ISSN:0003-2654    

DOI:10.1039/AN97398FX013

出版商:RSC    

年代:1973

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97398BX015

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

iv THE ANALYST [April, 1973THE ANALYSTE DlTO RIAL ADVlSO RY 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. Srahl (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.N O T I C E TO SUBSCRIBERSSubscriptions for The Analyst, Analytical Abstracts and Proceedings should beThe Chemical Society, Publications Sales Office,Blackhorse Road, Letchworth, Herts.Rates for 1973(other than Members of the Society)sent to:(a) The Analyst, Analytical Abstracts, and Proceedings, with indexes . . . . €37.00(b) The Analyst, Analytical Abstracts printed on one side of the paper (without(c) The Analyst, Analytical Abstracts printed on one side of the paper (withindex), and Proceedings .. . . . . . . . . . . . . f38.00index), 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 index) f 35.00(f) The Analyst, and Analytical Abstracts printed on one side of the paper (with. . . . . . . . . . . . . . . . . .index) €42-00 . . . . . . . . . . . . . . . . . .(Subscriptions are NOT accepted for The Analyst and/or for Proceedings aloneApril, 19731 SUMMARIES OF PAPERS IN THIS ISSUESummaries of Papers in this IssueThe Determination of Bis(tri-n-butyltin) Oxide and Di-n- butyltinOxide in Preserved Softwood by Atomic-absorptionSpectrophotometry and PolarographyMethods are described for the determination of the total organotincompounds by atomic-absorption spectrophotometry and for the specificdetermination of bis(tri-n-butyltin) oxide and di-n-butyltin oxide by atomic-absorption spectrophotometry and polarography.Bis(tri-n-butyltin) oxideand di-n-butyltin oxide are extracted from the wood with hydrochloricacid - ethanol solution and separated from each other and from wood extrac-tives, fungicides and insecticides by adsorption on to Amberlite CG-120cation-exchange resin followed by elution with solutions containing differentconcentrations of hydrochloric acid in ethanol.The procedures have been used to determine bis(tri-n-butyltin) oxide anddi-n-butyltin oxide in Scots pine, Corsican pine, Western hemlock, Japaneselarch, Sitka spruce, Douglas fir and Western red cedar.(The late) A.I. WILLIAMSDepartment of the Environment, Building Research Establishment, Princes Ris-borough Laboratory, Princes Risborough, Aylesbury, Buckinghamshire, HP17 9PX.Analyst, 1973, 98, 233-242.An Improved Method for the Determination of Whole Blood Leadby Using an Atomic-absorption TechniqueThe Delves technique for measuring whole blood lead has been con-siderably improved by wet ashing with aqua regia instead of simply dryingwith hydrogen peroxide. Smoke is eliminated, and the reproducibility ofthe method is improved.G. ALAN ROSE and ELIZABETH G. WILLDENDepartment of Pathology, St.Peter’s Hospitals, Endell Street, London, W.C.2.Analyst, 1073, 98, 243-246.Bis(6-Methyl-2-pyridyl)glyoxal Dihydrazone as a SpectrophotometricReagent for the Rapid Determination of Copper in Alkalis, Milk and BrineThe synthesis, characteristics and analytical applications of bis (6-methyl-2-pyridyl) glyoxal dihydrazone are described. This compound producescoloured solutions selectively with copper(1) ions (Amax. = 440 nm, =8.7 x lo3 1 mol-l cm-l) that can be extracted into various organic solvents;it behaves as a cuproine-type reagent. The 1: 1 orange - yellow copper(1)complex has been used for the spectrophotometric determination of traceamounts of copper in saturated brine, alkalis and milk. The most importantadvantage of using this reagent is the total recovery of copper that is possiblefrom ammoniacal solutions.M. VALCARCEL and F. PIN0Department of Analytical Chemistry, University of Seville, Seville, Spain.Analyst, 1973, 98, 246-250.An Electrical Conductivity Detector for Paper, Thin-layerand Column ChromatographyAn electrical conductivity detector has been developed for applicationto the paper, thin-layer and column chromatography of ionic substances.Two arms of an a.c. Wheatstone bridge comprise two pairs of electrodesapplied directly on to the separating layer so that a signal ensues from thebridge when a chromatographic spot passes one electrode pair while the otherpair is traversed by the eluting agent. The sensitivity is about 1 pg and theprecision is about 2 to 3 per cent.V. DI STEFAN0 and P. MARINICentro Sperimentale Metallurgico, Via di Caste1 Romano, Rome, 1 taly.Analyst, 1973, 98, 251-256.

ISSN:0003-2654    

DOI:10.1039/AN97398FP037

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

April, 19731 THE ANALYST ixLECTURES AND COURSES APPOINTMENTS VACANTTHE UNIVERSITY OFMANCHESTER INSTITUTE OFSCIENCE AND TECHNOLOGYAdvances in Chemistry SeriesSPECTROSCOPICIDENTIFICATIONOF ORGANIC COMPOUNDSwill be held in the Institute from2nd to 6th July, 1973.This course is intended for chemists of graduate level, in industry orteaching, who have had little or no experience in interpreting infraredor nuclear magnetic resonance spectra.Further particulars, with form of application, can be obtained fromthe Registrar, UMIST, P.O. Box No. 88, Manchester M60 1QDPlease mentionTHE ANALYSTwhen replying to advertisementsstBOOKSMONOGRAPHSREPRINTSorders for all publications ofthe Society (except journals)odd be sent direct or througha bookseller to-THE SOCIETY FORANALYTICAL CHEMISTRYBook Department9/10 Savile Row,London, WIX IAFTHE EAST AFRICANPESTICIDESCONTROLORGANISATIONrequireAN ANALYSTt o establish a small laboratory a t Arusha for theverification of pesticide contents of selectedtechnical and formulated pesticides, and, as amember of E.A.P.C.O.t o assist in (a) promotingthe safe and effective use of pesticides and (b)enforcing conditions for the sale and proper useof efficient and appropriate pesticide-productsin East Africa.Candidates must have a good Honours degree inChemistry plus at least 4 years post-graduateexperience in Pesticide Chemistry. Knowledgeof method analysis of major pesticides i s essential.Salary is in a range of approximately f2890 -4050 a t present rate ofexchange.Appointmentis for 2 years in the f i r s t instance. A substantialgratuity is payable on completion of service.The post described is partly financed by Britain’sprogramme of aid t o the developing countriesadministered by the Overseas DevelopmentAdministration of the Foreign and Common-wealth Office.Apply to:CROWN AGENTSM. Division, 4, Millbank, London, SWI P 3JDfor application form and further particularsstating name, age, brief details of qualifi-cations and experience and quotingreference number M 3 E/720450/AA DBINDINGHave your back numbers of The Analystbound in the standard binding case.Send the parts and the appropriateindex(es) together with a remittancefor f2.40 t o :W.HEFFER & SONS LTD.,CAMBRIDGE, ENGLANx SUMMARIES OF PAPERS I N THIS ISSlilSThe Identification and Semi-quantitative Assay of Some Fat-soluble[April, 1973Vitamins and Antioxidants in Pharmaceutical Products andAnimal Feeds by Thin-layer ChromatographyA thin-layer chromatographic method for the identification and semi-quantitative assay of vitamin A (alcohol), its acetate and palmitate, vitamin D,a-tocopherol, a-tocopheryl acetate, BHA (butylated hydroxyanisole ; 2-t-butyl-4-methoxyphenol), BHT (butylated hydroxytoluene ; 2,6-di-t-butyl-4-methyl-phenol) and ethoxyquin in vitamin preparations is described. The samplesolutions are applied to thin layers of silica gel ancl the vitamins and anti-oxidants are separated by using n-hexane - ethyl methyl ketone - di-n-butylether (34 + 7 + 6) as the developing solvent.llccomposition of vitaminsA and D when applied to the adsorbent layer is inhibited by the presenceof triethylamine in the spotting solvent. The compounds are identifiedby means of their Rp values, their appearance in ultraviolet radiation ofwavelengths 254 and 360 nm and their response to iron(II1) chloride - bipyridyland iron(II1) chloride - potassium hexacyanoferrate(II1) spray reagents ;they are assayed by visual comparison with standards. The method hasbeen applied to gelatin-protected vitamin beads, animal feed additives, multi-vitamin tablets, oily vitamin concentrates and halibut-liver oil samples.A simple colour test for distinguishing vitamin I), from vitamin 11, afterremoval of vitamin A and its esters is also described.G.W. JOHNSON and C. VICKERSAnalytical Research, Quality Control, Thc Boots Company I,td., T’cnnyfoot Strcct,Nottingham.Analyst, 1973, 98, 257-267.Gas - Liquid Chromatographic Determination of Vitamin Din Cod-liver OilA gas - liquid chromatographic method for the determination of vitaminD (cholecalciferol) in cod-liver oil is described. It involves saponificationof the oil, extraction of the unsaponifiable matter, removal of interferencessuch as cholesterol and retinol (vitamin A) by precipitation, and columnchromatography on Sephadex LH-20 and Florisil ; the final determination ofcholecalciferol is carried out by gas - liquid chromatography. A determinationcan be completed in less than 2 days.J. G.BELL and A. A. CHRISTIEDepartment of Trade ancl Industry, Laboratory of the Govcrnnicnt Chemist,Cornwall House, Stamford Street, London, SIC1 YNQ.Analyst, 1973, 98, 268-273.The Determination of Trace Amounts of Cobalt and Other Metalsin High-purity Water by Using Ion-exchange MembranesMicrogram amounts of cobalt, chromium, copper, iron, nickel and zincare concentrated on ion-exchange resin impregnated membranes from largevolumes of reactor cooling waters. Atomic-absorption measurement on theacid-extracted membranes has permitted the determination of cobalt downto 0.01 pg 1 in water samples, with an analytical precision of rf- 12 per cent.a t the 96 per cent. confidence limit, based on twelve replicate observations.Garnma-spectrometric measurement of the nuclide cobalt-ti0 present in thereactor cooling waters has enabled membrane efficiency to be determined ;at cobalt levels of 0.01 and 0.1 p g l-l, the efficiencies arc- shown to bc 85 percent.and greater than 99 pcr cent., rcspectivclly.H. JAMESAtomic Energy Establishment, Winfrith, I>orchestcr, Dorset.Analyst, 1973, 98, 274-288xii SUMMARIES OF PAPERS IN THIS ISSUE [April, 1973Indirect Complexometric Titration of Barium and Strontiumafter Stepwise Precipitation as Sulphate fromHomogeneous SolutionAn indirect method has been developed for the stepwise titration ofbarium and strontium with EDTA, based on precipitation of the elementsas sulphate from a homogeneous solution. Barium, strontium and othermultivalent cations are complexed by titration with EDTA a t pH 10.0 byusing a mixed indicator comprising Eriochronie black T, Titan yellow andNaphthol green B.Barium is then selectively replaced from its EDTAcomplex and precipitated as the sulphate with an excess of magnesiumsulphate solution. The excess of magnesium is determined by titration withEDTA and the equivalent concentration of barium is calculated. Strontiumis determined in the same solution in the same way, with an excess of zincsulphate solution.The optimum conditions for precipitation and the interferences due tovarious anions and cations have also been studied. Copper, nickel, cobaltand iron interfere and a second method for the determination of bariumalone has been derived wherein the interfering elements are complexed withtriethanolamine, potassium cyanide and ascorbic acid.Phosphate andchromate interfere and require to be separated.B. C. SINHA and S. K. ROYCentral Glass and Ceramic Research Institute, Calcutta-32, India.Analyst, 1973, 98, 289-292.Molecular Interaction Errors in Phase-solubility AnalysisResults are presented for the assay by phase-solubility analysis of ibufenac(4-isobutylphenylacetic acid) and of saccharin in the presence of variousimpurities. The results for impurities obtained for ibufenac mixtures werealmost twice those expected, while those for saccharin mixtures were normal.From a consideration of these results, the effect of compound formation bymolecular interaction is discussed.D.THORBURN BURNS, J. B. GALLAGHER, R. J. STRETTONDepartment of Chemistry, University of Technology, Loughborough, Leicestershire,L E l l 3TU.and J. S. WRAGGAnalytical Research, Quality Control, The Boots Company Ltd., Pennyfoot Street,Nottingham.Analyst, 1973, 98, 293-296.A Critical Examination of Procedures for the Assay ofSodium FluorideReport prepared by the Fluorine Sub-committee.ANALYTICAL METHODS COMMITTEE9/10 Savile Row, London, W1X 1AF.Analyst, 1973, 98, 297-302April, 19731 THE ANALYST xiiiSELECTED ANNUALREVIEWSof theAN A LY T I CAL SC I EN C ESVolume 2 - 1972Just publishedCONTENTSThe Techniques and Theory of ThermalAnalysis Applied to Studies on lnorganicMaterials with Particular Reference t oDehydration and Single Oxide Systems- D.DollimoreDevelopments in Ion Exchange -F. VernonThermometric and Enthalpimetric Titri-metry - L. S. Bark, P. Bate and J. K.GrimePp. vi + 149 f5.00; U.S. $13.00Obtainable from-The Society for Analytical ChemistryBook Department,9/10 Savile Row, London, W I X IAFMembers of The Chemical Society may buy person;copies at the special price of f3.00; U.S. $8.00SPECIALIST ABSTRACTJOURNALSpublished bySCIENCE AND TECHNOLOGY AGENCYAtomic Absorption and FlameEmission Spectroscopy AbstractsVol. 5,1973, bimonthly €24X-Ray Fluorescence SpectrometryAbstractsVol. 4, 1973, quarterly ’ €24Thin-Layer Chromatography AbstractsVol. 3, 1973, bimonthly €24Gas Chromatograph y-MassSpectrometry AbstractsVol.4, 1973, quarterly €37Nuclear Magnetic ResonanceSpectrometry AbstractsVol. 3, 1973, bimonthly €30Laser-Raman Spectroscopy AbstractsVol. 2, 1973, quarterly €30X-Ray Diffraction AbstractsVol. 1-2, 1973, quarterly €30Neutron Activation Analysis AbstractsVol. 2-3, 1973, quarterly €30Electron Microscopy AbstractsVol. 1, 1973, quarterly €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 932xiv THE ANALYST [April, 1973(CARLO ERBAAnalytical Standards forTrace Elements AnalysisDIVISIONE CHIMICA INDUSTRIALE / VIA C.IMBONATI 24 I20159 MILAN0Modern trace analysis techniques more and more frequently nall for the use of referencestandards of metals.Spectrography, Atomic Absorption Spectrophotometry, Emission Spectrophotometry, X rayFluorescence are techniques which particularly require the use of these standards.It is however necessary to make a distinction between application of such techniques towater, or to other solutions whatever the basic solvent, oil or hydrocarbon.In fact if one uses the same technique on an aqueous solvent, one must use an aqueoussolution. If one uses a non-aqueous solvent the standards used must be soluble in thissolvent.Standards for atomic absorptionshould actually be called standard solutions for metaltrace anlysis, where the metal is in an aqueoussolution acidified by nitric acid, and may therefore beused as a standard for any analytical techniquerequiring it.Atomic absorption spectrophotometry is now beingused more and more in analysis in both research andindustrial laboratories, as this is the fastest andeasiest independent method for metal determinations.it may be applied to any soluble matrix.As for any instrumental technique, it is important tohave available standards of the metals involved,to set both the method and apparatus, and to revealany interference or positive or negative effects(caused by the matrix, solvent, etc.).In any case a control against a standard is advisablewhen plotting calibration curves.In fact in atomicabsorption spectrophotometry, the theoretical linearrelationship between absorbance and concentration,known as Beer’s law, is effective only within verynarrow limits.It will now be clear how important it is to haveavailable solutions with a known content, at least forthe most frequently determined metals.Carlo Erba STANDARDS for Atomic Absorption are thefollowing concentrated solutions of metal nitratewhich, when diluted to 1000 ml with distilled water,give a slightly acidic solution (about 0.1% HNO,) at aconcentration of metalAluminium STANDARDBarium STANDARDCadmium STANDARDCalcium STANDARDChromium STANDARDCobalt STANDARDCopper STANDARDiron STANDARDLead STANDARDion of 1000 ppm:Lithium STANDARDMagnesium STANDARDManganese STANDARDNickel STANDARDPotassium STANDARDSilver STANDARDSodium STANDARDStrontium STANDARDZinc STANDARDSpecial booklet available on requestnMetallorganic standardsThese compounds are in fact improperly calledmetallorganic, as they are generally metal salts ofcarboxylic organic acids or organic metal complexes;but this expression has been chosen because it givesa more immediate idea of the metal atom being linkedto an organic radical which eases solution in oils,even when the substance involved is not an alkyl oran aryl.They are used as oil-soluble standards in thespectrographic analysis of traces of metals in oils andfats, in petroleum derivatives and in lubricatingagents.The analysis of metals in non-aqueous media iscarried out with spectographs and atomic absorptionspectrophotometers using samples of known contentas controls. Therefore it has been necessary to studyand develop organometallic compounds and organicsalts of metals, having a known metal content.The stability is obtained by the use of solubilisingagents such as 2-l-Ethylhexanoic acid,6-Met h I y-2,4- heptan d i o ne, 2-Et h y l-h exy lam i n e, andbis-(2-Ethyl hexy1)dithiocarbamicacid-bis-(2-ethylhexyl)arnmonium salt, with Xylene.Thus, clear and stable solutions in an oil base areobtained, with concentrations up to 500 ppm of metal.It is also possible to prepare solutions containingmore than one metal, bearing in mind that mixtures ofmetals are more soluble than the individualconstituents.Carlo Erba metallorganic standards available in 5 g.vials concern the following elements:Aluminium, Barium, Bismuth, Boron, Cadmium,Calcium, Chromium, Cobalt, Copper, iron,Lanthanum, Lead, Llthlum, Magnesium, Manganese,Nickel, Phosphorus, Potassium, Siiicium, Silver,Sodium, Strontium, Tin, Vanadium, Zinc

