ISSN:0003-2654    

DOI:10.1039/AN97196FX013

出版商:RSC    

年代:1971

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97196BX015

出版商:RSC    

年代:1971

数据来源: RSC

摘要:

iv THE ANALYST [April, 1971THE ANALYSTEDITORIAL ADVISORY BOARDChairman: A. A. Smales, O.B.E. (Harwell)YT. Allen (Bradford)*L. S. Bark (Solford)R. Belcher (Birmingham)L. J. Bellamy, C.B.E. (Waltham Abbey)L. S. Birks (U.S.A.)E. Bishop (Exeter)A. 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.)W. Kemula (Poland)*G. F. Kirkbright (London)*G. W. C. Milner (Harwell)G. H. Morrison (U.S.A.)*G. Nickless (Bristol)S. A. Price (Tadworth)D. 1. Rees (London)E. B. Sandell (U.S.A.)W. Schoniger (Switzerland)H. E. Stagg (Manchester)E.Stahl (Germany)A. Walsh (Australia)*T. S. West (London)P. Zuman (U.S.A.)*Members of the Board serving on the Executive Committee.~NOTICE TO SUBSCRIBERS(Other than members of the Society)Subscriptions for The Analyst, Analytical Abstracts and Proceedings should besent through a subscription agent or direct to:The Chemical Society, Publications Sales OfFice,Blackhorse Road, Letchworth, Herts.(a) The Analyst, Analytical Abstracts, and Proceedings, with indexes . . ..index), and Proceedings . . .. .. .. .. .. ..index), and Proceedings . . .. .. .. .. .. ..(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 (withThe Analyst and Analytical Abstracts without Proceedings-(d) The Analyst and Analytical Abstracts, with indexes .. . . .. ..index) . . . . . . . . . . . . . . . . . . . .index) . . . . . . . . . . . . . . . . . . . .(e) The Analyst and Analytical Abstracts printed on one side of the paper (without(f) The Analyst and Analytical Abstracts printed on one side of the paper (withf 27-59 $66.00f 28-50 $69.00f34.75 $84.00€25.00 $60.00f 26.00 $63.00f32.25 $78.00(Subscriptions are NOT accepted for The Analyst and/or for Proceedings alone)Members should send their subscriptions to the Hon. TresureSUMMARIES OF PAPERS I N THIS ISSUE [April, 1971Summaries of Papers in this IssueDetermination of Eleven Metals in Small Samples of Blood bySequential Solvent Extraction and Atomic-absorptionSpectrophotometryA method is described for the determination of eleven metals in a 1-mlsolution of an oxidised blood sample. The metals, iron, copper, bismuth,zinc, cadmium, lead, cobalt, nickel, manganese, strontium and lithium areselectively extracted into small (0.30 to 0.50 ml) volumes of isobutyl methylketone as their chelates or ion-association complexes, and are determined inthe organic phases by atomic-absorption spectrophotometry .The enhance-ment effect of the organic solvent combined with the extraction and concen-tration of the metals results in average sensitivity increases of seven timesthat obtained by a direct determination on the aqueous solutions.The recovery of the metals added to blood is quantitative and, withtwo exceptions (lead and bismuth), a precision of better than 8 per cent.can be achieved at the 0.1 p.p.m.level.The results are given of an application of the method to a study of theproblem of metal ingestion by children who have pica.H. T. DELVES, G. SHEPHERD and P. VINTERDepartment of Chemical Pathology, The Hospital for Sick Children, Great OrmondStreet, and the Institute of Child Health, University of London, 30 Guilford Street,London, W.C.l.AnaZyst, 1971, 96, 260-273.Quantitative Determination of Taurine by ano-Phthalaldehyde - Urea ReactionTaurine is made to react with o-phthalaldehyde in the presence of ureaand phosphate ions, and on acidifying the mixture with acetic acid a purpleproduct is formed with an extinction maximum at 560 nm.A method basedon this reaction is described for the quantitative determination of taurineand it is applied to the determination of this amino-acid in rat brain afterpassage of the tissue extract through ion-exchange resins. The effect of otheramino-acids on the accuracy of the method is discussed.M. K. GAITONDE and R. A. SHORTMedical Research Council Neuropsychiatry Unit, Woodmansterne Road , Carshalton,Surrey.Analyst, 1971, 96, 274-280.A Spectrofluorimetric Method for the Determination of SmallAmounts of Sulphate IonThe enhancement of the fluorescence of the binary complex of zirconiumwith Calcein blue at pH 1-9 is used to determine sulphate ion in the range0.2 to 12 mg (2 to 12 000 p.p.m.). Excitation and fluorescence maxima occur a t350 and 410 nm, respectively.The fluorescence is stabilised immediatelyand remains unchanged for over 4 hours. Fluoride present in low con-centrations gives rise to high results and must be absent. Other anions thatform complexes with or precipitate zirconium, e.g., oxalate, phosphate,tartrate and tungstate, cause low results, but there is a high tolerance towardsmost cations except iron(II1) and cobalt(I1).LAY HAR TAN and T. S. WESTChemistry Department, Imperial College, London, S.W.7.Analyst, 1971, 96, 281-285xii THE ANALYST(. CARL@ ERBA[April, 1971Divisione Chimica lndustriale I Via C. lmbonati 24 / 20159 MilanoRS solvents forUV and IRsaectr OD hot ornetr VAcetone UV and IRAcetonitrile UV and IRBenzene UV and IRCarbon tetrachlorideUV and IRChloroform UV and IRCyclohexaneUV and IRN-N-DirnethylformamideUV and IRDichloroethane IRDimethylsulfoxide UVDioxane UVEthyl acetate IREthyl alcohol UVEthyl ether UVn-Heptane UVn-Hexane UVlsooctane UV and IRlsopropyl alcohol UVMethilene chlorideUV and IRMethyl alcohol UVn-Pentane UVPotassium bromide 1RTetrachloroethylene I RTetrahydrof uranUV and IRToluene IRTrichloroet hylene I

ISSN:0003-2654    

DOI:10.1039/AN97196FP057

出版商:RSC    

年代:1971

数据来源: RSC

摘要:

April, 19711 THE ANALYST xiiiFOR SALE~ ~~~Spectrographic System consisting of 3.4 metre Hilger Jaco EbertGrating Spectrograph Source Unit FS131, Arc and Spark Stand FS55,Comparator Microphbtometer L99, and a selection of accessories.Inspection and iurther information can be arranged by contactingThe Laboratory Secretary, C.E.G.B., Berkeley Nuclear Laboratories,Berkeley, Glos.LECTURES AND COURSESUNIVERSITY OF BRISTOLDEPARTMENT OF INORGANICCHEMISTRYM.SC. COURSE IN ANALYTICAL CHEMISTRY(WITH SPECIAL REFERENCE TOINSTRUMENTAL METHODS)OCTOBER 1971-1972Applications are invited from persons whohold or expect to be awarded, an appropriatehonours degree or equivalent qualification.The course offers a complete background tothe principals and practice of InstrumentalAnalytical Chemistry.The aim is to develop a research approach toAnalytical Chemistry and investigations ofRelevant Analytical Problems includingPollution Studies will form a major part of thecourse.Applications and enquiries should be sent tothe Registrar, The University, Senate House,Bristol, 2, as soon as possible.UNIVERSITY OFLONDONM.Sc. Course inAnalytical ChemistryApplications are invited from persons whohold or expect to hold an appropriatehonours degree or equivalent qualifications,e.g. G.R.I.C.The Science ResearchCouncil has given a ‘Priority’ rating to thecourse for tenure of its Advanced CourseStudentships. These will be allocatedjointly by the two Colleges.The course is given at Chelsea College andImperial College.Following a core ofcommon lectures, separate options aregiven at each College.registered at one of the two collegesaccording to the expression of their interestsand the availability of space.and enquiries should be addressed to theRegistrar at either College.There is specialisation in organic analysis atChelsea College and in trace analysis atImperial College.Students will beApplicationsChelsea College, Imperial College,Manresa Road, South Kensington,London S.W.3. London S.W.7.BINDINGHave your back numbersof The Analyst bound inthe standard binding case.Send the parts and theappropriate index(es) to-gether with a remittancefor f I *90to:W. Heffer & Sons Ltd.Cambridge EnglandIf you have a problem i n standardising ananalytical procedure, the use of a solidreagent i n tablet form may be your answer.We have been manufacturing“ANALOID”COMPRESSED REAGENTSfor over 50 years.Our experience i s at your service and weshall be pleased to consider making tabletsfor your specific requirements if thesecannot be met from our standard range.Send your enquiries to:-RIDSDALE & CO.LTD.Newham Hall, Newby,Middlesbrough, Teesside TS8 9EATelephone: 0642 372 I SUMMAKIES OF PAPERS IN THIS ISSUEPotentiometric Method for the Determination of AromaticMonothiosemicarbazonesA method has been developed for the quantitative determination ofaromatic monothiosemicarbazones, in which addition of silver nitrate to asolution of the organic compound leads to complex formation.The hydrogenion liberated on complex formation is determined by potentiometric titration.M. J. M. CAMPBELL, R. GRZESKOWIAK and I. D. M. TURNERDepartment of Chemistry, Thames Polytechnic, London, S.E. 18.[April, 1971Analyst, 1971, 96, 286-287.The Determination of Glycerol by the I.U.P.A.C. Form of theMalaprade MethodThe accepted periodate m-thod for the determination of glycerol hascome under review by the International Organisation for Standardisation(Sub-committee ISO/TC47/GT2) and certain modifications have been sug-gested with regard to the different pH end-points for the sample and blankand the possible loss of formic acid from the system by volatilisation. Thepresent paper summarises the view of the U.K.delegates to ISO/TC47/GT2,which are :(i) The choice of pH about 8.0 for the sample is justified on the groundthat it contains formic acid in addition to strong acids. The blank, whichcontains only strong acids, should, on general grounds and by calculation,be titrated to pH 7.0. The use of pH 6-5 instead of 7.0 for the blank is anempirical correction designed to compensate for some lack of stoicheiometryor other bias in the procedure and to bring the results into agreement withthose obtained by independent moisture and specific gravity determinations.This “correction” amounts to 0.03 per cent.(ii) There is a potential loss of formic acid by volatilisation from thesystem (estimated variously to be equivalent to 0.01 to 0.04 per cent.ofglycerol), which partly explains the apparent lack of stoicheiometry.(iii) Modifications have been proposed to minimise this loss of formicacid and (by adding formate ions to both solutions) to unify the end-pointsat or about pH 8.0. Unless these modifications lead to a pronounced improve-nieiit in reliability, it seems doubtful whether further extensive trials on aninternational basis would be justified.R. F. BARBOURSewcastle Technical Centre, Procter & Gamble Ltd., Newcastle upon Tyne.and J. DEVINEUnilever Research Laboratory, IJnilever Ltd., Port Sunlight, Cheshire.Analyst, 1971, 96, 288-295.The Separation and Determination of Pentachlorophenol inTreated Softwoods and Preservative SolutionsA method is described for the extraction, separation and determination ofpentachlorophenol or its sodium salt in softwoods and preservative solutions.Pentachlorophenol is separated from lower chlorophenols and wood extrac-tives by adsorption on to Bio-Rad AG 2-x8 anion-exchange resin, eluted withglacial acetic acid, extracted into chloroform, and determined by spectro-photometric measurement of the blue 4-aminophenazone - pentachlorophenolcomplex.The effect of time and temperature on the oxidation - condensationreaction of 4-aminophenazone with pentachlorophenol has been investigated.The procedure is particularly useful for the study of the distribution ofpentachlorophenol-containing preservatives in wood.A.I. WILLIAMSDepartment of the Environment, Forest Products Research Laboratory, PrincesRisborough, Bucks.AndJ’st, 1971, 96, 296-305xviii SUMMARIES OF PAPERS I N THIS ISSUE [April, 1971The Determination of Ethanol in Paints, Inks and Adhesives byGas ChromatographyA gas-chromatographic method for the determination of ethanol inpaints, inks, adhesives and similar composite products has been developedby which the time required for the analysis of samples has been greatlyreduced.The ethanol is distilled azeotropically from the sample in thepresence of toluene, and the distillate is examined directly by gas chromato-graphy without any further treatment other than mixing with an internalstandard. The problem of the longer retention time of toluene, comparedwith those of the lower aliphatic alcohols, has been overcome by the use ofback-flushing. From the results obtained by the analysis in duplicate ofa series of samples, including a number prepared in the laboratory containingknown amounts of ethanol, the accuracy and precision of the method havebeen established.J. R.HARRISDepartment of Trade and Industry, Laboratory of the Government Chemist, Corn-wall House, Stamford Street, London, S.E.l.Analyst, 1971, 96, 306-309.The Determination of Residues of Dichlorvos and Malathionin Wheat Grain by Gas - Liquid ChromatographyA procedure is described for the determination of malathion and di-chlorvos in grain. After extraction with methanol, and clean-up on a charcoalcolumn, if required, the pesticide residues are determined by gas - liquidchromatography on Apiezon L and butane-1,4-diol succinate columns witha phosphorus-sensitive detector.Between the concentrations of 0-25 and 10 p.p.m.both pesticides wererecovered from spiked samples with between 87 and 99 per cent. efficiency.S. CRISP and K. R. TARRANTDepartment of Trade and Industry, Laboratory of the Government Chemist, Corn-wall House, Stamford Street, London, S.E.l.Analyst, 1971, 96, 310-313.The Microbiological Assay of the Vitamin B, Complex(Pyridoxine, Pyridoxal and Pyridoxamine) with Kloeckera BrevisA complete re-investigation of the microbiological assay of the vitaminB, complex (pyridoxine, pyridoxal and pyridoxamine) with the yeast Kloec-ksra brevis has been made. The superiority of the proposed method over otherse.g., those involving Saccharowzyces carlsbergensis 4228 (ATCC 9028), theprotozoan, Tetrahywzena pyri forwzis and the X-ray mutant Neurosfiorasitophila 299, is discussed and suitable methods of extraction are described,particularly for blood, liver, human and rat brain, and urine.Kloeckera brevis has also been found to be a more suitable organismfor the cup-plate assay of the vitamin B, complex than S.carlsbergensis.The zones of exhibition are sharp and clear and background growth is reducedto a minimum, which is frequently not so with S. carlsbergensis.E. C. BARTON-WRIGHTGalloway and Barton-Wright, Haldane Place, London, S.W. 18.Analyst, 1971, 96, 314-318.Potential Hazards by Formation of Silver Acetylide upon AspiratingSolutions Containing High Concentrations of Silver to anAtomic- absorption Spectrophotometer when Acetylene is Used as FuelCommunicationR.J. REYNOLDS and D. S. LAGDENEvans Electroselenium Research Laboratories, Braintree, Essex.Analyst, 1971, 96, 319xx THE ANALYST [April, 1971Microfiche Editions of The Andyst and Andytical AbstructsAs announced on page 88 of the current (April) issue of Proceedings of the Society for Analyticalchemistry, the Society proposes to produce a microfiche edition of The Analyst, and possibly ofAnalytical Abstracts also.The Analyst for 1969 (volume 94) will be published on microfiche shortly, price f 15; $36(reference: a).A reduced price (f7.50; $18) will be charged t o customers who subscribed during 1969 t othe (spaper” edition of The- Analyst.The future programme of microfiche production depends on the response to this initial offer.Customers, o r potential customers, are therefore urged t o write t o the publishers stating if theyare interested in ordering-(6) The Analyst for 1970 (volume 95) on microfiche;(c) Analytical Abstracts for I969 (volumes I6 and 17) ;(d) Analytical Abstracts for I970 (volumes I 8 and 19) ;(e) Current numbers, as published during 1971, of both journals.The prices of items (6) (c) and (d) are expected t o be similar t o that fixed for item ( a ) ; theprice of a current subscription (item a) i s expected t o be €25 ($60) t o subscribers not taking thestpaper” edition; o r f 12-50 ($30) to subscribers taking the “paper” edition.......................................................................................................................................................................................ORDER FORMTo the Society for Analytical ChemistryBook Department9/10 Savile Row, London WIX IAF(a) Please supply I set of microfiche containing The Analyst for 1969, volume 94, at the full priceof € I5/$36* a t the special reduced price of f7.50/$18*Tot .........................................................................................................................................................................................* Delete parts that do not apply.i If the special reduced price is t o be paid, this address should be identical with that t o which the “paper”subscription was sent in 1969; if the customer has moved in the meantime, the former address should also be giveni n a note sent with this order.Please tickappropriate box(es) I will pay on receipt of invoice(b) I am interested in obtaining later volumes of The Analyst... . . . . . . . . . . .(c, d) I am interested in obtaining Analytical Abstracts on microfiche. . . . . . . . . . . 0(e) I am interested in a current subscription (from January 1971) t o both Analyst and( f ) I am considering purchasing a microfiche edition; please send a specimen fiche. . . . .Analytical Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . April, 19711 THE ANALYSTAn ‘impossible’ analytical problem?xxitwo minutes from nowyou could be on the way to solving itActivation analysis is a fast-developing technique particularlyhelpful in solving difficult problems of trace element analysis.It offers a unique combination of extreme sensitivity with unam-biguous identification of an impurity. Sample contamination andreagent ‘blank’ errors are avoided and the technique can often beused non-destructively. An Activation Analysis Unit has now beenestablished at Harwell in collaboration with the Analytical Re-search & Development Unit. If you would like further details, orwould like the opportunity to discuss ways in which we can helpto solve your particular problems, complete and post the couponor ring Abingdon 4141, Ext.3085.To :Activation Analysis Unit, Harwell, Didcot, Berks.I am interested in the services of the Activation Analysis Unit.I should like to :Receive further information by post c] Discuss my problem with youI am also interested in assistance with :c] IR spectrometry 0 mass spectrometry 17 NMR spectrometryc] computer applications c] on-line analysisother analytical techniques (tick as a~~romiate)Name ,AddressAA 1

