5 0 0 5 * . 34 32 0 0 32 34 33 0 0 33 .. 7 7 0 0 7 - 7 0 0 7 - - - - 110 Recovery of added a-tocopherol, per cent. r i d Colorimetry chromatography 98 98 92 92 - 94 100 97 92 95 - - - - 90 98 80 90 90 - - - * Thin-layer chromatography indicated the presence of tocopherol dimers, which were removed by dry column chromatography before the colour reaction. Values for vitamin E before the removal of dimers were: corn oil 740, peas 8 to 12, baked beans 11 and lettuce 15 to 16 pg g-1. DISCUSSION Owing to the instability of the tocopherols towards oxidising agents in hot alkaline solutions, great care has to be taken during the saponification stage.An inert atmosphere of nitrogen was used during saponification and the addition of various antioxidants to the liquor was tried. Ascorbic acid was found to be the most suitable; the others, which included hydroquinone, butylated hydroxyanisole (2-t-butyl-4-methoxyphenol) and P-acetamido- phenol, were ineffective or undesirable as they caused interference in subsequent processes. An investigation of some of the variables of the saponification stage (temperature, time and alkali concentration) showed that a short saponification time was beneficial and that consistent recoveries of 96 per cent. were obtained from 2 g of dried egg by using 10ml of ethanol, 5 ml of 60 per cent. m/V aqueous potassium hydroxide solution and a reflux time of 15 minutes.Both methods may be affected by interfering substances, such as carotenoids, retinol and cholesterol. The colorimetric method can tolerate at least 25 mg of cholesterol without interference in the determination of 50 pg of a-tocopherol, but can be seriously affected by some carotenoids and other coloured substances, and, if added, antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene (2,6-di-t-butyl-+-cresol). On the other hand, the gas - liquid chromatographic method is very susceptible to the presence of trace amounts of cholesterol (see Table 11) but is largely unaffected by carotenoids and by antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene. Various adsorption and partition columns were examined in attempts to clean up the extracts and to reduce inter- fering substances to an acceptable level.The most effective column was one that contained alumina, de-activated with 10 per cent. of water, with a 7 per cent. solution of diethyl ether166 CHRISTIE ef aE.: DETERMINATION OF VITAMIN E IN FOOD [Analyst, Vol. 98 in cyclohexane as the eluting agent. This column separated the tocopherols from carotenoids, retino1 and cholesterol fairly well and allowed the subsequent colorimetric determination to be made without serious interference. Alumina, however, did not remove tocopherol dimers, which, if present, had to be separated from the tocopherols by dry column chromatography5 before the colorimetric method was applied. In the gas - liquid chromatographic method, the trimethylsilyl ethers of cholesterol and a-tocopherol have similar retention times, 2.31 and 2.40, respectively, relative to squalene.This similarity gives rise to overlapping peaks in the chromatogram and makes the removal of trace amounts of cholesterol essential. By using a Celite - digitonin column,6 it was found that 2 mg of cholesterol could be completely separated from 200pg of tocopherol, 99 per cent. of the vitamin being found in the first 10ml of eluate and the cholesterol not appearing even after elution with a further 60ml of n-hexane. Several tocopherol derivatives were examined for their suitability as internal standards for the gas - liquid chromatographic method. Two commercially available derivatives, a-tocopheryl acetate and a-tocopheryl hydrogen succinate, were found to be unsuitable because the acetate obscured the peak of a-tocopherol trimethylsilyl ether and the succinate gave an asymmetrical peak, probably owing to decomposition.The propionyl and butyryl derivatives were therefore prepared and examined as possible standards. As shown in Table 11, the relative retention time of the butyrate was too long but a-tocopheryl propionate had the characteristics required of a standard for the quantitative determination of several toco- pherols. From published values of retention times,7 it is possible that a-tocopheryl propionate would obscure any a-tocotrienol that may be present. However, no evidence for the presence of this compound was found in any of the foods given in Table I. The internal standard is added after chromatography on the alumina column and before the removal of sterols on a Celite - digitonin column, as the propionate accompanies a-tocopherol in this separation. TABLE I1 RETENTION TIMES OF TOCOPHEROL AND CHOLESTEROL DERIVATIVES RELATIVE TO SQUALENE = 1-00 Relative Compound retention time Relative Compound retention time &Tocopherol trimethylsilyl ether 1.27 a-Tocopherol trimethylsilyl ether 2-40 y-Tocopherol trimethylsilyl ether 1-62 a-Tocopheryl acetate . .. . 2.54 a-Tocopheryl hydrogen succinate 2-14 a-Tocopheryl propionate . , 3.37 Cholesterol trimethylsilyl ether 2.31 a-Tocopheryl butyrate . . .. 4*34 Preliminary work confirmed that 0.1 pg each of a-, y- and &tocopherol, injected as their trimethylsilyl ethers so as to prevent decomposition and tailing, were readily separated and detected on a 1.8-m glass column containing 2.5 per cent.of SE-30 on acid-washed Chromo- sorb G, with nitrogen as carrier gas, a column temperature of 225 "C and a hydrogen flame- ionisation detector. Later, by using 2.7-m columns containing 3 per cent. of OV-1 on Gas- Chrom Q at 235 "C, a ten-fold increase in sensitivity was obtained with improved separation of peaks, which was especially useful for vegetable products for which some small interfering peaks may be encountered. The results obtained for the vitamin E content of uncooked foods, given in Table I, show that agreement was obtained between the colorimetric and gas - liquid chromatographic methods for animal products, all of which contained only a-tocopherol.Reasonable agreement between the two methods was also obtained for vegetable products after the removal of dimeric tocopherols in the samples of corn oil, baked beans and lettuce. Some non-tocopherol peaks were present in the gas chromatogram for lettuce and, although these peaks did not interfere in the determination, an improved clean-up procedure for this type of material would be desirable. According to T ~ e n , ~ a disadvantage of the colorimetric method is that the various tocopherols do not give the same absorbances on either a mass or a molar basis and, by using a-tocopherol as the standard, an error would be introduced for those samples which contain tocopherols other than in the a-form. However, the maximum error from this source would not be greater than +14 per cent.if the sample contained only 8-tocopherol.4March, 19731 BY COLORIMETRY AND GAS -LIQUID CHROMATOGRAPHY 167 Gas - liquid chromatography gave only 75 per cent. of the tocopherol content found by colorimetry and 80 per cent. for the recovery of added a-tocopherol. Examination of the lipid fraction of peas by thin-layer chromatography gave three spots, one for y-tocopherol, an unknown spot that ran more slowly than that of a-tocopherol, and a fast-running spot, probably a dimer. Two-dimensional thin-layer chromatography did not indicate the slower running spot to be a tocotrienol, but an unknown phenol has been reported in this fraction from peas.8 Purification of the extract by dry column chromatography left only y-tocopherol, but after the subsequent removal of sterols by precipitation and passage through a Celite - digitonin column, dimeric products reappeared. The low yield of y-tocopherol from peas obtained by gas - liquid chromatography may therefore be due partly to the formation of dimeric products during the analytical operations. This difficulty was not experienced with other samples and the methods were found to be satisfactory for the range of foods examined, with the exception of peas. In our experience, the simple colorimetric procedure is applicable to most types of food; when significant interference is encountered, for example from added antioxidants , or when know- ledge of the individual tocopherols is required, the gas - liquid chromatographic procedure is used. Anomalous results were obtained with frozen peas. The proposed methods have not been applied to animal feedstuffs. This paper is published with the permission of the Government Chemist. 1. 2. 3. 4. 6. 6. 7. 8. REFERENCES Beaton, C . H., and McHenry, E. W., “Nutrition-A Comprehensive Treatise,” Volume 2, Academic Analytical Methods Committee, Analyst, 1959, 84, 356. Ames, S. R., J , Ass. Off. Analyt. Chem., 1971, 54, 1. Tsen, C. C., Analyt. Chem., 1961, 33, 849. Dean, A. C., Chem. 6 Ind., 1971, 677. Schwartz, D. P., Brewington, C. R., and Burgwald, L. H., J . Lipid Res., 1967, 8, 64. Slover, H. T., Valis, R. J., and Lehmann. J., J . Amer. Oil Chem. Soc.. 1968, 45, 680. Gaunt, J. K., and Stowe, B. B., Plant Physzol., 1967, 42, 851. Press, New York, 1964. Received July 26th, 1972 Accepted September 18th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800161