ISSN:0003-2654    

DOI:10.1039/AN97398BP043

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

APRIL, 1973 THE ANALYST Vol. 98, No. I165 The Determination of Bis(tri-n-butyltin) Oxide and Di-n-butyltin Oxide in Preserved Softwood by Atomic -absorption Spectrophotometry and Polarography BY (THE LATE) A. I. WILLIAMS (Department of the Environment, Building Research Establishment, Princes Risborough Laboratory, Princes Risborough, Aylesbury, Buckinghamshire, HP17 9PX) Methods are described for the determination of the total organotin compounds by atomic-absorption spectrophotometry and for the specific determination of bis(tri-n-butyltin) oxide and di-n-butyltin oxide by atomic- absorption spectrophotometry and polarography. Bis(tri-n-butyltin) oxide and di-n-butyltin oxide are extracted from the wood with hydrochloric acid - ethanol solution and separated from each other and from wood extrac- tives, fungicides and insecticides by adsorption on to Amberlite CG-120 cation-exchange resin followed by elution with solutions containing different concentrations of hydrochloric acid in ethanol.The procedures have been used to determine bis(tri-n-butyltin) oxide and di-n-butyltin oxide in Scots pine, Corsican pine, Western hemlock, Japanese larch, Sitka spruce, Douglas fir and Western red cedar. THE fungicidal properties of organotin compounds were first described in the literature in 1954 by van der Kerk and Luijten.1 In more recent years, bis(tri-n-butyltin) oxide has found increasing use for the protection of timber against fungal a t t a ~ k . ~ ? ~ Bis(tri-n-butyltin) oxide is known in the timber preservation industry as tributyltin oxide or TBTO.The usual form of treatment is carried out by impregnating seasoned wood with solutions of the preservative in organic solvents either by the double vacuum process4 or the Drilon proce~s,~ or by application to the surface of the timber by brushing, dipping or deluge. Some pre- servative solutions contain only TBTO, but in others the organotin may be formulated together with other constituents, e.g., pent achlorophenol, gamma-benzene hexachloride, dieldrin, copper naphthenate, zinc naphthenate, polychloronaphthalene, monochloronaph- thalene, o-phenylphenol, lauryl pentachlorophenate or water-repellent compounds. It is necessary, therefore, to be able to determine TBTO in the presence of these compounds and wood extractives. TBTO is a reactive compound and readily forms TBTX compounds with acids, where X is an anion.Wood contains natural phenolic and acidic extractive constituents and it is possible that in treated wood the anions of these compounds replace the oxide radical. Therefore, in this work the tributyltin radical is determined and the results are expressed as TBTO. This approach also applies to di-n-butyltin oxide (DBTO). The chemical determination of TBTO is needed for the study of the loading anddistribution of the preservative achieved by treatment with different processes and preservative formula- tions and to investigate the permanence of the preservative under various service conditions. Also, because bis(tri-n-butyltin) oxide may be converted into di-n-butyltin oxide in situ (e.g., this conversion is known to be caused by ultraviolet light), it is important, for research studies on the permanence of the preservative, to be able to determine separately tributyltin and dibutyltin.In laboratory tests, DBTO was shown to be ten times less toxic than TBTO to fungi6 Existing methods for the determination of TBTO are mostly based on the deter- mination of inorganic tin after the decomposition of the organometallic compound. Methods involving the use of X-ray fluorescence spectr~metry,~~~ polar~graphy,~J~ gas - liquid chro- matography,11J2 thin-layer chromatography,13 radioactivation analysis,14 atomic-absorption spectrophotometry,lS colorimetric techniques16J7' and titration proceduresl8 have been des- cribed in the literature for the determination of organotin compounds, but most of these methods are not suitable for the determination of TBTO in wood. Some of the methods 0 SAC; Crown Copyright Reserved.233234 WILLIAMS : DETERMINATION OF ORGANOTIN COMPOUNDS IN SOFTWOODS [ A TZdySt, VOl. 98 are not specific and give inaccurate results and others are insensitive. Also, because it is difficult to decompose TBTO completely, procedures that involve wet-ashing techniques are slow and tedious. The initial problem in the development of procedures for the determination of TBTO in timber is the extraction of the organotin compound from wood. Recent work has shown that some preservative chemicals can be rapidly leached from thin sections of ~ o o d ~ ~ - ~ or from sawdust.22 It has now been found that leaching with a 0.05 per cent.V/V solution of concentrated hydrochloric acid in ethanol followed by an atomic-absorption spectrophoto- metric finish affords a rapid method for the determination of total tin in preserved wood. This procedure is suitable for the routine determination of organotin compounds in treated wood when a non-specific method is required. Because atomic-absorption spectrophotometry is non-specific, an alternative technique was sought for the separate determination of TBTO and DBTO. It is known that organotin compounds are reduced directly at the dropping-mercury e l e ~ t r o d e , ~ ~ , ~ * which offered the possibility of using a polarographic method. Unfortunately, wood extractives, which are also extracted from the wood during leaching, interfere and it is necessary to separate the organotin compounds from wood extractives before polarographic analysis.This separation was achieved by adsorption of the organotin compounds as chlorides (TBTC1 and DBTCl,) on Amberlite CG-120 cation-exchange resin, and by using suitable solvents the TBTCl and DBTCl, could be eluted separately. Atomic-absorption spectrophotometry is a more common technique for the determination of preservatives and the polarographic procedures were developed principally as a means of checking the ion-exchange eluates to confirm that separation of TBTCl and DBTC1, had taken place. Also, they were used to check the results obtained by the atomic-absorption spectrophotometric procedures. EXPERIMENTAL PREPARATION OF STANDARD SAMPLES AND SAMPLING- Standard samples were prepared by impregnating wood with dioxan solutions containing known amounts of technical grade TBTO, and the full cell process26 and freeze-drying2e were used so as to prevent re-distribution and loss of the preservative.The sample blocks were freeze-dried at 0 "C to a residual solvent content of about 6 per cent. and no TBTO was detected in the dioxan condensate. From the observed mass of treating solution retained in the blocks after impregnation, the percentage of TBTO, based on the oven-dry mass of wood, was found by calculation to lie in the range 0.023 to 1.34 per cent. Despite these precautions, the distribution of TBTO in the treated blocks will not be uniform owing to the anatomical structure of wood. Concentration gradients of TBTO can occur across the annual rings, more being present in the spring or early wood, as the void space is greater, than in the summer or late wood.Therefore, for development work on the procedures, it was decided to use radial sections (cut across the annual rings), as they are more representative of the bulk of the wood. Microtome sections 0.1 mm thick were taken at intervals through the dry block and combined to make one sample for analysis. Adjacent thin sections were taken in order to make up replicate samples. The amounts of the samples taken for analysis were in the range 0.3 to 1 g. SEPARATION OF ORGANOTIN COMPOUNDS- Initially, TBTO was extracted from standard samples with ethanol for polarographic analysis and with isobutyl methyl ketone for atomic-absorption spectrophotometry.Although TBTO was completely recovered from freshly treated timber with these solvents, it was not possible to recover all of the organotin compounds from aged samples and up to 50 per cent. remained in the wood. For complete recovery, the organotin compounds were extracted as the chlorides with a 0.05 per cent. V/V solution of hydrochloric acid in ethanol. This solvent quantitatively removed the organotin compounds from aged samples and was found to be suitable for atomic-absorption spectrophotometry and cation-exchange procedures.* The direct polarographic determination of TBTCl and DBTC1, in the above hydrochloric acid in ethanol leach solutions was not possible owing to interference of the reduction wave * The ethanol used throughout this work was of 97.6 per cent.concentration.April, 19731 BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY AND POLAROGRAPHY 236 by wood extractives. In order to separate the TBTCl and DBTC1, from wood extractives, the organotin compounds were adsorbed on Amberlite CG-120 cation-exchange resin (chro- matographic grade, 200 mesh). Direct application of TBTCl and DBTCl, in a 0.05 per cent. V/V solution of hydrochloric acid in ethanol to columns of resin resulted in only 20 per cent. of the organotin compounds being adsorbed. However, on diluting the leach solution with water (10 ml of water to 20 ml of leach solution), more than 99 per cent. of the organotin compounds was retained. The wood extractives passed through in the initial eluate. The possibility of separating TBTCl and DBTC1, on the resin column was also investi- gated, The elution of the organotin compounds was monitored by polarographic analysis of fractions of the eluate.Separation could not be effected by using different concentrations of hydrochloric acid in ethanol, but it was achieved by varying the water content of the eluting solution. Under the conditions described in the method, it was possible to remove the TBTCl from the resin in less than 10 ml of a solution containing 10 per cent. V/V of water and 0.3 per cent. V/V of hydrochloric acid in ethanol. In solutioiis that contained 10 per cent. or more of water, only TBTCl was eluted. DBTCl, was subsequently eluted with 5 per cent. V/V of hydrochloric acid in ethanol. In a solution of this concentration it was possible to remove the DBTCl, from the resin in less than 10 ml of eluting agent.If a solution containing less hydrochloric acid in ethanol is used, much greater volumes of eluting agent are required in order to elute DBTC1, from the resin. It is important that the volume of eluate obtained is kept to a minimum so as to avoid loss in sensitivity during atomic-absorption studies with these solutions. The use of this cation-exchange procedure also provides a means of concentrating TBTCl and DBTCl, from dilute extracts. The separation of wood extractives, TBTCl (expressed as TBTO) and DBTCl, (expressed as DBTO) is shown in Fig. 1. 2400 2000 131 1600 3 --. al +-' .- g 1200 L w X W 800 400 0 240 - A 200 - 131 3. ;S 160- kl n 7 120- z ([J 0 I- 80- h 16 24 32 40 E luate/m I in C,H,OH k- Leach 4- 33% of H,O 110% of H,O k 0 .3 " ,"' HCI 4 C,H,OH- in C,H,OH --+ in C,H,OH Solution 10% of H,O Fig. 1. Elution of (A) wood extractives, (B) TBTCl (expressed as TBTO) and (C) DBTC1, (expressed as DBTO) ATOMIC-ABSORPTION SPECTROPHOTOMETRY- The use of hydrochloric acid in leaching procedures and in ion-exchange separations made it necessary to examine the effect of different concentrationslof hydrochloric acid in ethanol in the use of these solutions as media for atomic-absorption spectrophotometry. The absorbance was recorded for solutions containing 20 pg ml-l of TBTO and increasing amounts of hydrochloric acid in ethanol. The results showed that maximum absorbance occurred with solutions containing less than 2 per cent. V/V of hydrochloric acid. Similar236 WILLIAMS : DETERMINATION OF ORGANOTIN COMPOUNDS IN SOFTWOODS [AndySt, VOl.98 results were obtained for DBTO. Hence the leach solutions consisting of 0.05 per cent. V/V of hydrochloric acid in ethanol used for the extraction of total organotin give approximately maximum sensitivity. A solution of 5 per cent. V/V of hydrochloric acid in ethanol was used to elute DBTC1, from the cation-exchange column. With a solution with this acid concentration a small but acceptable loss in sensitivity occurs. The use of water in the ion-exchange procedure required that the effect of increasing concentrations of water in a 0.3 per cent. V/V solution of hydrochloric acid in ethanol on the absorbance signal for tin in TBTO be studied. The results showed that the absorbance signal for tin decreased with increasing concentration of water, but the sensitivity obtained by using a solution of 10 per cent.V/V of water and 0-3 per cent. V/V of hydrochloric acid in ethanol was adequate for the levels of TBTO encountered in wood. Wood contains calcium, potassium, sodium and strontium and some samples of certain species contain lithium. These elements enhance the tin absorbance signal, lithium much more so than the others. This interference is usually observed with Western red cedar, but it occasionally occurs with pines. The interference was effectively overcome by the addition of an excess of lithium (1000 pg ml-l) to the test and calibration solutions in the direct method. The presence of calcium, potassium, sodium and strontium in solutions con- taining an excess of lithium ions did not affect the tin absorbance signal. The interfering elements are separated from TBTCl and DBTCl, in the ion-exchange procedure and, therefore, do not influence the equilibrium in the flame between atoms and ions of tin during the specific determination of TBTCl and DBTC1,.POLAROGRAPHY- A 2-ml volume of the TBTCl eluate was diluted to 10ml with a support electrolyte, consisting of 0.94 per cent. V/V of hydrochloric acid and 2-5 per cent. V/V of ethanol in 1 M aqueous potassium chloride solution, for polarographic analysis. A similar volume of the eluate containing DBTC1, was diluted with 1 M aqueous potassium chloride solution for analysis. This dilution ensured that in both test solutions the concentrations of ethanol, hydrochloric acid and potassium chloride were similar.In this electrolyte, DBTC1, gave two reduction waves at peak potentials of -0.64 and -0.75 V, and TBTCl gave one reduction wave at a peak potential of -0.85 V. Owing to interference to the -0-75 V DBTC1, reduction wave by the TBTCl reduction wave, it was not possible to use the TBTCl wave for the determination of TBTCl in the presence of DBTC1,. It was possible to determine DBTC1, in the presence of TBTCl by using the -0.64 V peak. EFFECT OF OTHER FUNGICIDES AND INSECTICIDES- Commercial formulations of TBTO wood preservative solutions may also contain other constituents. The effect of the presence of such compounds on the determination of TBTO by atomic-absorption spectrophotometry and polarography was examined.Solutions con- taining 20 pg ml-l of TBTO and 400 pg ml-l each of pentachlorophenol, lauryl pentachloro- phenate, copper naphthenate, zinc naphthenate, o-phenylphenol, monochloronaphthalene, polychloronaphthalene and water-repellent waxes, or 40 pg ml-l each of lindane and dieldrin, were examined by the proposed procedures. In the atomic-absorption and polarographic procedures, after cation-exchange separation no interference occurred and complete recovery of TBTO was achieved. Only copper and zinc, from copper and zinc naphthenates, were adsorbed on the resin during cation-exchange separation of the organotin compounds. The ions of these two elements had a more negative reduction potential than TBTCl and caused no interference to the polarographic waves of DBTC1, or TBTCl.The presence of copper and zinc ions also caused no interference during atomic-absorption spectrophotometry. Copper and zinc naphthenates caused interference during the direct determination (without ion exchange) of total organotin compounds in leach solutions by the atomic- absorption procedure by enhancing the absorbance signal of tin. The extent of the interference was investigated by preparing two series of solutions, one series containing 20 pg ml-1 of TBTO plus increasing amounts of copper naphthenate and the other containing 30 pg ml-l of TBTO $us increasing amounts of zinc naphthenate. All of the solutions were made up with 0.05 per cent. V/V of hydrochloric acid in ethanol. The solutions were aspirated and the recorded absorbances plotted against concentrations of either copper naphthenate orApril, 19731 BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY AND POLAROGRAPHY 237 zinc naphthenate (Fig.2). In both instances the tin signal was enhanced and reached a plateau; similar results were obtained with DBTC1,. This interference can be overcome if an excess of lithium ions is added to both the test and calibration solutions. 