ISSN:0003-2654    

DOI:10.1039/AN97196BP067

出版商:RSC    

年代:1971

数据来源: RSC

摘要:

APRIL, 1971 THE ANALYST Editorial Vol. 96, No. I141 The Impact of Instrumentation on the Analytical Laboratory THAT we live in a time of change is nowhere more evident than in the analytical laboratory. While this has probably been true for most of the 97 years our Society has been in existence, the development of highly specialist instrumentation to supplement, extend or replace “wet” analytical methods of analysis has given the past 30 years a special character. Broadly speaking, the 1940s and the 1950s saw the steady development of hardware incorporating the scientific discoveries of the previous 100 years and the 1960s saw the consolidation of such equipment in our analytical laboratories as well as the advent of high-cost items resulting from the development of better devices to meet the demands for better diagnostic procedures and for faster routine methods.Hence we now have, for example, infrared spectrophotometry, mass spectrometry and nuclear magnetic resonance spectrometry in conjunction, if necessary, with gas or thin-layer chromatography as an extremely powerful set of procedures in the organic field, while in the inorganic area we have been able to add the electron-probe analyser to an already impressive set of diagnostic and measurement techniques. Great benefits have accrued to the analyst, and thence to the world at large, as a result of these developments. Not only can he give a faster service to his customers, but often a better service in the sense that the information he supplies is better in quality and, not infrequently, he can supply information when hitherto such information would have been impossible to obtain.For example, the studies of many pollution problems have needed the development of suitable analytical methods before they could become meaningful. These benefits from developments in instrumentation have not been gained cheaply. They are affecting the cost of running our laboratories, the shape of the laboratories them- selves, and the staff we need to employ. So although the future shows every promise of further rich gains in new or improved analytical techniques, it is perhaps pertinent to consider the effects of these repercussions and any steps we may need to take to deal with them. The comments that follow have been generated from a necessarily limited environment and are offered more as a basis for discussion rather than any attempt to be dogmatic.The cost of analytical equipment probably began to be most sharply felt in the late 1950s with the coming of gas chromatographs at about El000 each. To use gas chromato- graphy was to realise its potential and one soon needed several instruments; it was not un- common, however, to find laboratories not able to afford such equipment. Infrared and ultraviolet spectrophotometers could be regarded a little differently because, usually, one of each would be enough and they would last for many years; new models of gas chromatographs appeared with the frequency of new cars. Other instruments began to appear with prices in the thousands rather than hundreds of pounds ; atomic-absorption spectrometers, polaro- graphs, the do-it-yourself infrared spectrophotometers and X-ray fluorescence spectrometers and, as we moved through the 1960s, E30 000 was becoming commonplace for mass spectro- meters, nuclear magnetic resonance equipment and electron-probe analysers.Now, looking ahead, we can visualise a proliferation of improved instruments of all kinds, many of which are becoming virtually automatic in operation (and consequently perhaps a little less versatile !) . Inevitably, most laboratories are producing more data and, inevitably, many are turning to computers for help with the difficulties thus created. It is also inevitable, as we seek to acquire better instrumentation for our laboratories, that management will require some assurance of getting a good return on the capital involved; 257258 EDITORIAL [Analyst, Vol.96 fortunately, if the equipment expenditure has been well conceived, there is little difficulty here. Sooner or later, however, the successful use of high-cost equipment is likely to stimulate more work than can be conveniently carried out in the normal working day, and requests for extra equipment are likely to be met with the observation that at 35 to 40 hours per week the existing equipment is hardly overworked. If the laboratory is concerned mainly with process control, a tradition of shift working probably exists already, so that not too much difficulty is experienced in operating the expensive equipment on a shift basis. However, if it is located in a research laboratory, then the possibility of shift working may not be one that commends itself to the staff for whom the normal working day is traditional.It should, of course, be added that not all equipment is amenable to continuous shift working, although up to 12 to 16 hours per day can probably be achieved with most. There is another vital and inevitably expensive factor to be considered: analytical equipment no longer lasts for ever. The wise laboratory manager must now reckon the life of most of his laboratory instruments as something like 5 years, regarding anything in excess of that as a bonus; he will also keep in mind that an elderly instrument that still produces results may perhaps be more expensive to operate and possibly provides less information than its more modern counterpart.As a result, his annual forecast of capital expenditure must now contain a realistic sum for replacing such equipment as this becomes necessary. We are, of course, always reluctant to throw away instruments, but this is clearly something we shall have to get used to. However, with the increasing cost of equipment, it may even- tually prove worthwhile to hire equipment rather than buy it outright; this is a relatively new possibility for analytical chemists, and it remains to be seen how far it becomes feasible. Yet another factor affecting our laboratory costs is maintenance. With the increasing complexity of our tools, we can no longer allow the local do-it-yourself enthusiast to handle any but the simplest of difficulties, and even in the larger laboratories the resident instrument engineer cannot be expected to cope with the many varieties of equipment now used. Hence, we are having to rely more and more on the expensive service engineer from the instrument manufacturer to keep much of our equipment in operation; of course, the quality of the service thus obtained will play a significant part in our original choice of such equipment.Many of our laboratories are still much the same shape as they were 20 years ago, except that the benches are now covered with gas chromatographs, atomic-absorption spectrometers, and so on. However, we usually site the larger items of equipment in empty rooms and design the laboratory facilities around them, but we are rarely fortunate enough to be able to plan for any future extension at the same time-and so the seeds of future frustration are sown.When the laboratories are located in the factory, we are finding that a significant part of our process control has been moved out of the laboratory on to the plant, either to be carried out by on-line equipment working continuously or by process operators with semi-automatic devices. Indeed, whenever possible, today’s plant designers will be seeking not only to have as much testing as possible on-line, but to have information from such equipment fed back to the control room to play its part in continuous plant control; the days of producing results for process control testing that are studied at leisure are surely numbered. Finally, the increase in instrumentation has had its effect on the inhabitants of our laboratories.In our quest for accuracy and speed at lowest cost, life can be interesting and often exciting for the research analyst. However, it is becoming more likely that with any particular problem he will now only need to prove the usefulness of a particular technique, leaving the design and construction of the hardware to the instrument manufacturer, who now undertakes much of the development work on the more commonly used techniques. When automatic equipment operates on-line on the plant, responsibility for its continuous operation, its maintenance and the provision of back-up services can involve the analyst in local politics and public-relations exercises for which his analytical training may not have equipped him, and he may need to tread warily.When the equipment sits in the laboratory, especially when routine process control is the main objective, life could become dreadfully dull for the technicians operating it unless some imagination is brought to bear on the arrangement of work schedules. As our laboratories become more highly rnechanised, so the staff employed make less and less use of wet-chemical methods and, unless we are very careful, the point might be reached when there is nobody about who can carry out such methods; as the older analysts retire, this is something that needs to be watched carefully. The increase in instru-April, 19711 EDITORIAL 259 mentation is undoubtedly causing changes in the activities of those working in our labora- tories and bringing, in turn, changes in the way they need to be trained o r re-trained, a subject that is being actively pursued by our new Education and Training Group. Analytical work has always been interesting but, such has been the impact of modern instrumentation, we are now able to do so much more than was possible in the past. There- fore the future looks bright for the analytical scientist, but it could be fraught with difficulty if he does not learn to use his new tools wisely. A. G. JONES

ISSN:0003-2654    

DOI:10.1039/AN9719600257

出版商:RSC    

年代:1971

数据来源: RSC

摘要:

260 Analyst, April, 1971, Vol. 96, $9. 260-273 Determination of Eleven Metals in Small Samples of Blood by Sequential Solvent Extraction and Atomic-absorption Spectrophotometry BY H. T. DELVES, G. SHEPHERD AND P. VINTER (Department of Chemical Pathology, The Hospital for Sick Children, Great Ormond Street, and the Institute of Child Health, University of London, 30 Guilford Street, London W.C.1) A method is described for the determination of eleven metals in a 1-ml solution of an oxidised blood sample. The metals iron, copper, bismuth, zinc, cadmium, lead, cobalt, nickel, manganese, strontium and lithium are selectively extracted into small (0-30 to 0.50 ml) volumes of isobutyl methyl ketone as their chelates or ion-association complexes, and are determined in the organic phases by atomic-absorption spectrophotometry.The enhance- ment effect of the organic solvent combined with the extraction and concen- tration of the metals results in average sensitivity increases of seven times that obtained by a direct determination on the aqueous solutions. The recovery of the metals added to blood is quantitative and, with two exceptions (lead and bismuth), a precision of better than 8 per cent. can be achieved at the 0.1 p.p.m. level. The results are given of an application of the method to a study of the problem of metal ingestion by children who have pica. TRACE-ELEMENT survey analysis of biological materials has shown that in addition to those metals known to be essential to man a further 20 metals are consistently present in human tissues.lS2 Many of these “non-essential” metals do not have any known or suspected bio- chemical function and are thought to be environmental contaminant^.^ The contamination of tissues with metals is a greater problem with children than with adults because normally developing children mouth and chew unfamiliar objects as a way of examining them.For some children the desire to chew such objects is uncontrollable, and these children are said to have pica. Such children have a higher incidence of lead poisoning,4 because of an increased oral ingestion of lead-containing materials, than children who do not have pica. In view of the wide range of metals used in the manufacture of materials, such as paints, plastics, rubbers and paper, likely to be chewed by children, it is possible that metals other than lead are excessively ingested by children.Multi-metal determination in blood samples taken from one group of children with pica and another group without pica would be of value in estab- lishing whether the excessive ingestion of metals, other than lead, is a problem in young children. The analytical methods that have been used successfully for multi-metal determination in biological materials are d.c. arc emission spectroscopy,l spark-source mass spectrometry5 and neutron-activation analysis.6 These techniques are, however, expen~ive,~ 9 6 difficult to apply to the routine analysis of a large number of ~arnples~.~ and require pre-concentration from large sample sizes1 Although simultaneous multi-element determination by atomic- absorption spectrophotometry is not yet feasible with commercially available instruments, the technique has good sensitivity for many elements and is easily adapted to routine analysis, and the basic equipment for single-element determinations is relatively cheap.This paper describes a method for the determination of eleven metals in a 1-ml sample solution of oxidised blood by sequential solvent extraction and atomic-absorption spectrophotometry. The metals determined were iron, copper, bismuth, zinc, cadmium, lead, cobalt, nickel, manganese, strontium and lithium. When investigating biochemical lesions in young children, it is wise to take the minimum amount of blood possible for the determinations required. Any method developed must therefore be capable of providing multi-metal determinations with as little as 1 to 2-ml samples for each of two duplicate determinations. 0 SAC and the authors.DELVES, SHEPHERD AND VINTER 261 With the exception of iron, zinc and copper, the normal physiological concentrations of the above metals are so low (0.005 to 0.05 p.p.m.) that a solvent-extraction and concentration stage is essential to obtain atomic-absorption signals that are accurately distinguishable from the background signals.A single extraction and 2-fold concentration from a 1-ml sample solution of oxidised blood would yield only 0.5 ml of solvent for the atomic-absorption deter- minations. Even by reducing the sample uptake rate of the nebuliser to one half of that recommended by the manufacturer (a Perkin-Elmer 303 instrument was used), this volume would be sufficient for only two determinations (Fig.1). The determination in duplicate of the above eleven metals by using a single “universal” extractant, if available, would therefore require more than 9ml of blood. However, a sequential separation of the metals would enable them to be determined by using 1 ml of sample solution and could therefore provide the basis of a method for multi-metal determination on the small samples of blood that can be taken from children. EXPERIMENTAL DEVELOPMENT OF A SEQUENTIAL EXTRACTION SCHEME- The solvents that can be used for atomic-absorption spectrophotometry with air-supported flames are limited to those which support the combustion processes of the flames. Fortunately, isobutyl methyl ketone, which has been shown by Allan’ to be an excellent solvent for flame atomic-absorption determinations, has also been proved useful for the extraction of metal chelates.8~~ This solvent was therefore used for each extraction stage in the separation scheme described. The extraction and concentration of metals from 1 ml of an aqueous sample solution into 0.5 ml of solvent yielded sufficient solution for one single atomic-absorption determination by using the sample uptake rate of the nebuliser recommended by the manufacturer, vDix., 5.6 ml minute-l of isobutyl methyl ketone. When this was reduced to 2.8 ml minute-l, the reduction in sensitivity was 14 per cent.relative, but determinations could be made with as little as 0.20ml of solvent. It was then possible to make determinations of two different elements with 0.5 ml of extract with little loss in sensitivity or precision (Fig.1). An extrac- tion scheme was therefore devised to separate the metals to be determined into groups of h 50- hl - 60- S 0 .- v) .- $, 70- 2 c-’ Q, m 80- Q) ? Q. A f l 9 0 1 #. B C D E F Fig. 1. Absorption signals from a continuous 20-s aspiration of a 0.2 pg ml-1 cadmium solution in isobutyl methyl ketone (A), and discrete aspira- tions (B to F) from 1.0 ml of the same solution until the entire sample was consumed; 1.0 ml of the solution was sufficient for four determinations (B to E) but not for the fifth deter- mination (F)262 DELVES, SHEPHERD AND VINTER : DETERMINATION OF [Analyst, Vol. 96 100 80 P E .w 2 60 al Ol (u 2 40 Y B 20 1 .o 2 .o 3 .O 0 Hydrochloric acid, N 0.2 0.4 0.6 0.8 1.0 DDDC in isobutyl methyl ketone, 1 per cent.w/v Hydrochloric acid, N PH PH :Fig. 2. The extraction of metals from aqueous solutions of oxidised blood samples: (a), cupferron; (b), diethylammonium diethyldithiocarbamate from 2 N hydrochloric acid ; (c), triisooctylamine ; (4, ammonium pyrrolidinedithiocarbamate ; (e) , 8-hydroxyquinoline ; and (f) , 2-thenoyl-3,3,3-tri- fluoroacetoneApril, 19711 ELEVEN METALS I N SMALL SAMPLES OF BLOOD Aqueous phase Organic phase Add 0.50 ml of 0.175 per cent. w/v DDDC in isobutyl methyl ketone and extract for 1 minute. Remove supernatant liquid 263 B Copper, loo% bismuth, loo% ; _3 (lead, 3y0) To 1.0 ml of sample solution (in 2.2 N HC1*) in a 7-ml polypropylene centrifuge tube, add 0.05 ml of freshly prepared 10 per cent. w/v aqueous cupferron solution.Mix and extract for 15 s with 0.50 ml of isobutyl methyl ketone. Remove and retain super- natant liquid, repeat twice with 0.5 ml of isobutyl methyl ketone for each extraction. Combine the three organic extracts I Extract with 0.50ml of 15 per cent. v/v T I 0 in isobutyl methyl ketone. Centrifuge and remove supernatant liquid I I A Iron, 100%; tin, 94y0;t --+ molybdenum, 94% ;t thallium, 100% ;t (copper. 2%) Add 0.3 ml of the 6 N ammonia solution - 5 per cent. w/v ammonium citrate reagent, 0.05 ml of bromophenol blue indicator solution and sufficient N ammonia solution to produce a blue colour without a red background (pH 2.8 to 3.2). Add 0.1 ml of 1 per cent. w/v of aqueous APDC solution, mix, add 0.50ml of isobutyl methyl ketone and extract for 1 minute.Remove supernatant liquid I D -+ + I I Add 0.1 ml of 6 N ammonia solution to bring pH to between 9.0 and 9.5. Extract for 1 minute with 0.5ml of 0.2 M HTTA in isobutyl methyl ketone. Centrifuge if necessary and remove super- natant liquid F __f C + Cadmium, 100%; zinc, 100%; lead, 84% Cobalt, 100% ; nickel, 100% Add 0.2 ml of 6 N ammonia solution to bring pH to between 8.5 and 9.0. Mix and extract for 1 minute with 0.30 ml of 0.1 M 8-hydroxyquinoline in isobutyl methyl ketone. Remove supernatant I liquid -1: Manganese, 100% Strontium, 100% ; lithium, 92% ; calcium, 100% ; t magnesium, 97 % t * The additions of aqueous cupferron solution reduce the concentration to 2 N hydrochloric acid.t Metals that are extracted but not determined in the final method. The precise adjustment of the pH of the aqueous phase for the APDC extraction (pH 2.8 to 3.2) enables the required pH values for the 8-hydroxyquinoline and HTTA extractions to be attained by adding the volumes of 6 N ammonia solution indicated. No further adjustment is necessary. HTTA undergoes hydrolysis into trifluoroacetate and acetylthiophene a t pH values greater than 8.0. The organic extract with this reagent must be removed as quickly as possible after extraction to avoid hydrolysis, which results in precipitation in both phases. This precipitate does not contain strontium or lithium but can cause trouble by blocking the nebuliser capillary if allowed to form or to remain in the organic phase.All the solutions of the organic reagents should be prepared immediately before use. Fig. 3. Procedure for the sequential separation of metals264 DELVES, SHEPHERD AND VINTER: DETERMINATION OF [Analyst, Vol. 96 two. The number of metals extracted in any one group could be increased to include iron or zinc, or both, as their high concentrations in blood (500 and 10 p.p.m., respectively) would enable determinations of them to be made with a dilution of a small, e.g., 50-p1, portion of the extract. Seventeen elements that are either essential or toxic to man, or are general environ- mental contaminants, were investigated and a scheme was developed for the quantitative extraction and separation of 16 of them. Five metals were not included in the final method: molybdenum, tin and thallium, because of their low sensitivity by atomic absorption; and calcium and magnesium, because they were not of interest in the present pica studies.The extraction efficiencies of these metals may be of interest to workers in the field of trace metals and biological systems and are shown in Figs. 2 and 3. The extraction systems investigated are shown in Fig. 2 (a to f). In each instance, the aqueous phase was prepared from a hydrochloric acid solution of the inorganic residues from a blood sample that had been wet oxidised with nitric, perchloric and sulphuric acids and evaporated to dryness. The volume of hydrochloric acid solution used to dissolve the inorganic residues was one-fifth of the original volume of blood. This five times concentrated solution was used to prepare the aqueous phases for the extraction studies, so that each aqueous phase contained all of the inorganic constituents of blood at their normal physio- logical concentrations.This took into account any effects that these constituents, e.g., phosphate ions, may have had on the extraction equilibria. Ammonium citrate solution (to give a final concentration of 1 per cent. w/v of ammonium citrate) was added to those aqueous phases the pH values of which were adjusted to greater than 1.0 for the extraction studies. This gave some buffering action and prevented the precipitation of metal hydroxides at high pH values. A 2: 1 ratio of the volumes of the aqueous to organic phases was used to ensure that a concentration stage was obtained.A single 60-s extraction was used for all systems except that with cupferron (Fig. 3 A) for which three successive 15-s extractions were necessary to remove completely the high concentration of iron present in blood. The metals to be investigated were added to the aqueous phases a t concentrations that were sufficiently high (2 to 10 pg ml-l) to permit their accurate determination, before and after extract ion, by at omic-absorpt ion spect ropho t ometry . The experimental conditions giving the optimum separation of the metals are indicated by the dotted lines intercepting the axis of abscissae in Fig. 2 (a tof). The procedure for the sequential separation of the metals prior to their determination is given in Fig. 3. Iron, which is present at high concentrations in blood (450 pg rnl-l), was separated in the first extraction stage (Fig.2 a) by extracting with cupferron and isobutyl methyl ketone- This extraction stage was used to remove iron from the aqueous solution of oxidised blood prior to investigating the other extraction systems (Fig. 2, b to f). This eliminated any interference from the high concentrations of iron in these solutions that could react with,. and thus remove, most of the added reagent or form chelates that are insoluble in isobutyl methyl ketone, such as the iron(II1) - ammonium pyrrolidinedithiocarbamate (APDC) chelate and iron(II1) 8-hydroxyquinolinate. Group A-Three successive extractions with isobutyl methyl ketone from an aqueous phase that was 2 N in hydrochloric acid and contained 0.05 per cent.w/v of cupferron gave a quantitative extraction of iron(II1) , molybdenum(VI), thallium(II1) and tin(I1) , with only 2 per cent. extraction of copper(I1). Group B-Quantitative extraction of copper and bismuth was easily achieved during preliminary tests with diethylammonium diethyldithiocarbamate (DDDC) in isobutyl methyl ketone, but it was not possible to separate bismuth from lead, or bismuth and lead from the other metals. This extraction system was therefore investigated by using a 6 x 4 factorial arrangement of six different reagent concentrations in isobutyl methyl ketone, and four different hydrochloric acid concentrations (0.5, 1.0, 2.0 and 4-0 N) in the aqueous phases. The extraction from 2 N hydrochloric acid solution into a 0.175 per cent.w/v solution of the reagent in isobutyl methyl ketone gave the best separation of copper(I1) and bismuth(III), both of which were completely extracted, from lead(II), 3 per cent. of which was extracted. Cadmium(I1) and all of the other metals studied were not extracted under these conditions. Group C-Triisooctylamine (TIO) fonns a quaternary salt in hydrochloric acid solutions, the chloride ion of which can undergo anion exchange with chloride ion-association complexesApril, 19711 ELEVEN METALS I N SMALL SAMPLES OF BLOOD 265 of metals to yield extractable species. Zinc(II), cadmium(I1) and lead(I1) were separated with this reagent from those metals which survived the first two extraction stages. Grou@ D-Ammonium pyrrolidinedithiocarbamate has poor selectivity but was used to separate quantitatively cobalt(I1) and nickel(I1) from manganese(I1) and the other metals remaining after the extractions for groups A, B and C.Groz@ E-Manganese( 11) was quantitatively extracted and separated from the alkali metals and the alkaline earths at pH 8-5 to 9.0 with 0.1 M 8-hydroxyquinoline in isobutyl methyl ketone. Grou@ F-Lithium(I), strontium(II), calcium(I1) and magnesium(I1) were all extracted with 0.2 M thenoyltrifluoroacetone (HTTA) in isobutyl methyl ketone (IBMK) at pH 9 to 9.5. The quantitative extraction of lithium( I) was unexpected but has been reported by HealylO Metal Fe c u Bi Cd Pb Zn co Ni Mn Sr Li 20 40 60 0 0 bservat ion heig ht/mm I I 3.0 4.0 CZH2 flow/l minute-' O 1 /PO 210 Fig. 4. Effect of (a) observation height and (b) acetylene flow-rate on flame absorbance for organic solutions of metals TABLE I EXPERIMENTAL CONDITIONS FOR ATOMIC ABSORPTION A/nm 302.0 324.7 223.1 228.8 283-3 213-9 240.7 232.0 280* 460.7 670.8 Slit Band Range width/mm pass/nm expansion 0.3 1.0 0.3 1.0 1.0 1.0 0.3 0.3 1.0 1.0 1.0 0.2 0.7 0.2 0.7 0.7 0.7 0.2 0.2 0.7 1.3 1.3 1 1 3 3 3 1 3 3 3 10 10 Observation height/mm 10.0 10.0 7-5 10.0 10.0 12.0 7.5 10.0 10.0 10.0 15.0 Acetylene flow/l minute-l 2-9 2.9 2.6 2-6 2-9 2.6 2.7 2.7 2-9 2.1 2.1 *Unresolved triplet 279.5, 279.8 and 280.1 nm.Sample uptake rate 2-8 ml minute-l of isobutyl methyl ketone. Air flow 22.8 1 minute-1, 30 p.s.i. ; acetylene pressure, 5 p.s.i. Noise suppression 2, i.e., 2-s time constant in amplifier output circuit.266 DELVES, SHEPHERD AND VINTER: DETERMINATION OF [Analyst, Vol. 96 to be the result of adduct formation, with isobutyl methyl ketone acting as a neutral donor ligand according to the reaction OPTIMUM FLAME CONDITIONS FOR THE ATOMIC-ABSORPTION DETERMINATION OF METALS IN The optimum observation height, i.e., the vertical distance between the optical axis of the monochromator and the top of the burner head, and the optimum fuel-to-oxidant flow ratio were determined for the eleven metals under investigation.An air - acetylene flame and a triple-slot (Boling) burner were used for the investigations. The acetylene and air flow meters of the Perkin-Elmer 303 instrument were calibrated in free litres per minute at 15 "C in accordance with the recommendations of Mansfield and Wine- fordnerll and Kirkbright and Sargent,12 who have criticised the publication of arbitrary and meaningless flow-rates of gases in atomic-absorption methods.The optimum flame conditions established from these observations and other instrumental data for the atomic-absorption determination of the metals are given in Table I. Strontium and lithium were the only metals that showed any marked dependence of free-atom concentration in the flame on observation height and acetylene-to-air ratio. Both metals form hydroxide and oxide species in air-supported flames and the reduction of these species depends on the atomic hydrogen concentration in the flame. The maximum absorption signals were obtained with observation heights just above the primary reaction zone, where the atomic hydrogen concentration is known to be greatest.l3 The response of the other metals (Fig.4, a and b) indicated varying degrees of oxidation in the higher regions of the flame and increased reduction to free metal atoms with fuel-rich flames. Li&) + HTTA,o,g) + BIBMK,, + LiTTA(IBMK),,,) + H&) ISOBUTYL METHYL KETONE SOLUTIONS- The results are given in Fig. 4 (a and b). METHOD APPARATUS- A Perkin-Elmer 303 atomic-absorption spectrophotometer fitted with a 10-cm long triple-slot air - acetylene burner head (Boling) was used. Hollow-cathode lamps were used as radiation sources for each of the metals determined. The absorption signals were recorded on a 10-mV chart recorder, Rikadenki B-24X. The instrumental settings for the atomic- absorption determinations are given in Table I.Silica conical $asks-25-ml capacity. Stoppered glass t.ubes-5-ml capacity, graduated in O-l-ml divisions. Glass tubes-10-ml capacity, 125 x 15 mm i.d. Polypropylene tubes-7-ml capacity, 100 x 100 mm id., and 3-ml capacity, 75 x 7 mm Pasteur pipettes. Glass syringes-2-ml capacity. Rotamixer-Made by Hook and Tucker. Sand-bath-Thermostatically controlled up to 300 "C. The concentrated acids and ammonia solution used were Aristar grade reagents. All Nitric acid, concentrated, sp.gr. 1.42. Perchloric acid, sp.gr. 1.54-This substance should be handled cautiously, and the wet Sulphuric acid, concentrated, sp.gr. 1-84. Dilute sulphuric acid, (1 + 4 v / v ) . Hydrochloric acid, concentrated, sp.gr. 1-18. Dilute hydrochloric acid solutions-Prepare 10 and 4.5 N hydrochloric acid solutions by diluting the concentrated acid with de-ionised water, and standardise by titration.Prepare a 1 + 1Ov/v dilute hydrochloric acid solution. Ammonia solution, concentrated, sp.gr. 0.88. Dilute ammonia solutions, 6 N and 1 N (approximately)-Prepare by diluting 42 and 7 ml, i.d. REAGENTS- the other reagents used were of analytical-reagent grade unless otherwise specified. ashing should be carried out in suitably designed fume cupboards. respectively, of the concentrated reagent solution to 100 ml with de-ionised water.April, 19711 ELEVEN METALS IN SMALL SAMPLES OF BLOOD 267 Phenol red indicator solution, 0.02 per cent. w/v. Bromophenol blue indicator solution, 0.04 per cent, w/v. Ammonia - ammonium citrate solution, 5 per cent.w/v ammonium citrate in 6 N ammonia solution-Dissolve 40 g of citric acid in about 200 ml of de-ionised water. Add 2 drops of phenol red indicator solution and sufficient concentrated ammonia solution (about 100 ml) to produce a red colour. Remove trace metals from this solution by extraction with a 0-1 per cent. w/v solution of dithizone in chloroform as previously described,14 add 5ml of con- centrated nitric acid solution and dilute to 500 ml with de-ionised water. Dilute 50 ml of this 10 per cent. w/v ammonium citrate solution to 100 ml with 42 ml of ammonia solution (sp.gr. 0.88) and de-ionised water. Isobutyl methyl ketone saturated with water. Cupferron, 10 per cent. w/v solution in de-ionised water-Dissolve 1.0 g of the reagent in 10 ml of water.Diethylammonium diethyldithiocarbamate, 0.176 per cent. w/v solution in is0 butyl methyl ketone-Dissolve 0-044 g of the reagent in 25 ml of isobutyl methyl ketone. Triisooctylamine, 15 per cent. v / v solution in isobutyl methyl ketofine-Dilute 4.5 ml of the reagent to 30 ml with isobutyl methyl ketone. Ammonium Pyrrolidinedithiocarbamate, 1.0 per cent. w/v aqueous solution-Dissolve 0.1 g of the reagent in 10 ml of de-ionised water. 