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

168 Analyst, March, 1973, Vol. 98, pp. 168-171 Chemical Composition of Alleppy Cardamom Oil by Gas Chromatography BY A. K. S. BARUAH, S. D. BHAGAT AND B. K. SAIKIA (Regional Research Laboratory, Jorhat, Assam, India) An isothermal gas-chromatographic technique used to investigate the chemical composition of cardamom oil is described. A total of twenty-one components were detected by our method. Most of the peaks, representing 98.1 per cent. of the oil, have been identified. CARDAMOM oil was obtained by the steam distillation of capsules of the Alleppy variety of Elettaria cardamomuum Maton var. MinuscuZa BurkiZZ, belonging to the family Zingiberaceae, which is cultivated in South India. The physicochemical properties of the oil have been previously reported.112 Lewis, Nambudiri and Philip3 determined the chemical composition of the oil by using different methods.The determination of the composition of the oil by fractionation and column chromatography has also been r e p ~ r t e d . ~ In the present study a gas - liquid chromatographic technique is described for the rapid determination of the chemical composition of cardamom oil. EXPERIMENTAL The composition of the complex volatile oil was studied by gas - liquid chromatography on a Beckman GC-2A gas chromatograph, with a thermal conductivity detector operated at 120 mA. The output was recorded with a Bristol Dyna-master recorder at a chart speed of 30 inches per hour. The sample was injected by using a Hamilton 10-pl syringe. A 18 7 & 6 5844 I I I I 25 20 15 10 5 Time/minutes 90 80 v, CI 70 ' E 60 3 2 -!? I-' .- 50 $ 40 3 z 30 $ 8 20 v, 0 L 10 Fig.1. Chromatogram of oil obtained from Alleppy cardamom capsules. Chromatograph, Beckman GC-2A; column, 6 per cent. Carbowax 20M (12 feet long and 4 inch diameter tube) ; temperature, 140 "C; carrier gas, hydrogen; flow-rate, 60 ml min-1; and detector, thermal conductivity. Identified peaks are : 3, a-pinene; 4, sabinene; 6 , myrcene; 6, limonene; 7, cineole; 8, p-cymene; 9, methylheptenone; 13, linalool; 14, linalyl acetate; 15, 6-terpineol ( ? ) ; 17, a-terpineol; 18, a-terpinyl acetate; 20, citronellol; and 21, geraniol 0 SAC and the authors.BARUAH, BHAGAT AND SAIKIA 169 stainless-steel column (12 feet x & inch), packed with Carbowax on 60 to 80-mesh Chromosorb white (hexamethyldisilane treated), was used with hydrogen as the carrier gas.In this work, unbleached dry capsules of the Alleppy variety of cardamom were used. Two hundred grams of partially crushed capsules (the whole fruit, seeds and pod together) were steam distilled in duplicate for 5 hours in a Clevenger5 glass distillation apparatus. A colourless, volatile oil was obtained with a yield of 7 per cent. V/m on a fresh mass basis. The oil used for gas - liquid chromatographic examination had the following properties- Specific gravity at 19 "C . . .. . . 0.928 Refractive index at 19 "C . . .. . . 1.4636 Optical rotation at 20 "C . . .. . . +26" Acidvalue .. .. .. .. .. 4 Ester value . . .. .. .. .. 128 Solubility in 70 per cent. ethanol at 22 "C . . 1 volume of oil is soluble in 2-5 to 4 volumes GAS-CHROMATOGRAPHIC EXAMINATION- Different packed columns were investigated in order to choose suitable stationary phases for the optimum resolution of the components of the oil.Of the various polar and non-polar phases investigated, Carbowax is the most suitable liquid phase, as it is capable of eluting symmetrical peaks in a reasonably short period. After testing different grades of Carbowax for column efficiency, 5 per cent. Carbowax 20M and 30 per cent. Carbowax 1000 have been found to be satisfactory. Figs. 1 and 2 show considerable differences in the separating ability of the two types of Carbowax; Carbowax 20M column material was intermediate in polarity and was found to be the most suitable for our purposes. With an isothermal operating temperature of 140 "C and 7 0 , 10 5 Time/minutes Fig.2. Selectivity of Carbowax 1000 column. Sample, cardamom oil; column, 30 per cent. Carbowax 1000 (6 feet long and 2 inch diameter tube) ; temperature, 100 "C; carrier gas, hydro- gen; flow-rate, 60 ml min-l; and de- tector, thermal conductivity. Identi- fied peaks are: 3, a-pinene; 4, sabinene; 5, myrcene; 6, limonene; 7, cineole; and 8, p-cymene170 BARUAH, BHAGAT AND SAIKIA: CHEMICAL COMPOSITION OF [Af%dySt, VOl. 98 a hydrogen flow-rate of 60 ml min-l, effective separation of most of the components was achieved within 22 minutes. Fig. 1 represents the chromatogram of the oil obtained from Alleppy cardamom capsules. A 30 per cent. Carbowax 1000 column has an advantage in resolving the compounds of low boiling-point, whereas its efficiency is poor for components of the oil of higher boiling-point, in comparison with Carbowax 20M.Fig. 2 shows the specific selectivity of the column in resolving sabinene from myrcene and limonene from cineole. A total of twenty-one components were resolved with our system of analysis, of which fourteen major constituents were identified by comparing their retention times with those of authentic samples on polar and non-polar columns, vuiz., Carbowax 20M, Carbowax 10oO and SE-30. The retention times (in minutes) of the various components in the chromatogram were measured. The identities and retention times of the components separated on the Carbowax 20M column are given in Table I. TABLE I SUMMARY OF THE RETENTION TIMES, CONCENTRATIONS AND IDENTITIES OF TWENTY-ONE COMPONENTS DETECTED IN ALLEPPY CARDAMOM OIL Peak Retention Concentration, number timelminutes per cent.Component 1 0.04 0.2 - 2 0.27 0.4 - 3 0.66 1.6 a-Pinene !] 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 0.98 1-49 1.92 2.44 3.30 3.93 4-40 5.77 6.14 7.56 9.84 11.10 12-36 13.46 14-92 21-34 4.9 1.9 2.5 34-2 0.5 0.1 0.1 0.4 0.2 6.4 3.1 1-7 0.2 1.7 37.7 0.4 0-5 1.2 Sabinene Myrcene Limonene Cineole p-Cymene Methylheptenone c - Linalool Linalyl acetate /3-Terpineol a-Terpineol Terpenyl acetate Citronellol Geraniol - - The concentrations shown in Table I indicate the relative percentage of each individual component based on peak area integration (height x width at half-height). In five different determinations, the concentration of any individual component varied only by 1 to 2 per cent.The time of distillation plays an important r6le in controlling the proportions of the con- stituents of the oil. In Table I1 the variation in concentration of two major constituents and of the yield of oil with respect to distillation time are shown. TABLE I1 VARIATION OF YIELD AND RELATIVE PROPORTIONS OF CONSTITUENTS WITH TIME OF DISTILLATION Distillation Yield of oil, Cineole, Terpenyl acetate, Fraction time/minutes per cent. V/wi per cent. per cent. 1 0 to 30 2-93 47 30 2 30 to 60 1.83 37 37 3 60 to 90 0.80 26 48 4 90 to 180 1.00 18 58 5 180 to 270 0.50 17 58 RESULTS AND DISCUSSION Gas - liquid chromatographic analysis of the essential oil derived from Elettaria carda- momum Maton (Alleppy variety) revealed the presence of 34.2 per cent.of cineole and 37.7 per cent. of terpenyl acetate as major constituents. Other components present in theMarch, 19731 ALLEPPY CARDAMOM OIL BY GAS CHROMATOGRAPHY 171 oil were cc-pinene , sabinene, myrcene, limonene, p-cymene , methylheptenone , linalool , linalyl acetate, p-terpineol, cc-terpineol, citronellol and geraniol. Identification of the various com- ponents was carried out by examining authentic samples of the components under operating conditions identical with those used for the actual samples. The area of each peak was used for calculating the proportion of that constituent in the oil. Figs. 1 and 2 indicate that better separation of components of the oil with low boiling- points, particularly sabinene, myrcene, limonene and cineole, can be achieved by use of a 30 per cent.Carbowax 1000 column. The separation of components of higher boiling-point is carried out with a 5 per cent. Carbowax 20M column. Table I1 shows that the time of distillation has a direct influence on the proportions of the constituents of the oil. As the distillation proceeds the proportion of terpenyl acetate in the oil obtained increases, whereas that of cineole decreases. CONCLUSIONS After scanning various types of stationary phase, Carbowax 1000 and 20M were selected as they gave the more symmetrical peaks and more efficient separation. Under isothermal conditions these two columns have been used to analyse a complex oil containing a wide range of organic compounds. From this work it is clear that the choice of column packing material is important in order to attain the best possible accuracy and precision. The work described in this paper shows that gas chromatography constitutes a rapid method for the determination of the chemical composition of cardamom oil. The authors are grateful to Dr. M. S. Iyengar, Director of the Institute, for his keen interest in this work. REFERENCES 1. Guenther, E., “The Essential Oils,” Volume 5, Van Nostrand and Co. Inc., New York, 1952, 2. 3. 4. 5. pp. 85 to 105. “The Wealth of India,” Volume 3, C.S.I.R., New Delhi, 1952, pp. 149 to 169. Lewis, Y. S., Nambudiri, E. S., and Philip, T., Perfum. Essent. Oil Rec., 1966, 57, 623. Nigam, M. C., Nigam, I. C., Handa, K. L., and Levi, L., J . Pharm. Sci., 1966, 54, 799. Guenther, E., op. cit., p. 317. Received April 17th, 1972 Accepted September 28th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800168