1 1 I 1 I 1 0 40 80 120 160 200 Copper n'aphthenate or zinc naphthenate/pg ml- ' Fig. 2. Interference to tin absorbance signal. Curve A, interference by zinc naphthenate; and curve €3, inter- ference by copper naphthenate ANALYSIS OF TECHNICAL TBTO- Technical TBTO may contain tri-n-butyltin chloride, di-n-butyltin compounds and solvent. The purity of the technical TBTO used in this work was checked by a polarographic procedure that differed from that previously described.Approximately 0.1 g of TBTO, accurately weighed, was dissolved in ethanol and the solution diluted to 100 ml with ethanol. A 10-ml aliquot of this solution was diluted to 100 ml with a support electrolyte consisting of 6.7 ml of ammonia solution (sp. gr. 0+380), 5.7 ml of glacial acetic acid and 2 ml of 1 per cent. m/V Triton X-100 solution, diluted to 1 litre with water. Polarograms were recorded with a Southern Analytical KlOOO cathode-ray polarograph with the following instrument settings- Start potential/V Peak potmtial/V Ris(tri-n-butyltin) oxide . . .. - 1.05 - 1.35 Tri-n-butyltin chloride . . . . - 0.70 - 0.97 Di-n-butyltin dichloride .. .. - 0.80 - 1.07 n-Butyltin trichloride . . .. - 0.45 -0.71 Di-n-butyltin oxide (insoluble) . . - - No DBTC1, and only trace amounts of TBTCl were detected in the test solution. No reduction wave was observed for DBTO, probably owing to its high insolubility. In order to detect DBTO, another sample was dissolved in a 5 per cent. V/V solution of hydrochloric acid in ethanol and the solution examined for DBTC1, by the polarographic procedure described later, but only a trace amount of DBTO was found. Therefore, it was concluded that the TBTO used in the preparation of the standard samples was pure and did not introduce any errors in the calculated content of the standards. RESULTS The procedures outlined above were used to determine the loading of organotin compounds in standard samples of treated wood.All of the results were based on the oven-dry mass of wood and were expressed as TBTO or DBTO content. The results, given in Table I, were in good agreement with each other and with the calculated TBTO content. Some of the standard samples were examined immediately after they had been freeze-dried. DBTO was not detected in these samples. The remaining samples were examined 6 months after impregnation, and these samples contained both TBTO and DBTO. The standard deviation, based on seven determinations at the 0.20 per cent. level, was &0.0039 per cent. for the direct determination of organotin compounds in leach solutions. The standard deviations for TBTO and DBTO, based on seven determinations at the 0.07 per cent.level for TBTO and the 0.0035 per cent. level for DBTO, for the atomic-absorptionta w 00 3 E TABLE I +I w 5 After cation-exchange separation La r * E .. M LOADING OF ORGANOTIN COMPOUNDS IN STANDARD SAMPLES COMPARED WITH LOADING CALCULATED FROM SOLUTION RETENTIONS 1: Time of examina- Species tion Scots pine.. .. Directly Corsican pine . . after Western hemlock . . treat- Sitkaspruce . . ment Western hemlock.. Japanese larch . . Western red cedar After Scots pine . . . . storage Corsicanpine .. Western hemlock . . Sitka spruce .. Western red cedar Western hemlock . . Japanese larch . . Scots pine . . .. Douglas fir.. .. Scotspine .. .. Douglas fir.. .. Calculated TBTO content, per cent. 0.78 0.39 0.60 1-12 0.038 0.030 0.024 0.026 1-34 0-75 0.36 0.62 1.18 0.040 0.038 0.028 0.023 0-027 Organotin content by atomic-absorption spectrophotometry, expressed as TBTO, per cent.1.35 0.74 0.36 0-61 1-15 0.043 0.039 0.026 0-024 0.028 A I \ G Atomic-absorption spectrophotometry Polarograph y 2 1 ------7 1: TBTO DBTO DBTO expressed as TBTO DBTO DBTO expressed as o 0 w 0 b Z - 0 1: 0 0 0 C U r L content, content, TBTO equivalent, content, content, TBTO equivalent, per cent. per cent. per cent. per cent. per cent. per cent. - - - Not - - d Not - 0.040 detected - 0.039 detected - 0.027 - 0.028 - 0.026 - 0,026 - 0.028 - 0.027 - 0.92 0.32 0.38 0.92 0.32 0.38 1: 0.72 0,021 0.025 0.72 0.021 0.025 0.35 0.009 1 0.01 1 0.35 0.0088 0.01 1 1.10 0.033 0.040 1.10 0.03 1 0.037 0.0056 0.035 0-0046 0.0055 0.034 0.0047 0-025 0.0024 0.0029 0.024 0.0024 0.025 0.0029 0-0035 0.025 0.0029 0.0035 is;' v) 0.67 0.036 0.043 0.57 0.036 0.043 !2 0.029 0-097 0.012 0.030 0.010 0.012 r 0.017 0.0050 0.0060 0.018 0*0050 0.0060 0 cn 0 z 0 U 0-0029 v) W 00Apd, 19731 BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY AND POLAROGRAPHY 239 determination after cation-exchange separation were &O-OOlO per cent.for TBTO and &O.O0OlO per cent. for DBTO. The standard deviations, based on seven determinations at the 0-20 per cent. level for TBTO and the 0.06 per cent. level for DBTO, for the polaro- graphic technique were 50.0036 per cent. for TBTO and &040025 per cent. for DBTO. The atomic-absorption sensitivities were 2 pg ml-l for TBTO in 0.05 per cent. V/V of hydrochloric acid in ethanol, 2.5 pg ml-l for DBTO in 5 per cent.V/V of hydrochloric acid in ethanol and 3 pg ml-1 for TBTO in 0-3 per cent. V/V of hydrochloric acid and 10 per cent. V/V of water in ethanol. To demonstrate the potential value of the proposed atomic-absorption procedure, the distribution of TBTO was investigated in double vacuum treated Scots pine sapwood and in Corsican pine sapwood that had been dip-treated for 3 minutes. Specimens, with surface dimensions of 25 x 25 mm and depth 20 mm, were sawn from the bulk of the treated wood and sections were taken through the radial face in the tangential direction. For the double vacuum treated specimen, starting at the surface, ten thin sections 0.1 mm in thickness were cut on a microtome to form one sample for analysis. The sampling process was repeated to a depth of 12 mm from the surface of the specimen.For the Corsican pine, the first three samples were made up of five thin sections, 0.1 mm in thickness, and the next three samples of ten thin sections, 0-1 mm in thickness. The total organotin content, expressed as TBTO, was plotted against depth of sample. The curves, given in Fig. 3, showed that it is possible to evaluate the distribution of preservative over small areas. 0 - 4 I a 1 6 I a 1 i o I Depth from surface/mm Fig. 3. Distribution of organotin, ex- pressed as TBTO, in (A) double vacuum treated Scots pine sapwood and (B) dip- treated Corsican pine sapwood The amounts involved would permit the analysis of each 0-1-mm section of the double vacuum treated wood and the first millimetre of dip-treated timber separately so as to obtain a more close distribution pattern.It is also possible to evaluate the conversion of TBTO and DBTO and the distribution of these compounds by using the cation-exchange procedures. METHODS APPARATUS- A tomic-absor$tiora s$ectro$h.otometry-The atomic-absorption equipment consisted of a Pye Unicam, Model SPSOA, Series 2, single-beam spectrophotometer fitted with an EM1 No. 9662A photomultiplier and a Pye Unicam, Model AR25, linear recorder. A Cathodeon tin hollow-cathode lamp for use at a wavelength of 224.4 nm was used.24-0 WILLIAMS : DETERMINATION OF ORGANOTIN COMPOUNDS I N SOFTWOODS [Analyst, Vol. 98 Polarography-Polarograms were recorded with a Southern Analytical Instruments KlOOO cathode-ray polarograph and a mercury-pool reference electrode.Solutions were de- oxygenated with oxygen-free nitrogen prior to measurement a t 25 & 0.25 "C. REAGENTS- Ethanol-97.5 per cent. Use for the preparation of reagents. Hydrochloric acid in ethanol, 5 per cent. V/V solution-Dilute 50ml of concentrated hydrochloric acid to 1 litre with ethanol. Hydrochloric acid in ethanol, 0.05 per cent. V/V solution-Dilute 10 ml of the 5 per cent. V/V solution of hydrochloric acid in ethanol to 1 litre with ethanol. Hydrochloric acid and water in ethanol, solution containing 0.3 per cent. V/V of hydrochloric acid and 10 per cent. V/V of water-Dilute 3 ml of concentrated hydrochloric acid and 100 ml of water to 1 litre with ethanol. Water in ethanol, 10 per cent. V/V solution-Dilute 100 ml of water to 1 litre with ethanol.Water in ethanol, 33 per cent. V/V solution-Dilute 333 ml of water to 1 litre with ethanol. Hydrochloric acid solution, 20 per cent. V/V-Dilute 20 ml of concentrated hydrochloric acid to 100ml with water. Lithium chloride solution, 5000 pg ml-l-Dissolve 3-06 g of anhydrous lithium chloride in and dilute to 100 ml with the 0.05 per cent. V/V solution of hydrochloric acid in ethanol. Support electrolyte 1-Dissolve 7.4600 g of potassium chloride in water and dilute to 100 ml with water. Support electrolyte 2--Dissolve 7.4600 g of potassium chloride in water, add 0.94 ml of concentrated hydrochloric acid and 2.5 ml of ethanol, and dilute to 100 ml with water. Cation-exchange resin-Amberlite CG-120 chromatographic resin, 200 mesh. Bis(tri-n-butyltin) oxide standard solution 1-Dissolve 0.1000 g of bis(tri-n-butyltin) oxide in ethanol, add 5 ml of the 5 per cent.V/V solution of hydrochloric acid in ethanol and dilute to 500ml with ethanol. 1 ml of solution = 200 pg of TBTO. in ethanol and dilute to 500 ml with ethanol. 1 ml of solution = 200 pg of TBTO. Bis(tri-n-butyltin) oxide standard solution 2-Dissolve 0.1000 g of bis(tri-n-butyltin) oxide Di-n- butyltin oxide standard solution-Dissolve 0.0500 g of di-n-butyltin oxide in 50 ml of a warm solution of 5 per cent. V/V of hydrochloric acid in ethanol, cool, and dilute to 500 ml with the 5 per cent. V/V solution of hydrochloric acid in ethanol. 1 ml of solution = 100 pg of DBTO. Atomic-absorption method for determining organotin compounds-The instrument operating conditions were as follows- Wavelength .. .. Lamp current . . Burner height .. Slit width . . .. Attenuator setting . . Scale expansion . . Burner . . . . Aspiration rate . . Acetylene flow-rate . . Nitrous oxide flow-rate .. . . 224-4nm . . . . 0.05 mm . . .. 1 .. . . 7mA . . .. u p to x10 . . . . Nitrous oxide - acetylcnc .. . . 0.7 cm . . . . 3 to 4 ml min-1 .. .. . . . . 3800 ml min-' a t a pressure of 0.7 kg cm-2 5 1 min-l a t a pressure of 2.1 kg cm-2 CALIBRATION SOLUTIONS Transfer by pipette, with suitable precautions, 1, 2, 3, 5, 10, 15, 20 and 25 ml of TBTO standard solution 1 into 100-ml calibrated flasks containing 20 ml of lithium chloride solution, dilute to the mark with the 0.05 per cent. V/V solution of hydrochloric acid in ethanol and mix. The solutions contain 2, 4, 6, 10, 20, 30, 40 and 50 pg ml-l of TBTO, respectively.PROCEDURE- Transfer the weighed sample into a 50-ml distillation flask. Add 30 ml of the 0-05 per cent. Tr/V solution of hydrochloric acid in ethanol and fit a reflux distillation condenser to the flask. Boil the solution for 10 minutes, cool it to room temperature, decant the leach solution from the wood into a flask, fit a stopper and allow any particles of wood to settle.April, 19731 BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY AND POLAROGRAPHY 241 Transfer 8ml of the leach solution into a 10-ml calibrated flask, dilute to the mark with lithium solution and mix. Use the operating conditions given above and aspirate a suitable range of calibration solutions followed by the sample solution. Do not disturb the sediment during aspiration of the sample solution.Check the calibration solutions after the last sample has been run. Aspirate the 0.05 per cent. V/V solution of hydrochloric acid in ethanol between each test or calibration solution. Plot a calibration graph of the concentration (pgml-1) of TBTO against absorbance. To determine the TBTO equivalent of the organotin compounds in the sample solution, compare the absorbance reading with the calibration graph. The volume of the 0.05 per cent. V/V solution of hydrochloric acid in ethanol for leaching the organotin compounds from the wood can be varied according to the amount of sample taken for analysis and its organotin content. CATION-EXCHANGE SEPARATION OF WOOD EXTRACTIVES, TBTO AND DBTO CHROMATOGRAPHIC COLUMN- is used.The reservoirs have a capacity of 50 ml. PREPARATION OF CHROMATOGRAPHIC COLUMN- Soak the cation-exchange resin in water for 24 hours. Slurry sufficient resin into the column to form a bed 2-5 cm deep when the solids settle down. Elute the column sequentially with 50 ml of 2 M sodium hydroxide solution, water until the eluate is free from alkali, 50 ml of 2 M hydrochloric acid, water until the eluate is free from acid, and finally 20 ml of ethanol. It is necessary to agitate the resin after eluting it with ethanol so as to remove air bubbles. The resin column is now ready for use. To regenerate the column after each run, elute it successively with 20 ml of 20 per cent. V/V hydrochloric acid solution, water until the eluate is free from acid, and 20 ml of ethanol. PROCEDURE- Add 20ml of the 0-05 per cent.V/V solution of hydrochloric acid in ethanol and fit a reflux distillation con- denser to the flask. Boil the solution for 10 minutes, add 10ml of water and continue to boil the mixture for 2 minutes. Cool the contents of the flask to room temperature, transfer a suitable aliquot (up to 25ml) into the chromatographic column reservoir and elute at the rate of 1 drop per 2 s. Rinse the reservoir and elute the resin with 2 volumes of 5 ml of the 33 per cent. V/V solution of water in ethanol, then 5 ml of the 10 per cent. V/V solution of water in ethanol. Elute the TBTCl with the solution of 0.3 per cent. V/V of hydrochloric acid and 10 per cent. V/V of water in ethanol, discard the first 1 ml of eluate and collect the next 10ml in a 10-ml calibrated flask.Wash the reservoir and elute the resin with 5 ml of ethanol. Elute the DBTC1, with the 5 per cent. V/V solution of hydrochloric acid in ethanol, discard the first 0.5 ml of eluate and collect the next 10 ml in a 10-ml calibrated flask. The solutions are ready for examination by atomic-absorption spectrophotometry or polarography. A quick semimicro-scale column, of 10 cm effective length and 1 cm bore, with a tap Weigh the sample and transfer it into a 50-ml distillation flask. DETERMINATION OF TBTO BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY CALIBRATION SOLUTIONS- Transfer by pipette, with suitable precautions, 1, 2, 3, 5, 10, 15, 20 and 25 ml of TBTO standard solution 2 into 100-ml calibrated flasks containing 50 ml of ethanol, 10 ml of water and 6 ml of the 5 per cent.V/V solution of hydrochloric acid in ethanol, dilute to the mark with ethanol and mix. The solutions contain 2, 4, 6, 10, 20, 30, 40 and 50 pg ml-l of TBTO, respectively. PROCEDURE- Continue as described in the second paragraph of the Procedure (p. 240) for the atomic- absorption method for determining organotin compounds. Aspirate the solution of 0-3 per cent. V/V of hydrochloric acid and 10 per cent. V/V of water in ethanol between each test or calibration solution.242 WILLIAMS DETERMINATION OF DBTO BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY CALIBRATION SOLUTIONS- Transfer by pipette, with suitable precautions, 1, 2,3,6,10,18, 20 and 26 ml of DBTO standard solution into 100-ml calibrated flasks, dilute to the mark with the 6 per cent.V/V solution of hydrochloric acid in ethanol and mix. The solutions contain 1, 2, 3, 6, 10, 16, 20 and 25 pg ml-1 of DBTO, respectively. PROCEDURE- Continue as described in the second paragraph of the Procedure (p. 240) for the atomic- absorption method for determining organotin compounds. Aspirate the 6 per cent. V/V solution of hydrochloric acid in ethanol between each test or calibration solution. DETERMINATION OF TBTO BY POLAROGRAPHY Transfer 2 ml of the TBTO eluate into a 10-ml calibrated flask, dilute to the mark with support electrolyte 2 and mix. Transfer 5 ml of the test solution into a polarographic cell containing a mercury-pool electrode, de-oxygenate for 10 minutes with oxygen-free nitrogen and record the peak current at -0.85 V with a start potential of -0.06 V.To obtain the TBTO content of the test solution, compare the peak current with a calibration graph. Prepare a calibration graph by using the TBTO atomic-absorption standards and the polarographic technique described above. DETERMINATION OF DBTO BY POLAROGRAPHY Transfer 2 ml of the DBTO eluate into a 10-ml calibrated flask, dilute to the mark with support electrolyte 1 and mix. Transfer 6 ml of the test solution into a polarographic cell containing a mercury-pool electrode, de-oxygenate for 10 minutes with oxygen-free nitrogen and record the peak current at -0.64 V with a start potential of -0450 V. To obtain the DBTO content of the test solution, compare the peak current with a calibration graph. Prepare a calibration graph by using the DBTO atomic-absorption standards and the polaro- graphic technique described above. 1. 2. 3. 4. 6. 6. 7. 8. 9. 10. 11. 12. 13. 14. 16. 16. 17. 18. 19. 20. 21. 22. 23. 24. 26. 26. REFERENCES van der Kerk, G. J. M., and Luijten, J. G. A., J . APpZ. Chem., Lond., 1964, 4, 314. Hof, T., and Luijten, J. G. A., Timb. Technol., 1969, 67, 83. Hof, T., J . Inst. Wood Sci., 1969, 23, 19. Levi, M. P., Wood, 1969, 34, 39. Smith, C. S., and Watson, R. W., Ibid., 1967, 32, 62. Nishimoto, K., and Fuse, G., Q. JZ Tin Res. Inst., 1966, 70, 3. Guenther, F., Geyer, R., and Stevenz, D., Neue Hutte, 1969, 14, 663. Ishii, Y., Kawamura, H., and Yagi, S., Bunseki Kagaku, 1968, 17, 3. Bork, V. A., and Selivokhin, P. I., Plast. Massy, 1969, 10, 60. Booth, M. D., and Fleet, B., Analyt. Chcm., 1970, 42, 826. Geissler, H., and Kriegsmann, H., 2. Chemie, Lpz., 1904, 4, 364. Tonge, B. L., J . Chromat., 1966, 19, 182. Herold, B., and Droege, K. H., 2. analyt. Chem., 1969, 245, 296. Owaki, H., Maeda, H., and Wada, N., Nugayashi Kogyo Kenkyusho Kenkyn Hokoku, 1960, 24, 92. Freeland, G. N., and Hoskinson, R. M., Analyst, 1970, 95, 679. Hardon, H. J., Brunink, H., and van der Pol, E. W., Ibid., 1960, 85, 847 Adamson, J. H., Ibid., 1962, 87, 697. Chromy, V., and Vrestal, J., ChemickL Listy, 1966, 60, 1637. Williams, A. I., Analyst, 1968, 93, 111. - , Ibid., 1968, 93, 611. -, Ibid., 1969, 94, 300. -, Ibid., 1970, 95, 498. Costa, G., Gaza. Chim. Ital., 1960, 80, 42. -, Annali Chim., 1961, 41, 207. Cartwight, K. St.G., and Findlay, W. P. K., “Decay of Timber and its Prevention,” Second Smith, D. N. R., and Cockcroft, R., Nature, Lond., 1961, 189, 163. Edition, H.M. Stationery Office, London, 1968, p. 286. Received Seftember 1 l f h , 1972 Accepted Novembcv 2Yh, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800233