8-Hydroxyquinoline, 0.1 M solution in isobutyl methyl ketone-Dissolve 0-36 g of the reagent in 25 ml of isobutyl methyl ketone. Standard solutions ofthe metals-Prepare from the pure metal, or a suitable salt, standard solutions of each metal, containing 5 mg ml-l for iron and 1 mg ml-l for the other metals, in 1 per cent.w/v hydrochloric acid. Mixed standard solution of metals-For each of the metals except iron, prepare dilute standard solutions containing 100 pg ml-l of metal in 1 per cent. v/v hydrochloric acid solution. Place the following volumes of the 100 pg ml-l standard solutions of the metals indicated into a 100-ml calibrated flask and dilute to volume with de-ionised water: lithium, 0.10 ml; cadmium, cobalt, nickel, manganese and strontium, 0.50 ml; lead and bismuth, 3.00 ml; copper, 5.00 ml; and zinc, 25-0 ml. Working standard solutions-To a series of six 10-ml calibrated flasks, add 0, 0.5, 1.0, 2.0, 3.0 and 4.0 ml of the mixed standard solution of metals. Add, in the same order, 0, 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the 5 mg ml-l standard iron solution. Add 2.2 ml of 10 N hydro- chloric acid solution to each flask and dilute to volume with de-ionised water.These solutions are 2-2 N with respect to hydrochloric acid and contain: Solution I A \ Metal 1 2 3 4 5 6 0 0.5 1.0 2.0 3.0 4.0 Cd, Co, Ni, Mn and Sr 0 2.5 5.0 10.0 15.0 20.0 BiandPb pgper100ml 0 15 30 60 90 120 c u 0 25 50 100 150 200 Zn 0 125 250 500 750 1000 0 100 200 300 400 500 Fe, pg ml-l Li 1 PROCEDURE- With a pipette introduce 2.0 ml of haemolysed whole blood into a 25-ml silica conical flask. Rinse down the sides of the flask with water (about 2 ml) and add 5 ml of concen- trated nitric acid, 1.0 ml of perchloric acid (sp.gr. 1.54) and 0-1 ml of the dilute sulphuric acid (1 + 4) solution. Mix and allow to stand for 15 minutes. Place the flask on a sand-bath (or hot-plate) at a temperature of 150 "C.Watch care- fully for any signs of vigorous reaction and frothing and remove the flask if the frothing becomes excessive. When the initial reaction has subsided, leave the flask on the sand-bath until a clear yellow - brown solution is obtained. Increase the temperature to 200 "C and evaporate to fumes of perchloric acid, then increase the temperature to about 300 "C and evaporate to dryness. For convenience this can be done overnight. Remove the flask from the sand-bath. When cool add 0.5 ml of the 4-5 N hydrochloric acid solution and return it to the sand-bath at a temperature of 150 "C. Evaporate the solution just to dryness and remove the flask from the sand-bath. When cool, the residue may become yellow as a result of residual hydrochloric acid vapour in the flask, but no268 DELVES, SHEPHERD AND VINTER : DETERMINATION OF [Analyst, Vol.96 Fe, pg rnl-' 0 TOO 200 3@0 400 500 I I 1 I I i Cu, Zn, pg per 100 ml 100 - 80 - w 07 r .- 2 60- Y a a 40 - 20 - Mn, Cd, Sr, Li, Bi, Co, Pb, Ni, 1-19 per 100 ml Fig. 5. Calibration graphs (lithium concentration was one-fifth of that shown; zinc concentration was 5 times that shown; and lead and bismuth concentrations were 6 times those shown) liquid should remain. Add 1.00 ml of 4.5 N hydrochloric acid solution to the flask and place it on a sand-bath at a temperature of about 150 "C for about 2 minutes. Remove the flask from the sand-bath and allow it to cool. A clear yellow solution should be obtained. Transfer it to a 5-ml graduated tube, rinsing the flask three times with 0-2 to 0.3 ml of water, dilute to 2-00 ml with de-ionised water and mix.With a pipette place a 1.00-ml aliquot of the solution in a 7-ml polypropylene centrifuge test-tube for the sequential extraction and separation of the metals by the procedure given in Fig. 3. Mix the phases by holding the tubes against the rubber pad of a vibrator - mixer. When the phases have separated, transfer the upper organic phase into a 3-ml polypropylene tube with a syringe-controlled Pasteur pipette. After each extraction stage, determine the concentrations of the metals in the organic phases by using the experimental conditions given in Table I. For the determination of iron and zinc dilute 0.05ml of the appropriate organic phase with 2.00ml of isobutyl methyl ketone in a 7-ml polypropylene tube.Determine all other metals directly in the organic phases. Determine duplicate reagent blanks with each batch of samples by using the method as described, except that water is substituted for haemolysed whole blood. Establish calibration graphs by transferring with a pipette 2.00 ml of the mixed working standard solutions into 10-ml glass tubes and carry out the separation scheme as described but with twice the given volumes of the reagents. This provides enough solution for the atomic- absorption determinations before and during a run of samples. To obtain the best results with the method described it is necessary to exercise a little more care than would normally be required for atomic-absorption determinations with larger volumes of solution.The nebulisation of 0.2 ml from a 0.5-ml fraction is judged on a time basis (about 4 s) and some practice is needed to enable a new operator to become skilled at making (at least) four determinations with 1 ml of solvent. Losses of isobutyl methyl ketone by volatilisation from the extraction tubes are negligible during the time required for analysis (about 0.7 per cent. loss after 30 minutes).April, 19711 ELEVEN METALS IN SMALL SAMPLES OF BLOOD 269 RESULTS Calibration graphs and the results of recovery tests are given in Fig. 5 and Table 11. Recovery tests were carried out for both synthetic aqueous standards and for solutions of metals added to blood samples. With the former, the solution of the inorganic residues from the oxidation stage was divided into two portions.A l-ml portion was subjected to TABLE I1 RECOVERY OF METALS ADDED TO BLOOD ANALYSED BY THE DESCRIBED PROCEDURE Concentration Mean per cent. recovered of metal added, A --, Number r pg per 100 ml Bi Cd Co Ni Mn Sr 5 104 79 100 94 112 88 10 118 98 96 89 113 103 20 120 108 104 91 111 109 30 107 102 98 89 106 104 40 120 101 107 90 106 105 50 108 91 98 89 103 98 20 40 60 80 100 120 Mean per cent. recovered r Pb Cu Znt Fei 110 73 95 105 90 98 101 105 97 104 97 110 105 108 101 101 110 106 99 103 108 102 100 103 * Li added at one-fifth of concentration shown. t Zn added at 10 times concentration shown. 1 Fe added at 500 times concentration shown. Li* of tests 88 4 105 4 110 4 101 5 103 3 103 5 the separation scheme and the metals determined in the organic extracts (Table 111); the remaining 4-ml portion was analysed directly by atomic-absorption spectrophotometry for the same metals. The correlations between the concentrations found by both techniques are given in Table IV.TABLE I11 RECOVERY OF SYNTHETIC STANDARD SOLUTIONS OF METALS AFTER OXIDATION Results are expressed as regression equations of the form : concentration found = (concentration added) m + c. Ideally m should approach 1-000 and c should approach zero AND EXTRACTION Constants in regression equation (pg per 100 ml) Concentration 7 range studied, f A Metal* m G pg per 100 ml c u 0.9 19 - 2.2 10 to 400 Bi 1-075 - 4.399 2-5 to 100 Cd 1.009 + 0.806 2.5 to 100 Zn 1.001 -0.218 25 to 1000 Pb 0.945 + 2.086 2.5 to 100 c o 0.992 +0-115 2.5 to 100 Ni 0,889 +3*17 2.5 to 100 Sr 0.996 -0.138 2.5 to 100 Li 1.037 +0-515 0.5 to 20 * Only one result was obtained for manganese because of mechanical loss Iron was not of sample: added 100 p g per 100 ml; found 102 pg per 100 ml. included in these studies.Results for precision are given in Table V. The detection limits given in Table VI are twice the standard deviation of the results of replicate blank determinations. Iron and zinc are not included in this table because they are determined on dilutions of the organic extracts.270 DELVES, SHEPHERD AND VINTER: DETERMINATION OF [Amalyst, Vol. 96 TABLE IV CORRELATION COEFFICIENTS FOR CONCENTRATIONS FOUND AFTER EXTRACTION AND Metal . . . . . . Cu Bi Cd Zn Pb Co Ni Sr Li Correlation coefficient.. 0.998 * 0.998 0.992 * 0.994 0.990 0.994 0.993 BY DIRECT ANALYSIS OF AQUEOUS SOLUTIONS * No results for direct determinations because of insensitivity of atomic-absorption spectrophotometry for these metals in aqueous solutions. The average increase in sensitivity resulting from solvent extraction and a 2-fold con- centratlon was 6.7 times (range 5.9 t o 7.1) that obtained by direct analysis of the corre- sponding aqueous solution. The increased sensitivity factor was 9.1 for manganese, which resulted from a %fold concentration stage. APPL I c ATI ON- The method described was applied to the analysis of small samples of blood taken from two groups of children. One group had a history of pica whereas the other group did not. For brevity they are referred to as the “pica group” and the “control group,” respectively.The results are given in Tables VII and VIII. Metal Fe * c u Bi Cd Pb Zn co Ni Mn Sr Li Metal TABLE V PRECISION OF THE METHOD Mean concentration found, Standard Sample pg per 100 ml deviation Blood . . . . . . . . . . 343-3 16.49 Blood . . . . . . . . .. 81 2.73 Blood plus 100 pg of Cu per 100 ml 7-42 Blood . . 2.1 1.63 Blood plus 60’;g of Bi per i60 ml ’ * 2.63 Blood . . . . . . . . . . 2-4 0.22 Blood plus 10 pg of Cd per 100 ml. . 0.22 Blood . . .. . . . . .. 10.0 3.1 Blood plus 60 pg of Pb per 100 ml. . 1.8 Blood . . 382 27.4 Blood plus 50b’pg of‘ i n pe; i 0 0 ml’ 821 52.0 Blood . . .. . . . . . . 0-6 1 0.28 Blood plus 10 pg of Co per 100 ml 0-6 1 Blood . . . . . . 10.0 0.78 Blood plus 10 pg of Ni per ib0 ml’ ’ 1.84 Blood .. . . . . . . . . 1.37 0-17 Blood plus 10 pg of Mn per 100 ml 0.60 Blood . . . . . . . . .. 4.7 0.37 Blood plus 10 pg of Sr per 100 ml . . 0.36 Blood . . . . . . . . 0.45 0.09 Blood plus 2.0 pg of Li per 100 ml’ ’ 0-15 181 66.0 12.3 71.4 10.1 19.8 11.6 15.6 2.06 * Fe concentrations are in micrograms per millilitre. Coefficient of variation, per cent. 4.8 3-3 4.1 4.0 9.2 1-8 2.5 7.2 6.3 6.0 7.8 9.3 12.4 5.2 7.9 2.3 7.3 78 31 46 20 TABLE VI LIMITS OF DETECTION . Cu Bi Cd Pb Co Ni Mn Detection limit, p g per 100 ml . . 0.58 3.2 0.74 4.0* 0.44 4.2 0.2 Number of tests 19 10 9 10 9 10 10 10 10 10 10 10 9 10 9 10 10 10 9 6 10 Sr Li 0.56 0*1* * The blank signals were not distinguishable from the flame background signals. These values were twice the standard deviation of the flame background signals.April, 19711 ELEVEN METALS I N SMALL SAMPLES OF BLOOD TABLE VII CONCENTRATIONS OF METALS FOUND IN BLOOD SAMPLES FROM THE CONTROL GROUP COMPARED WITH OTHER REPORTED VALUES Found in this study From literature r A ’I Mean,* Mean, Standard Number Metal p g per 100 ml pg per 100 ml deviation of tests Fe 475 x 102 381 x lo2 47.2 88 c u 107 97 21-8 82 Bi 1.2 0.9 1.7 44 Zn 650 509 140 83 Ccl 0.7 0-5 0-5 88 Pb 20 11 6.5 37 co 0.03 0.4 0.6 65 Ni 4.6 2.2 2-2 76 Mn 2.6 1.2 0.9 90 Sr 0.95 2-9 2.5 75 Li 2.0 0.3 0.4 70 * Data from B ~ w e n .~ 27 1 TABLE VIII RAISED CONCENTRA4TIONS* OF METALS FOUND I N BLOOD SAMPLES Pb Zn Mn Fe Sr Cd Cu Bi Li Ni Co Upper concentration Number of children limit, pgper 100ml 36 930 3.8 240 x 102t 10.5 1.9 160 6.1 1-6 8.7 2-3 with raised concen- trations of metals in : Control group ., 0 1 1 0 2 0 0 1 3 1 1 Picagroup . . 60 22 19 10 9 7 1 3 3 0 0 * Greater than the mean plus three times the standard deviation of the mean of the control group. t Abnormal concentrations of iron in whole blood will be lowered, e.g., in anaemia. This lower concentration limit is the mean less three times the standard deviation of the mean of the control group. Samples listed are those below 240 p g ml-l of iron. There were 194 children in the “pica” group. DISCUSSION No investigations were made into the effects of the hollow-cathode lamp current or slit width on the intensity of the absorption signals. The primary aim in these analyses was to obtain both a sufficiently intense and stable emission of radiation from the hollow-cathode lamp, which would permit a range expansion of up to ten times and at the same time keep the photomultiplier gain to a minimum.This enabled a small time constant to be used in the smoothing circuit of the recorder amplifier, which allowed determinations to be made satisfactorily with 0.20 to 0.30 ml of the organic extract (Fig. 1). The lamp currents and slit widths used fulfilled these requirements, and were usually those recommended by the manufacturer. No study of potential interferences of sample concomitants on the determinations was made because the extraction equilibria were established in the presence of the inorganic constituents of blood $Zus large numbers of added metal ions at concentrations up to about 1000 times those normally present in blood.Further, the high specificity of atomic-absorption spectrophotometry would be complemented by the selective solvent extraction of the metals to be determined. A check for spectral interference between metals extracted together was negative, as expected. The number of operations in the extraction scheme was kept to a minimum, so that the method could easily be applied to large numbers of samples. For example, the hydro- chloric acid concentration of the aqueous solutions of oxidised blood was fixed at 2.2 N, and this enabled the first three extractions to be carried out without any direct adjustments of the acidity of the aqueous phases. Only one accurate adjustment of the pH was necessary. By adjusting the pH of the aqueous phase for the fourth extraction stage to 3.0 0.2 the pH values of the aqueous phases for the subsequent stages were obtained simply by adding the stated volumes of 6 N ammonia solution.272 DELVES, SHEPHERD AND VINTER: DETERMINATION OF [Analyst, Vol.96 For routine application to large numbers of samples the extractions were carried out batchwise over a period of 3 days. In this way two people could in 1 week analyse fifteen samples in duplicate for each of the eleven metals. This amounted to about 440 deter- minations including reagent blanks and standards. The recovery tests for synthetic aqueous standard solutions of metals were quantitative (Table 111) and good correlations were observed between the concentrations found by direct analysis of these solutions and of the extracted organic phases (Table IV).The latter results showed that the extraction procedure, which was established for solutions of oxidised blood samples, was also quantitative for synthetic aqueous standards. The recoveries of metals added to whole blood (Table V) were quantitative for all of the metals determined. The coefficients of variation were better than +S per cent. relative at a concentration of 10 pg per 100 ml for nine of the metals (Table V). With bismuth and lead, however, the precision was poor at this low concentration, which approached the detection limits for these metals. At higher concentrations of about 60 pg per 100 ml the precision was better, being 4.0 per cent. for bismuth and 2.5 per cent. for lead.The precision for lithium was good, being 7-3 per cent. even at the low concentration of 2.00 pg per 100 ml (Le., 0.02 p.p.m.). The extraction - concentration stages resulted in increased atomic-absorption sensitivities of about seven times that obtained by direct analysis of aqueous solutions, and eliminated interferences from any non-specific absorption signals from the constituents of oxidised blood solutions, and from the effect of phosphate ions on strontium. The increase in detection limits was of the same magnitude as the increased sensitivity because the variations in the flame background signals were about the same when aqueous or organic solvents were burned. It would have been possible to improve on the detection limits obtained (Table VI) by using increased range expansion and noise suppression.However, this would have required larger volumes of solution and hence larger sample sizes to avoid dilution. The method described recommends that 2 ml of blood be taken for analysis but only 1 ml is used for the extraction - atomic absorption determinations. In our study of children with pica the remaining 1 ml was used for the determination of chromium, which is not reported here. The sensitivity of the method could be improved by using the whole of the 2 ml of oxidised blood solution for the determinations. On the other hand, smaller volumes of blood, Le., 1 ml or less, could be used for the preparation of the oxidised blood solution, if only a small sample is available. The results for the “control group” (Table VII) agreed with published results for normal persons where these were available.It must be pointed out that the children from this group were those undergoing surgery and were assumed to be biochemically normal. A study of their clinical histories is necessary to confirm this. The abnormally high concentra- tions given in Table VIII showed that, in addition to lead, the metals zinc, manganese, stron- tium, cadmium and lithium could be excessively ingested by children with pica. A detailed survey of all of the results is being carried out, and the clinical histories of the children con- cerned are being evaluated by medically qualified colleagues. CONCLUSION The method described provides the means of trace-metal survey analysis of large numbers of blood samples at a capital cost far below that of other available techniques such as spark- source mass spectrometry, neutron-activation analysis, etc. The sequential separation of metals from a l-ml portion of oxidised blood solution, as described in this paper, enables eleven metals to be determined in 2 ml or less of blood, which is of value when the samples are taken from a child who is young or sick, or both. We thank Professor B. E. Clayton, consultant chemical pathologist at the Hospital for Sick Children for her interest in this work, and The Wellcome Trust for financial support. REFERENCES 1. 2. 3. Tipton, I. H., in Seven, M. J., and Johnson, L. A., Editors, “Metal Binding in Medicine,” J. B. Butt, E. M., Nusbaum, R. E., Gilmour, T. C., Didio, S. L., and Sister Mariano, Arch. Emir. Hlth, Bowen, H. J. M., “Trace Elements in Biochemistry,” Academic Press, London and New York, Lipincott Co., Philadelphia, Montreal, 1960, p. 27. 1964, 8, 60. p. 159.April, 19711 ELEVEN METALS IN SMALL SAMPLES OF BLOOD 273 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Bicknell, J., Clayton, B. E., and Delves, H. T., J . Ment. Defic. Res., 1968, 12, 282. Evans, C. A., jun., and Morrison, G. H., Analyt. Chem., 1968, 40, 869. Samsahl, K., Brune, D., and Wester, P. O., Int. J . Appl. Radiat. Isotopes, 1965, 16, 273. Allan, J. E., Spectrochim. Acta, 1961, 17, 467. Starv, J., “The Solvent Extraction of Metal Chelates,” Pergamon Press, Oxford, 1964, p. 76. Sandell, E. B., “Colorimetric Determination of Traces of Metals,” Third Edition, Interscience Healy, T. V., J . Inorg. Nucl. Chem., 1968, 30, 1025. Mansfield, J. M., and Winefordner, J. D., Analytica Chim. Acta, 1968, 40, 357. Kirbbright, G. F., and Sargent, M., Analyst, 1968, 93, 552. Hermann, R., and Alkemade, C. Th. J., “Chemical Analysis by Flame Photometry,” Second Delves, H. T., and Vinter, P., J . Clin. Path., 1966, 19, 504. Received July 3rd, 1970 Accepted October 22nd, 1970 Publishers Inc., New York, 1959, p. 60. Edition, Interscience Publishers Inc., New York and London, 1963, p. 32.