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

172 AnaZyst, March, 1973, Vol. 98, $9. 172-175 Fungicide Residues Part III.* The Determination of Residues of Binapacryl in Selected Fruits by Gas Chromatography BY P. B. BAKER AND R. A. HOODLESS (Department of Trade and Industry, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SEl 9NQ) A simple method for determining residues of binapacryl in selected fruits is presented. After extraction with a mixture of hexane, diethyl ether and dimethylformamide, binapacryl is separated from interfering co-extractives by chromatography on a silica gel column and is quantitatively determined by electron-capture gas - liquid chromatography. A confirmatory chemical test for binapacryl is also described. BINAPACRYL [2-( l-methyl-n-propyl)-4,6-dinitrophenyl2-methylcrotonate, I3 is a non-systemic acaricide and fungicide that is used for the control of red spider mites and powdery mildews of top fruit, and that has been shown to be very active against powdery mildews of app1es.l Binapacryl is included in the list of Approved Products for Farmers and Growers,2 but it can be used only on apples or non-edible crops in the United Kingd~m.~ The methods of determination of residues of binapacryl have been reviewed by Buxton and M ~ h r .~ The principal methods described are colorimetric5~s and pho~phorimetric.~ An electron-capture gas-chromatographic method is also mentioned by Buxton and Mohr but no details are given. The report8 of the Food and Agriculture Organization and World Health Organisation Joint Meeting on Pesticide Residues in Food in 1969 expressed a need for the development and evaluation of a gas-chromatographic method suitable for regulation purposes.The procedure described in this paper has been devised to provide suitable extraction, clean-up and gas-chromatographic conditions for the detection, identification and determination of residues of binapacryl in selected fruits. TABLE I RETENTION TIMES OF BINAPACRYL AND OTHER PESTICIDES Compound Binapacryl . . .. .. .. Quintozene . . .. .. .. Y-BHC.. .. .. .. .. Heptachlor epoxide . . .. .. pp'-TDE . . .. . . .. pp'-DDE . . .. .. .. Pp'-DDT . . .. .. .. p-BHC .. .. .. . . op'-DDT .. .. .. ,. 1, l-Di( 4-chlorophenyl)-2-chloroethylene Dieldrin . . .. .. . . Dinoseb acetate . . .. .. Endrin .. .. .. .. Dinoterb acetate .. .. .. Dinobuton .. .. .. . . Captafol . . .. . . .. Retention time on column 1 (binapacryl = 1.00) .. 1*oot .. 0.10 .. 0.14 . . 0-27 .. 0.29 .. 0.29 .. 0.33 . . 0-36 . . 0-38 . . 0.41 .. 0.45 .. 0-45 .. 0-46 .. 0.48 .. 0-67 .. 1.08 t Retention time for binapacryl w 9 minutes. # Retention time for binapacryl M 10 minutes. Retention time on column 2 (binapacryl = 1.00) 0.22 0.19 1-45 1.10 0.86 0.17 0-83 1-15 0.94 1.00: - - - I - 1-44 * For Part I1 of this series, see Analyst, 1972, 97, 713. @ SAC; Crown Copyright Reserved.BAKER AND HOODLESS 173 EXPERIMENTAL The samples used in this work were obtained from retail sources and therefore had un- known histories of treatment. The sample is macerated with a mixture of hexane, diethyl ether and dimethylformamide.The extract obtained is shaken with water and the organic layer is subjected to column chromatography on a silica gel column. After concentration of the eluate, its binapacryl content is determined by means of gas - liquid chromatography with an electron-capture detector. Of the columns tested, that containing 3 per cent. GE-XE 60 on Chromosorb W (column 1) was found to be the most suitable for quantitative determina- tions. Captafol had a retention time similar to that of binapacryl on this column but these compounds can be separated by using a column containing 15 per cent. DC200 on Gas- Chrom Q (column 2). The retention times of binapacryl and other pesticides are given in Table I. The presence of residues of binapacryl may be confirmed, in the absence of dinoseb acetate and dinobuton, by the application of a simple chemical transformation, which consists in hydrolysis of binapacryl (I) with methanolic potassium hydroxide solution to give the free phenol (dinoseb, 11) and then methylation with diazomethane to give the ether (III).g The formation of the ether can be confirmed by gas - liquid chromatography.I II I l l METHOD REAGENTS- Analytical-reagent grade materials should be used whenever possible. Hexane-Distil light petroleum (boiling range 60 to 80 "C) from sodium hydroxide pellets and, to check the suitability of this hexane fraction, concentrate a 50-ml volume to 1 ml and examine the product by gas - liquid chromatography. Diethyl ether-Distil, and examine as for hexane. Dimethylformamide-Laboratory-reagent grade.Celite-Heat Celite 545 at 500 "C for 10 hours, cool and store it at 130 "C. Silica geLHeat silica gel MFC (Hopkin and Williams) at 500 "C for 4 hours, cool and Sodium sul$hate-Heat anhydrous, granular sodium sulphate at 500 "C for 10 hours, Diazomethane-Prepare from N-methyl-N-nitrosotoluene-4-sulphonamide and use in Methanolic potassium hydroxide solution-Dissolve 2 g of potassium hydroxide in 100 ml Eluting solvent-Prepare a mixture of 4 volumes of hexane and 1 volume of diethyl ether. Binapacryl standard solution-Dissolve 20.0 mg of binapacryl in hexane and dilute to 100 ml with hexane. Dilute 0.5 ml of this solution to 100 ml with hexane. This dilute solution contains 1 pg ml-l of binapacryl. store it at 130 "C. cool and store it at 130 "C. situ.10 of methanol.Sulphuric acid, 1 N. APPARATUS- An isothermally operated gas chromatograph, fitted with a tritium-foil electron-capture detector,ll was used. The detector was housed in the chromatograph oven and thus main- tained at the same temperature as the column. The applied detector potential was 24 V and the columns used were as follows. Column 1 was a glass column, 180 x 0.4 cm id.,174 BAKER AND HOODLESS: FUNGICIDE RESIDUES. PART 111 [Analyst, Vol. 98 packed with 3 per cent. GE-XE 60 on silanised Chromosorb W, 80 to 100 mesh. The column temperature was 200 “C, the carrier gas was nitrogen and the flow-rate was 300 ml min-l, measured at room temperature. Column 2 was also a glass column, 180 x 0.4 cm i.d., but packed with 15 per cent. DC 200 on Gas-Chrom Q, 100 to 120 mesh.The column temperature was 204 “C, the carrier gas was nitrogen and the flow-rate was 375 ml min-1, measured at room temperature. The samples were injected directly on to the column. Homogeniser-The homogeniser was fitted with a 150-ml vortex beaker. Evaporator-A Kuderna-Danish 500-ml capacity evaporator was used for large volumes of solvents while a glass micro-Snyder column was used for small volumes of solvents. Chromatographic columns-The columns were glass, 30 cm in length x 1-3 cm i.d., fitted with glass stopcocks. Filtration apparatus-This apparatus comprised a Biichner funnel fitted with a glass sinter of porosity 3 (Quickfit No. SF3A33*) and an adaptor with a side-arm for connection to a suction pump (Quickfit No. MF18/33*). PROCEDURE- Quantitative procedure-To 20g of sample (taken from a 200 to 250-g batch of well mixed substrate) add 5 g of Celite, 20 ml of dimethylformamide and 50 ml of eluting solvent.Homogenise the mixture in a 150-ml vortex beaker for 3 minutes. By using the filtration apparatus, filter the mixture directly into a 250-ml separating funnel fitted with a 24/29 ground-glass socket. Wash the vortex beaker with 5 ml of eluting solvent, then wash the residue in the Biichner funnel with a further 5 ml of eluting solvent and add the washings to the filtrate. Shake the filtrate and washings with 200 ml of water and 10 ml of saturated sodium sulphate solution and allow the layers to separate. Discard the lower aqueous layer. Pour a slurry of l o g of silica gel in eluting solvent into a chromatographic column, allow it to settle and drain until the solvent level reaches the top of the silica gel.Run the organic layer slowly from the separating funnel on to the column and allow the liquid to pass through the column under the influence of gravity, collecting the eluate in the Kuderna- Danish evaporator. Wash the separating funnel with 5 ml of eluting solvent and add the washings to the column. Pass a further 50ml of eluting solvent through the column and collect the eluate in the Kuderna-Danish evaporator. Examine a 5-p1 portion of the eluate by gas - liquid chromatography and compare the peak height obtained with that from a 5-pl injection of a standard solution containing 1 pg ml-1 of binapacryl. Evaporate the combined eluates so that the peak heights are comparable and then calculate the amount of binapacryl present in the sample.A calibration graph, prepared by injecting 5-p1 volumes of standard solutions and plotting the resultant peak heights against the masses of binapacryl taken, showed that the