ISSN:0003-2654    

DOI:10.1039/AN96691FX005

出版商:RSC    

年代:1966

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN96691BX007

出版商:RSC    

年代:1966

数据来源: RSC

摘要:

iv SUMMARIES OF PAPERS I N THIS ISSUE [February, 1966Summaries of Papers in this IssueSome Sensitive and Selective Reactions in InorganicSpectroscopic AnalysisA Special LectureT. S. WESTChemistry Department, Imperial College, London, S. W. 7.Analyst, 1966, 91, 69-77.Recent Developments in the Measurement of Nucleic Acidsin Biological MaterialsA Supplementary ReviewSUMMARY OF CONTENTSIntroductionPreparation of tissue samples for nucleic acid determinationsPrecautions during removal of tissuesExtraction of acid-soluble compoundsExtraction of lipidsMethods of determining nucleic acids in the tissue residue1. The procedure of Schmidt and ThannhauserThe use of alkaline hydrolysis to separate RNA from DNAThe RNA fraction of the alkaline digestThe DNA fraction of the alkaline digestRecommendations for the use of the Schmidt - Thannhausermethod2.The Schneider procedure3.4. Other proceduresThe procedure of Ogur and RosenGeneral recommendations for nucleic acid determinationH. N. MUNRO and A. FLECKDepartment of Biochemistry, The University of Glasgow.Analyst, 1966, 91, 78-88.REPRINTS of this Review paper will soon be available from the Secretary,The Society for Analytical Chemistry, 14 Belgrave Square, London, S.W. 1,a t 5s. per copy, post free.A remittance for the correct amount, made out t o The Society for-4nalytical Chemistry, MUST accompany every order; these reprints are notavailable through Trade Agents.A Sensitive and Selective Spectrophotometric Procedurefor the Determination of PhosphorusPhosphorus as phosphate is determined by an amplification procedurein which the heteropoly acid H,PO,(MOO,)~, is formed and extracted awayfrom excess of molybdate reagent.The 12 molybdate ions associated withthe phosphate are then determined spectrophotometrically a t 710 mp as thegreen molybdenum(v1) complex with 2-amino-4-chlorobenzenethiol in chloro-form. Depending on the procedure used, the effective molar absorptivityfor phosphorus is 96,900 or 359,000. The proposed procedure is thereforemuch more sensitive than previously described methods for phosphorus.Amounts of phosphorus down to 0.2pg (0-008 p.p.m.) may be determined.Large excesses of silicon, germanium, arsenic or antimony do not interfere.A simple masking procedure obviates any interference from up to a 30-foldexcess of tungsten(v1).V. DJURKIN, G.F. KIRKBRIGHT and T. S. WESTChemistry Department, Imperial College, London, S W.7.Analyst, 1966, 91, 89-93February, 19661 THE ANALYST Vprogress in productionBecause BDH is a world supplier its progress in the production of laboratorychemicals reflects the international development of the applications of chemistryand biochemistry in education, research, medicine and industry over the last fiftyyears. It has been remarkable progress, whether considered in relation to methods,equipment or scale of manufacture.Advances in the classical methods have gone hand in hand with the employment ofcompletely new techniques of manufacture and purification ; these, and the cori-tributions of modern chemical engineering, have made it possible for the BDH out-put of reagents to increase by more than a hundred times in the past half-century.This in turn has required equivalent developments in analytical resources, inspec-tion services, distribution facilities and commercial organisation.BDH output in1965 calls for the packaging of several million containers a year of thousands ofwidely diverse and often hazardous chemicals. They reach the laboratories of overa hundred countries in every part of the world.POOLE * LONDON * BRISTOL * LIVERPOOL * MIDDLESBROUGHBOMBAY * TORONTO * JOHANNESBURG * SYDNEY * AUCKLAND LC/ P46SUMMARIES OF PAPERS IN THIS ISSUEThe Determination of Residues of Dimethoate withMulti- band Chromatoplates[February, 1966A method is proposed for the determination of dimethoate residues invegetable material.Tripartite multi-band plates are prepared and used toseparate the dimethoate from co-extracted materials ; spot-area measurementis used to give a quantitative determination of the amount of pestcide.D. C. ABBOTT, MRS. J. A. BUNTING and J. THOMSONMinistry of Technology, Laboratory of the Government Chemist, Cornwall House,Stamford Street, London, S.E.l.Analyst, 1966, 91, 94-97.The Solvent-extraction and Absorptiometric DeterminationIron(I1) and iron(m) can be completely extracted from M sulphuric acidinto a 0.02 M solution of Z-mercaptopyridine- l-oxide and determined absorp-tiometrically