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

Analyst, April, 1973, Vol. 98, pp. 243-245 243 - - - - - - An Improved Method for the Determination of Whole Blood Lead by Using an Atomic-absorption Technique -0 10 20 30 CI s - 4 0 2 W n 3 -60 C e! t- -50 .E ‘5 70 80 90 BY G. ALAN ROSE AND ELIZABETH G. WILLDEN (Department of Pathology, St. Peter’s Hospitals, Endell Street, London, W.C.2) I The Delves technique for measuring whole blood lead has been con- siderably improved by wet ashing with aqua regia instead of simply drying with hydrogen peroxide. Smoke is eliminated, and the reproducibility of the method is improved. VJ IN a recent survey, Keppler et a2.l found that less than half of the clinical laboratories that were reported to be undertaking blood lead determinations were doing so with a reasonable degree of accuracy.This situation was improved by Delves,* who used atomic-absorption techniques in conjunction with what is now called the “Delves technique.” In this method whole blood was placed in a cup together with 100-volume hydrogen peroxide and, after drying the mixture, the cup was introduced directly into an air - acetylene flame with an absorption tube assembly attached. Unfortunately, the recorded smoke trace produced when using Delves’ method can be very misleading and difficulties in the interpretation of the results can lead to inaccuracies. N Pb 1 Y 1 @ SAC and the authors.244 ROSE AND WILLDEN : IMPROVED METHOD FOR THE DETERMINATION [Analyst, VOl. 98 We have found considerable advantages in using aqua regia instead of hydrogen peroxide. Firstly, the smoke produced on introduction of the sample can be eliminated, and secondly, the reproducibility can be greatly increased.The original tube and cups, which were made of nickel, had to be replaced by similar apparatus made of fused quartz (obtainable from Thermal Syndicate Limited, Wallsend-on-Tyne) . In each instance, 0.010 ml of blood was subjected to wet ashing by adding the aqua regia and evaporating to dryness on a hot-plate, care being taken to avoid spluttering. Measurements were carried out on a Perkin-Elmer 290B spectrophotometer with the modified Delves’ absorption tube attachment. Results obtained by use of the Delves technique, in which whole blood (0.010 ml) and 100-volume hydrogen peroxide (0.020 ml) were mixed together in the cup, have been compared with our results, which were obtained by using aqua regia instead of hydrogen peroxide. As can be seen from Fig.1, the two methods give comparable results, but the interpretation of the results was facilitated, and their reproducibility considerably enhanced, by using our improved method. It will be noted that when 0.600 ml of aqua regia is added to the sample, the smoke traces are very small, and are negligible with 0.800 ml. Different volumes of aqua regia were tried. I - TABLE I COMPARISON OF RESULTS OBTAINED BY USING THE TWO METHODS ON REPLICATE BLOOD SAMPLES Number Mean/ Range/ Standard Method of assays pg per 100 ml pg per 100 ml deviation Delves’ method(with the original matched cups) 15 09-26 59 to 79 4.32 Delves’ method (with quartz cups) . . . . 15 67-1 3 47 to 79 8.96 Aqua regia method (with matched cups) .. 20 08-25 64 to 72 2.49 Aqua regia method (with unmatched cups) . . 20 08-60 60 to 77.5 4.93 10 20 c; f 30- 0 L 40- c .$ 50 m .- $, 60- $ 70- C m 80 90 A blood sample with a high lead content was examined by the two methods and the results subjected to statistical analysis. The comparison is shown in Table I, from which it can be seen that oxidation of the blood with hydrogen peroxide is unsatisfactory when the quartz cups are used. The aqua regia method with unmatched quartz cups is almost as accurate as the original Delves method with matched nickel cups, but is considerably improved when the quartz cups are matched in terms of length of time in use. - - - - - A 01 ~ 75 50 25 100 I 75 50 25 Pb concentration/pg per 100 ml Fig. 2.Comparison of pure lead stan- dards, with (peaks A) and without (peaks B) added electrolytesApril, 19731 245 Fig. 2 shows typical peaks for pure lead standards over the range required for deter- minations on whole blood samples, both with and without addition of electrolytes at levels comparable with those found in whole blood. The differences between the two sets of results are very small and can be attributed to normal experimental errors. Calibration graphs of aqueous standards and of whole blood containing increasing amounts of lead are shown in Fig. 3. This method of addition, involving the use of whole blood of known lead content spiked with various amounts of lead solution, gave very similar results to those obtained with the aqueous solutions.Thus, the two lines are almost parallel and the error introduced by the deviation from absolute parallelism was 3 per cent., i.e., recoveries of added lead were 100 -j= 3 per cent. OF WHOLE BLOOD LEAD BY USING AN ATOMIC-ABSORPTION TECHNIQUE D Added lead content / pg per 100 ml Fig. 3. Comparison of whole blood and aqueous lead standards. A, whole blood with added lead standards; and B, aqueous standards In order to establish that the smoke was being removed satisfactorily, measurements were taken on a nearby non-resonant wavelength by using blood with a lead content of 50 pg per 100 ml. An absorption signal equivalent to 3 to 5 pg per 100 ml was obtained. Thus, the non-specific absorption accounts for up to 10 per cent. of the whole blood lead. This value compares favourably with that found by Delves’ original method.2 The method has proved to be reliable for whole blood lead values both in the normal and the abnormally high ranges. The equipment was donated by the Institute of Urology, London. REFERENCES 1. 2. Keppler, J. F., Maxfield, M. E., Moss, W. D., Tietjen, G., and Linch, A. L., Amer. Ind. Hyg. Delves, H. T., Analyst, 1970, 95, 431. Received April 27th, 1972 Acccpted November 21st, 1972 Ass. J., 1970, 31, 412.

ISSN:0003-2654    

DOI:10.1039/AN9739800243

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

246 Artalyst, April, 1973, Vol. 98, PP. 246-250 Bis( 6 - methyl-2 = pyridy1)glyoxal Dihydrazone as a Spectrophotometric Reagent for the Rapid Determination of Copper in Alkalis, Milk and Brine BY M. VALCARCEL AND F. PIN0 (Department of Analytical Chemistry, University of Seville, Seville, Spailz) The synthesis, characteristics and analytical applications of bis(6-methyl- 8-pyridyl) glyoxal dihydrazone are described. This compound produces coloured solutions selectively with copper(1) ions (Amax. = 440 nm, E = 8.7 x 103 1 mol-1 cm-l) that can be extracted into various organic solvents; it behaves as a cuproine-type reagent. The 1: 1 orange-yellow copper(1) complex has been used for the spectrophotometric determination of trace amounts of copper in saturated brine, alkalis and milk.The most important advantage of using this reagent is the total recovery of copper that is possible from ammoniacal solutions. THE work described in this paper forms part of an investigation into the use of hydrazones for trace-metal analysis. The ferroin-type reagents, 2,2'-bipyridylglyoxal dihydrazone, di- acetyl dihydrazone and phenyl 8-pyridyl ketone hydrazone, have been applied1 to the spectrophotometric determination of trace amounts of iron while 6-methylpicolinaldehyde hydrazone was used2 as a selective cuproine-type reagent for the determination of copper. In the present work, bis(6-methyl-2-pyridyl)glyoxal dihydrazone was used for the selec- tive determination of trace amounts of copper in different materials. EXPERIMENTAL SYNTHESIS OF REAGENT- Five millilitres of 99-9 per cent.m/m hydrazine hydrate were added to 6 g of bis(6-methyl- 8-pyridy1)glyoxal (Fluka) dissolved in 150ml of hot absolute ethanol and the mixture was refluxed for 24 hours. On cooling to 0 "C, the crystals that separated out were filtered off and recrystallised twice from hot ethanol. The crystals finally obtained, dried at 60 "C under pressure, were white and melted at 154 to 155 "C. On analysis their elemental content was found to be: C 62.5, H 5-9 and N 31.5 per cent.; the content calculated for Cl4HI6N6 was: C 62.67, H 5.96 and N 31.34 per cent. APPARATUS- meters, equipped with l-O-cm glass or quartz cells. SOLUTIONS- Spectrofikotometers-Unicam SP800, Unicam SP600 and Beckman DU spectrophoto- Digital fiH meter-Philips, PW9408, with glass - calomel electrodes.All solvents and reagents were of analytical-reagent grade. Bis(6-methyl-2-pyridyl)glyoxal dihydrazone reagent solutions-Solutions of concentration Standardised solutions of copper(II). Ascorbic acid, 2 per cent. m/V aqueous solution (as reducing agent). Acetic acid - sodium acetate bzc$er solution, pH 4.8. Q SAC and t h e authors. 0.1 per cent. m/V in ethanol and 0.05 per cent. m/V in nitrobenzene.VALCARCEL AND P I N 0 247 Extraction solution 1-Dissolve 5 g of sodium perchlorate monohydrate and 3 g of ascorbic Extvaction solution 2-Dissolve 5 g of sodium perchlorate monohydrate and 3 g of ascorbic Trichloroacetic acid, 50 per cent. m/V aqueous solution. acid in 250 ml of the above buffer solution. This solution remains stable for 1 week.acid in 250 ml of distilled water. This solution also remains stable for 1 week. RECOMMENDED PROCEDURES ALKALIS- To 10 ml of extraction solution 2 in a separating funnel add 50 ml of alkali, followed by 10 ml of 0.05 per cent. m/V reagent solution in nitrobenzene. Shake the mixture vigorously for 2 minutes, allow the phases to separate and transfer the lower, organic, layer into a flask containing anhydrous sodium sulphate. Measure the absorbance of this solution at 440 nm against the reagent solution in nitrobenzene. Prepare a calibration graph in a similar manner by adding appropriate amounts of copper to 2 M sodium hydroxide solution. SATURATED BRINE- Method A-To 25 ml of saturated brine in a 50-ml calibrated flask, add 10 ml of acetic acid - sodium acetate buffer solution, 2 ml of 2 per cent.m/V ascorbic acid solution and 5 ml of 0.1 per cent. m/V ethanolic reagent solution, and dilute to the mark with distilled water. Measure the absorbance of the solution at 440nm against a reagent blank prepared in a similar manner. Method B-To 10 ml of saturated brine in a separating funnel add 10 ml of extraction solution 1 and 10 ml of 0.05 per cent. m/V reagent solution in nitrobenzene. Shake the funnel vigorously for 1 minute, allow the phases to separate and transfer the lower layer into a flask containing anhydrous sodium sulphate. Measure the absorbance at 440 nm against the reagent solution in nitrobenzene. Calibration graphs are constructed from standard solutions treated in the same way. MILK- To 100 ml of milk in a 300-ml Erlenmeyer flask, add 25 ml of 50 per cent.m/V trichloro- acetic acid solution, slowly and with constant shaking. Shake the flask vigorously, place it in a boiling water bath for 16 minutes and cool it in ice - water to 10 "C. Transfer 25 ml of the supernatant liquid into a separating funnel. Add 7 ml of 2~ sodium hydroxide solution, 10ml of extraction solution 1 and 10ml of 0.05 per cent. m/V reagent solution in nitrobenzene. Shake the funnel vigorously for 1 minute, allow the phases to separate and transfer the lower layer into a flask containing anhydrous sodium sulphate. Measure the absorbance of this solution at 440 nm against the reagent solution in nitrobenzene. Obtain the content of copper, in micrograms, from a standard graph prepared by substituting the appropriate copper solutions and distilled water for the milk sample.RESULTS AND DISCUSSION BIS(6-METHYL-2-PYRIDYL)GLYOXAL DIHYDRAZONE REAGENT- The ultraviolet spectrum for the reagent shows a bathochromic shift in an acidic medium (Amax. 347 and 268 nm) compared with an alkaline medium (Am,,, 295nm, with a shoulder at 266 nm), with two isosbestic points at 277 and 313 nm. The Phillips and Merritt3 method is used for the determination of the ionisation constant; the average pK value is 4.82. This behaviour may be caused by protonation of the nitrogen atoms in the pyridine rings; the pK value is very similar t o that of pyridine and its derivatives. The reagent is resistant to hydrolysis in a strongly acidic medium (6 M hydrochloric acid).This property is usual with a-diimines, in contrast with the corresponding imines ; 6-methyl- picolinaldehyde hydrazone2 hydrolyses in 2 M hydrochloric acid at 20 "C in 30 minutes. The reaction of the reagent with thirty cations at various pH values was investigated; it reacts only with copper(1) and palladium, and the absorption spectra of solutions of these metals are shown in Fig. 1. It acts as a cuproine-type reagent in that methyl groups adjacent to the nitrogen atom in pyridine produce the well known blocking effect.248 VALCARCEL AND P I N 0 : BIS(6-METHYL-2-PYR1DYL)GLYOXAL DIHYDRAZONE [A?ZaZy!yst, VOl. 98 " 400 450 500 550 600 650 Wavelengthhm Fig. 1. Absorption spectra of solutions of COIII- plexes formed with reagent: 1, 10 p.p.m. of copper ( 1 ) ; 2, 10 p.p.m.of palladium; and 3, reagent alone (water blank) REACTION WITH COPPER(I)- Aqueous media-The orange - yellow 1 : 1 copper(1) complex (ratio found by the Job absorptiometric method, by isolation of the perchlorate complex, [Cu(C,,H,,N,)]ClO,, in the solid state and elemental analysis*) of the reagent is formed completely over the pH range from 4.5 to 11.2 in aqueous solution (see Fig. 2). The effect of other experimental variables was determined. The system conforms to Beer's law over this pH range, the molar absorptivity ( E ) being 8.7 x lo3 1 mol-l cm-l. The optimum concentration range, evaluated by Ringbom's method, is 2 to 5 p.p.m. of copper. The colour intensity of the solutions of the complex remains constant for several hours. The relative error (P = 0.05) of the method is &0-13 per cent.The colour reaction is selective for copper. Silver, mercury( 11), mercury(I), cadmium, zinc, iron(II), cobalt(II), thallium, lead, tin(II), uranium(VI), calcium, strontium and barium do not interfere at the 300 p.p.m. level in pH 4.8 buffer. Nickel does not interfere at the I 1 Fig. 2. Effect of pH on the formation of the copper (I) complex: 1, in water; 2, in pent- anol; 3, in nitrobenzene; and 4, in chloroform * The results of the analysis were C 38.7, H 3.5 and N 19.1 per cent. ; calculated values, C 38.96, H 3-71 and N 19-48 per cent.April, 19731 AS A SPECTROPHOTOMETRIC REAGENT FOR THE DETERMINATION OF coprm 249 50 p.p.m. level, while iron(III), bismuth, antimony(II1) and manganese( 11) precipitate at the 100 p.p.m.level. The most serious interferences are from palladium, gold(III), EDTA and oxalate. At pH 8.7 interferences are greater. If an excess of reagent instead of ascorbic acid is used as reducing agent, the same results are obtained over the pH range 4.5 to 9. Extraction-The coloured complex formed in aqueous solution can be extracted into various organic solvents, such as pentanol, chloroform and isobutyl methyl ketone, with no perchlorate, but in the pH range from 9 to 12 the absorbance of the organic layer is not stable with time (Fig. 2). Copper(1) ions are completely extracted with either nitrobenzene or chloroform, with perchlorate in the aqueous phase. The optimum pH range is 3.2 to 5.7 (nitrobenzene) and 4-5 to 5.7 (chloroform); from pH 9 to 12 the absorbance remains lower but stable with pH and time (Fig.2). Beer’s law is adhered to at pH 4.8 and 10.9 with both of these solvents. The coloured organic solutions are stable for several hours. The extraction has a high relative error with chloroform but the relative error (P = 0.05) of the method with nitrobenzene is &0.36 per cent. The optimum concentration range evaluated by Ringbom’s method is 2 to 6 p.p.m. of copper. After the extraction the effect of the ions mentioned above can be investigated. Those ions which did not interfere with the colour reaction do not interfere when using this technique at pH 4.8 or 11. Interference from nickel, iron(III), bismuth and manganese(I1) is now suppressed up to a level of 300 p.p.m. Palladium, gold(TIT), EDTA and oxalate also interfere in this method.DETERMINATION OF COPPER- Typical results for the determination of copper in sodium hydroxide, potassium hydroxide and ammonia solutions are given in Table I. The method is very useful because the recovery TABLE I DETERMINATION OF COPPER IN ALKALIS Each result is the average of three separate analyses Solution Copper added/pg g-1 Copper recovered/pg g-’ liecovery, per cent. Sodium hydroxide. . .. 10.0 10.26 102.5 Potassium hydroxide . . 14.3 14.6 102.1 Ammonia .. .. .. 24.1 24.1 100.0 from ammonia solutions is complete. The present reagent is more suitable than 6-methyl- 2-pyridylphenyl ketoxime, proposed by Pemberton and Dieh14 in 1969 as a reagent for the determination of copper in alkalis, because with the latter the recovery of copper from am- monia solutions is only partial (32 per cent.).Trace amounts of copper in saturated brine can be determined accurately in homo- geneous media (Method A) and with the extraction technique (Method R). Typical results for the determination of copper are given in Table 11. TABLE I1 DETERMINATION OF COPPER IN SATURATED BRINE Each result is the average of three separate analyses Copper r - Copper found/pg ml-1 Recovery, per cent. added/pg ml-l Method A Method €3 Method A Method B 2.0 4.0 6.3 6.8 2.0 4.0 6-2 6.8 1.95 100 97.6 4.0 100 100 6.3 98.6 100 6.7 100 99 The reagent is also suitable for the rapid determination of the copper content of milk. The results (Table 111) are comparable with those obtained by Smiths in 1967 when using the same method with a 0.05 per cent. m/V solution of zinc dibenzyldithiocarbamate in toluene.260 VAZCARCEL AND PIN0 TABLE I11 DETERMINATION OF COPPER IN MILK Number Average concentration of Confidence limits Commercial sample of determinations copperlpg per 100 ml (96 per cent.) 1 6 34.6 f3-6 2 6 8 f 2.3 3 6 12 f 2.6 REFERENCES 1. 2. 3. 4. 6. Graciani, E., Ph.D. Thesis, University of Seville, 1969 (and unpublished work). Valcarcel, M., and Pino, F., Infcidn. Quim. Analit. Pura APl. Ind., 1972, 26, 116. Phillips, J . P., and Merritt, L. L., J. Amer. Chem. SOC., 1948, 70, 410. Pemberton, J. R., and Diehl, H., Talanta, 1969, 16, 393. Smith, A, C., J. Dairy Sci., 1967, 50, 664. Received July 12th. 1972 Accepted November loth, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800246