detector response was linear over the range from 1.0 to 8.0 ng. With a signal to noise ratio of 3: 1, the limit of detection was 200 pg of binapacryl. Hydrolysis of binapacryl and methylation of the resulting phenol (dinoseb)-Evaporate 5 ml of the final solution from the quantitative procedure to about 1 ml by using a micro-Snyder column and then to dryness with a gentle current of air. Add 1 ml of methanolic potassium hydroxide solution and warm the mixture on a steam-bath for about 2 minutes before adding 1 ml of sulphuric acid and extracting twice with 2 6 m l portions of diethyl ether, Pass the extracts through a small column of anhydrous sodium sulphate contained in a Pasteur pipette and collect the eluate in a test-tube. Add 0.5 ml of methanol and bubble diazomethane into the mixture for 5 minutes as described in the CIPAC Handbook, Method 35.2, “Methylation in situ with diazomethane.”1° CAUTION-Diazomethsne is toxic and potentially explosive.Then, bubble nitrogen through the sample solution for 10 minutes to remove excess of diazomethane. Inject 5p1 of the sample solution into the gas chromatograph with column 1 in position. The presence of binapacryl in the sample is indicated by the formation of 2-( l-methyl-n-propyl)-4,6-dinitroanisole (111), which has a retention time of 0.29 relative to binapacryl.The structure of the compound was confirmed by gas chromatography combined with high-resolution mass spectrometry. * James A. Jobling and Co. Ltd., ST15 OBG. Laboratory Division, “Quickfit” Works, Stone, Staffordshire,March, 19731 BAKER AND HOODLESS: FUNGICIDE RESIDUES. PART 111 175 RESULTS The recovery of binapacryl from samples of apples, cherries, peaches, pears and plums was checked by adding known volumes of a standard solution to 20-g portions of the chopped samples, followed by removal of the solvent with a gentle stream of air. The samples were then treated as described under Procedure. The results obtained by using column 1 for the gas-chromatographic determination are shown in Table 11, TABLE I1 RECOVERY OF BINAPACRYL FROM FRUITS WHEN USING COLUMN 1 Binapacryl recovered Recommended tolerance Fruit leve17/mg kg-l Apple .. .. 0.6 Cherry .. .. 1.0 Peach . . .. 1.0 Pear. . .. .. 0.5 Plum .. .. 0.3 Binapacryl added/mg kg-l 0.06 0-1 0.26 1.0 10.0 1.0 1.0 0.6 0.3 Mean of three determinations, per cent. 83 89 87 91 83 79 86 76 84 Range, per cent. 79 to 85 83 to 96 81 to 90 84 to 98 79 to 86 76 to 82 84 to 88 71 to 78 79 to 92 Recovery experiments were also carried out on some fruit samples with column 2 in use for the gas-chromatographic determination. The results are shown in Table 111. Blank determinations carried out on untreated samples were below the limit of detection for binapacryl. Any breakdown of binapacryl to the phenol, dinoseb, on the fruit will not be observed when using the proposed method as dinoseb is not detected under the gas - liquid chromato- graphic conditions described.Dinoseb acetate or dinobuton, if present, will invalidate the confirmatory test for binapacryl as both of these substances will also give rise to 2-(l-methyl- n-propyl)-4,6-dinitroanisole. TABLE I11 RECOVERY OF BINAPACRYL FROM FRUITS WHEN USING COLUMN 2 Binapacryl Binapacryl recovered by a single Fruit added/mg kg-l determination, per cent. Apple . . .. .. 0.5 98 Cherry .. .. 0.6 80 Pear . . .. .. 0-5 87 The authors thank the Government Chemist for permission to publish this paper and Dr. K. S. Webb for carrying out the mass spectrometry. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 1 . REFERENCES Doma, S., Clifford, D. R., and Byrde, R. J. W., Pestic. Sci., 1972, 2, 197. Ministry of Agriculture, Fisheries and Food, “1972 List of Approved Products and Their Uses for Farmers and Growers,” H.M. Stationery Office, London, 1972. Ministry of Agriculture, Fisheries and Food, “Recommendations for Safe Use in the United Kingdom of Chemical Compounds Used in Agriculture and Food Storage,’’ Recommendation Sheet No. 568, 1969. Buxton, R. W., and Mohr, T. A., in Zweig, G., Editor, “Analytical Methods for Pesticides, Plant Growth Regulators and Food Additives,” Volume V, Academic Press, New York, 1967, p. 235. Haller, H. L., and Schechter, M. S., Ind. Engng Chem. Analyt. Edn, 1944, 16, 324. Cheng, K. W., and Kilgore, W. W., J . Agric. Fd Chem., 1963, 11, 477. Martin, E. A., Can. J . Pharm. Sci., 1970, 5, 13. I ‘ 1969 Evaluations of Some Pesticide Residues in Food,” Food and Agriculture Organization and Howard, S. F., and Yip, G., J . Ass. Off. Analyt. Chem., 1968, 51, 24. Raw, G. R., “Analysis of Technical and Formulated Pesticides,” CIPAC Handbook, Volume I , de Faubert Maunder, M. J., Egan, H., and Roburn, J., Analyst, 1964, 89, 167. World Health Organisation, Rome, 1970, p. 3. CIPAC Ltd., Harpenden, 1970, p. 776. Received September 15th, 1972 Accepted November 7th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800172

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

176 Anahst, March, 1973, Vol. 98, pp. 176-180 The Rapid Determination of the Organophosphorus Pesticides Diazinon and Dichlorvos in Blood by Gas Chromatography BY A. F. MACHIN, M. P. QUICK AND D. F. WADDELL (Biochemistry Department, Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Weybridge, Surrey) A rapid method for the gas-chromatographic determination of diazinon and dichlorvos in blood is described. Protein is precipitated by mixing the blood with an excess of acetone containing an internal standard and the supernatant liquid is injected, directly or after concentration, into the gas chromatograph. Recoveries are satisfactory from 0.1 ml of blood containing 0-1 p.p.m. of the pesticide without concentration of the extract. If a 1-ml sample is used and the extract concentrated, 0.02 p.p.m.can be determined. I t is suggested that the method should be applicable to a wide range of organophosphorus pesticides. The method is also shown to be effective for determining diazinon in tissues and organs, but it is not recommended for this purpose. A thermionic phosphorus detector is used. THE gas-chromatographic method described by Machin and Quick,l referred to in this paper as the elution method, has proved to be satisfactory for determining several organophosphorus compounds of low volatility in blood and tissues. The procedure consists in triturating the sample with sand and sodium sulphate, eluting the mixture with ether or methanol and injecting a portion of the eluate after concentration and addition of an internal standard.A faster method, suitable also for more volatile compounds such as dichlorvos, was needed for determinations in blood. In most of the published the blood is extracted with an immiscible solvent ; in one procedure8 plasma or haemolysed blood is injected directly into the gas chromatograph. It seemed that extraction methods suitable for the more polar pesticides would be too slow and that direct injection would be inconvenient in routine use because of the need to replace columns frequently and to clean syringes carefully after each injection. In the method described, blood is mixed with an excess of acetone containing an internal standard and a portion of the acetone solution is injected into the chromatograph. The method has been verified with diazinon and dichlorvos.Dichlorvos is hydrolysed too rapidly to be found often in the blood of exposed animals, but it is a useful compound for assessing the versatility of the method because it is one of the more polar and the most volatile of the commonly used organophosphorus pesticides. EXPERIMENTAL Preliminary work showed that about four volumes of acetone or methanol were required to precipitate blood proteins, and that separation was sharper with acetone. As in previous work,l diazinon was chromatographed successfully on an XE-60 stationary phase with thermionic detection. With triisobutyl phosphate as the internal standard, calibration solu- tions in acetone gave a linear response ratio over a concentration range of diazinon from 0.02 to 10 pg ml-1.The same column could be used for dichlorvos but peaks from this compound showed slight tailing. Results were better when using a column containing 2 per cent. m/m of diethylene glycol adipate polyester and 0.2 per cent. m/m of orthophosphoric acid on Aeropak acid-washed silanised Celite. The phosphoric acid did not cause an excessive back- ground current with the thermionic detector. With triethyl phosphate as the internal standard the response ratio was linear from 0.05 to 10pgml-l of dichlorvos. When diazinon was dissolved in blood, good recoveries were obtained by adding the blood to four volumes of acetone containing triisobutyl phosphate and chromatograms of @ SAC; Crown Copyright Reserved.MACHIN, QUICK AND WADDELL 177 unfortified blood showed no interfering peaks. Dichlorvos could not be dissolved in blood owing to its rapid enzymatic hydrolysis, but recoveries were satisfactory when blood was added to solutions of dichlorvos in acetone containing triethyl phosphate.As there were again no interfering peaks in chromatograms from unfortified blood it was thought that the