ISSN:0003-2654    

DOI:10.1039/AN9719600260

出版商:RSC    

年代:1971

数据来源: RSC

摘要:

274 Analyst, April, 1971, Vol. 96, 99. 274-280 Quantitative Determination of Taurine by an o-Phthalaldehyde - Urea Reaction BY M. K. GAITONDE AND R. A. SHORT (Medical Research Council Neuropsychiatry Unit, Woodmansterne Road, Carshalton, Surrey) Taurine is made to react with o-phthalaldehyde in the presence of urea and phosphate ions, and on acidifying the mixture with acetic acid a purple product is formed with an extinction maximum at 560 nm. A method based on this reaction is described for the quantitative determination of taurine and it is applied to the determination of this amino-acid in rat brain after passage of the tissue extract through ion-exchange resins. The effect of other amino-acids on the accuracy of the method is discussed. AMINES react with o-phthalaldehydel s 2 to give coloured products and this reaction has also been used for their detection on paper chromatograms.Curzon and Giltrow3 used the reaction for identifying spots of taurine and other amino-acids and they also noted that different products resulted if the reaction was performed in the presence of urea. These observations have been made the basis of a specific quantitative method for the determination of taurine in tissue extracts. METHOD MATERIALS AND REAGENTS- Amino compounds were obtained from Koch-Light Laboratories Limited (Colnbrook, Buckinghamshire) or from California Corporation for Biochemical Research (Los Angeles, California). A sample of homotaurine (3-aminopropane-1-sulphonic acid) was kindly supplied by Dr. J. C. Watkins. o-Phthalaldehyde, urea, hydrochloric acid (sp.gr.1-18), acetic acid (99.6 per cent.), phosphoric acid (spgr. 1.75) and other reagents were products of B.D.H. (Chemicals) Limited, Poole, Dorset. Urea solution-A 50 per cent. urea solution was prepared by dissolving 50g of urea in water and diluting to 100 ml at room temperature (21 "C). o-Phthalaldehyde reagent-A saturated solution of o-phthalaldehyde (0-6 per cent.) was prepared by suspending 2 g of o-phthalaldehyde in 100 ml of water in a loosely stoppered conical flask and warming it in a boiling water bath for 2 to 3 minutes; the flask was then removed from the bath and stoppered tightly, and the contents were shaken vigorously. By repeated warming and mixing most of the residue in the flask dissolved in the water, although a small amount of yellow oily material remained adhered to the surface of the flask.The contents were then mixed on a mechanical mixer at room temperature for 1 to 2 hours and filtered through Whatman glass-fibre paper (GF/C). The clear filtrate was used directly or stored in a brown bottle for several weeks in the cold room a t about 8 to 10 "C until required. QUANTITATIVE DETERMINATION OF TAURINE WITH 0-PHTHALALDEHYDE- To 1-ml aqueous samples (containing 0.1 to 1.0pmole of taurine) in stoppered tubes, 1 ml of 0.02 M sodium phosphate buffer (pH 6.8) was added and the contents were mixed. The tubes were then placed in an ice-bath for 5 to 10 minutes, 1 ml of freshly prepared 50 per cent. urea solution was added consecutively to all tubes and the contents were mixed.The saturated (0.6 per cent. w/v) ice-cold solution of o-phthalaldehyde was then added in 1-ml amounts to all tubes. After mixing of the contents, the tubes were stoppered and placed in the ice-bath. After 5 minutes, 0.5 ml of 99.6 per cent. acetic acid was added, the contents were mixed immediately by repeatedly inverting the stoppered tubes, and the tubes were again placed in the ice-bath. The solution in the tubes was transferred to a 1-cm cell and the extinction at 560 nm measured against water until it reached a maximum value, which was then recorded. All optical measurements were made within 40 minutes of the addition of the acetic acid, 0 SAC and the authors.GAITONDE AND SHORT 275 and the time required for the extinction to reach the maximum value was between 2 and 3 minutes for those samples taken from the ice-bath 30 to 40 minutes after the addition of acetic acid.For rapid measurement of the highest extinction given by each sample, a limited number of tubes were removed from the ice-bath and left at room temperature, while the previous set of samples were investigated with the spectrophotometer to find their maximum extinctions. The amount of taurine in the sample was determined by reference to a calibration curve. The extinction at 560 nm was proportional to the amount of taurine in the range 0.2 to 1.0 pmole, and that at the isosbestic point at 490nm to the range of taurine from 0.05 to 0.7 pmole (Fig. 1). Taurine/pmole The relationship between the amount of taurine with o-phthalaldehyde when using 50 per cent.urea solution and 0.02 M phosphate buffer (pH 6.8), and the extinction a t 560 nm (-@-.-) and at the isosbestic point, 490 nm, (- 0- 0-) of the purple product formed after the addition of acetic acid Fig. 1. Concentration of o-p h t ha1 a1 de h yde, per cent. The effect of concentration of o-phthalaldehyde solution on the extinction a t 560 nm of the product formed on reaction with taurine (0.5 pmole) when using 0.02 M phosphate buffer (pH 6.8) and 50 per cent. urea solution Fig. 2. RESULTS FACTORS AFFECTING THE REACTION OF TAURINE WITH O-PHTHALALDEHYDE- Concentration of o-phthalaldehyde-The saturated 0.6 per cent. solution of o-phthalalde- hyde was diluted with water. The diluted reagent solutions gave lower extinction values than the saturated solution of the reagent when allowed to react with taurine in the presence of urea and phosphate (Fig.2). Concentration of urea-The typical purple product was formed from taurine after its reaction with o-phthalaldehyde only in the presence of urea. The extinction a t 560nm of the purple product formed on reaction of taurine (0.5 pmole) with o-phthalaldehyde in- creased with increasing concentration of urea, readings being 0.60, 0.63, 0-64, 0.65, 0-65 and 0.67 when using 10, 20, 25, 30, 40 and 50 per cent. urea solution, respectively. However, the stability of the purple product decreased with increasing concentration of urea solution (Fig. 3). The acid reagent for development of the coloztr-The intensity of the colour was greater on acidifying the reaction mixture with acetic acid than with hydrochloric, phosphoric or sulphuric acids.Temperature of incubation-The product formed on acidification of the reaction mixture was purple, provided that the temperature of the reaction mixture was between 0 and 39 "C. At higher temperatures the reaction product was yellow. The purple product was formed at pH values between 2.0 and 2.9.276 GAITONDE AND SHORT : QUANTITATIVE DETERMINATION OF [Analyst, Vol. 96 0.7 0.6 0.5 0.4 0.3 - 410 450 500 550 600 0 10 20 30 40 50 60 Wavelength/nrn Time after adding acetic acid/minutes Fig. 4. The optical spectra of the reac- tion products of taurine (1 pmole) with The effect of concentration of added o-phthalaldehyde when using 50 per cent. urea solution on the extinction a t 560 nm of the urea solution and 0.02 M phosphate buffer product formed on reaction of taurine (0.5 pmole) (pH 6.8) a t 0 "C.The spectra were recorded with o-phthalaldehyde in the presence of 0.06 M using a Unicam SPSOO spectrophotometer phosphate buffer (pH 6.6). A, 10 per cent. urea a t 0.5 minute (A) and a t 8-5 minutes (B) solution and B, 50 per cent. urea solution after the addition of acetic acid Fig. 3. Samples pre-incubated at 0 "C and subsequently acidified gave an orange product , with an extinction peak at 470 nm, which gradually changed into a purple product with maximum extinction at 560 nm (Fig. 4). The development of the purple product was almost complete in 40 to 50 minutes at 0 "C and could be accelerated on warming to between 16 and 21 "C (Fig. 5). The purple colour was stable for 2 to 3 minutes on reaching its maximum extinction at 560 nm (see Fig.3) and was stable for 10 minutes at the isosbestic point between 490 and 500 nm. Samples pre-incubated at higher temperatures (21 to 36°C) gave the purple product with maximum extinction at 560 nm immediately on acidification, its extinction increasing during the first 2 minutes and then showing a slow decrease. 0 20 40 60 80 100 120 140 Time/minutes Fig. 5. Rate of development of purple colour after reaction of taurine (1 pmole) with o-phthalaldehyde when using 0-06 M sodium phosphate (pH 6.4) and 50 per cent. urea solution. Curve A gives the extinction a t 560 nm recorded directly after withdrawal of the reaction mixture from the ice-bath a t different times after the addition of acetic acid.Curve B gives the extinction at 560 nm of the same samples on allowing thc development of the purple colour up to its maximum value in the spectrophotometer .- t; 0.2 *G 0.1 U O 0.02 0.04 0.06 0.08 0.10 0.20 0-30 Molarity of sodium phosphate (pH 6.6) Fig. 6. The effect of phosphate ions (pH 6-6) on the extinction a t 560 nm of the product formed on reaction of taurine (0.5 pmole) with o-phthalaldehyde when using 50 per cent. urea solution (A), and 25 per cent. urea solution (R)April, 19711 TAURINE BY AN O-PHTHALALDEHYDE - UREA REACTION TABLE I EXTINCTION OF THE PRODUCTS OF AMINO COMPOUNDS ON TREATMENT WITH O-PHTHALALDEHYDE Amino compound Taurine . . . . . . Homotaurine .. .. H ypotaurine .. .. Glycine . . . . . . Lysine . . . . ..6-Hydroxylysine . . . . y-Aminobutyric acid . . /3-Alanine . . . . . . Ethanolamine . . .. Ornithine . . . . . . Cysteamine . . . . . . Cystamine . . .. .. Tyramine . . . . . . 3-Hydroxytyramine (dopamine) Phenvlethvlamine . . Tryptamiie . . . . 5-Hydroxytryptamine 5-H ydroxytryptophan Histamine . . . . N-E-Methyllysine . . a-Alanine . . .. Arginine . . .. Aspartic acid . . Asparagine . . . . Citrulline . . .. Cysteine hydrochloride S-Methylcysteine , . Penicillamine .. Cysteic acid .. Cysteine sulphinic acid Cystine . . . . Cystine disulphoxide Homocysteic acid . . Glutathione, GSH . . ,, GSSG . . L-Cystathionine . . Methionine . . . . Methionine sulphoxide Methionine sulphone Glutamic acid . . Glutamine . . . . Histidine . . . . l-Methylhistidine . . 3-Methylhistidine .. Proline . . . . Serine .. .. Homoserine . . . . Threonine . . . . Leucine . . . . Isoleucine . . . . Valine . . .. Diaminopimelic acid Phenylalanine . . Tyrosine . . . . Ammonium chloride Ergothioneine . . Glucosamine .. Tryptophan . . .. Homocystine .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. .. .. .. . . .. . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. . . . . .. .. .. Amax 560 560 500 560 560 560 560 560 560 560 560 560 520 to 570 530 to 630 510 to 560 480 to 495 480 450 475 560 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Extinction a t 560 nm - a t o oc 1.24 0.75 0.32 0-97 1-05 1.00 0.32 0-58 0.40 0.26 0.27 0.46 0.27 0.57 0.41 0.17 0.33 0.07 0.15 0.12 0.08 0.05 0.06 0-04 0.05 0.05 0.04 0.09 0.05 0.04 0.02 0.04 0.11 0.04 0.04 0.10 0.11 0-04 0-05 0.04 0.04 0-04 0.02 0.02 0.02 0.05 0.06 0.04 0.04 0.03 0.06 0.06 0.04 0.04 0.03 0.02 0.02 - - a t 36 "C 1-13 0.66 0.23 0.22 0.72 0.28 0.12 0-24 0-14 0.30 0.65 0.69 0.25 0.34 0.16 0.11 0-26 0-16 0-28 0.2 1 0-33 0.17 0.1 1 0-27 0.14 0.09 0-30 0.34 0.27 0.27 0-29 0.20 0.36 0-17 0-20 0.2 1 0.3 1 0.12 - - - - - - - - - 0.12 1.19 0.13 - - - - 0-26 0.34 0-05 0.02 - Each compound (1 pmole) was treated with 0-6 per cent.of o-phthalaldehyde solution while using 50 per cent. urea solution and 0.06 M sodium phosphate solution (pf-I.6.4) a t 0 "C for 5 minutes or a t 36 "C for 30 minutes.The reaction mixture was acidified with acetic acid and the maximum extinction value was recorded. 277278 GAITONDE AND SHORT : QUANTITATIVE DETERMINATION OF [Arcalyst, Vol. 96 Molarity and $H of the phosphate bu$er--The presence of small amounts of phosphate ions was necessary for the formation of the purple product but phosphate buffer solutions of higher molarity (greater than 0-1 M phosphate) gave a considerable decrease in the extinction of the purple solution (Fig. 6). The extinction was highest if the reaction mixture was incubated at a pH between 6.5 and 7.2 (Fig. 7 ) . A reagent of 0.02 M phosphate buffer (pH 698) was chosen for use in the quantitative determination of taurine. 0.7 0.6 PH Fig. 7. The effect of 0.02 M and 0.06 M sodium phosphate buffers the extinction a t 560 nm of the product formed on reaction of taurine (Oi5 .pmole) with o-phthalaldehyae when using 50 per cent.urea solution. A, 0 . 0 2 ~ sodium phosphate buffer and B, 0.06~ sodium phosphate buffer The presertce of chloride iouts-Samples of taurine solutions up to 0.05 M in sodium chloride However, higher concentrations of the did not show any appreciable interference effect. chloride ions gave a decrease in the extinction of the purple product. REACTION OF AMINO COMPOUNDS WITH 0-PHTHALALDEHYDE IN THE PRESENCE OF UREA- Several amino compounds were treated with o-phthalaldehyde and 50 per cent. urea solution at 0 or 36 "C. After 5 minutes, the reaction mixture was acidified with acetic acid, and the extinction maximum of the product and its extinction at 560nm were recorded (Table I).The first twelve amino compounds, except hypotaurine, listed in Table I gave products with extinction maxima a t 560 nm. Hypotaurine gave an extinction maximum at 500 nm, with an extinction of 1.26 under the conditions described in Table I. The products of other amino compounds had extinction maxima below 490 nm or had a wide absorption band between 510 and 630nm. For the quantitative determination of taurine, the reaction with o-phthalaldehyde a t 0 "C was preferred to that at 36 "C because (i) under the conditions used several a-aminocar- boxylic acids gave a yellow product ( E ~ ~ ~ = 2 250) at 36 "C but no coloured product at 0" C. The extinction of the products at 560 nm was less when these a-aminocarboxylic acids were reacted at 0 "C than at 36 "C; (ii) the calibration curve was a straight line; and (iii) the procedure is applicable under certain conditions to the determination of lysine, hydroxylysine and glycine (e.g., in protein hydrolysates). Lysine gave a brownish-black product (A,,, 450) and glycine a yellowish green product (A,,, 450) on treatment with o-phthalaldehyde.EXTRACTION OF THE REACTION PRODUCTS WITH CHLOROFORM- After optical measurement the reaction mixture was equilibrated with 4 ml of ch1oroform.l The purple products formed from glycine, cysteamine and cystamine were found to be extractable with chloroform, in which the products of the two sulphur-containing amino-acids were stable for 16 to 20 hours. This property can be utilised in the detection of these acids in the presence of other sulphur-containing amino-acids.The reaction products of tryptamine and phenylethylamine were extractable into the chloroform phase but those given by 5-hydroxytryptamine, tyramine and 3-hydroxytyramine remained predominantly in the aqueous phase.April, 19711 TAURINE BY AN 0-PHTHALALDEHYDE - UREA REACTION 279 MECHANISM OF REACTION OF TAURINE WITH 0-PHTHALALDEHYDE IN THE PRESENCE OF UREA- Taurine also gave rise to a purple product if urea solution in the reaction mixture was replaced by thiourea solution (10 per cent. w/v), acetamide solution (50 per cent. w/v) or glutamine solution (5 mM), but not if urea solution was replaced by acetone or ethyl methyl ketone. It was found that under conditions in which a mixture of urea and phosphate was pre- incubated for 5 minutes with taurine and o-phthalaldehyde, or with taurine and subsequently o-phthalaldehyde, or with o-phthalaldehyde and subsequently taurine, the reaction product formed on the addition of acetic acid was yellow - orange, changing with time into a purple product.The purple product was not formed if phosphate was omitted from the incubation mixture, or if taurine was omitted from the incubation mixture but added to the acidified reaction mixture. The following mechanism, which is consistent with the experimental findings, is proposed : (i) urea + phosphate + o-phthalaldehyde -+ [A] (ii) [A] + taurine--+ [B] (iii) [B] + acid -+ [C] -+ [D] -+ [El orange purple yellow It may be assumed that urea, thiourea and glutamine form addition products [A] similar to those reported for a~etamide.~ 0:;: + NHzCONH;, - Q - F H N-CO-NH2 0”H ‘H 0-p ht ha lalde h yd e urea N-arnido-1,3 -dihydroxyisoindoline The compound [A] is then converted into the purple product according to the mechanism postulated in reactions (ii) and (iii) above.Phosphate probably acts as a catalyst in the formation and stabilisation of the reaction product [A]. In view of the fact that glutamine can replace urea in the reaction it appears that only one amid0 group of urea is reacting with o-phthalaldehyde. In general, several amino compounds of the type NH2-CH2-R, where R is -COOH or an aliphatic carbon chain (e.g., taurine, homotaurine, lysine, glycine, cystamine, cysteamine, y-aminobutyric acid, p-alanine, ornithine, ethanolamine or butylamine) , gave purple products on treatment with o-phthalaldehyde at 0 “C; hyptotaurine gave a pink product.An intense purple product was also given by isopropylamine. a-Aminocarboxylic acids, except those mentioned above, gave no purple products on acidification of their reaction products with o-phthalaldehyde. In experiments in which urea was not present during the incubation but was added at the end of 5 minutes of incubation and then allowed to react for 1 to 5 minutes, no purple product resulted on subsequent acidification. This observation, which showed that taurine also reacts with o-phthalaldehyde in the absence of urea, was also found to be dependent on the presence of phosphate ions in the incubation mixture.The reaction mechanism may be similar to the one postulated above for urea. The addition compound so formed is probably responsible for fluorescent products reported for several amines (and a-aminocarboxylic acids) by earlier workers5 to lo APPLICATION OF THE METHOD TO TISSUE EXTRACTS The method can be applied directly to the determination of taurine (and homotaurine) in the in vitro assay of enzymes involved in metabolism with methionine and other sulphur- containing amino-acids. Taurine is present in large amounts in rat brain and certain other tissues. Its concentration in tissue extracts was determined by the present method after removal of y-aminobutyric acid, glycine and lysine, which interfere to some extent, depending on their concentration in the tissue extract.Rat brain was homogenised in ice-cold 5 per cent. w/v perchloric acid or 10 per cent. w/v trichloroacetic acid. The suspension was centrifuged and the clear extract decanted, after which the tissue residue was washed once with the same acid. The washing was combined with the main extract, which was then neutralised to precipitate perchlorate ions or washed three times with ether to remove tri-280 GAITONDE AND SHORT chloroacetic acid. The aqueous extract was filtered into a calibrated cylinder and made up to a suitable volume (e.g., 25ml). A fraction containing taurine that was free from inter- fering amino-acids was obtained from the extract by one of the following two procedures. In the first, a portion of the aqueous extract (10 to 20 m1) was passed through a two- column assembly consisting of Zeo-Karb 225 (Hf form, 10 cm) fitted on top of Dowex 1 X-10 (carbonate form, 12 cm).After the passage of the sample, the column assembly was washed with 50ml of water. The Dowex 1 column was disconnected from the assembly; taurine adsorbed on this resin was eluted with 0.1 N acetic acid under pressure of nitrogen gas until all the carbonate was exchanged with acetate and free acid emerged from the column. The neutral eluate was evaporated to dryness and the residue dissolved in 5 ml of water. In the alternative procedure a portion of the aqueous tissue extract (10 to 20ml) was passed through a two-column assembly consisting of Zeo-Karb 225 (H+ form, 10 cm) fitted on top of Dowex 1 X-10 (acetate form, 12 cm).The effluent emerging from the column assembly contained taurine and neutral compounds, which were collected quantitatively by washing the column assembly with water (50 ml) . The effluent (@us washings) was evaporated to dryness and the residue dissolved in 5 ml of water. Because chloride ions inhibit the o-phthalaldehyde - urea reaction, the substitution of Dowex 1 in the acetate form with a Dowex 1 column in the chloride form should be avoided. If it nevertheless occurs, the chloride ions (washed out as hydrochloric acid) should be removed by repeated evaporation of the effluent fraction. Homotaurine, when present in the tissue extracts, was found in the taurine fraction on ion-exchange chromatography. The taurine fraction obtained by these two procedures con- tained phosphoethanolamine and, probably, glycerylphosphorylethanolamine.They were resolved on paper chromatograms developed with acetone - ethyl methyl ketone - water (2 + 2 + l), which gave relative RF values, with respect to that of taurine, of 0.32 for phospho- ethanolamine and 0-55 for glycerylphosphorylethanolamine. Moreover, the fraction obtained by the second procedure also contained free sugars. None of these compounds interfered in the determination of taurine in the tissue extracts. A mean value of 6.76 +_ 0.09 (s.e.m.) prnole of taurine per gram of brain was obtained for Wistar albino rats of 100-g body weight. From the same tissue extracts taurine was isolated by combined ion-exchange and paper Chromatography. Taurine was eluted from paper chromatograms and the eluate, after removal of interfering volatile materialll and reaction with ninhydrin,12 gave a mean value of 6.88 0.16 (s.e.m.) pmole of taurine per gram of brain.The present value may be compared with a value of 6.94 pmole per gram of brain for rats of 73-g body weight.13 An inspection of the values reported for taurine content of brains of rats of different body weights shows a wide scatter (this has been reviewedl4). This suggests that, besides age, other factors such as strain and nutritional state of the animal may also affect the taurine content of the brain. As all the interfering amino compounds are adsorbed on the cation-exchange resin (Zeo-Karb 225, H+ form), the use of a second column of anion-exchange resin is not necessary in enzyme assay systems. The effluent emerging from the cation-exchange resin is either assayed for taurine directly, or after concentration of the material by evaporation. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. REFERENCES Klein, G., and Linser, H., Hoppe-Seyler’s 2. physiol. Chem., 1932, 205, 251. Patton, A. R., J . Biol. Chem., 1935, 108, 267. Curzon, G., and Giltrow, J., Nature, 1954, 173, 314. Reynolds, R. D., and Conboy, R. J., J . Org. Chem., 1965, 30, 2251. Shore, P. A., Burkhalter, A., and Cohn, V. H., jun., J . Pharmac. Exp. Ther., 1959, 127, 182. Juhlin, L., and Shelley, W. B., J . Histochem. Cytochem., 1966, 14, 525. Cohn, V. H., jun., and Shore, P. A., Analyt. Biochem., 1961, 2, 237. Kremzner, L. T., Ibid., 1966, 15, 270. Rogers, C. J., Chambers, C. W., and Clarke, N. A., Ibid., 1967, 20, 321. Sen, N. P., Somers, E., and O’Brien, R. C., Ibid., 1968, 26, 457. Gaitonde, M. K., Dahl, D. R., and Elliott, K. A. C., Biochem. J., 1965, 94, 345. Sorbo, B., Clinica Chim. Acta, 1961, 6, 87. Garvin, J. E., Archs. Biochem. Biophys., 1960, 91, 219. Gaitonde, M. K., in Lajtha, A., Editor, “Handbook of Neurochemistry,” Volume 111, Plenum Received August 3rd, 1970 Accepted September 30th, 1970 Press, New York, 1970, p. 225.