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

ArtaZyst, April, 1973, Vol. 98, $9. 251-256 251 An Electrical Conductivity Detector for Paper, Thin-layer and Column Chromatography* BY V. DI STEFAN0 AND P. MARINI (Ccntro Spcrimentale Metallurgico, V i a di Caste1 Romano, Rome, Italy) An electrical conductivity detector has been developed for application to the paper, thin-layer and column chromatography of ionic substances. Two arms of an a.c. Wheatstone bridge comprise two pairs of electrodes applied directly on to the separating layer so that a signal ensues from the bridge when a chromatographic spot passes one electrode pair while the other pair is traversed by the eluting agent. The sensitivity is about 1 pg and the precision is about 2 to 3 per cent. THE problem involved in the quantitative determination of the components of a mixture after a chromatographic separation has been carried out is still difficult and complex.While the identification of the individual components can be simplified to measurement of R, values, the quantitative analysis is carried out by using techniques and methods that are often exclusively applicable to only one or other of the components of a mixture and great care must be exercised to avoid interference effects from the chromatographic support. The methods capable of more general use can be classified as direct and indirect methods. The former permit quantitative analysis to be carried out directly on the chromatographic medium without other manipulation, while indirect methods require a series of manipulations after the development of the chromatogram. Examples of the application of such methods can be found in the literat~re.l-~ With indirect methods, quantitative analysis needs to be performed by highly qualified personnel and by use of rather complex instrumentation, while chromatographic separations are carried out simply and often with very simple apparatus.However, there are relatively few direct methods of quantitative analysis of general applicability, especially in paper and thin-layer chromatography. The present work is intended as a contribution towards pro- viding a direct detection method that is useful in many instances. A description with illustrations is given of an electrical conductivity detector applied to paper, thin-layer and column chromatography, which is capable of detecting small amounts of any substance that is a charge carrier and that can be eluted by substances that exhibit a certain electrical conductivity .CONFIGURATION OF THE SYSTEM- The simplest form of such a detector as originally assembled consists of two parallel, narrow, thin gold plates between which is enclosed a strip of paper, thus forming a con- ductivity cell, which is used as an arm of a Wheatstone bridge; the bridge is connected to a differential amplifier whose output is fed into a chart recorder. One end of the paper strip is immersed in the eluting agent and a flow of eluting agent is established along the strip. When this flow becomes constant, the signal from the Wheatstone bridge is also constant and the bridge can be set to zero. A solution of a conducting substance in the eluting agent used is prepared and 2 to lop1 of this solution are applied with a precision microsyringe close to the immersed end of the paper strip, which results in a spot being formed which travels in the flow of eluting agent towards the conductivity cell.When the spot reaches the electrodes a conductivity peak is recorded. More elaborate arrangements were subsequently designed and the electrodes were applied to thin layers of silica, alumina and cellulose, as well as to a special type of column. In Fig. 1 is shown the best arrangement attained at present. Four electrodes are now used, forming a working cell and a reference cell. They consist of thin, narrow platinum strips, 1 mm wide x 1 mm thick and. 35 mm long, placed 1-5 mm apart, embedded in a PTFE support on which the thin layer is deposited in the usual way.The eluting agent is fed in at the upper end and the sample is applied at a suitable distance from the feeding point * Presented at the Third SAC Conference, Durham, July 12th to 16th, 1971. Q SAC and the authors.252 DI STEFAN0 AND MARINI : AN ELECTRICAL CONDUCTIVITY DETECTOR [AndySt, VOl. 98 of the eluting agent, either with a microsyringe or by using a narrow strip of polished gold on which the substances to be determined have been deposited from solution by evaporation of the solvent. The conveniently supported gold strip is brought into contact with the layer and contact maintained until a further sample is required to be applied. One end of the thin layer, which is kept in a horizontal position, is exposed to the air so as to enable the eluting agent to evaporate; the other parts of the chromatographic system are suitably sealed in order to prevent its evaporation; efforts are at present being made to embed the electrodes in a glass plate, glass being a more satisfactory material in this respect than PTFE.The above electrode arrangement yielded the most satisfactory results, but other arrange- ments were also tried, as shown in Fig. 2. In Fig. 2 (a), an arrangement is shown in which one electrode is embedded in the support and the other gently applied over the thin layer. We found that this system produced cells the apparent volume of which extended too far within the layer, thus giving rise to enlarged peaks. In Fig. 2 ( b ) , a comb-like type of electrode is shown, which gave rise to a slight perturbation of the flow and to tailed peaks.r 1 5 4 2 Fig. 1. Detector system: 1, support; 1 a~itl 3, electrodes; 4, leads; and 5, thin layer Fig. 2. Different arrangements of elec- trodes In Fig. 3 is shown the design of a special column, which was developed in order to allow the conductivity detector to be applied to column systems. A large conventional column could easily accommodate at least one pair of electrodes, but is not suitable for the separation and detection of micro-amounts of substances. A capillary column does not afford enough room for the electrodes, so that the annular space enclosed between two concentric columns, which may be regarded as a thin-layer column, was used. This space is currently 1 mm wide but could be further reduced, possibly to 0.3 to 0.4 mm, thus obtaining a cylindrical layer suitable for micro-analytical purposes.Filling such a column does not present particular difficulties and can be achieved by carefully pouring the substance chosen as the chromatographic support, in the form of a slurry, from the top of the column while the latter is maintained in a vertical position and in contact with a plate vibrating at about 50 Hz and with moderate amplitude. The two conductivity cells, applied to the separating layer, constitute two adjacent arms of a Wheatstone bridge to which an oscillator feeds a 2-V, 2000-Hz signal. The output of the bridge is fed into a differential amplifier, which could be replaced by a lock-in amplifier for greater accuracy.This amplified signal is fed both into an integrator and into a chart recorder. If the cells are arranged in such a way that one electrode of one of the cells is short-circuited with one of the electrodes of the other cell, while the signal from the oscillator is applied to the other two electrodes, the zero line is almost independent of variations in the flow of the eluting agent and changes in the environmental conditions. Electrodes can be set side by side in pairs located symmetrically with respect to the cylinder axis. Samples are applied with a microsyringe through neoprene diaphragms located upstream at convenient distances from the electrodes.April, 19731 FOR PAPER, THIN-LAYER AND COLUMN CHROMATOGRAPHY 253 I Fig. 3. Chromatographic column THEORY OF THE RESPONSE- across its terminals when a chromatographic spot is flowing through the working cell is-- It can easily be demonstrated that the response of the bridge in terms of the potentid kX (C--CZ+ .. . ) Valkh (0 + aJ2 + 0 1 P = where p is the instantaneous root mean square of the potential across the bridge; I/ the root mean square of the supply potential; a1 the conductivity of the arm adjacent to the working cell; a the conductivity of the working cell before and after the spot has passed, whose value is usually very close to a,; k the constant of the working cell, a function of the geometry of the electrodes and of the electrical path between them; h the equivalent con- ductivity of the spot material x 103; and C the instantaneous equivalent concentration of the spot material.As a first approximation the response is proportional to concentration, which has been confirmed experimentally as will be shown later. The higher the supply potential the higher the response; it was found that, as with most conductimeters, 2 V are sufficient to obtain a good and clean response. The response is higher if the conductivity of the eluting agent is low, but the conductivity of the latter cannot be too low or the stability of the zero line and linearity of response with varying concentration will be affected. In a series of experiments with a diethyl ether based eluting agent, which gave rise to a resistance across the electrodes of 250 kQ, it was found that trace amounts of moisture in the sampling device are capable of rendering the entire system unstable for a long period of time.Thus, if the conductivity of the eluting agent is reduced to very low values, the electronics of the device must be suitably designed and great care should be used in handling the apparatus. If we set (0 + aJ2 * = Vqkh it can be seen that t,b is the constant of the detector and as a first approximation c = #P The complete response with time, that is, the chromatogram, is a peak, and the amount of the substance under examination is proportional to the area of the peak.264 DI STEFAN0 AND MARINI AN ELECTRICAL CONDUCTIVITY DETECTOR [Anat!ySt, VOl. 98 RESULTS AND DISCUSSION The logical first application of such a detector is to separation systems for inorganic ions and the work carried out on some of these systems is reported below.In the course of experiments performed with the various types of electrodes described in this paper it was found that better results were obtained with cell configurations of the type shown in Fig. 1 and interest was accordingly concentrated on these cells. In Fig. 4 (a) a separation of Fe3+ from Zn2f ions in a mixture of these ions on a thin layer of silica gel G is reported; the eluting agent used was acetone - 12 N hydrochloric acid - 30 20 10 0 - I I I I I I I I I I 0 10 20 30 40 50 60 70 80 30 - (4 (4 Fe3+ - - I I IApril, 19731 FOR PAPER, THIN-LAYER AND COLUMN CHROMATOGRAPHY 265 hexane-2,5-dione (100 + 1 + 0.5). The sample analysed consisted of 10 pl of a solution containing 0.2 pg p1-1 of both iron and zinc ions.On the y-axis of the graph the output of the amplifier is given in millivolts. The true resistance of each cell was about 400 SZ. In Fig. 4 (b) the chromatogram for the mixture of Fe3+ and Mn2+ ions, which was obtained under the same conditions as for the previous chromatogram, shows that the Fe3+ peak appears after almost exactly the same time as the Fe3+ peak in Fig. 4 (a). The same is true of Fig. 4 (c), which relates to the separation of a mixture of Fe3+ and Co2+ ions carried out under the same conditions as before. With the mixture of Mn2+, Co2+ and Zn2+ ions, separa- tions were not attempted as efforts were devoted more to testing the properties of the detector than to studying separation techniques. The sensitivity of the entire system depends largely on the characteristics of the elec- tronics used and the level obtained with the prototype is shown in Fig.4 (c), for which the sample taken amounted to only 0.75 pg of Co2+. The chromatogram shown in Fig. 4 (a) is the result of an attempted separation of a mixture of Fe3+, Cr2f and Co2+ ions, by using as eluting agent acetone - pentyl acetate - pentane-2,4-dione - 6 N hydrochloric acid (150 + 10 + 1.5 + 1.5). For every substance it is necessary to plot a calibration graph and consequently care should be taken to maintain constant experimental conditions. No difficulties arose and thermostatic control of the system was found to be unnecessary. The only precautions necessary were the maintenance of a sufficient supply of eluting agent to the layer and prevention of evaporation of the standard solutions prepared with the same eluting agent.The calibration graph for iron is plotted in Fig. 5. On the abscissa is the area originally obtained in millivolts per minute, but it has been plotted in square centimetres, with an approximation of 10 per cent., in order to demonstrate the extension of the peak. Standard solutions with different concentrations were used in order that samples should be at constant volume, but it was noticed that the volume of the eluting agent had no efkect and all of the straight lines obtained could be extrapolated to zero, which was further confirmed with a sample of the pure eluting agent. Nevertheless, we found that a higher dispersion was obtained with samples at constant concentration and with variable volume, which was caused by the graduation and calibration of the microsyringe, and we therefore finally resorted to the use of samples of constant volume.Peak area/cm2 Fig. 5 . Calibration graphs for different ions: A, Co*+; 13, I:@+; and C, Zn2+ The calibration graphs for Zn2+ and for Co2+ ions are also shown in Fig. 5. From day to day, or after any interruption of the flow of eluting agent through the chromatograph, we256 DI STEFAN0 AND MARINI noticed some variations in the angular coefficients of the calibration graphs, so that for analytical purposes it is advisable to introduce a standard sample at the beginning of each analytical cycle. In order to observe the effect of time upon the angular coefficient of the calibration line, we examined, once every hour for 7 hours, a standard volume of 4 pl of a solution containing about 1 pg p1-1 of iron.The result of this experiment is shown in Table I. The mean value was 4.11 pg, the standard deviation 0.25 pg and the coefficient of variation 6.2 per cent. TABLE I EFFECT OF TIME ON ANGULAR COEFFICIENT OF CALIBRATION LINE FOR IRON Tirne/hours . , .. 0 1 2 3 4 5 6 7 Amount of ironlpg . . 4.2 4-0 4.2 3.9 4.1 4.3 4.0 4.2 CONCLUSION A conductivity detector consisting of one or two electrode pairs applied to a chromato- graphic layer appears to be a simple device that is capable of minimising difficulties and manipulations associated with quantitative liquid chromatography. For paper and thin-layer chromatography, the best results were obtained by applying, in contact with the layer, narrow strip electrodes embedded in the supporting material. With column chromatography, good results were obtained by filling the annular space between concentric columns with the solid phase, while the electrodes were supported by the glass of the column. Samples from a solution in the eluting agent of the substances to be analysed are applied directly to the layer with a precision microsyringe. In order to obtain accurate results, analyses should be made and calibration graphs obtained by using sample solutions with a constant volume of eluting agent. 1 . 2 . 3. 4 . 5 . 6. 7 . 8 . 9 . REFERENCES Petrowitz, H. J., Mitt. dt. Ges. Holzforsch., 1962, 48, 57. Fisher, R. B., Parson, D. S., and Morrison, G. A., Nature, Lond., 1948, 161, 764. Seher, A., Nahrung, 1960, 4, 466. -, Mikrochim. Acta, 1961, 308. Zollner, N., Wolfram, G., and Amin, A., KZin. Wschr., 1962, 40, 273. Neubauer, D., and Mothes, K., Planta Med., 1961, 9, 466. Hefendehz, F. W., Ibid., 1960, 8, 65. Ganshirt, H., and Morianz, K., Arch. Farmaz., 1906, 293, 1066. Schaulze, P. E., and Wenzel, M., Angew. Chem., 1962, 74, 777. Received May 31st, 1972 Accepted November 7th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800251