method would be effective in practice, unless dichlorvos is bound so strongly to constituents of the blood that it is not released during the analytical procedure. Boyero showed that some vinyl phosphate insecticides were sorbed by blood proteins and, to a similar extent, by bovine serum albumin. The possible effect of such binding was therefore tested by comparing the recoveries of dichlorvos from physiological saline buffered at pH 7.0 in the presence and absence of bovine serum albumin (5 per cent.m/V). The results (Table I) show that there was no interference from sorption by the protein. (Chromatograms of the unfortified solutions showed no interfering peaks.) TABLE I EFFECT OF PROTEIN ON THE RECOVERY OF DICHLORVOS FROM SALINE SOLUTIONS Dichlorvos recovered,* p.p.m., from- Dichlorvos added, r A -l p.p.m. saline saline plus 6 per cent. of albumin 0.50 0-51, 0.57 0.47, 0.49 1.0 1.1, 0-94, 0.94, 0.94 0.94, 1.0, 1.1 5.0 6.4, 4.9, 4.9 6.1, 4.9, 6.4, 6.4 * Each value represents the mean of duplicate injections of a separate solution. Duplicates agreed within 5 per cent. of their mean. Procedures for increasing the sensitivity of the method by concentrating the solution before injection were examined.As a useful increase should be obtained by evaporating off the acetone and injecting the aqueous residue, the response of the thermionic detector to solutions of the test compounds and their internal standards in mixtures of acetone and water was determined. Fig. 1 shows the change in this response with increasing water content of the solution. It can be seen that the response to dichlorvos and triethyl phosphate was only slightly affected and that the ratio of the signals from the two compounds hardly varied. The response to both diazinon and triisobutyl phosphate, however, increased sharply as the water content exceeded about 80 per cent. The results show that it should be possible to increase the sensitivity of the determination of dichlorvos by concentrating the acetone I I I I 1 I I 1 I 1 I 0 20 40 60 80 100 Water content, per cent.Fig. 1. Effect of water content of aqueous acetone solutions on response of thermionic detector: 1, response to triethyl phosphate; 2, response to diazinm; 3, response to triisobutyl phosphate; and 4, response to dichlorvos178 MACHIN, QUICK AND WADDELL: RAPID DETERMINATION OF DIAZINON [Analyst, vol. 98 extract. With diazinon , concentration would reduce the accuracy and reproducibility, but the results might be adequate for residue work. Attempts were made to increase the sensitivity factor by the following procedure. Ten millilitres of blood, fortified as described previously, were mixed with 40 ml of acetone, most of the water was frozen out by chilling in a mixture of solid carbon dioxide and acetone and the ice was removed by centrifugation at -15 "C.The decanted supernatant solution was concentrated to about 2 ml and a portion injected into the chromatograph. Satisfactory recoveries were obtained, but the procedure was too slow and needed too much careful attention to be convenient for routine use. A modification of the method was compared with the elution method for determining diazinon in the liver, kidney, brain and muscle of dosed sheep. Each tissue (0.1 g) was added to an equal amount of anhydrous sodium sulphate in a centrifuge tube, acetone con- taining triisobutyl phosphate was added and the tissue compressed into the salt and the mixture dispersed in the acetone with a glass rod. Fat was similarly dispersed in acetone but without sodium sulphate.Table I1 shows the residues of diazinon found by the two methods in sheep that had been given large oral doses (450 to 600 mg k g l ) . Although the range of levels in some tissues was too restricted to constitute an adequate test of the proposed method, the close agreement between the two sets of results is evidence that acetone is an effective extractant. In practice, however, the elution method was found to be more con- venient and the following method is suggested for determinations in blood, but not in tissues or organs. TABLE I1 RESIDUES OF DIAZINON FOUND IN TISSUES OF DOSED SHEEP BY THE PROPOSED AND ELUTION METHODS Tissue or organ ( Liver . . .. .. Muscle . . .. .. Fat . . .. .. Brain . . .. .. Kidney cortex . . . . Kidney medulla.. .. Diazinon found by Number elution method, 3 27 to 32 5 2 to 37 8 15 to 330 6 2.5 to 26 6 0.3 to 17 6 0.2 to 17 sf samples p.p.m. (range) Diazinon found by proposed method,* per cent. of that by elution method 113, 101, 87 119, 105, 93, 104, 103 83, 106, 83, 94, 90, 107, 93, 97 93, 110, 83, 85, 91 89, 98, 101, 121, 96 101, 105, 110, 97, 75 * Each value represents the mean of duplicate injections of a separate solution. Duplicates agreed within 5 per cent. of their mean. METHOD APPARATUS- Gas chromatograph-The gas chromatograph, detector and gas flow-rates were as pre- viously described.1 Glass columns, 5 feet x inch 0.d. (1.5 mm i.d.), were packed with 1.5 to 2 per cent. XE-60 on 100 to 120-mesh Aeropak 30 for the determination of diazinon, and with 2 per cent.diethylene glycol adipate polyester and 0.2 per cent. orthophosphoric acid on the same support for the determination of dichlorvos. The column temperatures were 170 "C for diazinon and 120 "C for dichlorvos; the detector temperature was 220 "C and the injector temperature 200 "C for both compounds. Injections were made directly on to the column. Centrijkge tubes-These were 10-ml graduated tubes, with stoppers. REAGENTS- Acetone-Dry some analytical-reagent grade acetone with anhydrous potassium car- bonate and distil it. Subject it to gas chromatography after concentrating it to one hundredth of the original volume. Internal standard solutions-Triisobutyl phosphate (Koch-Light Laboratories Ltd. , pure grade) 0.5, pg ml-l in acetone. Use for diazinon. Triethyl phosphate (Hopkin and Williams), 0-5 pg ml-l in acetone.Use for dichlorvos. Calibration solutions-Diazinon, 0.02 to 10 pg d - l , in acetone containing 0.5 pg ml-l of triisobutyl phosphate. Dichlorvos, 0.02 to 10 pg ml-l, in acetone containing 0.5 pg ml-l of trie t hyl phosphate.March, 19731 AND DICHLORVOS I N BLOOD BY GAS CHROMATOGRAPHY 179 PROCEDURE- Add 0.1 ml of blood by pipette to 0.4 ml of the appropriate internal standard solution in a centrifuge tube. Stopper the tube, mix the contents with a vortex mixer, and allow the precipitate to settle. Inject duplicate 0.5-pl volumes into the gas chromatograph. Measure the ratio of the peak height of the test compound to that of the internal standard and compare it with the corresponding ratio obtained from calibration solutions of similar concentrations.Typical retention times are : diethyl phosphate 1 a 0 minute, dichlorvos 3.1 minutes, triisobutyl phosphate 1-5 minutes and diazinon 3.9 minutes. If the peak from the compound of interest is too low to be measured reliably, proceed as follows. Mix the contents of the tube, and add (by pipette) 1.0 ml of blood. Mix with a vortex mixer and centrifuge the mixture a t 2000 r.p.m. for 5 minutes. Decant the supernatant liquid into a second centrifuge tube and evaporate the acetone under a stream of dry nitrogen. Remove the tube when evaporation becomes very slow; about 0.4ml of aqueous solution remains. Centrifuge again if necessary, inject 0.5 or 1-p1 portions in duplicate into the chromatograph, and complete the determination as described above.Add 4 ml of acetone to 0.2 ml of internal standard solution in a centrifuge tube. RESULTS Recoveries of diazinon dissolved at various concentrations in blood from sheep or cattle are shown in Table 111, and of dichlorvos from blood - acetone mixtures in Table IV. Each value is the mean of duplicate injections from a single sample. Samples that were concen- trated before injection are indicated. Blood from sheep given oral doses of 450 to 600 mg k g l of diazinon was analysed by the proposed method and by the elution method. Table V shows the results obtained by the former method as percentages of those by the latter. TABLE I11 RECOVERY OF DIAZINON DISSOLVED IN BLOOD Diazinon recovered, per cent. A f \ Diazinon, Extract chromatographed Extract concentrated added, p.p.m.without concentration before chromatography 0.02 - 127, 125 0.05 - 148, 138, 120 0-1 89 130, 120, 130 0.2 100, 84.6 113, 117, 119 0.5 106, 111, 109 99, 116, 113 1 87, 93, 92 - 2 108, 102, 107 - 5 98,100,100 - 10 96, 89, 90 - All levels, mean f s.d. 9743 f 8.27 95 per cent. confidence limits 93.7 to 101.9 122.6 f 11-96 116.6 to 129.4 DISCUSSION The results in Tables I11 and IV show that both diazinon and dichlorvos were determined satisfactorily in the recovery experiments. Comparison of the recoveries of diazinon when determined by direct injection of the acetone solution and by injection after concentration shows that results were generally too high and more variable when the solution was concen- trated. Recoveries of dichlorvos from concentrated and unconcentrated extracts were equally good.Determinations of diazinon by the proposed and the elution methods (Table V) show that the former can be expected to give slightly lower results (the mean value of 96.4 per cent. for all the samples is significantly below 100 per cent. at the 0.05 probability level) but the difference is unimportant in residue determinations. The standard deviations of the values in Table V are rather large but appear to be acceptable because errors in two inde- pendent methods contributed to the imprecision. The two methods gave similar results when used to determine diazinon in tissues and organs (Table 11) and this similarity confirms that acetone is a good extractant. The method could not be tested so critically for dichlorvos180 MACHIN, QUICK AND WADDELL TABLE IV RECOVERY OF DICHLORVOS FROM ACETONE AFTER ADDITION OF BLOOD Dichlorvos recovered, per cent.Dichlorvos added, p.p.m. 0.02 0.04 0.1 0-2 0-5 1 2 20 Solution chromatographed without concentration - 108, 102, 81, 85 100, 106, 106, 90 90, 83 93, 98, 104, 99 97,98 100,102 All levels, mean & s.d. 96-8 f 8-10 95 per cent. confidence limits 92.7 to 100.8 - 3 Solution concentrated before chromatography 110,118 102’99 101, 102, 75, 104 96, 101 93, 94 104, 105 - - 100.3 f 9-67 94.7 to 105.9 but the demonstration that sorption by albumin does not interfere (Table I) provides indirect evidence that the good recoveries from acetone solutions in the presence of blood, shown in Table IV, would be realised in practice.It is concluded that the proposed method is satisfactory for determining diazinon and dichlorvos in blood at levels above about 0.1 and 0.02 p.p.m., respectively. As little as 0.02 p.p.m. of diazinon can be determined, but with reduced accuracy and precision. The method is very rapid and should be suitable for most pesticides and their non-ionic metabolites that contain phosphorus and are amenable to gas chromatography, at levels that give adequate peaks without concentration of the extract. Higher sensitivities would be dependent upon the effect of water on the response of the thermionic detector to both the compound being determined and its internal standard. A simplified form of the method, without an internal standard, should also be useful as a semi-quantitative screening test.The enhancement by water of the response of the detector to diazinon suggests that for some compounds such a test might be very sensitive. TABLE V RESIDUES OF DIAZINON FOUND IN BLOOD OF DOSED SHEEP BY THE PROPOSED AND ELUTION METHODS Diazinon found by elution method, p.p.m. (range) 0.1 to 0.5 0-51 to 2.0 2.1 to 5.0 5.1 to 11 Total . . Diazinon found by proposed method, per cent. of that by elution method Number 95 per cent. Standard of samples Mean Range confidence limits deviation 6 98 81 to 112 85 to 110 12.1 9 106 93 to 125 98 to 113 9.4 24 97 76 to 113 93 to 101 9.8 24 92 75 to 112 89 to 96 8.6 63 96 75 to 125 94 to 99 10.2 A I > The authors thank Mr. L. Rampton for experimental work and Miss C. N. Hebert for statistical analysis of the results. They are grateful to Fisons Ltd., Agrochemicals Division, and Shell Research Ltd., for gifts of diazinon and dichlorvos, respectively. REFERENCES 1 . 2. 3. 4. 5. 6. 7. 8. 9. Machin, A. F., and Quick, M. P., Analyst, 1969, 94, 221. Jain, N. C., Fontan, C. R., and Kirk, P. L., J . Pharm. Pharmac., 1965, 17, 362. Baumler, J., and Rippstein, S., Arch. Tox., 1969, 25, 57. Girenko, D. B., and Klisenko, M. A., Gig. Sanit., 1970, 35, 77. Vukovich, R. A., Triolo, A. J., and Coon, J. M., J . Agric. Fd Chem., 1969, 17, 1190. Schultz, D. R., Marxmiller, R. L., and Koos, B. A., Ibid., 1971, 19, 1238. Greenhalgh, R., Dokladalova, J., and Haufe, W. O., Bull. Envir. Contam. Toxic., 1972, 7, 237. Boelcke, G., Arch. Tox., 1970, 26, 161. Boyer, A. C., J . Agric. Fd Chem., 1967, 15, 282. Received September 15th, 1972 Accepted December 13th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800176

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

Analyst, March, 1973, Vol. 98, $$. 181-189 181 The Examination of Tetrachlorvinphos and its Formulations for the Presence of Tetrachlorodibenzo-p-dioxins by a Gas = Liquid Chromatographic Method BY T. J. N. WEBBER AND D. G. BOX (Shell Research Limited, Woodstock Agricultural Reseamh Centre, Sittingbourne, Kent) A gas-chromatographic method for the examination of the insecticide tetrachlorvinphos [Gardona, the 2- or trans-styrene isomer of 2-chloro- 1-(2,4,5-trichlorophenyl)vinyldimethyl phosphate] for the presence of tetra- chlorodibenzo-p-dioxins is described. Procedures are described both for the extraction and clean-up of the sample and for the final analysis to determine tetrachlorodibenzo-@-dioxins down to 0.025 p.p.m. or less by gas-liquid chromatography with electron- capture detection.Tetrachlorodibenzo-@-dioxins were not detected in various samples of tetrachlorvinphos and its formulations, either before or after accelerated storage for 2 weeks a t 55 "C. INITIAL experiments by the Bionetics Research Laboratories in the U.S.A. to examine the toxicology and teratogenicity of a range of pesticides suggested that 2,4,5-trichlorophenoxy- acetic acid was a powerful teratogen. However, it was later found1 that the sample of 2,4,5-trichlorophenoxyacetic acid used in this study contained about 27 p.p.m. of 2,3,7,8-tetra- chlorodibenzo-9-dioxin (2,3,7,8-TCDD) and, from further w ~ r k , ~ ~ ~ it now seems probable that this dioxin impurity was primarily responsible for the effects noted. Additional work4+ has confirmed the highly toxic properties of chlorodioxins.Dioxins are formed by the condensation of chlorinated phenols under conditions of heat and alkali nit^^-^ and although the precise conditions have not been experimentally deter- mined, it is theoretically possible for any ortho-chlorinated phenol to undergo this reaction. Consequently, any pesticide with a chlorinated phenoxy nucleus or which is derived from a chlorinated phenol precursor could contain chlorinated dioxins, and the Agricultural Research Service, U.S.D.A., has already initiated a programme1* to enable the significance of such impurities in currently registered pesticides of this type to be assessed. Trichlorophenols are not used in the synthesis of tetrachlorvinphos (2-isomer)ll and, moreover, the conditions used in its manufacture do not seem severe enough, with respect to either alkalinity or temperature, to lead to the formation of dioxins from any trichloro- phenols formed as by-products.However, as certain samples of tetrachlorvinphos have been found to contain small amounts (less than a total of 200 p.p.m.) of 2,4,5- and 2,3,6-trichloro- phenol, it was decided to confirm experimentally the absence of dioxins. [Tetrachlorvinphos, Gardona," is the 2- or tram-styrene isomer of 2-chloro-l-(2,4,5-trichlorophenyl)vinyldimethyl phosphate.] Procedures have been reported for the determination of 2,3,7,8-TCDD in 2,4,5-trichloro- phenoxyacetic acid,12J3 in fats and oils,l4 in corn oil,l5 in commercial chlorophenolsl6 and in selected pesticides derived from chlor~phenols.~~ This paper describes a quantitative method for the determination of TCDDs and its application to the examination of samples of tetrachlorvinphos and its formulations for the presence of the six TCDDs that could result from the interaction of 2,4,5- and 2,3,6-trichloro- phenol.Methods of clean-up involving the use of aluminium oxide and aluminium oxide - sulphuric acid, as proposed p r e v i o ~ s l y , l ~ ~ ~ ~ - ~ ~ were found to be unsatisfactory because in- sufficient clean-up was achieved to permit the required sensitivity to be attained. However, sequential use of silica gel chromatography and aluminium oxide column chromatography followed by treatment of the resultant eluate with concentrated sulphuric acid made it possible to determine the level of TCDDs down to a limit of about 0.025 p.p.m.The synthesis and characterisation of the five TCDDs that were not available as pure materials are also described. * Shell registered trade mark. @ SAC and the authors.182 WEBBER AND BOX : EXAMINATION OF TETRACHLORVINPHOS FOR [Autaiyst, Vol. 98 EXPERIMENTAL SAFETY PRECAUTIONS- 2,3,7.8-TCDD is extremely toxic and is capable of causing a severe delayed response upon minimal contact with the skin. Consequently, samples containing or suspected to contain this compound or any other dioxin should be handled by using the same techniques as those used in handling radio- active materials, so as to prevent any contact with the skin, inhalation or ingestion. n-Hexane-Laboratory-reagent grade, distilled. Benxene-Analytical-reagent grade.Diethyl etker-Analytical-reagent grade. The suitability of different batches of these solvents for use in the procedure below was checked as follows: 20 ml of hexane and 5 ml each of benzene and diethyl ether were evaporated to 1 ml and 10-pl aliquot portions were examined by gas chromatography under the conditions described later. Those batches of solvent which gave residues that showed peaks with the same retention times as the TCDDs were rejected. Sulphuric acid, concentrated-Purest grade available. The Arist ar grade supplied by BDH Chemicals Ltd. was found to be suitable. Silica gel-Chromatographic grade. Sorbsil M60 (J. Crosfield and Sons Ltd.) was