ISSN:0003-2654    

DOI:10.1039/AN9719600274

出版商:RSC    

年代:1971

数据来源: RSC

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Analyst, April, 1971, Vol. 96, pp. 281-285 28 1 A Spectrofluorimetric Method for the Determination of Small Amounts of Sulphate Ion BY LAY HAR TAN AND T. S. WEST (Chemistry Department, Imperial College, London, S. W.7) The enhancement of the fluorescence of the binary complex of zirconium with Calcein blue a t pH 1.9 is used to determine sulphate ion in the range 0.2 to 12 mg (2 to 12 000 p.p.m.). Excitation and fluorescence maxima occur at 350 and 410 nm, respectively. The fluorescence is stabilised immediately and remains unchanged for over 4 hours. Fluoride present in low con- centrations gives rise to high results and must be absent. Other anions that form complexes with or precipitate zirconium, e.g., oxalate, phosphate, tartrate and tungstate, cause low results, but there is a high tolerance towards most cations except iron(II1) and cobalt(I1). THE most widely used methods for the determination of moderately large amounts of sulphate involve precipitation of barium sulphate followed by a gravimetric or titrimetric procedure.Smaller amounts can be measured turbidimetrically or nephelometrically after precipitation. Several indirect spectrophotometricl and fluorimetric2 procedures have also been described, based on the bleaching or quenching action of sulphate ion on some highly coloured or fluorescent metal - dyestuff complexes. Jones and Letham3 have described another novel indirect method based on the use of chloroaminobiphenyl. Recently a kinetochromic pro- cedure has been used to provide a direct absorptiometric method based on the catalytic effect of sulphate ion on the reaction between methylthymol blue and a partially polymerised solution of zirconyl ions.4 A similar fluorimetric procedure has been devised for sulphate ion in which the fluorescent reagent morin is used instead of methylthymol blue.5 Although the last two methods mentioned above are very sensitive they are kinetically controlled and require to be applied under rigidly maintained conditions.In this paper, we describe a direct fluorimetric determination that depends on the enhancement of fluorescence produced by allowing sulphate ion to react with an unpolymerised zirconium solution and the fluorescent reagent Calcein blue. We have recently described a similar reaction for fluoride ion6 based on the zirconium - Calcein blue system and have described the probable mechanism of the reaction resulting from the formation of a ternary complex between the three reactants.The sulphate reaction is much weaker than the fluoride reaction and it was found necessary to use a considerable (100 to 1000 mole ratio) amount of sulphate ion to obtain a linear calibration graph. Nevertheless, although this requirement completely vitiated our efforts to establish the existence of a similar ternary complex involving sulphate ion, zirconium and Calcein blue, we were easily able to establish linear relationships between the increase of fluorescence and the amount of added sulphate. M solution of Calcein blue (curves A and A’, respectively), an exactly formulated 1 : 1 zirconium - Calcein Fig.1 shows the excitation and emission spectra of a 0 SAC and the authors.282 TAN AND WEST: A SPECTROFLUORIMETRIC METHOD FOR [A~alyst, Vol. 96 '1 'IExcitation 60 50 - 40 - 300 350 & 400 \ I I I I I I I I I I I I 400 450 500 Wavelength/nm Fig. 1. Excitation and emission spectra Curves A and A': Curves B and B' : 1 0 - 6 ~ 1 : 1 Calcein blue - zirconium Curves C and C': 10-'JM 1 : 1 Calcein blue - zirconium Excitation spectrum Emission spectrum M Calcein blue + 200-fold excess of sulphate A: emission at 450 nm B: emission at 410 nm C: emission at 410 nm A': excitation at 325 nm B': excitation a t 350 nm C': excitation at 350 nm 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 lo-* M Calcein blue/ml Fig. 3. Effect of concentration of Calcein blue: curve A, zirconium - Calcium blue - sul- phate [2 ml of lo-* M zirconium(1V) in 3 M hydro- chloric acid + 4 ml of 10-2 M potassium sulphate] ; curve B, zirconium- Calcein blue (as A but without potassium sulphate) ; and curve C, ?effect caused by sulphate ion (B-A) 70L 6orio 50 :: 20 l o t ' 1-8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 1 1 1 1 I I I I 1 1 I PH Fig.2. Effect of pH on sensitivity: curve A, zirconium - Calcein blue - sulphate [2 ml of M Calcein blue + 2 ml of lo-* M zirconium(1V) in 3 M hydrochloric acid + 4 ml of 10-2 M potassium sulphate in 100 ml) ; and curve B, zirconium - Calcein blue (as A but without potassium sulphate) n Fig. 4. Attempted continuous vari- ation graph: broken line, net effect, i.e., corrected curve; n = millilitres of 10-2 M potassium sulphate added to (10-n) ml of M zirconium - Calcein blueApril, 19711 283 blue solution (curves B and B', respectively) and an exactly formulated 1 : 1 zirconium - Calcein blue solution with a 200-fold molar excess of sulphate ion (curves C and C', respec- tively), all at pH 1-9.These spectra are not corrected for the spectral response charac- teristics of the gratings of the monochromators or of the photomultiplier, nor do they account for changes in the lamp emission with wavelength. As in the previously reported reaction with fluoride, sulphate ions enhance the fluorescence of the zirconium-Calcein blue complex, but produce no changes in the wavelengths of excitation or fluorescence maxima. Fig. 2 shows the effects of varying the pH of the reaction of a solution containing Calcein blue, zirconium and potassium sulphate (curve A).Curve B is for an exactly similar solution but without sulphate ions. The fluorescence of the sulphate-enh anced system reaches a maximum at pH 1-9. The pH adjustments were made by the addition of small amounts of concentrated ammonia solution. The fluorescence intensities of solutions with pH values greater than 3 are not given because their fluorescence decreases rapidly on standing. It may, however, be relevant to observe that the fluorescence intensities of the zirconium - Calcein blue solution and the sulphate-enhanced solution increases with increasing pH above 3 when measurements are made immediately. The effect of varying the concentration of Calcein blue is shown in Fig. 3. Curve A is for zirconium and potassium sulphate, curve B is for a similar solution without sulphate and curve C represents the difference between the two solutions, i.e., the net effect of the sulphate ion.It is apparent that the optimum ratio of Calcein blue to zirconium is about 1.3. This is the same ratio as that previously found for the reaction with fluoride6 and can be interpreted as evidence for a true ratio of 1 : 1 because of the low assay of the Calcein blue, which could not be obtained or purified to any greater extent in these studies. A continuous variation graph was made (Fig. 4) to investigate the probable nature of a ternary complex formed between sulphate, zirconium and Calcein blue. This was done by varying a 10-2 M sulphate solution against an exactly formulated M solution of 1 : 1 zir- conium-Calcein blue in the usual way.Such a graph requires the use of approximately equimolar solutions of zirconium - Calcein blue and sulphate but, as explained previously, the analytical procedure requires the presence of about a 100-fold molar excess of sulphate ion to obtain reasonable sensitivity. Consequently it was not possible to obtain a sharp maximum. The broad maximum obtained suggests the probable existence of a 1 : 1 : 1 ternary complex, but cannot be interpreted with any confidence as definite evidence. THE DETERMINATION OF SMALL AMOUNTS OF SULPHATE ION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. .. .. . . TABLE I INTERFERENCE EFFECTS Molar excess in brackets Percentage change Ion added . . .. .... . . .. .. .. . . .. .. .. . . .. .. . . . . .. .. .. .. .. .. . . 0 0 - 58 - 46 0 Quenched - 19 0 Quenched - 75 - 56 - 16 0 0 - 77 - 63 - 46 - 45 - 37 -8 + 67 0 + 33 0 Sulphite Fluoride Aluminium Beryllium Arsenic(V) Nickel Copper (I I) Lead Calcium Cobalt Zinc Cadmium Magnesium Iron (I1 I) (100) . . (10) . . (1) - * (0.1) . . (0.01) . . (100) . . (100) .. (100) 1 . (10) .. (1) ' - (100) . * (100) . . (100) .. (100) . . (100) . . (10) . . (1) * * (100) . . (100) . . (100) . . (100) . . (1) ' . Percentage change .. .. .. .. .. .. . . .. .. . . . . .. .. . . . . . . . . . . .. .. .. .. + 100 + 52 + 13 + 100 + 14 0 0 - 42 -7 0 0 0 0 0 - 76 - 49 0 0 0 0 - 72 - 50284 TAN AND WEST: A SPECTROFLUORIMETRIC METHOD FOR [Analyst, Vol. 96 The effects of foreign ions on the determination of sulphate ion by this procedure are shown in Table I.Each solution contained 4 mg of sulphate ion in a final volume of 100 ml. The solutions were prepared by the recommended procedure and the foreign ions were added in 100-fold, 10-fold or equivalent amounts to the sulphate solution before the addition of the other reagents. It will be seen that the only serious cationic interference observed in the range of metals studied is that caused by iron(II1). None of the comnion anions interferes, except those which form more stable complexes than sulphate with the zirconium ion, e.g., oxalate and tartrate, or which tend to form insoluble compounds, e.g., tungstate and phos- phate. These anions cause low results whereas fluoride, exceptionally, gave rise to a positive result.Large amounts of fluoride, however, break down the zirconium - Calcein blue complex by formation of ZrFG2-, etc. The positive interference caused by sulphite, and to a lesser extent thiosulphate, can almost certainly be attributed to oxidation to sulphate in these dilute solutions and are not regarded as reaction of these ions per se. EXPERIMENTAL REAGENTS- Potassium sulphate solution, 10-2 M. Zirconium oxychloride solution, M-Prepare by dissolving 0.032 2 g of ZrOC1,.8H20 in 100 ml of 3 M hydrochloric acid. Prepare a M solution with 3 M hydrochloric acid. M-Dissolve 0.016 g of Calcein blue in a few drops of 0.1 M potassium hydroxide and dilute to 500ml with distilled water. The solution must be dis- carded after 2 toe3 days. Ammonia solution, 8 per cent.APPARATUS- Fluorescence measurements were made with a double monochromating spectrofluorimeter (Farrand Optical Co. Catalogue No. 104244) fitted with a 150-W Xenon arc lamp (Hanovia Division Catalogue No. 901 C-1) and an RCA IP28 photomultiplier. A Honeywell Brown recorder was used in conjunction with the spectrofluorimeter. Fused quartz cells (10 x 20 x 50 mm) were used throughout and a pH meter was used to adjust the pH. PROCEDURE- Calibration graph (2 to 12 mg or 20 to 12 000 p.p.m.)-Transfer 2 to 12 ml of 10-2 M potassium sulphate solution at suitable volume intervals into a series of 100-ml calibrated flasks and add, in the following order, 2 ml of loe4 M Calcein blue, 3 to 4 ml of 8 per cent. ammonia solution and 2 ml of Make up to the mark.Measure the fluorescence of the solutions at 410nm with an excitation wavelength of 350 nm. Deduct the fluorescence of the blank solution containing all of the reagents except the sulphate solution. These measurements can be made immediately or within 4 hours of preparation. As the volume of reagents added is less than 10m1, unknown test solutions containing down to 20 p.p.m. of sulphate can be measured if 90 ml of sample are available, or a solution as concentrated as 12 000 p.p.m., if the test aliquot is restricted to 1 ml. Calibration graph (200 to 1 000 pg or 2 to 1 000 P.p.m.)-Repeat the above procedure by using 2 to 10 ml of M Calcein blue, 3 to 4 ml of 8 per cent. ammonia solution and 2 ml of M zirconium oxychloride in 3 M hydrochloric acid. In this instance solutions as dilute as 2 p.p.m.can be analysed if 90 ml of test solution are available. M solution by 10-fold dilution of the Calcein blue solution, M zirconium oxychloride in 3 M hydrochloric acid. M potassium sulphate solution, 2 ml of CONCLUSIONS A fluorimetric method has been established for the determination of sulphate ion in the range 200 pg to 12 mg, or concentration range 2 to 12000 p.p.m., assuming a sample solution availability of 90 and 1 ml, respectively. The method is rapid and simple and has a repro- ducibility equal to or better than 5 per cent. Phosphate, oxalate and tartrate cause low recoveries and should be absent. Fluoride in small amounts produces a much greater sensitisation,6 gives rise to high results and must be absent. Large Tungstate also interferes.April, 19711 285 amounts of fluoride destroy the fluorescence of the zirconium - Calcein blue complex com- pletely.Sulphur species, which are easily oxidised in aqueous solution, interfere by sulphite formation, but the procedure shows a high tolerance towards the cations examined, except cobalt(I1) and iron(II1). The mechanism whereby the increase in fluorescence is produced is not apparent from these experiments, and although ternary complex formation may possibly be involved, the complex is too weak to allow unequivocal evidence to be adduced. The analytical method is moderately sensitive (lower limit of 2 p.p.m.) and it has the fairly wide range characteristic of many fluorimetric procedures. It furnishes a potentially useful spectroscopic analytical procedure in addition to those very few which are currently available for the determination of sulphate ion. We are grateful to the Agricultural Research Council for the provision of a grant in support of this work, and to the Science Research Council for the provision of the spectro- fluorimet er . REFERENCES THE DETERMINATION OF SMALL AMOUNTS OF SULPHATE ION 1. 2. 3. 4. 5. 6. Bertolacini, R. J., and Barney, J. E., Analyt. Chem., 1957, 29, 281. Guyon, J. C., and Lorah, E. J., Ibid., 1966, 38, 155. Jones, A. S., and Letham, D. S., Analyst, 1956, 81, 15. Hems, R. V., Kirkbright, G. F., and West, T. S., Talanta, 1969, 16, 789. Hems, R. V., Ph.D. Thesis, Imperial College, 1969. Tan, Lay Har, and West, T. S., Analyt. Chem., in the press. Received September 7th, 1970 Accepted November 25th, 1970