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

A.naZyst, April, 1973, Vol. 98, @. 257-267 257 The Identification and Semi-quantitative Assay of Some Fat-soluble Vitamins and Antioxidants in Pharmaceutical Products and Animal Feeds by Thin-layer Chromatography BY G. W. JOHNSON AND C. VICKERS (Analytical Research, Quality Control, The Boots Company Ltd., Pennyfoot Street, Nottingham) A thin-layer chromatographic method for the identification and semi- quantitative assay of vitamin A (alcohol), its acetate and palmitate, vitamin D, a-tocopherol, a-tocopheryl acetate, BHA (butylated hydroxyanisole ; 2-t-butyl- 4-methoxyphenol), BHT (butylated hydroxytoluene ; 2,6-di-t-butyl-4-methyl- phenol) and ethoxyquin in vitamin preparations is described. The sample solutions are applied to thin layers of silica gel and the vitamins and anti- oxidants are separated by using n-hexane - ethyl methyl ketone - di-n-butyl ether (34 + 7 + 6) as the developing solvent.Decomposition of vitamins A and D when applied to the adsorbent layer is inhibited by the presence of triethylamine in the spotting solvent. The compounds are identified by means of their RB- values, their appearance in ultraviolet radiation of wavelengths 254 and 360 nm and their response to iron(II1) chloride - bipyridyl and iron(II1) chloride - potassium hexacyanoferrate(II1) spray reagents ; they are assayed by visual comparison with standards. The method has been applied to gelatin-protected vitamin beads, animal feed additives, multi- vitamin tablets, oily vitamin concentrates and halibut-liver oil samples. A simple colour test for distinguishing vitamin D, from vitamin D, after removal of vitamin A and its esters is also described.VITAR~IN A (retinol) and its esters, vitamin D, (ergocalciferol), vitamin D, (cholecalciferol) and vitamin E (a-tocopherol) are usually added to vitamin preparations either in the form of gelatin-protected beads, in which the vitamins are dispersed in a starch-coated matrix of gelatin and sugar, or in the form of concentrated solutions in oil. In both of these con- centrates, the vitamins are stabilised by the presence of antioxidants, such as a-tocopherol, 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole, BHA), 2,6-di-t-butyl-4-methylphenol (butylated hydroxytoluene, BHT) and 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline (ethoxy- quin). Rapid and reliable methods for the examination of the vitamin concentrates and their formulated products were required, and the use of thin-layer chromatography for this purpose was investigated. Many thin-layer chromatographic methods for the separation, identification and assay of vitamins A, D and E have been reported; these methods have been reviewed by Bolligerl and Strohecker and Henning2 The main difficulty in the assay of vitamins A and D by thin-layer chromatography arises from the instability of the vitamins on dry chromatographic adsorbents.Bolliger and Konig3 described a method in which the decomposition of vitamin D on silica gel layers was minimised by developing the chromatogram immediately after applying the vitamin extract (i.e., while the adsorbent was still saturated with the spotting solvent).Hanewald, Mulder and Keuning4 reduced the extent of decomposition of vitamin D by adding squalane to the vitamin solution and BHT to the developing solvent, while Ponchon and Fellers5 used a method in which the vitamin D solution was applied to the adsorbent layer in an atmosphere of nitrogen. Attempts to apply the published methods were often unsuccessful. None of the chromato- graphic systems gave a satisfactory separation of all the vitamins and antioxidants, and some decomposition of the vitamin A took place when it was applied to the adsorbent layers, even though the recommended precautions had been taken. The procedures described in this paper enable the vitamins and antioxidants to be applied to silica gel layers without decomposition and to be separated, identified and assayed in a single chromatographic system.0 SAC and the authors.258 JOHNSON AND VICKERS : THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION [Analyst, VOl. 98 When administered to chicks,6 vitamin D, has only 1 or 2 per cent. of the antirachitic activity of vitamin D, and it is therefore essential to ensure that the vitamin D beads used in poultry feed supplements contain vitamin D, and not vitamin D,. The two vitamins have been differentiated by infrared and nuclear magnetic resonance spectroscopy,2J by thin-layer partition chromatography1 and by the difference in colour of their spots on silica gel plates after spraying with concentrated sulphuric acid.l The instrumental methods require the use of large amounts of the vitamins in a pure form and are therefore unsuitable for the examination of formulations.The partition chromatographic and colour-test procedures require less sample and are less sensitive to the presence of impurities, but in our hands they did not prove very reliable. A simple colour test, which can be applied in the presence of many of the compounds likely to occur in formulations, has therefore been developed. EXPERIMENTAL APPARATUS AND REAGENTS- 0.25-mm adsorbent layer and activate the plates by heating them at 110 "C for 1 hour. 60 and 62 "C. Chromatographic plates-Prepare Kieselgel (Merck) HF,,, plates, 20 x 20 cm, with a Diethyl ether-Anaesthetic ether B.P. Chloroform-Re-distil chloroform B.P., collecting the fraction that distils between Methanol-Analytical-reagent grade.Di-n-butyl ether-Shake 2 litres of di-n-butyl ether with 100 ml of 10 per cent. aqueous sodium metabisulphite solution and allow it to stand for 24 hours. Run off the aqueous layer and wash the di-n-butyl ether with two 50-ml volumes of 1 N sodium hydroxide solution and then with water until the washings are neutral to litmus. Dry the di-n-butyl ether with anhydrous sodium sulphate and apply the peroxide test described for anaesthetic ether in the British Pharmacopoeia 1968. If no peroxides are detected, distil the di-n-butyl ether until its volume has been reduced to about 250 ml, collecting the fraction that distils between 138 and 141 "C. Store the di-n-butyl ether under nitrogen in a cool dark place and check that it is peroxide-free immediately before use.Vitamin and antioxidant standards-Use pure crystalline samples of vitamin A and D with the following activities: vitamins D, and D,, 40 000 000 i.u. g-l; vitamin A (alcohol), 3 300 000 i.u. g-l; vitamin A acetate, 2 900 000 i.u. g l ; and vitamin A palmitate, 1 818 000 i.u. g-l. The BHA and BHT used should comply with the requirements of the British Pharmacopoeia 1968. The other compounds used as standards should be of the highest commercially available quality. Standard solution of vitamins and antioxidants-Dissolve 5 mg each of vitamin D,, vitamin A (alcohol), vitamin A acetate, BHA and ethoxyquin, and 10mg each of a-toco- pherol, BHT, vitamin A palmitate and a-tocopheryl acetate, in cyclohexane - triethylamine (9 + 1) and dilute the solution to 20 ml with the same solvent mixture.Store the solution in a refrigerator. Standard solutions of vitamin A acetate, vitamin A palmitate, vitamin D, BHA, BHT and ethoxyquin-Dissolve 18.75 mg of vitamin D, (= 750 000 i.u.), 41.2 mg of vitamin A palmitate (= 75 000 i.u. of vitamin A), 25.8 mg of vitamin A acetate (= 75 000 i.u. of vitamin A), 25 mg of BHA, 25 mg of ethoxyquin and 50 mg of BHT in 50 ml of cyclohexane - triethylamine (9 + l), and dilute 2, 3,4, 5 and 6 ml of the solution to 10 ml with the same solvent mixture. Store the solutions in a refrigerator. Standard solutions of u-tocopherol and u-tocopheryl acetate-Dissolve 25 mg of a-tocopherol and 25 mg of a-tocopheryl acetate in 25 ml of cyclohexane - triethylamine (9 + 1) and dilute 2, 3,4, 5 and 6 ml of the solution to 10 ml with the same solvent mixture. Store the solutions in a refrigerator.Iron(III) chloride - potassium hexacyanoferrate(III) reagent-Dissolve 1.3 g of iron(II1) chloride (FeCl,.GH,O) in 100 ml of 2 N hydrochloric acid. Dissolve 0.7 g of potassium hexacyanoferrate(II1) in 100 ml of water. Mix equal volumes of the freshly prepared solutions immediately before use. Iron(III) chloride - potassium hexacyanoferrate(III) reagent (strongly acidic)-Mix two volumes of freshly prepared iron(II1) chloride - potassium hexacyanoferrate(II1) reagent with one volume of concentrated hydrochloric acid immediately before use.April, 19731 259 Iron(II1) chloride - bipyridyl reagent-Dissolve 06 g of 2,2'-bipyridyl in 100 ml of ethanol. Dissolve 0-2g of iron(II1) chloride (FeC1,.6H20) in 100ml of ethanol.Mix equal volumes of the freshly prepared solutions immediately before use. Standard vitamin D, solution-Dissolve 5 mg of vitamin D, (= 200 000 i.u.) in 5 ml of chloroform immediately before use. Standard vitamin D, solution-Dissolve 5 mg of vitamin D, (3 200 000 i.u.) in 5 ml of chloroform immediately before use. Developing solvent-n-Hexane - ethyl methyl ketone - di-n-butyl ether (34 + 7 + 6). PROCEDURE FOR THE IDENTIFICATION AND ASSAY OF VITAMINS AND ANTIOXIDANTS (SEE Extraction of vitamins and antioxidants from formulations-For the examination of low- potency vitamin formulations, transfer an amount of sample equivalent to 30 000 i.u. of vitamin D (see Note 2) to a low-actinic glass separator and disperse it in 120 ml of water at a temperature of 70 "C.Cool the mixture to ambient temperature, add 20 ml of methanol and extract the vitamins and antioxidants with 100 ml of diethyl ether, by gently shaking the separator. Allow the mixture to stand until the aqueous and diethyl ether layers have separated, run off the aqueous phase into a second separator and decant the diethyl ether layer, leaving any emulsified material in the first separator. To the residual emulsified material, add 20 ml of methanol, mix and add 100 ml of diethyl ether. Add the mixture to the aqueous phase in the second separator and shake the mixture gently so as to extract the vitamins and antioxidants. Again allow the phases to separate, run off the aqueous layer and decant the diethyl ether phase, adding it to that obtained from the first extraction.Repeat the extraction procedure until no vitamins or antioxidants are detected when the extracts are evaporated to dryness and examined by thin-layer chromatography (three or four extractions should suffice). Wash the bulked diethyl ether extracts by gently shaking them with two successive 20-ml volumes of water. Discard the washings, filter the diethyl ether solution through a cotton-wool plug and evaporate it to dryness under vacuum in a rotary film evaporator with the water-bath at a temperature of 50 "C. Dry the residue by dissolving it in successive 10-ml volumes of acetone and re-evaporating the solutions. If the volume of the residue from the extraction is negligible, dissolve it in exactly 4 ml of cyclohexane - triethylamine (9 + 1).Otherwise, dissolve the residue in a small volume of cyclohexane and transfer the solution to a 10-ml measuring cylinder, washing out the container with further small volumes of cyclohexane. Evaporate the cyclohexane a t 50 "C in a stream of nitrogen, until the volume has been reduced to about 3 ml, then add 0.4 ml of triethylamine and dilute the mixture to 4 ml with cyclohexane. For the examination of vitamin beads and high-potency formulations, carry out a similar procedure, but reduce the volumes of water, diethyl ether and methanol used in the extraction to one quarter of those specified for low-potency vitamin formulations. For the examination of solutions of vitamins in oil, dilute the sample with cyclohexane - triethylamine (9 + 1) until the solution contains 7500 i.u.ml-l of vitamin D and apply it directly to a thin-layer plate. OF VITAMINS AND ANTIOXIDANTS I N PHARMACEUTICALS AND FEEDS NOTE 1)- NOTES- 1. The vitamins should be protected from light a t all times. Excessive heating and exposure to atmospheric oxidation should also be avoided. 2. The amount of sample to be taken for the assay can be calculated by reference to any of the vitamins or antioxidants, depending on the relative amounts of each present. For most of the samples examined, it has been found best to calculate the amount of sample by reference to the vitamin D content. Identijcation of the extracted vitamins and antioxidants-Line a chromatographic tank with filter-paper, pour the developing solvent into the tank, saturating the filter-paper lining, and allow the tank to equilibrate for 30 minutes.Apply 2 pl of the sample solution and 2 p1 of the standard solution of vitamins and antioxidants to each of two chromatographic plates and score the surfaces of the plates 15 cm from the spots. Develop the chromatograms in the dark until the solvent fronts reach the scored lines, remove the plates from the tank and allow them to stand in a stream of cold air until most of the solvent has evaporated. Without delay, spray one of the plates with the strongly acidic iron(II1) chloride - potassium hexacyanoferrate(II1) reagent and heat it in an oven at 40 "C for 15 minutes.260 JOHNSON AND VICKERS : THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION [Analyst, vol. 98 Inspect the second plate successively in ultraviolet radiation of wavelengths 254 and 360 nm; then spray the plate with iron(II1) chloride - bipyridyl reagent and allow it to stand in the dark until the spots reach maximum intensity.Identify the vitamins and antioxidants by comparing the sample and standard chromatograms. Semi-q.uantitative assay of vitamins and antioxidants-By diluting suitable volumes of the sample solution with cyclohexane - triethylamine (9 + l ) , prepare solutions containing 7500 i.u. ml-l of vitamin D, 750 i.u. ml-l of vitamin A as the acetate or palmitate, 0.5 mg ml-1 of BHT, tocopherol and tocopheryl acetate, and 0.25 mg ml-l of BHA and ethoxyquin. Apply 2 pl of the appropriate sample solutions and 2 pl of each of the standard solutions of vitamin A acetate, vitamin A palmitate, vitamin D, BHA, BHT and ethoxyquin to a chro- matographic plate and score the surface of the plate 15 cm from the line of spots.Develop the chromatogram, in the dark, in a tank that has been equilibrated with the developing solvent for 30 minutes. When the solvent front reaches the scored line, remove the plate from the tank and allow it to stand in a stream of cold air until most of the solvent has evaporated. Then, without delay, spray the plate with iron(II1) chloride - potassium hexacyanoferrate(II1) reagent and assay the vitamins and antioxidants in the sample by comparing the sizes and intensities of the sample spots with the standards, which correspond to 0.6, 0-9, 1.2, 1.5 and 1.8 i.u. of vitamin A as the acetate, 0.6,0.9, 1.2, 1.5 and 1.8 i.u.of vitamin A as the palmitate, 6,9, 12, 15 and 18 i.u. of vitamin D, 0.2,0.3,0.4,0.5 and 0.6 pug each of BHA and ethoxyquin, and 0.4, 0-6, 0.8, 1.0 and 1.2 pg of BHT. For the assay of tocopherol and tocopheryl acetate, apply 2 p1 of the appropriate sample solutions and 2 pl of each of the standard solutions of a-tocopherol and a-tocopheryl acetate to a second chromatographic plate and develop the chromatogram as previously described. If tocopherol alone is present, use iron(II1) chloride - potassium hexacyanoferrate( 111) as the spray reagent ; if tocopheryl acetate is present, use the strongly acidic iron(II1) chloride - potassium hexacyanoferrate(II1) reagent. Assay the tocopherol and tocopheryl acetate in the sample by comparing the sizes and intensities of the sample spots with the standards, which correspond to 0-4,0.6,0.8, 1.0 and 1.2 pg each of a-tocopherol and x-tocopheryl acetate.PROCEDURE FOR DIFFERENTIATING VITAMIN D, FROM VITAMIN D,- Extract the vitamins and antioxidants from the sample by the method previously described. If the sample contains no vitamin A, dissolve the residue from the extraction in sufficient chloroform to give a theoretical vitamin D content of 40 000 i.u. ml-l and transfer 0.1 ml of this solution and 0-1 ml of each of the standard vitamin D solutions to three test- tubes. Evaporate the solutions to dryness in a stream of nitrogen at room temperature and dissolve the residues in 0.1 ml of glacial acetic acid. To each tube, add 2 ml of 72 per cent. m/m perchloric acid solution (see Note 3), mix the solutions well and immediately heat the tubes in a water-bath at 70 "C, with constant agitation, until the colour of the vitamin D, standard reaches a maximum (heating for about 60 s will usually suffice).Cool the solu- tions rapidly, add 1 ml of chloroform to each tube and shake the tubes vigorously. The presence of vitamin D, is revealed by the red or purple colour of the chloroform layer; if vitamin D, alone is present, the chloroform layer is coloured greenish yellow. NOTE 3-Perchloric acid should be handled with care. It is a powcriul oxidising agent, which may If the sample contains vitamin A, dissolve the residue from the extraction in sufficient cyclohexane - triethylamine (9 + 1) to give a theoretical vitamin D content of 40 000 i.u.ml-l and apply 0.1 ml of this solution, in the form of a narrow band, to a chromatographic plate. At one end of the band, apply 5 pl of standard vitamin I), solution to act as a marker, and score the surface of the plate 15 cm from the band. Develop the chromatogram in the n-hexane - ethyl methyl ketone - di-n-butyl ether (34 + 7 + 6) developing solvent until the solvent front reaches the scored line, remove the plate from the tank and inspect it in ultra- violet radiation of wavelength 254nm. Score the surface of the plate around the edge of the vitamin D band and, without delay, scrape the enclosed area of silica gel from the plate and transfer it to a small chromatographic column containing 10 ml of chloroform. Elute the vitamin D with chloroform (about 60ml) and evaporate the eluate to dryness under vacuum in a rotary-film evaporator with the water-bath at a temperature of 50 "C.Dissolve react violently if allowed to come into contact with strong reducing agents.April, 19731 261 tlie residue in a small volume of chloroform and transfer it to a test-tube. Place 0.1 nil of standard vitamin D, solution and 0.1 ml of standard vitamin D, solution in two other test- tubes and continue by the method previously described, starting from “Evaporate the solutions to dryness in a stream of nitrogen at room temperature. . , .” EXTRACTION OF VITAMINS AND ANTIOXIDANTS- Attempts to extract the vitamins and antioxidants from solid formulations by shaking aqueous dispersions of the samples with water-immiscible solvents usually resulted in the formation of emulsions.A preliminary digestion of the samples with