at 550 mp.The only significant interference is from copper(I1);the sensitivity of the method is 0.02 p g cm-2.J. A. W. DALZIEL and M. THOMPSONDepartment of Chemistry, Chelsea College of Science and Technology, ManresaRoad, London, S.W.3.Analyst, 1966, 91, 98-101.of Iron with 2-Mercaptopyridine-1 -oxideObservations on the Use of Titan Yellow for the Determinationof Magnesium with Special Reference to Soil ExtractsExamination by paper and thin-layer chromatography of several prepara-tions of titan yellow from different manufacturers has shown them to becomplex mixtures.By using a titan yellow selected chromatographically, it has been possibleto improve the stability and sensitivity of the coloured complex producedwith magnesium. Studies have been made of the effects of alkalis, interferingions and protective colloids, and the use of gelatin with a lithium hydroxide -glycine buffer is proposed for the determination of magnesium in soil extracts.Results have been compared with those obtained by atomic-absorptionspectroscopy and show close correlation.R.J. HALL, G. A. GRAY and L. R. FLY"Ministry of Agriculture, Fisheries and Food, National Agricultural Advisory Service,Kenton Bar, Newcastle upon Tyne.Analyst, 1966, 91, 102-112.An Automatic Method for the Determination of AnionicSurface- active Material in WaterAn automatic version of the methylene-blue procedure for determiningalkylbenzenesulphonates in fresh and saline waters on the AutoAnalyzer isdescribed. Results have been examined statistically and compared with thoseobtained by the manual methylene-blue method.Student's t-test indicatesmore than 90 per cent. probability that the manual and automatic methodswill give the same value. The sampling rate is 14 per hour, and the standard de-viation is 0.04 p.p.m. of Manoxol OT in the range 0 to 4 p.p.m. of Manoxol OT.Whilst chloride, sulphide and sulphate showed no interference when tested,peaty water did interfere with the method. However, the results obtainedshowed a correlation to the colour (in p.p.m. of platinum) of the waterexamined. Emulsification, which causes considerable error in the manualmethod, is completely avoided in the automatic method.A. SODERGRENThe Norwegian Institute for Water Research, Oslo, Norway.Analyst, 1966, 91, 113-118viii SUMMARIES OF PAPERS I N THIS ISSUE [February, 1966Automatic Methods for the Determination of Nitrogen, Phosphorusand Potassium in Plant MaterialMethods are described, for use with the Technicon AutoAnalyzer, thathave been successfully used in the determination of nitrogen, phosphorusand potassium in plant materials. Nitrogen is determined as the indophenol-blue complex on an aliquot of solution after digestion by a micro-Kjeldahltechnique.Alternative methods are suggested for the final determinationof nitrogen dependent on the range of values expected. The “bias” methodis extremely sensitive to small variations in nitrogen levels, and, because ofthis high sensitivity, the recorder signal-to-noise ratio must be high.Methodsare suggested for obtaining a maximum signal-to-noise ratio. Phosphorusand potassium are analysed at the same time on the same sample of ashsolution with a single manifold. Phosphorus is determined colorimetricallyby the yellow phospho-vanadate complex, and potassium by flame photo-metry by using an internal standard of lithium nitrate. Nitrogen deter-minations can be carried out a t a rate of 40 samples per hour, and phosphorusand potassium together a t a rate of 60 samples per hour. The accuracy ofthese determinations is better than those obtained by other recognisedtechniques.J. A. VARLEYChemara Research Station, Seremban, Malaysia.AnaZySt, 1966, 91, 119-126.The Analysis and Composition of Potable Spirits : Determinationof C,, C, and C, Alcohols in Whisky and Brandy byDirect Gas ChromatographyThe determination of the principal C, to C, alcohols in potable spiritsby direct gas chromatography is discussed. Application of this methodto 78 samples of various types of brandy and whisky gives results indicatingthat the total higher alcohol content is not characteristic of the type of spirit,but that the proportions of the alcohols to each other fall into distinct rangestypical of the particular spirituous beverage.D. D. SINGERMinistry of Technology, Laboratory of the Government Chemist, Cornwall House,Stamford Street, London, S.E. 1.Analyst, 1966, 91, 127-134.The Detection of Nanogram Amounts of Fluoride IonShort PaperA. D. WILSON and J. R. COOKEMinistry of Technology, Laboratory of the Government Chemist, Cornwall House,Stamford Street, London, S.E. 1.Analyst, 1966, 91, 135