found to be suitable. AZuminium oxide-Chromatographic grade, neutral, Brockmann activity I , 100 to 240 mesh.A suitable material is "Camag MFC" type as supplied by Hopkin and Williams. These adsorbents were oven dried for 48 hours at 120 "C and a sample was cooled to room temperature in a desiccator immediately before use. APPARATUS- Two gas chromatographs, equipped with electron-capture detectors (400-mCi tritium sources), were used. One chromatograph was fitted with an aluminium column, 1.8m x 3-0mm id., packed with 3 per cent. m/m OV-225 on 100 to 120-mesh Gas-Chrom Q and operated at an oven temperature of 200 "C with a nitrogen flow-rate of 0.05 1 min-l; the other chromatograph was fitted with a glass column, 0.9 m x 3.0 mm i.d., packed with a mixture of 3 per cent. m/m phenyl diethanolamine succinate (2 parts) and 3 per cent. m/m OV-17 (3 parts), both on 100 to 120-mesh Gas-Chrom Q, and operated at an oven temperature of 195 "C with a nitrogen flow-rate of 0.1 1 min-l.Under these conditions the retention times of 2,3,7,8-TCDD were 16.5 and 9.5 minutes, respectively. All glassware was cleaned with chromic acid, washed thoroughly with distilled water, dried and rinsed with n-hexane before use. The use, in the later stages of the clean-up, of glassware that had previously contained concentrated solutions of tetrachlorvinphos was avoided. REFERENCE MATERIALS- A reference sample of 2,3,7,8-TCDD, the single isomer that would result from the con- densation of 2 ,4,5-trichlorophenolJ was obtained from the Food and Drug Administration , USA. Condensation of 2,3,6-trichlorophenol could result in three additional isomers, 1,2,6,7-, 1,2,6,9- and 1,4,6,9-TCDD, while two further isomers, 1,2,7,8- and 1,4,7,8-TCDD, could result from the reaction of 2,4,5- with 2,3,6-trichlorophenol.The structure of dibenzo- $-dioxin is as follows- REAGENTS- Reference samples of these materials were not available and a mixture of them was therefore synthesised by the following method. PREPARATION OF SODIUM TRICHLOROPHENOLATES- 2,3,6-Trichlorophenol of purity greater than 95 per cent. (2.4 g) was dissolved in 10 ml of ethanol and 15 ml of a freshly prepared 0.825 mmol ml-l solution of sodium ethoxide in ethanol was added. The ethanol was removed in a rotary evaporator and the residueMarch, 19731 TETRACHLORODIBENZO-$-DIOXINS BY GAS - LIQUID CHROMATOGRAPHY 183 dried under vacuum over silica gel. The theoretical amount of 2.7 g of the dry sodium salt was obtained. By using the same procedure, a sample of sodium 2,4,5-trichlorophenolate was prepared from 2,4,5-trichlorophenol of purity greater than 95 per cent.PYROLYSIS OF SODIUM TRICHLOROPHENOLATES- An equimolar mixture of the sodium salts of 2,4,5- and 2,3,6-trichlorophenol (500 mg) was intimately mixed with 50 mg of fine copper powder and placed in the apparatus shown in Fig. 1. After evacuation of the reaction tube to 13-3 N mh2 (0-1 mm of mercury) pressure the mixture was heated gradually to approximately 280 "C and maintained at this tem- perature until the visible formation of a white sublimate, both in the reaction tube and on the cold finger, had ceased. Water -- Water - I ,Cold finger i n Reaction tube I- \ -+-- Electric hot-plate ' Coolin! bath at -78 C Fig.1. Apparatus for the pyrolysis of trichlorophenolates The crude product was removed, dissolved in benzene, washed three times with 2 M sodium hydroxide solution to remove unchanged trichlorophenol and then twice with distilled water. The resulting solution was dried over anhydrous granular sodium sulphate, evaporated to small volume and the residue was further purified by chromatography on aluminium oxide, as described later. On examination by gas - liquid chromatography the product appeared to contain (see Fig. 2) the six expected isomers of TCDD together with at least three additional components. Examination by combined gas chromatography - mass spectrometry resulted in the total ion current chromatogram shown in Fig.3, and it was possible to make the assignments shown in Table I to seven of the constituents of the mixture. The differences between the chromatograms shown in Figs. 2 and 3 were due to two factors. The inferior separation in the total ion current chromatogram (Fig. 3) was caused by the use of non-optimum gas - liquid chromatographic conditions for the gas-chromatographic - mass-spectrometric examination, while the difference in response arose from the type of detection system used. In the latter instance the measurement of total ion current (Fig. 3) indicates the relative amounts of each component present , whereas an electron -capture chromatogram (Fig. 2 ) depends on the characteristics of the particular compound as well as on the amount present. It was not possible to assign an unequivocal structure to each component in this solution but a radiochemical method has been proposed,18 which, if successful, would permit the184 WEBBER AND BOX : EXAMINATION OF TETRACHLORVINPHOS FOR [Analyst, VOl.98 assignment of the chlorine substitution pattern to each component without isolation or extensive purification of the pyrolysis product. p-dioxins 'etrachlorodibenzo- p-dioxins M Trichloro- di benzo- p-dioxins 40 30 20 10 0 Tirnehinutes Fig. 2. Electron-capture chromatogram of products resulting from the pyrolysis of a mix- ture of 2,4,5- and 2,3,6-trichlorophenolates: 20 ng total injected. Column packing 3 per cent. m/m OV-225 on 100 to 120-mesh Gas-Chrom Q; column temperature 200 "C; and carrier gas flow-rate 0.05 1 min-1 PROCEDURE- Extraction of technical materia&-About 1.25 g of technical tetrachlorvinphos was dissolved in 5 ml of benzene.Extraction of formzdations-About 5.0 g of the 5 per cent. m/m granules (previously ground to a fine powder in a small laboratory mill), 2-50 g of the 50 per cent. m/m wettable powder, 2-50 g of the 50 per cent. m/m dust concentrate or 1.85 g of the 75 per cent. m/m wettable powder were weighed, as appropriate, into 10-ml centrifuge tubes and 4 ml of benzene were added to each. The mixtures were shaken on a mechanical shaker for 15 minutes, centrifuged and the extracts removed by means of Pasteur pipettes. The extractions were repeated with a further 2-ml portion of benzene and the extracts were combined. SILICA GEL COLUMN CHROMATOGRAPHY- A column was prepared by filling a glass tube (0.4 m x 10 mm i.d., fitted with a tap and plugged with a small wad of cotton-wool) with n-hexane, adding 15 g of silica gel and allowing it to settle under gravity while gently tapping the column to ensure uniformity of packing.After allowing the excess of solvent to drain to the level of the adsorbent, the benzene solution from the extraction was transferred to the top of the column with a pipette. The column was eluted with n-hexane at the rate of 5 ml min-l, discarding the first 15-ml fraction and collecting the next 25 ml of eluate. This solution was evaporated to small volume byMarch, 19731 TETRACHLORODIBENZO-p-DIOXINS BY GAS - LIQUID CHROMATOGRAPHY 185 using a rotary vacuum still and the residue transferred to a 10-ml graduated centrifuge tube and made up to 1-25 ml with benzene.ALUMINIUM OXIDE COLUMN CHROMATOGUPHY- A column was prepared as above but by using l o g of aluminium oxide instead of the silica gel. An aliquot portion (1 ml) of the extract from the silica gel column was transferred by pipette on to the column and the column eluted at the rate of 2.5 ml min-l with 20 per cent. V/V diethyl ether in n-hexane. After discarding the first 30ml of solvent the next 30-ml volume of eluate was collected, evaporated to small volume, transferred to a graduated centrifuge tube and the volume was made up to 1 ml with n-hexane. Tetrac hlorod i benzo- p-d ioxi ns - 4 I p-dioxins 6 ’ Trichloro- dibenzo- p-dioxins Fig. 3. Total ion current chromatogram obtained in the combined gas-chromatographic - mass-spectrometric examination of products resulting from the pyrolysis of a mixture of 2,4,5- and 2,3,6-trichlorophenolates: 40 pg total injected SULPHURIC ACID TREATMENT- Concentrated sulphuric acid (2ml) was added to the contents of the centrifuge tube and the tube was shaken vigorously for 15 minutes on a mechanical shaker.After allowing the phases to separate, the organic layer was transferred by means of a Pasteur pipette to a 5-ml measuring cylinder; 4 ml of distilled water were added, the cylinder was shaken for a further 5 minutes and the phases were again allowed to separate. The organic layer contained the TCDDs, if present, and was termed the “test solution.” TABLE I GAS-CHROMATOGRAPHIC - MASS-SPECTROMETRIC EXAMINATION OF PRODUCTS RESULTING FROM THE PYROLYSIS OF A MIXTURE OF 2,4,5- AND 2,3,6-TRICHLOROPHENOLATES Peak m/e of Number of chlorine number* parent ion atoms present Identification 3 } Trichlorodibenzo-p-dioxin 1 286 2 286 3 3 320 4 4 320 4 5 320 4 Tetrachlorodibenzo-p-dioxin 6 7 364 364 5 6 * Refers to Fig.3. } Pentachlorodibenzo-#-dioxin186 WEBBER AND BOX: EXAMINATION OF TETRACHLORVINPHOS FOR [Analyst, Vol. 98 GAS - LIQUID CHROMATOGRAPHIC ANALYSIS- The optimum detector potential was determined by using 5 to 8-pl injections of a 0.1 pg ml-l