trypsin reduced the extent of emulsification caused by gelatin, but did not affect that produced by other con- stituents, such as wheat meal or maize meal. The most efficient extraction was obtained by using diethyl ether as the extracting solvent and adding small volumes of methanol during the extraction so as to break down emulsified material. Methanol was preferred to ethanol for this purpose as it caused smaller amounts of water and water-soluble impurities to be carried through with the diethyl ether to the final evaporation stage. The small amount of hydroquinone or propyl gallate present in anaestlietic ether B.P. to prevent peroxide formation does not interfere, either in the chromatographic separation (when it remains at the origin), or in the extraction procedure.It can be removed, if necessary, by passing the solvent through a column of activated alumina. OF VITAMINS AND ANTIOXIDANTS I N PHARMACEUTICALS AND FEEDS STABILITY OF VITAMINS A AND D DURING THE CHROMATOGRAPHIC EXAMINAIION- When solutions of vitamin A, vitamin A esters and vitamin D are applied to thin layers o f silica gel, rapid decomposition of the vitamins takes place, leading to a reduction in the size and intensity of the vitamin spots and to the appearance of spots corresponding to the decomposition products on the subsequent chromatogram. The addition of a small amount of triethylamine to the spotting solvent suppresses this decomposition and if the vitamins are applied in a cyclohexane - triethylamine (9 + 1) solution, they can remain in contact with the silica gel for up to 10 minutes before development, without any detectable decom- position taking place.The triethylamine remains at the origin during the chromatographic run and does not interfere in the separation. Although no evidence of decomposition of vitamins A and D during the chromatographic separation has been observed, decomposition can occur when the plates are dried, leading to a reduction in the size and intensity of the vitamin spots. This decomposition is inhibited by ethyl methyl ketone and di-n-butyl ether, and if the plates are allowed to remain moist with developing solvent and the chromatograms are examined as soon as possible after chromatography, no loss of vitamins or antioxidants can be detected.Bolliger and Konig’s quantitative assay procedure for vitamin D,3 in wliicli the vitamin is eluted from the silica gel adsorbent after chromatography and assayed colorimetrically by its reaction with antimony trichloride, has been carried out on solutions of pure vitamin L> with cyclohexane - triethylamine (9 + 1) as the spotting solvent and hexane - ethyl methyl ketone - di-n-butyl ether (34 + 7 + 6) as the developing solvent. In duplicate determina- tions, recoveries of 98.5 and 99-5 per cent. of vitamin D were obtained. I I)ENTIFICATION AND ASSAY OF VITAMINS AND ANTIOXIDANTS ON THE CHROMATOGRAM- As a general reagent for the detection of vitamins and antioxidants, tlie strongly acidic iron(II1) chloride - potassium hexacyanoferrate(II1) spray reagent is preferred.It reacts with all the compounds under investigation and it is one of the few reagents that will detect tocopheryl acetate without the need for a preliminary hydrolysis. For the assay of tlie vitamins and antioxidants (except tocopheryl acetate), the less acidic iron( 111) chloride - potassium hexacyanoferrate(II1) spray reagent is used, because it gives a lower level of back- ground colour on the chromatogram. Plates that have been sprayed with either of the two 1-cagents should be protected from excessive heat and light, which produce dark-coloured backgrounds. Although the various compounds can usually be identified by their R, values (see Table I), it may be necessary to confirm the identification by use of the more selective methods of detection and identification indicated in Table 11.The identification of vitamin A and its esters can be confirmed by the characteristic greenish yellow fluorescent spots observed when the chromatogram is viewed in ultraviolet radiation of wavelength 360 nm; under these262 JOHNSON AND VICKERS : THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION [AnU~ySt, VOl. 98 TABLE I Rp VALUES OF VITAMINS AND ANTIOXIDANTS Compound RF value Compound RF value Propyl, octyl and dodecyl gallates . . Hydroquinone . . .. . . 0.03 Vitamin A (alcohol) .. . . 0.17 0 Vitamin D, . . .. .. . . 0.22 Vitamin D, . . .. .. . . 0.22 Pre-vitamin D, . . .. . . 0.30 Pre-vitamin D, . . .. . . 0.30 BHA .. . . .. .. . . 0-32 Ethoxyquin . . ..a-Tocopherol .. Vitamin A acetate . . a-Tocopheryl acetate BHT . . .. .. Anhydrovitamin A . . Vitamin A palmitate 8-Carotene . . . . . . . . 0.42 . . . . 0.51 . . . . 0.58 .. . . 0.66 .. . . 0.75 . . . . 0.81 .. . . 0.85 ,. . . 0.88 conditions, ethoxyquin and anhydrovitamin A give bluish white and orange - brown spots, respectively. The antioxidants and tocopherol are distinguished by the red-coloured spots produced when the chromatogram is sprayed with the iron(II1) chloride - bipyridyl reagent. The D vitamins and their thermal isomerisation products, the pre-vitamins (Velluz, Amiard and Petits) are identified by spraying the chromatogram with trichloroacetic acid or antimony trichloride reagent and viewing it in ultraviolet radiation of wavelength 360 nm; all the compounds give greenish yellow fluorescent spots with the trichloroacetic acid reagent and pink fluorescent spots with the antimony trichloride reagent.If necessary, the selective methods of detection can be used for assay purposes. Because of the possibility of decomposition of the vitamins (particularly vitamin A) in ultraviolet radiation, chromatograms that have been examined by one of the ultraviolet procedures shcTi1.d not subsequently be examined by another method. TABLE I1 APPROXIMATE LIMITS OF DETECTION OF VITAMINS AND ANTIOXIDANTS* Method of detection used Vitamin A (alcohol) Vitamin D, . . Vitamin D, .. BHA .. . . Ethoxyquin . . Tocopherol . . Vitamin A acetate Tocopheryl acetate BHT .. .. Vitamin A palmitate A B C D E F G H' . . 0.05 - .. - . . - ... . 0.1 . . 0.05 - .. - . . - * . . . 0.1 0.1 0.06 0.1 0.05 0-1 0-05 - 0.1 0.25 0.25 0.2 0.25 0.2 - 0.2 0-25 0.2 - - __ 0.06 - 0.05 0.1 0.06 0.05 - 0.05 0.1 0.05 0.05 - 0.05 0.1 0.05 0.1 0-25 1.0 - 0.1 0.5 0.1 _- 0.05 0.2 0.4 0.1 - 0-1 0.26 0.05 0.2 0.2 0.2 0.2 - 0.2 0.5 0.2 1.0 - - - - - - - - - - A = Inspection in ultraviolet radiation of wavelength 360 nm. B = Inspection in ultraviolet radiation of wavelength 254 nm. C = Iron(II1) chloride - potassium hexacyanoferrate(II1) reagent. D = Iron(II1) chloride - potassium hexacyanoferrate(II1) reagent, strongly acidic. Heat a t 40 "C E = Iron(II1) chloride - bipyridyl reagent. I; = 25 per cent. solution of trichloroacetic acid in chloroform. Heat a t 120 "C for 10 minutes and inspect in ultraviolet radiation of wavelength 360 nm.G = 20 per cent. solution of antimony trichloride in chloroform. Heat a t 120 "C for 10 minutes. H = As for G, but inspect in ultraviolet radiation of wavelength 360 nm. * The figures show the lowest loading of the compound, expressed in micrograms, that can be detected on the chromatogram. Where no figure is given, the compound is not detected at a level of 2.0 pg. for 15 minutes. DIFFERENTIATION OF VITAMIN D, FROM VITAMIN D3- During attempts to distinguish between vitamins D, and D, by means of the coloured products obtained when vitamin D reacts with aromatic aldehydes in perchloric acid solution (SchalteggerO), the formation of a transient red colour on warming vitamin D, with per- chloric acid solution was observed. By choosing experimental conditions that favoured this reaction and stabilising the coloured product by extracting it into an organic solvent, a method of distinguishing vitamin D, from vitamin D, was developed.Tocopherol, tocopherylApril, 19731 263 acetate, BHA, BHT, hydroquinone, propyl gallate and ethoxyquin do not interfere, but vitamin A and its esters give an immediate blue - purple colour that rapidly fades to an intense golden yellow colour; any vitamin A (alcohol or ester) present must therefore be separated from the vitamin D by thin-layer chromatography before the colour test is applied. A thin-layer chromatographic separation of the vitamin D may also be necessary if the sample extract contains organic matter that chars badly on heating with perchloric acid. Pre-vitamin D, gives the same response as vitamin D,.Vitamin D, and pre-vitamin D, give greenish yellow colours. RESULTS AND DISCUSSION Recovery experiments were carried out by submitting a mixture of vitamins and anti- oxidants to the assay procedure, alone and in the presence of the appropriate amounts of gelatin, arachis oil and a mixture of gelatin, limestone, wheat meal, maize meal and B-group vitamins. A loading on the chromatographic plate of 15 i.u. of vitamin D, 1-6 i.u. of vitamin A as the acetate, 1-5 i.u. of vitamin A as the palmitate, 1 pg each of BHT, tocopherol and tocopheryl acetate, and 0.5 pg each of BHA and ethoxyquin was used and the results were assessed independently by four analysts. Mean recoveries for the individual compounds varied between 94 and 99 per cent.of those required by theory, depending on the compound and the type of formulation examined, with results from individual operators falling within &lo per cent. of the means. No difference in response between vitamin D, and vitamin D, could be detected and the same standard solutions (which contain vitamin D,) can be used for the assay of both compounds. The recommended procedures were applied to manufactured batches of several different vitamin preparations; the results obtained are shown in Tables I11 and IV. Except in one instance (the assay of vitamin D in a multivitamin syrup), no significant interference from other constituents of the formulations was encountered. In the samples examined, BHA and BHT were the antioxidants most generally used.Ethoxyquin was detected only in preparations destined for use in animal feeds; with minor exceptions, its use in foodstuffs is prohibited.1° Propyl, octyl and dodecyl gallates were not detected in any of the samples. In some vitamin A concentrates, BHT was detected at levels that were too low for it to have any significant antioxidant effect; in such samples, the BHT is probably derived from the vitamin A, which may contain small amounts intro- duced during the manufacturing process. Samples that had been stored for long periods were found to contain small amounts of unidentified impurities, apparently produced by decomposition of the vitamins and anti- oxidants. In samples containing vitamin A esters, compounds with R, values of 0.43 (from vitamin A acetate) and 0.92 (from vitamin A palmitate) were detected, A compound with an R, value of 0.65 was detected in samples containing ethoxyquin, and pre-vitamin D was found in some samples that contained vitamin D.Fortunately, the decomposition products either do not interfere in the assay, or the interference can be eliminated by the use of a selective method of detection. The impurities were shown to be decomposition products by dissolving the individual vitamins and antioxidants in hexane, exposing the solutions to ultraviolet radiation and examining the degradation products ; the same im- purities were detected in the artificially degraded vitamin A acetate, vitamin A palmitate, vitamin D and ethoxyquin as had been detected in the stored samples. The recommended assay procedure was applied to the determination of vitamin A in halibut-liver oil with synthetic vitamin A palmitate as the standard.Although the vitamin A in fish-liver oils is present as a mixture of long-chain fatty esters of the all-trans-, 13-cis-, 9-cis- and 9,13-di-cis-isomers of vitamin A,ll no separation of the isomers occurred and the single spot of vitamin A ester obtained from the halibut-liver oil was similar in appearance to and had the same Rp value as that obtained from the vitamin A palmitate. The quanti- tative results obtained by using iron(II1) chloride - potassium hexacyanoferrate(II1) reagent and ultraviolet radiation as the methods of detection, were comparable with those obtained by the official method of assay of the British Pharmacopoeia.12 Attempts to ascertain the vitamin A content of halibut-liver oil by hydrolysing the sample and determining the liberated vitamin A (alcohol) by thin-layer chromatography were unsuccessful, because two hydrolysis products, with RF values of 0.17 and 0.21, were obtained, which may correspond to mixtures of different isomeric forms of the vitamin. OF VITAMINS AND ANTIOXIDANTS IN PHARMACEUTICALS AND FEEDSTABLE I11 VITAMIN AND ANTIOXIDANT CONTENTS OF COMMERCIAL SAMPLES (SOLIDS) Sample* Gelatin-protected beads Vitamin A beads- Vitamin A, 325 000 i.u.g-l . . . . Vitamin -4, 500 000 i.u. 8-l . . .. Vitamin D, beads- Vitamin D, 850 000 i.u. g-l . . . . Vitamin D, 400 000 i.u. g-l . , . . Vitamin D, beads- Vitami+z E acetate beads- Tocopheryl acetate, 25 per cent. .. Vitamin A and D, beads- } } Vitamin A, 325 000 i.u. g-l Vitamin D, 108 000 i.u. g-l Vitamin A, 500 000 i.u. g-l Vitamin D, 125 000 i.u. g-I Animal feed additives Formula 1- 1 Vitamin A, 9640 i.u. g-1 Vitamin D, 2372 i.u. g-l Vitamin E, 0.43 per cent. with B- group vitamins and acetomenaph- thone in a wheat meal and limestone base J Formula 2- Vitamin A, 15 120 i.u. g-1 Vitamin D, 3770 i.u. g-' with B-group vitamins in a limestone base Vitamin D/ i.u. 6-1 1150 000 450 000 430 000 410 000 105 000 102 000 144 000 125 000 130 000 2600 2300 2600 4300 4400 Vitamin A Vitamin A Ethoxy- Tocopheryl ' (as ace- (as palmi- BHA, BHT, quin, Tocopherol, acetate, & tate)/i.u. 6-l tate)/i.u. g-1 per cent. per cent. per cent. per cent. per cent. tl 340 000 340 000 338 000 545 000 465 000 400 000 6400 330 000 340 000 630 000 580 000 320 000 9800 10 000 9900 15 700 15 700 17 000 13 000 200 000 450 450 450 680 730 0.65 1.6 0.38 0.32 9 9 0.4 0.75 0-65 1.3 Trace 6 1.4 9 0.6 3-2 0.5 0.25 0.25 0.25 0.2 1 0.35 0.35 0.37 6 9 c 0, W 00Sample* Vitamin D/ i.u.g-l Formula 3- Vitamin A, 9600 i.u. g-l Vitamin D, 2350 i.u. g-l with ribo- flavine in a maize meal base Formula 4- base Vitamin tablets 2300 } 2500 As for formula 3, but in a limestone Calciferol Tablets, Strong, B.P.- Vitamin D, 152 000 i.u. g-' . . . . 150 000 150 000 Calcium with Vitamin D Tablets, B.P.C.- Vitamin D, 1000 i.u. g-l . . . . 990 1050 1350 Vitamirz A , D and C pellets- Vitamin A, 11 100 i.u. g-' Vitamin D, 1460 i.u. g-' with vitamin } E: C in a sugar base illultivitarnin pellets- 570 Vitamin A, 7300 i.u.8-l Vitamin D, 590 i.u. g-' with B-group vitamins, vitamin C and trace metals in a sugar-coated chocolate base } E Syrup Multivitamin syrupt- 40 67 Vitamin A, 750 i.u. 8-l Vitamin D, 75 i.u. g-' with B-group vitamins and vitamin C in a syrup base TABLE I1 I-continued Vitamin -4 Vitamin A tate)/i.u. g-l tate)/i.u. g-' (as ace- (as palmi- BHA, per cent. 7200 2900 7400 2900 9400 9100 Trace Trace 10 900 Trace 11 600 Trace 11 100 Trace 7950 Trace 7350 Trace 7200 Trace 750 Trace Trace 690 Ethoxy- BHT, quin, per cent. per cent. Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace 0.12 0.13 0.09 0.10 Tocopher yl Tocopherol, acetate, per cent. per cent. * The figures quoted for vitamin concentrates are those declared by the manufacturer; the figures quoted for formulations are based on the declared vitamin content of the concentrate used in their preparation.Vitamin concentrates usually contain an excess of the vitamins to allow for decomposition on storage. t.Because of solubility difficulties, acetone - triethylamine (9 + 1) was used as the spotting solvent for this formulation. The vitamin D was assayed quantitatively by Bolliger and Konig's pr~cedure,~ but with acetone - triethylamine (9 + 1) as the spotting solvent and hexane - ethyl methyl ketone - di-n-butyl ether (15 + 5 + 3) as the developing solvent; the vitamin D could not be assayed by visual comparison with standards because of distortion of the vitamin D spot.TABLE IV Sample* VITAMIN AND ANTIOXIDANT CONTENTS OF COMMERCIAL SAMPLES (OILS) Vitamin A Vitamin A (as palmitate)/ (B.P. assay1*)/ Vitamin D/ BHA, BHT, Tocopherol, per cent.i.u. g-l i.u. g-' i.u. g-l per cent. per cent. Vitamin A palmitate solution- Vitamin A, 1 000 000 i.u. g-l . . .. 1 030 000 1 015 000 0.48 Trace 1 120 000 1 068 000 0.51 Trace 980 000 1 010 000 0.7 Vitamin A and D concentrate- Vitamin A, 600 000 i.u. g-l 540 000 498 000 57 000 0.32 Trace Vitamin D, 50 000 i.u. g-l } 525000 507 000 57 000 0.30 Trace Halibut-liver oilt . . .. . . I . 24 800 22 800 Not determined 0.17 29 700 29 500 Not determined 0.18 26 400 25 200 Not determined 0.20 22 500 22 400 Not determined 0.17 As for Table 111. t Vitamin D is present in this material a t too low a level to be assayed by the recommended procedure. The tocopherol was assayed by applying an increased loading (5 pl) of sample and standard solutions to the chromatographic plate and using iron(II1) chloride - bipyridyl reagent for detection. 0 n 36 Y 0 W 00April, 19731 OF VITAMINS AND ANTIOXIDANTS IN PHARMACEUTICALS AND FEEDS 267 The procedures described in this paper have been successfully applied to the analytical control of vitamins and antioxidants in vitamin concentrates and formulated products. Because of their speed and simplicity, they enable a comprehensive control of manufacturing procedures to be maintained at minimum cost. The authors are grateful to Messrs. E. E. Taylor and C. Knewstubb for samples, technical information and helpful comment. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Bolliger, H. K., in Stahl, E., Editor, “Thin-layer Chromatography,” Academic Press, London, Strohecker, R., and Henning, H. M., “Vitamin Assay,” Academic Press, London, 1965. Bolliger, H. R., and Konig, A., 2. analyt. Chem., 1965, 214, 1. Hanewald, K. H., Mulder, F. J., and Keuning, K. J., J . Pharm. Sci., 1968, 57, 1308. Ponchon, G., and Fellers, F. X., J . Chromut., 1968, 35, 63. Stecher, P. G., Editor, “The Merck Index,” Seventh Edition, Merck and Co. Inc., Rahway, New Morris, W. W., Wilkie, J. B., Jones, S. W., and Friedman, L., Analyt. Chem., 1962, 34, 381. Velluz, L., Amiard, G., and Petit, A., Bull. SOL Chim. Fr., 1949, 601; Chem. Abstr., 1960, 44, 2999. Shaltegger, H., Helv. Chim. Acta, 1946, 29, 286. “The Antioxidant in Food Regulations 1966,” S.I. 1966 No. 1600, H.M. Stationery Office, London. Chesterfield, N. J., Australus. J . Pkarm., 1969, 50 (600), S97. “The British Pharmacopoeia 1968, 1966, p. 210. Jersey, U.S.A., 1960, p. 1100. The Pharmaceutical Press, London, 1968, p. 1359. Received June 6th, 1972 Accepted November 7th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800257