ISSN:0003-2654    

DOI:10.1039/AN9719600281

出版商:RSC    

年代:1971

数据来源: RSC

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286 Analyst, April, 1971, Vol. 96, $9, 286-287 Potentiometric Method for the Determination of Aromatic Monothiosemicarbazones BY M. J. M. CAMPBELL, R. GRZESKOWIAK AND I. D. M. TURNER (Deflartment of Chemistry, Thames Polytechnic, London, S.E.18) A method has been developed for the quantitative determination of aromatic monothiosemicarbazones, in which addition of silver nitrate to a solution of the organic compound leads to complex formation. The hydrogen ion liberated on complex formation is determined by potentiometric titration. THIOSEMICARBAZONES are compounds of great importance because of their pharmacological activity against tuberculosis,l viruses,2 protozoa,S smallpox4 and certain kinds of t ~ m o u r s . ~ During our investigation (unpublished work) of metal complexes of these compounds we observed that no reliable method for quantitative determination of these organic ligands was available.Koshkin’s method,6 which involves precipitation, filtration and back-titration, is time consuming and suffers from the disadvantage that it does not give a sharp end-point colour change; this change is, moreover, affected by the pH of the solution. In this paper we outline a simple and rapid method for the quantitative determination of monothiosemicarbazones by potentiometric titration of the hydrogen ion liberated on complex formation when silver ion is added to a solution of the organic compounds. EXPERIMENTAL REAGENTS- All reagents used were of analytical-reagent grade. Hydrochloric acid, 0.1 N-Prepared from standard volumetric acid. Sodium hydrogen carbonate solution, 0.1 N-About 8-4 g of sodium hydrogen carbonate were dissolved in 1 litre of distilled water.The solution was standardised with the above acid, the end-point being determined potentiometrically. APPARATUS- A Vibret Laboratory pH meter, Model 46A, was used in conjunction with a glass - calomel combined electrode, the results being plotted manually from the meter readings. Measurements were also carried out on the automatic instrument, Metrohm potentiograph (Series E436), fitted with a 10-ml automatic burette and a glass - calomel electrode. GENERAL PROCEDURE- Weigh accurately about 150 mg of benzaldehyde thiosemicarbazone and dissolve it in 35 ml of dioxan. To this solution add 80 ml of water and 5 ml of silver nitrate solution.Stir the mixture by using a magnetic stirrer and titrate with the standardised sodium hydrogen carbonate solution (Note). If the manual method is adopted, add 0-1-ml portions of the titrant near the end-point. A typical set of titration figures obtained by using the Vibret pH meter with manual plotting is given below. Titrant added/ml . . 0.0 6.4 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.6 7.8 pH of solution . . 2-01 2-57 2.68 2.78 2.90 3.03 3-18 3.40 4-16 4.46 4.60 4.72 4-80 Determine a blank under the same experimental conditions. The value found was about 0.02 ml of titrant. Silver nitrate solution, 5 per cent. 0 SAC and the authors.CAMPBELL, GRZESKOWIAK AND TURNER The percentage purity is easily calculated from the equation- 287 V x N x E w x 10 Percentage purity = where V is the volume of titrant in millilitres, corrected for the blank titre; N is the normality of titrant; E is the equivalent weight of organic compound in grams; and W is the weight taken in grams.NOTE-We found that the use of other basic reagents such as sodium hydroxide or sodium car- bonate causes precipitation of silver salts before the end-point is reached. RESULTS AND DISCUSSION Addition of silver nitrate to the thiosemicarbazone results in complex formation, in which an equivalent amount of hydrogen ion is released into the solution. ,NH2 ,N H2 /R + H+ c-s R + Ag+ - C-S-Ag ‘NH--N=C / nN-N=C \ ‘R, R1 The nature of the complexes formed is under investigation and the results will be reported at a later date. The determinations were carried out on ten different samples for each thiosemicarbazone studied and the results obtained were within the narrow range of 100 t 0.5 per cent.The method was also successful with thiosemicarbazones of the following carbonyl compounds : 9-isopropylbenzaldehyde (cutizone) ,2 salicylaldehyde, isatin and furfuraldehyde. With the thiosemicarbazones of alkyl ketones and acetophenone reduction of silver ion to silver took place, while with benzophenone no complex formation occurred. A satisfactory end-point could be obtained even for amounts as small as 20 mg; how- ever, the scatter of results increased to k 2 per cent. Erroneous results can be caused by the decomposition of complex on the electrode if the concentration of the solid in the mixture is too great. With the automatic titrator, con- centrations of 150mg of ligand in 25ml of dioxan and 25ml of water can be used, but greater dilution had to be used in the manual method.The potentiometric method has the advantage over the Koshkin procedure that it is rapid, even with the manual apparatus, and the end-point is easier to determine. However, Koshkin obtained good results even with alkyl thiosemicarbazones , such as acetone thiosemi- carbazone, that cannot be determined by the potentiometric method. The authors thank Burroughs Wellcome (Dartford) for the use of the Metrohm E436. REFERENCES 1. 2. Orlova, N. N., Aksensova, V. A., Selidovkin, I). A., Bogdanova, N. S., and Pershin, G. N., 3. 4. 5. 6. Koshkin, N. V., Zh. Analit. Khirn., 1963, 18, 1492. Domagk, G., Behenish, R., Mietzsch, F., and Schmidt, H., Naturwissenschaften, 1946, 33, 315. Farmakologiya i Toksikologiya, 1968, 31, 725; translated as Russ. Pharm. Toxic., 1968, 348. Butler, K., U.S. Patent No. 3 382 266, 1968. Bauer, D. J., St. Vincent, L., Kempe, C. H., and Dowine, A. W., Lancet, 1963, ii, 494. Petering, H. G., Buskirk, H. H., and Underwood, G. E., Cancer Res., 1964, 64, 367. Received May 13th, 1970 Accepted October lst, 1970