solution of 2,3,7,8-TCDD and an amplifier setting selected so that an injection of 0.8 ng of 2,3,7,8-TCDD gave a peak height of a t least 40 per cent. of full-scale deflection. Aliquot portions (5 to 8 pl) of the test solution were chromatographed on the OV-225 column interspersed with injections of the 0.1 pg ml-1 2,3,7,8-TCDD standard solution and of the mixed reference solution (see Fig.2). If a peak occurred at a retention time at or very close to that of a TCDD, then the test sample and standards were re-chromatographed on the mixed OV-17 - phenyl diethanolamine succinate column. RECOVERY SAMPLES- Results for the recovery of 2,3,7,8-TCDD were obtained by spiking samples of tetra- chlorvinphos and the particular formulated product under examination (amounts as given under Extraction of formdations) with 0.125-pg portions of the TCDD and analysing the spiked samples according to the method described. The recoveries obtained were normally greater than 80 per cent.PRECISION- It is difficult to give a reliable estimate of the precision of the method as none of the samples analysed contained any TCDD, but the recovery experiments indicated that the repeatability was within &0.015 p.p.m. at the 0.1 p.p.m level. RE s ULTS Twelve samples of tetrachlorvinphos representative of current production, and samples of the following types of formulated product: 75 per cent. m/m wettable powder, 50 per cent. m/m wettable powder, 50 per cent. m/m dust concentrate and 5 per cent. m/m granules, were selected for analysis. These particular formulations were chosen as they contained solid fillers that might provide the basic sites necessary for the formation of the phenolate ion. In addition, duplicate 6-g samples of six selected samples of tetrachlorvinphos and duplicate 10-g samples of the formulated materials were stored in small screw-capped bottles for 2 weeks at 55 "C prior to analysis.TABLE I1 ANALYSIS OF SAMPLES OF TETRACHLORVINPHOS AND ITS FORMULATIONS FOR THE PRESENCE OF TETRACHLORODIBENZO-$-DIOXINS Before storage After storage - 7 TCDD content, Recovery, TCDD content, Recovery, Sample type p.p.m. per cent. p.p.m. per cent. Technical material . . * . . . <Om05 90 to 96 (0.025 82 to 100 50 per cent. m/m wettable powder . . <Om01 96 to 100 <0.01 83 to 86 75 per cent. m/m wettable powder . . (0.02 - < 0.02 95 50 per cent. m/m dust concentrate . . (0.01 70 <om01 - 6 per cent. m/m granules . . . . <Om005 - < 0-005 100 Nom-The limits of detection are as given in the table; however, TCDDs were not detected in any sample.Table I1 shows the results obtained for the samples of tetrachlorvinphos. The lower limit of detection obtained on the after-storage samples was due to improved optimisation of the gas - liquid chromatographic conditions. The percentage figures are the recoveries obtained from the addition of 0.125 pg of 2,3,7,8-TCDD to the 1.25-g sample of tetrachlor- vinphos ( i t ? . , equivalent to 0.1 p.p.m. of TCDD) prior to analysis. Typical chromatograms resulting from the analysis of a sample of tetrachlorvinphos with and without the addition of 0.1 p.p.m. of 2,3,7,8-TCDD are shown in Figs. 4 and 5. Table I1 also summarises the analyses of the various formulated materials, the limit of detection being dependent on the initial amount of sample taken.In all samples a large impurity peak was observed with a relative retention time of 2.4 on the chromatograms obtained by using the OV-225 column (retention of 2,3,7,8-TCDDMarch, 19731 TETRACHLORODIBENZO-fi-DIOXINS BY GAS - LIQUID CHROMATOGRAPHY I - Hexachlorobenzophenone I 187 Ti me/m i nu tes Fig. 4. Electron-capture chromatogram of tetra- chlorvinphos after clean-up. Column packing 3 per cent. m/m OV-225 on 100 to 120-mesh Gas-Chrom Q; column temperature 200 "C; and carrier gas flow-rate 0.05 1 min-1 = 1-00>. This impurity was not removed or even reduced by further prolonged treatment with concentrated sulphuric acid, and its identification was considered to be essential. Hexachlorobenzophenone 0.1 p.p.rn. of 2,3,7,8-TCDD 1 I I I I I I I I 40 35 30 25 20 15 10 5 Time/rninutes Fig.5. Electron-capture chromatogram of tetra- chlorvinphos spiked with 0.1 p.p.m. of 2,3,7,8-TCDD, after clean-up. Column packing 3 per cent. m/m OV-225 on 100 to 120-mesh Gas-Chrom Q; column temperature 200 "C; and carrier gas flow-rate 0-05 1 min-1188 WEBBER AND BOX: EXAMINATION OF TETRACHLORVINPHOS FOR [AndySt, VOl. 98 To obtain sufficient sample for examination, 40 g of tetrachlorvinphos were subjected to the same clean-up procedure as that used for the analysis. Combined gas-chromatographic - mass-spectrometric examination of the residue showed a parent ion at an m/e value of 386 (containing six chlorine atoms) together with strong peaks at m/e values of 207 (trichloro- benzoyl) and 179 (trichlorophenyl) (see Fig.6), from which it was concluded that the impurity is hexachlorobenzophenone (about 1 to 2 p.p.m.). m/e Fig. 6. Mass spectrum of material causing a large impurity peak in all samples DISCUSSION AND CONCLUSIONS An analytical method has been developed for the detection and determination of 2,3,7,8- tetrachlorodibenzo-$-dioxin and the other TCDDs that could result from the condensation of 2,4,5- and 2,3,6-trichlorophenol in samples of tetrachlorvinphos and its formulations. Initial studies on the toxicology of 2,3,7,8-TCDDls dictated that the final procedure had to be capable of detecting TCDDs in tetrachlorvinphos at the 0.05 p.p.m. level. The sensitivity of the electron-capture detector avoided the need to use large amounts of sample but necessi- tated a rigorous clean-up procedure so as to remove interfering compounds with the same retention times as those of the TCDDs.Sequential use of silica gel column chromatography and aluminium oxide column chromatography, followed by treatment of the resulting eluate with concentrated sulphuric acid, was found to be satisfactory. The sulphuric acid treatment was essential as not only did it act as a further clean-up stage but its use avoided the need to reflux the adsorbents with chloroform in a Soxhlet apparatus so as to remove contaminants that otherwise interfered in the final gas - liquid chromatographic analysis. As 2,3,7,8-TCDD was the only pure material available, a solution containing six TCDDs (confirmed by gas-chromatographic - mass-spectrometric examination) resulting from the pyrolysis of a mixture of 2,4,5- and 2,3,6-trichlorophenol was used as reference. The presence of pentachlorodibenzo-@dioxins in this mixture could have resulted from small amounts of tetrachlorophenols in the starting materials, although it seems more likely that chlorine radicals produced during the pyrolysis were responsible.When using this reference solution it was assumed that the individual electron-capture responses of the various TCDD isomers were the same as that of 2,3,7,8-TCDD. Tetrachlorodibenzo-$-dioxins were not detected in any of the twenty-one samples of tetrachlorvinphos and its formulations that were selected for analysis, either before or after accelerated storage for 2 weeks at 55 "C. The authors thank their many colleagues who helped in this work, especially Mr.J. K. Wellby for carrying out much of the experimental work and Mr. P. A. Harthoorn for preparing the mixed dioxin reference solution. REFERENCES 1. 2. 3. 4. 6 . Nature, Lond., 1970, 226, 309. Sparschu, G. L., Dunn, F. L., and Rowe, V. K., Toxic. AppZ. Plzarmac., 1970, 17, 317. Nature, Lond., 1971, 231, 483. Jones, E. L., and Krizek, H., J . Invest. Derm., 1962, 39, 611. Kimmig, J., and Schultz, K. H., Dermatologica, 1967, 115, 640.March, 19731 TETRACHLORODIBENZO-$-DIOXINS BY GAS - LIQUID CHROMATOGRAPHY 189 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Higginbotham, G. R., Huang, A., Firestone, D., Verrett, J., Ress, J., and Campbell, A. D., Nature Milnes, M. H., Ibid., 1971, 232, 395. Sandermann, W., Stockmann, H., and Casten, R., Chem. Ber., 1957, 90, 690. Tomita, M., Ueda, S., and Narisada, M., J . Pharm. SOC. Japan, 1969, 79, 186. Kearney, P. C., Paper Presented before a Joint Meeting on Pesticides, United Kingdom, Canada Blackwood, J. E., Gladys, C. L., Loening, K. L., Petrarca, A. E., and Rush, J. E., J . Amer. Chem. Storherr, R. W., Watts, R. R., Gardner, A. M., and Osgood, T., J . Ass. Off. Analyt. Chem., 1971, Elvidge, D. A., Analyst, 1971, 96, 721. “Official Methods of Analysis of the Association of Official Analytical Chemists,’’ Eleventh Edition, Williams, D. T., and Blanchfield, B. J., J . Ass. Off. Analyt. Chew., 1971, 54, 1429. Firestone, D., Ress, J., Brown, N. L., Barron, R. P., and Damico, J. N., Ibid., 1972, 55, 85. Woolson, E. A., Thomas, R. F., and Ensor, P. D. J., J . Agric. Fd Chem., 1972, 20, 351. Harthoorn, P. A,, to be published. Brown, V. K. H., and Thorpe, E., to be published. Lond., 1968, 220, 702. and United States at Washington, D.C., November 5th, 1970. SOC., 1968, 90, 509. 54, 218. Association of Official Analytical Chemists, Washington, D.C., 1970, p. 468. Received August 3rd, 1972 Accepted Decembev 6th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800181

出版商:RSC    

年代:1973

数据来源: RSC