出版商:RSC    

年代:1973

数据来源: RSC

摘要:

268 Analyst, April, 1973, Vol. 98, pj4. 268-273 Gas - Liquid Chromatographic Determination of Vitamin D in Cod-liver Oil BY J. G. BELL AND A. A. CHRISTIE (Department of Trade and Industry, Laboratory of the Government Chemist, Covnwall House, Stamford Street, London, SE1 9NQ) A gas - liquid chromatographic method for the determination of vitamin D (cholecalciferol) in cod-liver oil is described. It involves saponification of the oil, extraction of the unsaponifiable matter, removal of interferences such as cholesterol and retinol (vitamin A) by precipitation, and colunin chroniatography on Sephadex LH-20 and Florisil ; the final determination of cholecalciferol is carried out by gas - liquid chromatography. A determination can be completed in less than 2 days. EXISTING methods for the determination of vitamin D in foodstuffs and natural products are not entirely satisfactory.Biological assays with rats and chickens are time consuming, expensive and lack precision ; chemical methods are insufficiently sensitive and may be seriously affected by the presence of vitamin A and cholesterol, both of which frequently occur with vitamin D and often in considerable excess. The well known reaction between vitamin D and antimony trichloride suffers also from the disadvantages that the resultant coloured product is highly sensitive to trace amounts of moisture and its intensity varies with time. Of the physicochemical methods, ultraviolet spectroscopy can detect as little as 2 pg of calciferol by absorption measurement at wavelength 265 nm, but many other substances absorb in this region of the spectrum and effectively restrict the value of the technique to the determination of vitamin D in concentrates. Infrared spectrophotonietry has been used to distinguish ergocalciferol from cholecalciferol on the basis of the additional double bond present in the former, but for quantitative work the technique lacks sensitivity, 250 pg of the vitamin being the minimum amount required.In 1960, the application of gas - liquid chromatography to vitamin D determinations gave the first indication that the use of more sensitive methods was possible1 and, in 1966, Murray, Day and Kodicek2 made a major advance by converting ergocalciferol and cholecalciferol into their isovitamins by treatment with antimony trichloride reagent in order to obtain single separate peaks that could be distinguished from those of small amounts of cholesterol.About this time, a need arose in our laboratory for a chemical method of determining vitamin D in products such as cod-liver oil and pharmaceutical preparations. This paper describes the development of a gas - liquid chromatographic method for determining vitamin D in cod-liver oil by saponification and extraction of unsaponifiable matter; removal of vitamin A and other interferences in two stages by partition and adsorption chromatography; precipitation of cholesterol; formation of trimethylsilyl derivatives of the isovitamins ; and gas - liquid chromatography. Ergo- calciferol is used as an internal standard in the method as vitamin I) is present as cholecalci- ferol in cod-liver oil.EXPERIMENTAL RIJAGBNTS AND MATERIALS- All reagents should be of analytical-reagent quality unless otherwise stated. Digitonin B.P.-The material supplied by Koch-Light has been found to be suitable. Sephadcx LH-20-Supplied by Pharmacia, Uppsala, Sweden. Florisil, 60 to 100 mesh. .4 ntimony trichlovide solution, 20 per cent. m/V in chloroform. Tartaric acid solution, 40 per cent. m/V. Bis(trinzethylsi1yl)acetamide-Supplied by Phase Separations, Queensferry, Flintshire. Ergocalcijerol (vitamin D,), pure. Cholecalciferol (vitamin D3), pure. Su-Clzolestane solution-Dissolve 1 mg of pure 5u-cholestane in 100 ml of cliloroform. @ SAC; Crown Copyright Reserved.BELL AND CHRISTIE 269 APPARATUS- Glass columns for chromatography-These were 1-5 and 2 cm in diameter, and of minimum length 40 cm, each fitted with a sintered-glass disc of porosity 2, Quickfit socket and PTFE key.Gas chromatograph-Pye, Model 104, with flame-ionisation detector and 2.7 m x 3 mm i.d. glass columns packed with 3 per cent. OV-17 on Gas-Chrom Q, 100 to 120 mesh. Recorder-Honeywell Electronik 19, 1-mV input, fitted with a disc integrator. PREPARATION OF CHROMATOGRAPHIC COLUMNS- Scphadex LH-20 partition column-A 90 + 10 methanol - water mixture was used as the stationary phase and 2,2,4-trimethylpentane as the mobile phase. Saturate each solvent with the other by shaking equal volumes together for 1 minute and then store them separately. Allow about 100 g of Sephadex LH-20 to swell in 90 per cent. methanol for at least 24 hours before use. During this period decant the supernatant liquor at least twice, re-shaking with fresh 90 per cent.methanol each time. Fit an extension column to the top of a 40 x 2-cm glass column and add the Sephadex slurry to a height of twice that of the column required, q., to 70 cm for a final column height of 35 cm. Open the tap at the bottom of the column and allow the Sephadex to pack by settling. When the flow from the column drops to the rate of 1 ml mi+, close the tap, wash the sides of the extension column with 90 per cent. methanol and allow the Sephadex to settle by gravity until the supernatant liquid is com- pletely clear. After opening the tap, apply pressure to the top of the column extension by means of a blowball and gently compress the Sephadex until no noticeable further reduction in column height is observed.Adjustments to the height of the column can be made by adding or withdrawing slurry but at all times the Sephadex must be kept covered with liquid. Pass at least one bed volume of 2,2,4-trimethylpentane through the Sephadex, after which the column is ready for use. Remove the extension tube for sample addition and replace it for elution. Cool and weigh 2OOg into a clean, dry, screw-capped bottle, add 20ml of water and shake the mixture vigorously until it is thoroughly mixed and the powder is free flowing. Place the bottle on a roller mixer for 2 to 3 hours and allow the bottle to stand overnight. Meanwhile, prepare the eluting agent, a 1 + 4 mixture of diethyl ether and 2,2,4-trimethylpentaneJ add anhydrous sodium sulphate, shake the mixture and allow it to stand overnight in a stoppered flask. Fill a column, 40 x 1.5 cm, about half-full with 2,2,4-trimethylpentane.Weigh 30 g of the prepared Florisil into a 100-ml separating funnel that has been fitted with a cone and con- nected to the top of the column. Open the tap of the separating funnel and allow the Florisil to run smoothly into the solvent in the column, tapping the side of the column occasionally to ensure even packing of the Florisil and the release of any entrained air bubbles. As the packing proceeds it may be necessary to run off some of the 2,2,4-trimethylpentane from the column so as to prevent the liquid level from reaching the stem of the separating funnel. When all of the Florisil has been added, remove the separating funnel and add a 2-cm layer of anhydrous sodium sulphate to the top of the column.Lower the liquid level in the column until it just covers the solid material. The column is now ready for use. FZorisil column-Heat 210 to 220 g of Florisil for 3 hours at 160 "C. CALIBRATION OF COLUMNS- Determine the eluting characteristics of both the Sephadex and Florisil columns before use by transferring approximately 100 pg of vitamin D in the appropriate solvent to the top of each column and collecting fractions of the eluate. For the Sephadex column, collect 5-ml fractions after the appearance of the solvent front by using a flow-rate of approximately 30 to 40 ml h-1; for the Florisil column collect 20-ml fractions at a flow-rate of approximately 60 to 80 ml h-l.All the fractions are scanned on an ultraviolet spectrophotometer at 265 nm to locate the vitamin D peak and thus obtain the eluting position of vitamin D for each column. On Sephadex, vitamin D is normally found in the 20 to 50-ml fraction; on lilorisil, the 140 to 210-ml fraction usually contains the vitamin D. PROCEDURE Carry out all operations in artificial light.270 BELL AND CHRISTIE : GAS - LIQUID CHROMATOGRAPHIC [ANdySt, VOl. 98 SAPONIFICATION AND EXTRACTION- Weigh 20 g of potassium hydroxide pellets into a 250-ml twin-necked round-bottomed flask. Add about 10 ml of water followed by 140 ml of absolute ethanol, shake the mixture and warm it until the solid is completely dissolved. Prepare a solution of ergocalciferol in absolute ethanol, 1 ml of which is equivalent to 50 pg of the vitamin.Add 2 ml of this solution to the flask as an internal standard followed immediately by 5Og of cod-liver oil. Reflux the mixture for 15 minutes while bubbling a slow stream of nitrogen through the liquid, then add 25 ml of water and cool to room temperature as quickly as possible. Transfer the contents of the flask to a 1000-ml separating funnel, dilute to 600 ml with water and add immediately 400ml of a mixture of equal volumes of diethyl ether and light petroleum (boiling range 40 to 60 "C). Allow the phases to separate completely, approximately 10 minutes being required, and run the aqueous layer into a second 1000-ml separating funnel. Repeat the extraction with a further 200 ml of the solvent mixture, discarding the aqueous layer and adding the solvent layer to the first separat- ing funnel.Wash the combined solvent extracts twice by spraying with 100ml of water from a wash-bottle. Do not shake the separating funnel but when the phases have separated discard the aqueous layer. Add 250 ml of water to the separating funnel, shake it gently, allow the phases to separate and discard the washings. Repeat this procedure twice more, shaking the funnel vigorously on the last occasion. At this stage the layers should be clear and completely separated. If not, repeat with a further washing. Run the solvent layer into a large rotary evaporating flask, add 20 ml of absolute ethanol and proceed to remove the solvent under low pressure. At the first sign of cloudiness or precipitation add more absolute ethanol (25 to 50 ml) and evaporate to a volume of about 10 ml, then transfer the solution to a smaller flask and remove all of the solvent.Shake the mixture thoroughly for 2 minutes. PRECIPITATION OF STEROLS- Dissolve the residue in the flask with small amounts of warm methanol and transfer the liquid to a 10-ml graduated centrifuge tube. Cool it at 0 "C for 30 minutes, centrifuge it at 1000 to 2000 r.p.m. for 5 minutes and return the supernatant liquor to the evaporating flask. Remove the solvent, add a few millilitres of 2,2,4-trimethylpentane and evaporate again. Dissolve the residue in 2 ml of 2,2,4-trimethylpentane. SEPHADEX COLUMN CHROMATOGRAPHY- With a pipette, transfer the extract of vitamin D on to the prepared Sephadex column, rinsing the flask with 2 ml of 2,2,4-trimethylpentane, and elute with solvent at the rate of approximately 20 to 40 ml h-l, collecting the fraction that contains vitamin D.Add a few millilitres of ethanol and evaporate to dryness on a rotary evaporator. REMOVAL OF STEROLS AS DIGITONIDES- Dissolve the residue in methanol, transfer the solution to a 10-ml centrifuge tube and dilute it to 9 ml. Add 1 ml of water, shake the mixture vigorously, cool it to 0 "C and allow it to stand for at least 30 minutes. Centrifuge it and pour the supernatant liquor into a 30-ml centrifuge tube containing a digitonin solution prepared by dissolving 0.4 g of digitonin in 10 ml of 90 + 10 methanol - water mixture. Stopper the centrifuge tube, shake it and allow it to stand overnight in a refrigerator.Centrifuge and decant the supernatant liquor through a Whatman No. 541 filter-paper into a 50-ml separating funnel. Shake the precipitate of digitonides with 10 ml of the methanol - water mixture, centrifuge the mixture and use the supernatant liquor to wash the filter-paper. Extract the liquid in the separating funnel with two 15-ml portions of carbon tetrachloride, collecting the extract in an evaporating flask. Remove the solvent by rotary evaporation and repeat the evaporation after the addition of a few millilitres of 2,2,4-trimethylpentane. F FLORISIL COLUMN CHROMATOGRAPHY- the extract to the prepared Florisil column. 60 to 80 ml h-l and collect the fraction containing vitamin D. Dissolve the residue in 2 ml of 1 + 4 diethyl ether - 2,2,4-trimethylpentane and transfer Elute with the solvent mixture at a flow-rate ofApril, 19731 DETERMINATION OF VITAMIN D IN COD-LIVER OIL 271 PREPARATION OF THE ISOVITAMINS AND FORMATION OF THE TRIMETHYLSILYL ETHERS- Remove the solvent on a rotary evaporator and dissolve the residue in 1 ml of the 5a-cholestane solution.Add 4 ml of the antimony trichloride solution, shake the mixture and allow it to stand for exactly 1 minute, then add 6 ml of tartaric acid solution, shake the mixture vigorously and transfer it to a 25-ml separating funnel. Rinse the flask with 10 ml of light petroleum (boiling range 40 to 60 "C), add it to the separating funnel and shake the funnel for about 15 s. Discard the lower layer and wash the light petroleum layer three times with an equal volume of water.Filter the light petroleum through a Whatman No. 541 filter-paper into a small flask, wash the paper with a small volume of solvent and evaporate to dryness. Add 0.1 ml of bis(trimethylsily1)acetamide to the residue and allow it to stand for 10 to 15 minutes. The sample is now ready for injection on to the gas chromato- graph as described below. The operating conditions of the gas chromatograph were: column oven temperature, 235 "C ; detector oven temperature, 250 "C; injection block temperature, 300 "C; carrier gas, nitrogen at the rate of 50 ml min-l, hydrogen at the rate of 50 ml min-l and air at the rate of 500 ml min-I. The recorder chart speed was 2 min cm-l. CALIBRATION OF THE GAS CHROMATOGRAPH- Prepare a standard mixture containing 100 pg of ergocalciferol, 100 pg of cholecalciferol and 10 pg of 5a-cholestane, isomerise the vitamins and prepare teh trimethylsilyl ethers as previously described.Inject between 1 and 2 p1 of this solution on to the gas chromatograph and adjust the sensitivity of the instrument so that responses of the vitamin are between half and full-scale deflection on the recorder. Calculate the areas under the ergocalciferol and cholecalciferol peaks and the retention times of the vitamins relative to 5a-cholestane. The approximate retention times for these conditions are 21 minutes for 5a-cholestane and 46, 61 and 68 minutes for the trimethylsilyl ethers of cholesterol, cholecalciferol isovitamin and ergocalciferol isovitamin, respectively.Inject the sample in the same way, measure the areas under the ergocalciferol and cholecalciferol peaks and calculate the amount of cholecalciferol present from the known addition of ergocalciferol and the ratio of the areas obtained from equal masses of the two forms of the vitamin. CALCULATION- Cholecalciferol (vitamin D,)/pg 8-1 - Peak area of cholecalciferol x ergocalciferol added (pg) Peak area of ergocalciferol x R x mass of sample (g) where R is the ratio of the peak areas of cholecalciferol to ergocalciferol for equal masses of these substances. - RESULTS AND DISCUSSION Saponification losses of vitamin D are minimised by heating the oil with a fairly con- centrated ethanolic solution of potassium hydroxide for a short period and by bubbling nitro- gen gently through the liquid.Extraction of the unsaponifiable matter with a 1 + 1 mixture of diethyl ether and light petroleum gives a cleaner separation than with diethyl ether alone. Most procedures for the removal of interfering substances involve the use of one or more adsorption columns of alumina, magnesium oxide or silicic acid. Celite impregnated with poly(ethy1ene glycol)3 and, more recently, Fluoropak 804 have also been used. Other systems rely on the conversion of retinol (vitamin A) into a derivative that is more readily separated from vitamin D than the parent compound.s~s In the proposed method, vitamin D is well separated from retinol and some other interfering substances on a partition column of Sepha- dex LH-20.7 This technique has the advantage that such columns are easy to handle and can.be readily regenerated. Separation of vitamin D from cholesterol is, however, incomplete and the residual sterol is removed by precipitation with digitonin before a final treatment on a column of Florisil to remove anhydrovitamin A and other unknown interfering compounds. By this means cholesterol present in cod-liver oil can be reduced to manageable proportions but, as shown in the chromatogram of a typical cod-liver oil in Fig. 1, a small amount still remains.272 BELL AND CHRISTIE: GAS - LIQUID CHROMATOGRAPHIC [Analyst, Vol. 98 I Fig. 1. Gas - liquid chromatogram of chole- calciferol in cod-liver oil: peak 1, added 5or-chole- stane ; peak 2, cholesterol trimethylsilyl ether; peak 3, cholecalciferol isovitamin trimethylsilyl ether; and peak 4, added ergocalciferol isovit- amin trimethylsilyl ether In our early work, a sensitivity of 1 pg of vitamin D was obtained on the gas chromato- graph when using glass columns containing Chromosorb G coated with 2-5 per cent.silicone elastomer 30. Later, increased sensitivity and superior separations were obtained with column supports and stationary phases specially designed for steroid analysis, such as 3 per cent. OV-17 on 100 to 120-mesh Gas-Chrom Q. Under these conditions, less than 0.1 pg of vitamin D can easily be determined by using a flame-ionisation detector. TABLE I RESULTS OF GAS - LIQUID CHROMATOGRAPHIC DETERMINATION OF CHOLECALCIFEROL I N REFINED COD-LIVER OILS Sample designation A B C D E F G 13 I J Cholecalciferol/pgg-l 2.8, 2.5, 2-9, 2.7, 3.5, 3.3, 3.3, 3.9, 3.7, 3.4, 3.2 2.8 3.5 3.3 3.5 3-5 3.5 3.9 3.5 3-8 Tables I, I1 and I11 show that the proposed method gives repeatable results that are in reasonable agreement with those given by the standard bioassay method.The procedure, which can be completed in less than 2 days, is suitable for the quality control of the vitamin D content of cod-liver oil. TABLE I1 CHOLECALCIFEROL CONTENT OF SOME COD-LIVER OILS : COMPARISON OF RESULTS BY THE BIOLOGICAL RAT ASSAY AND GAS - LIQUID CHROMATOGRAPHIC METHODS Cholecalciferol content f A > Mean diiTerence, -7 per cent. 10.2 i!i } +6 10-2 410 Fiducial Bioassay Gas - liquid chromatography range/ 1 Type of oil i.u. g-1 t% 8-l i.u. g-l High potency 309 to 466 9.6 382 9.8 Low potency 40 to 68 1.3 52 1.2 49 -6 s’o” ) 0 Veterinary * 56 to 99 1.9 76 1.8 62 to 95 1.9 77 2.0 64 to 93 2.0 78 2.1 83 66 to 102 2.1 83 - - Medicinal 72 to 100 2.1 85 2.3 92 +s *Prepared by adding to the low-potency oil 0-75 pg (30 i.u.) of cholecalciferol per gram.April, 19731 DETERMINATION OF VITAMIN D I N COD-LIVER OIL TABLE IT1 CHOLECALCIFEROL CONTENT OF REFINED COD-LIVER OILS : COMPARISON OF RESULTS BY THE BIOLOGICAL RAT ASSAY AND GAS - LIQUID CHROMATOGRAPHIC METHODS 273 Code number 91684 92485 94787 94787 93687 00588 00788 00788 01889 01889 Fiducial range/ i.u.per fl. oz. 2229 to 3170 3232 to 4322 2377 to 5250 2377 to 5250 2377 to 5250 2212 to 4931 2212 to 4931 2212 to 4931 2207 to 3394 2207 to 3394 Cholecalciferol content A \ Bioassay Gas - liquid chromatography w pg g-1 i.u. per A. oz. 2.6 2733 2.9 3067 3.6 3758 3.0 3090 3.4 3548 3.4 3623 3-4 3548 3.4 3500 3.4 3548 3.6 3772 3.5 3626 3.5 3681 3.5 3626 3.6 3726 3.5 3626 3.3 3480 2.6 2769 3.1 3249 2.6 2769 2.9 2999 Difference, per cent. $- 12 - 18 -1 -1 +6 i - 2 +3 -4 + 17 +9 Our thanks are due to the Marfleet Refining Company for the provision of samples of cod-liver oil and for determining their vitamin D content by the bioassay procedure of the British Pharmacopoeia. This paper is published with the permission of the Government Chemist. REFERENCES 1. 2. 3. 4. 5. 6. 7. Ziffer, H., Vanden Heuvel, W. J. A., Hashti, E. 0. A., and Horning, E. C., J. Amer. Chem. SOG., Murray, T. K., Day, K. C., and Kodicek, E., Biochem. J., 1966, 98, 203. Theivagt, J. G., and Campbell, D. J., Analyt. Chew., 1959, 31, 1375. Chen, P. S., Terepka, A. R., and Remsen, N., Ibid., 1963, 35, 2030. Barua, R. K., and Rao, M. V. K., Analyst, 1964, 89, 534. Said, F., Salah, M. K.. and Girgis, P., Ibid., 1966, 91, 459. Bell, J . G., Chem. G. Ind., 1971, 201. 1960, 82, 6411. Received November 7th, 1972 Accepted December 7th, 1972

ISSN:0003-2654    

DOI:10.1039/AN9739800268

出版商:RSC    

年代:1973

数据来源: RSC