ISSN:0003-2654    

DOI:10.1039/AN9719600286

出版商:RSC    

年代:1971

数据来源: RSC

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288 Analyst, April, 1971, Vol. 96, pp. 288-295 The Determination of Glycerol by the I.U.P.A.C. Form of the Malaprade Method* BY R. F. BARBOUR (Newcastle Technical Centre, Procter 6. Gamble Ltd., Newcastle upon Tyne) AND J. DEVIm ( Unilever Research Laboratory, Unilever Ltd., Port Sunlight, Cheshire) The accepted periodate method for the determination of glycerol has come under review by the International Organisation for Standardisation (Sub-committee ISO/TC47/GT2) and certain modifications have been sug- gested with regard to the different pH end-points for the sample and blank and the possible loss of formic acid from the system by volatilisation. The present paper summarises the view of the U.K. delegates to ISO/TC47/GT2, which are: (i) The choice of pH about 8.0 for the sample is justified on the ground that it contains formic acid in addition to strong acids.The blank, which contains only strong acids, should, on general grounds and by calculation, be titrated to pH 7-0. The use of pH 6.5 instead of 7.0 for the blank is an empirical correction designed to compensate for some lack of stoicheiometry or other bias in the procedure and to bring the results into agreement with those obtained by independent moisture and specific gravity determinations. This “correction” amounts to 0-03 per cent. (ii) There is a potential loss of formic acid by volatilisation from the system (estimated variously to be equivalent to 0.01 to 0.04 per cent. of glycerol), which partly explains the apparent lack of stoicheiometry. (iii) Modifications have been proposed to minimise this loss of formic acid and (by adding formate ions to both solutions) to unify the end-points at or about pH 8.0.Unless these modifications lead to a pronounced improve- ment in reliability, it seems doubtful whether further extensive trials on an international basis would be justified. THE accepted method of determining glycerol is based on oxidation by sodium periodate to formic acid and measurement of the latter by titration with standard alkali, which is a modification of that outlined by Malaprade.1 It involves the use of an end-point of pH 6.5 for the blank and of pH 8.1 for the sample. This procedure was originally put foward by the American Oil Chemists’ Society and was substantiated by the results of a series of inter- national collaborative trials conducted under its auspices in 1956-57.It was subsequently adopted by the International Union of Pure and Applied Chemistry (I.U.P.A.C.)2 and also by the British Standards Institution (B.S.I.) and other national bodies, and as such it has formed a satisfactory basis for commercial transactions in glycerine in all parts of the world. More recently, the method has come under review by the International Organisation for Standardisation (Sub-committee ISO/TC47/GT2), when it was stressed that the choice of pH values for the dual end-points has not been adequately clarified. The belief was expressed that this discrepancy existed to compensate for some consistent error or bias elsewherein the test, which should be further examined before adoption.As a result of experimental work by members of the Sub-committee, the following suggestions have been made: there * This paper was written a t the suggestion of the British Standards Institution Committee CIC/6- Glycerol by two of its members who are also U.K. delegates to the Working Group on glycerol of the International Organisation for Standardisation (ISO/TC47/GT2). It therefore represents the current U.K. attitude on the problem of the determination of glycerol, which is a t present engaging the attention of ISO. 0 SAC and the authors.BARBOUR AND DEVINE 289 is a slight loss of formic acid from the test sample by volatilisation, which could be a source of consistent erro1-39~; and a unified end-point could be achieved by addition of formate ions to both sample and blank.4 It is appropriate, therefore, to record the views of the U.K.delegates to the committee, which are summarised as follows. pH OF END-POINTS CURRENTLY USED- In the standard I.U.P.A.C. procedure, excess of periodic acid (a weak acid) is reduced before titration to iodic acid (a strong acid) so that its buffering action is eliminated. The acids being titrated are, therefore, for sample: sulphuric, iodic and formic acids, and for blank : sulphuric and iodic acids. Nominally, therefore, the end-points used for the titrations (pH 8.1 and 6.5) should accord with the equivalence points of formic acid and of the sulphuric acid - iodic acid mixture, respectively. It is conceded that it is unusual to subject a blank to a different procedure from that used for the sample and for this reason, although we retain the term here, it might be better referred to as a control.In the 1956 deliberations of the B.S.I. Sub-committee on Glycerine it was suggested that the result of carrying the pH of the sample to 8.1 was to over-titrate the strong acids contained therein, and that the blank should therefore also be titrated to pH 8.1 so that the errors in each instance should cancel out in the subsequent calculation. This suggestion was rejected on theoretical grounds and was not supported by practical tests in which solutions of formic acid and of a mixture of formic, sulphuric and iodic acids in concentrations equivalent to those of an actual glycerol determination were titrated potentiometrically under nitrogen with 0.1 N carbonate-free sodium hydroxide solution from pH 6.5 to 8.1.There was no significant difference in the amounts of alkali required by the two solutions unless there was contamination by atmospheric carbon dioxide (Lazarus, W., private communication), thereby indicating that in the mixture the alkali required to cover this pH range is determined effectively by the formic acid alone. No additional alkali is required by the presence of sulphuric and iodic acids, which are therefore not over-titrated. It is appropriate to consider what end-points for sample and blank might be predicted by strictly theoretical means. These are recorded in Appendix I, which gives estimates of the pH at the stoicheiometric end-points for solutions containing, for sample : sodium formate, iodate and sulphate, and for blank : sodium iodate and sulphate.The calculated pH values are 7.96 for the sample and 7.05 for the blank, respectively. This confirms the need for two different pH end-points for sample and blank and reasonably supports the choice of pH 8.1 for the sample titration, but not pH 6.5 instead of about 7 for the blank. Examination of the original correspondence in the period 1952 to 1955 between the United Kingdom Glycerine Producers' Association and the American Oil Chemists' Society reveals that the A.O.C.S. Glycerine Analysis Committee tried various pH values between 6.5 and 7-5 for the blank on a largely empirical basis; of these, pH 6-5 was selected as giving the best results, i.e., those most in conformity with the independent assay of the sample by specific gravity and moisture determinations.This latter method of assay was based on the belief that the sample of glycerol for assay was pure and conformed with that used for establishing the standard Bosart and Snoddy tables of specific gravity for glycerol - water solutions.5 The pH 7.0, which was the inflexion point actually found for the titration curve," was discarded, and as late as 1957 pH 6.5 was being erroneously quoted as the equivalence point. It is clear, therefore, that the pH of about 7.0 as the equivalence point for the blank is borne out by both theory and practice and that the selection of pH 6.5 in its place represents an empirical correction designed to allow for some slight imperfection in the over-all analysis, whether of stoicheiometry or otherwise.The effects of such pH differences as have been quoted are indicated by the p H - titration curves presented in Appendix B of the Minutes of the October 1965 Meeting of ISO/TC47/GT2, from which the following figures are taken. * It is recognised that inflexion point and equivalence point are not identical, but with strong elec- trolytes, as in the blank, the difference is negligible.6290 BARBOUR AND DEVINE : DETERMINATION OF GLYCEROL BY [Analyst, Vol. 96 Blank-A pH of 6.5 to 7-0 requires 0.012 ml of 0-125 N alkali solution equivalent to 0.03 per cent. of glycerol (at 0.41 g sample weight), and a pH of 6.5 to 8.1 requires 0.036 ml of alkali solution equivalent to 0-10 per cent. of glycerol. Sample-A pH of 6.5 to 7.0 requires 0.08 ml of alkali solution equivalent to 0.22 per cent. of glycerol, and a pH of 6.5 to 8.1 requires 0.16ml of alkali solution equivalent to 0.45 per cent.of glycerol. Thus, titration of the blank to pH 6.5 instead of 7.0 has the effect of raising the glycerol result by about 0.03 per cent. However, titration of both sample and blank to pH 7.0 introduces an error of about -0.26 per cent. of glycerol,* while titration of both to pH 8.1 introduces an error of about -0.10 per cent. of glycerol. In this consideration it is important that no significant amounts of other buffers should be present, e.g., carbon dioxide from the atmosphere or impurities in the sample. USE OF MODIFIED END-POINTS- As the presence of formic acid in the sample titration has the effect of raising the stoicheiometric or “equivalence” end-point to about pH 8, it was suggested that formic acid (or sodium formate) should be added to both blank and sample, which should be then titrated to pH 7.9 O*Z4 This addition would eliminate the need for different end-points and yield titration curves of similar character.Tests carried out in three laboratories, in which the results obtained in this way are compared with those by the normal I.U.P.A.C. method, are recorded in Appendix 11. They confirm that, within experimental error, the modification yields the same results as the normal blank to pH 6.5. This seems to remove the apparent undesirability of using different end-points for sample and “blank.” However, errors can arise in the procedure of adding the formate required to unify the end-points and more extensive tests would be necessary to assess the effect on reproducibility.VOLATILITY OF FORMIC ACID- Mormont and Gillet3 have pointed out that because of the exothermic reaction between glycerol and periodate there is a slight rise in the temperature of the solution and consequently some evaporation: this effect is made obvious by the slight condensation that occurs on the under surface of the clock-glass with which the beaker is closed. The condensate from the sample is acidic in contrast to that from the blank. This acidity led to the suggestion that there is a loss of formic acid from the vessel by volatilisation and that this loss is the source of the systematic error that is otherwise accounted for by the use of the empirically chosen value of pH 6.5 for the blank titration.In a model solution of formic and sulphuric acids similar in composition to that in a glycerol determination, a loss of 0.2 per cent. of its strength was demonstrated a t ambient temperature within 30 minutes in the open air (‘‘2 l’air libre”). This problem was examined in the U.K. in 1953 (Lazarus, W., Walley, G., and Wilkie, A. L., private communication) when an equivalent solution of formic acid was titrated, with and without a stream of nitrogen being bubbled through the solution for periods of up to 20 minutes prior to and during the titration: no significant loss of formic acid was detected. In a series of tests, variations observed in carrying out the glycerol determination at 0, 20 and 45 “C with the procedure then in use were not regarded as significant, and variations for a slight rise above room temperature certainly could not be regarded as significant. We have re-examined this problem in the light of Mormont and Gillet’s observation, taking into account the finer differences now being looked for, with the results given in Appendix 111.We agree that there is a rise of 4 to 5 “C in the temperature of the solution and have shown that the presence of formic acid can be specifically demonstrated by a colorimetric test in the condensate on the clock-glass from a single determination.’ While there is no doubt about the potential volatilisation of formic acid, the extent of the loss from the system in the average analytical procedure is more difficult to assess. Our recent tests indicate losses varying from zero up to 0.04 per cent.of glycerol, although some uncertainty must attach to those tests in which the solutions were left open to the atmosphere to exag- gerate the effect; the subsequent loss would be compensated for to some extent by absorption * This is close to the figure of “about 0.370” given in B.S. 2621-5 : 1964 as the correction that results from using the pH end-points 6.5 and 8.1. In our view the term “correction” should more properly be applied to the 0-03 per cent. difference that results from using pH 6.5 instead of 7-0 in the blank.April, 19711 THE I.U.P.A.C. FORM OF THE MALAPRADE METHOD 291 of atmospheric carbon dioxide. This tendency towards compensation must, of course, operate whatever the imperfection in the closure of the beaker may be.The potential volatilisation of formic acid has been re-examined also by Mormont, Gillet and Hei~~erth.~ The condensates from several tests were collected and tested by infrared spectroscopy, which again demonstrated the presence of formic acid. The con- densates on the clock-glasses from thirty-eight tests were also collected and combined for titration; the result obtained was equivalent to a mean recovery per test of 0.01 per cent. of glycerol on the clock-glass. The actual Loss by leakage from the system was thought to be in excess of this value, by a factor approaching 10, and the difference between the loss of formic acid and the gain in carbon dioxide, for a model mixture of formic and sulphuric acids, has been put at the mean figure of 0.2 per cent.per hour (Mormont, R., private com- munication). However, some doubt exists on whether this refers to open air conditions (“& l’air libre”) or conditions with a closed beaker (“en becher couvert”) since both these forms are used. The loss of formic acid from the system, found in terms of glycerol by two sets of investi- gators therefore varies from zero to upwards of 0.04 per cent. with one set, and approaches 0-2 per cent. with the other. The mean effect in our own tests is of the same order as the 0.03 per cent. of glycerol, which is equivalent to the gain accounted for by titrating the blank to pH 6.5 instead of 7.0. However, the following considerations make it inadvisable to equate the two effects at present: (a) it cannot be said whether it is normal practice to wash down the clock-glass before titration; (b) the formic acid in the condensate is not a measure of that lost from the system by volatilisation; and (c) the effective loss by volatilisation depends on: (i) the type of beaker (600m1, tall, in I.U.P.A.C.directions; 600m1, squat, in B.S. 2621-5: 1964), (ii) fit of the clock-glass, (iii) size of the beaker lip and (iv) absolute reaction temperature reached, which will depend partly on the initial ambient temperatures of the laboratory and of the reagents. It may be thought that there is reason to reconsider the equipment used, because originally the use of a beaker was presumably conditioned by the necessity of inserting separate elec- trodes.Present-day electrodes can be inserted as a single piece through a small ground-glass joint so that a suitable standard flask could be used with effective stoppering duringthe reaction period. Alternatively, a beaker without a lip and the top surface ground to take a ground-glass plate could be used. Even with the desire to retain as simple equipment as possible, the failure to close the vessel, in the light of errors ascribed to the passage of volatile components, appears to be a retrograde step. This still leaves a minor problem as to how much formic acid remains, after cooling for half an hour, in the vapour phase, some of which might disperse on opening the beaker to the atmosphere to conduct the titration. This amount should not be significant, especially if the simple expedient of immersing the reaction vessel in a bath of cold water is adopted.* The alternative approach chosen by Mormont et aZ.* is to damp down the exothermic effect, not by external cooling, but by adding the dilution water before the periodate reagent instead of at the end of the reaction.This is claimed to halve the error caused by the combined loss of formic acid and gain of carbon dioxide. However, it appears to be generally held by analysts familiar with the collaborative work in the U.K. in the 1950s that over-dilution affects the determination unfavourably, possibly because of the difficulty of excluding carbon dioxide, which affects the sample and blank to different extents. CONCLUSIONS The following aspects have been clarified by recent work on the standard procedure for the periodate determination of glycerol : (i) The use of two different pH end-points for the blank and sample is necessitated by the different nature of the acids being titrated, but whereas a pH of 8.1 for the sample is in reasonable conformity with the equivalence point of the formic acid, which is present only in the sample, the pH required for the blank, both as determined and for theoretical reasons, is about 7.0.The use of a pH of 6.5 in its place is not warranted except as a correction to account for an apparent lack of complete stoicheiometry in the reaction. This form of * Our calculation suggests a total vapour content equivalent to less than 0.02 per cent. of glycerol, under present standard conditions.292 BARBOUR AND DEVINE : DETERMINATION OF GLYCEROL BY [Autalyst, Vol.96 correction does not commend itself to all analysts; others accept it on the grounds that it is equivalent to only 0.03 per cent. of the glycerol present and that a correction of this magni- tude hardly warrants modification of an established method that has served industry well. (ii) There is a potential loss of formic acid from the system by volatilisation which, in principle, could account for the apparent lack of stoicheiometry. On the basis of the restricted amount of evidence available it is not yet possible to equate these two factors. (iii) Modifications to the standard procedure have been suggested4 to overcome these difficulties. They entail (a) adding the dilution water before instead of after the reaction, and (b) adding equal amounts of sodium formate to blank and sample, both of which are then titrated to the equivalence point of the formic ions, which is considered to be pH 7.9 0.2.The few tests carried out on item (b) give the same results as the standard I.U.P.A.C. pro- cedure, which it is designed to supplant as being based on sounder theoretical principles. Further extensive trials are envisaged to determine which of these procedures is preferable on grounds of accuracy and reproducibility. (iv) It is questionable whether further work to detect a bias of 0.03 per cent. of glycerol in a test with a standard deviation between laboratories that we estimate to be of the order of 0-22 per cent. is justifiable, as a very large number of tests would be required.But if this was thought desirable we suggest that an alternative approach, which should be considered, would be prevention of any formic acid loss by slight modification of the apparatus. This would only entail effectively sealing it and placing it in a bath of cold tap water during the reaction. It would then be appropriate to use the true equivalence points of pH 7.0 and about 8-0 for the respective titrations. This would have the advantage of placing the method on a sound theoretical basis without altering the nature of the solutions to be titrated and possibly introducing errors in the addition of sodium formate. The authors thank Procter and Gamble Limited and Unilever Limited for permission to publish this paper and for the necessary facilities provided. REFERENCES 1.2. 3. 4. 5. 6. 7. Malaprade, L., Bull. SOC. Chim. Fr., 1928, 43, 683. Standard Methods of the Oils and Fats Section of the I.U.P.A.C., Fifth Edition. Buttenvorths, Mormont, R., and Gillet, A. C., jun., XXXVIe Congrks Int. Chim. Ind., Bruxelles, September Mormont, R., Gillet, A. C., jun., and Heinerth, E., Talanta, 1969, 16, 701. Bosart, L. W., and Snoddy, A. O., Ind. Engng Chem., 1927, 19, 506. Marinenko, G., and Champion, C. E., Analyt. Chem., 1969, 41, 1208. Feigl, F., “Spot Tests in Organic Analysis,” Fifth Edition, Elsevier Publishing Company, Amster- Received August 13th, 1970 Accepted November 12th, 1970 London, 1966. 1966, Gr. X. S26, 691. dam, London, New York and Princeton, 1956, p. 340. Appendix I CALCULATION OF END-POINTS FOR SAMPLE AND BLANK Solution volume = 300 ml Periodate added = 3.0 g Molar concentration of iodate formed therefrom Glycerol taken = 0.41 g (at 100 per cent.glycerol) Molar concentration of formic acid (and formate) produced Dissociation constants at 25 “C, taken from the “Handbook of Chemistry and Physics,” Forty-fifth Edition, 1964 (Chemical Rubber Co.), are formic acid 1.77 x 10-4 = K,; iodic acid 1.69 x 10-1 = Kb; and water 10-14 = K,.April, 19711 THE I.U.P.A.C. FORM OF THE MALAPRADE METHOD 293 HB + B- + H+ + A- + HA With sodium formate and iodate present the equilibrium may be represented as- I t OH- where A- and B- are formate and iodate ions, respectively. Where Ca and c b are total molar concentrations of (formate ions + formic acid) and (iodate ions + iodic acid), respec- tively, this equilibrium gives- [H+] x [OH-] = Kw [H+] x [A-] =Ka [HA] [H+] X [B-] = Kb [HB] [B-] + [HB] = Cb = 0.047 [A-] + [HA] = Ca = 0.015 [H+l + [HA1 + [HBI + [H,OI = [OH-I + [H,OI.(In these dilute solutions the activity coefficients are taken as close to unity.) This last relationship is based on the fact that when only pure sodium formate and iodate are present, without added acid or alkali, the “free” plus “combined” H+ ionic concentration must equal the “free” plus “combined” OH- ionic concentration, which leads to the following relation- ship- [H+I4 + [H+I3 (Ka + Kb + ca + cb) + [H+I2 (KaKb + KaCb + KbCa - Kw) - [H’] (KaKw + KbKw) - KaKbKw = O . . .. .. * . (1) or, applying the above numerical values (2) A Sturm analysis indicates only one positive root ; and this may be approximated, iteratively, by initially ignoring the 3rd and 4th degree terms, solving the remaining 2nd degree relation for the positive root, inserting this in the 3rd and 4th degree terms, absorbing their values in the constant term, re-solving, re-inserting and proceeding to convergence.[H+I4 + 0-23 [H+I3 + 2.6 x [H+I2 - 1.7 x [H+] - 3.0 x 1O-l’ = 0 . . Thus, the 2nd degree solution is 1 1 Kw2 (Ka + Kb)2 [H+l = 2 (KaKb + KbC, [ KW (Ka + Kb) + + 4 KaKbKw (KaKb -t- KbCa + KaCb - Kw) -k KaCb - Kw) In this instance Kb is of the order of 1 000 x Ka; and hence in the above bracketed sums the only significant terms are those in Kb and KbCa. The above thus reduces to 1 = 1.1 x IH + 1 [ ~ w + J ~ w 2 + 4 ~wKa~a.1 - 2 c a applying the above numerical values. This gives pH = - log [la1 x 10-5 = 7.96.Inserting the above value in the 3rd and 4th degree terms yields a value of the order while the constant term is of the order 1O-l’. Thus the 3rd and 4th degree terms have negligible effect on the estimate, which remains at pH 7-96; and this indicates that a suitable pH end-point for the sample titration would be expected to be about 8. In the absence of iodate ions, c b = 0, and [H+] + Kb becomes a factor in equation (l), thereby cancelling and reducing this equation to- As before, ignoring the 3rd degree term, this yields- [H+I3 + [H+I2 (Ka + Ca) - [H+] Kw - KaKw = 0 = 1.1 x 10-8, applying the above numerical values.294 BARBOUR AND DEVINE: DETERMINATION OF GLYCEROL BY [Analyst, Vol.96 As before, the 3rd degree term remains negligible. This gives pH = 7-96, which is effectively the same as that when the iodate is present with the formate, as is to be expected from the form of the two expressions, where Ka is approximately 100 times smaller than Ca. Thus the iodate has practically no effect on the pH produced by the formate and, for the same reason, the effect of the sulphate can be ignored in the sample and blank titrations. In the blank titration formate is absent and, as above, the relationship becomes with Ca = 0, 1 r I 1 = 0.89 x 10-7, applying the above numerical values. Thus a suitable pH end-point would be expected to be about 7. This gives pH = -log (0.89 x lo-’) = 7.05. Appendix I1 COMPARISON OF I.U.P.A.C. PROCEDURE WITH THE “UNIFIED END-POINT” PROCEDURE The data are taken from document ISO/TC47/GT2-No.87; a record of tests performed in Brussels, Newcastle and London is given as follows. The method of test consisted in performing the normal I.U.P.A.C. glycerol determination to the usual end-points, viz., sample pH 8.1 and blank pH 6.5, at which stage equal amounts of formic acid solution were added to both sample and blank, and the titrations were con- tinued, both to pH 8.1. For this second stage the sample and blank titrations are taken as the sums of the titrations before and after formic acid addition, respectively. Bvussels*- Sample: to pH 8.1 Blank: to pH 6-5 to pH 8.1 (Sample less blank) (Sample less blank) (Sample less blank) Titration/ml I A \ (11) With added formic acid (1) I.U.P.A.C... . . . . 29-90 29-91 45.99 46.00 .. .. .. 4.89 4-89 - - . . . . 20.97 20.98 25.02 .. . . . . 25-01 25.02 (Mean). . . . 25.015 25.02 (Mean) (I - 11) - .. 25-02 - 0.005 For the amounts of sample taken this is equivalent to -0-018 per cent. of glycerol. * Reported by Mr. A. C. Gillet, jun., of Solvay et Cie, S.A., to whom our thanks are given. Titrationlml I L \ (11) With added formic acid London- (1) (with distilled glycerine) I.U.P.A.C. Sample: to pH 8.1 . . .. .. .. . . 42.65 62.37 topH 8.1 . . . . . . .. .. 24.56 (Sample less blank) . . . . . . .. .. 37.81 37.81 4.84 - Blank: to pH 6.5 . . . . .. .. .. (Sample less blank) (I - 11) . . . . . . 0.00 - Titration /ml r A 1 (11) With added formic acid Newcastle- (1) (three crude glycerines were used) Sample: to pH 8.1 .. . . . . 36.64 38.06 37.54 62-01 63.45 62.96 I.U.P.A.C. Blank: to pH 6-5 . . 4.78 4.78 4.76 4.78 - - - - . . . . to pH 8-1 . . . . . . - - - - 30.14 30.20 30-18 30-20 Mean blank . . . . . . . . 4.775 30-18 (Sample less mean blank) . . . . 31.865 33.285 32.765 31-83 33-27 32-78 (Sample less mean blank) (I - 11) + 0.035 +Ow015 -0.015 (Sample less mean blank) (I - 11) (Mean) +0.012 For the amounts of sample taken this is equivalent to +0-028 per cent of glycerol.April, 19711 SUMMARY- THE I.U.P.A.C. FORM OF THE MALAPRADE METHOD 295 Sample less blank (I - 11) (Mean) /ml Brussels . . . . . . - 0.005 London . . . . . . 0.00 Newcastle . . . . . . +0.012 Mean . . . . + 0.002 This indicates that, within experimental error of measurement, titration of the blank to pH 6.5 gives effectively the same result as addition of formic acid and titration of the blank to pH 8.1.This does not, however, measure the resulting reproducibility. Appendix I11 VOLATILISATION OF FORMIC ACID DURING THE DETERMINATION OF GLYCEROL (i) The condensate on the watch-glass from a single determination was treated with magnesium powder and dilute hydrochloric acid and then with sulphuric acid and chromo- tropic acid according to Feigl.' Production of a violet -pink colour indicated the presence of formic acid in the original sample." The following data are taken from Document ISO/TC 47/GT2-No. 66: (ii) The I.U.P.A.C. glycerol determination was carried out and the traces of condensate on the clock-glasses were collected separately and titrated. Two such tests showed an average acidity equivalent to 0.02 ml of 0.125 N sodium hydroxide, i.e., equivalent to about 0-04 per cent. of glycerol. (iii) The loss of forrnic acid to atmosphere from the test-reaction liquor was measured over 72 hours, and two such tests showed an average loss per hour (i.e., approximate duration of normal test) equivalent to 0.025 per cent. of glycerol. The loss during an actual deter- mination would probably be slightly higher, because during the first 0.5 to 1 hour the liquor is slightly warmer than it is later. (iv) Three tests similar to (ii) above, but with the condensate washings combined, showed a total equivalent to 0.02 ml of 0.125 N sodium hydroxide. This is equivalent to about 0.015 per cent. of glycerol per test. (v) A stream of nitrogen was passed through an aqueous solution of formic acid, of concentration similar to that in a glycerol determination, and the acid was subsequently titrated. No significant loss of formic acid was found. The above tests indicate that during the glycerol determination there could be an actual or potential loss of formic acid equivalent to 0.015 to 0-04 per cent. of glycerol. Stevenston, Ayrshire. * We are indebted for this test to Mr. S . M. Farrer of Imperial Chemical Industries Ltd., Nobel Division,

ISSN:0003-2654    

DOI:10.1039/AN9719600288

出版商:RSC    

年代:1971

数据来源: RSC