ISSN:0003-2654    

DOI:10.1039/AN96691FP025

出版商:RSC    

年代:1966

数据来源: RSC

摘要:

February, 19661 THE ANALYST xvT L r d e for classified advertisements is 5s. a line (or spaceequivalent of a liwe), with an extra charge of IS. for tkeuse of a Box Number. Semi-displayed classifiedadvertisements are 60s. per single-cotumn inch.BUILDING RESEARCH STATIONGarston, Watford, Herts.SPECTROGRAPHER (or trainee), graded ASSISTASTEXPERIMENTAL OFFICER/EXPERIMENTAL OFFI-CER, for long-tern1 programme of work on spcctrographic,flame-spectrographic and related methods of chemical analysis.QUALIFICATIOSS : Degree, Dip.Tcch., H.N.C., or equiva-lent in appropriate subject. Under 22, minimum qualificationis 2 G.C.E. “A” levels in Science and/or Maths. subjects.Experience of spectrography, photographic processing orelectronics useful, thongh not esscntial.SALARY: A.E.O.A568 (at 18)-&803 (at 22)-Llol7 (at 26or over)-L1243; E.O. (minimum age 26) L1365-Ll734.Prospects of pernianent pensionable appointmcnt.APPLICATION FORMS from the Director a t the aboveaddress quoting E/AF/lX. Closing date March lst, 1966.STAFFORDSHIRE COUNTY COUNCILCOUNTY CHEMICAL LABORATORYA4PPOINTMENT OF CHIEF ASSISTANT ANALYSTApplications are invited for the abovc-mentioned appoint-ment from persons having considerable experience in foodand drugs analysis preferably in a Public Analyst’s Labora-tory. Applicants’should havc the Branch E Diploma ofthe Royal Institutc of Chemistry or be qualified to take it.The salary will be 011 Lettered Grade “E” (@I 10 to A2450per annuni) and the appointment will be subject to theappropriate Superannuation Acts and Regulations.Assis-tance will be given towards removal expenses and there isa lodging allowance when a married man cannot transferhis home immediately.Applications, giving age, qualifications, experience andthe names and addresses of two referees and stating whetheror not the applicant is related to any member or seniorofficial of the County Council should be sent to the CountyMedical Officer of Health, County Buildings, Stafford, byMarch 7th, 1966.County Buildings,Stafford.T. H. EVANS,Clerk of the County Council.DERBYSHIRE COUNTY COUNCILANALYSTS required in the County Laboratory.Post 1 : Qualifications-Diploma of the Royal Instituteof Chemistry in Branch A, Branch E, or PhamaceuticalAnalysis. l h i s is a senior post, the duties include generalsupervision of junior staff and development of analyticalmethods.Salary L1770 rising by annual increments toA2110 (Grade C).Post 2 : Qualifications-Honours degree or G.R.I.C. anda t least two years post qualification experience in analysis ofFoods or Drugs. Duties will include some work on develop-ment of analytical methods. The work affords a good trainingfor the Branch E diploma. Salary L1495 rising by annualincrements to El745 (Grade A) with promotion to L1610-El940 (Grade B) for success in Branch E examination.Both posts are superannuable. Removal and Iodgingexpenses paid in certain circumstances. Houseloans available.Applications with names of two referees to The CountyAnalyst, County Offices, Matlock, Derbyshire.Canvassing disqualifies.FOOD ANALYSTA Senior Analyst is required by Bakers’ preparedmaterials manufacturers.Candidates should prefer-ably be of BSc. or G.R.I.C. standard, and must haveexperience in food analysis. Applications, includingdetails of age, qualifications and experience shouldbe sent to the Chief Chemist, Ch. Goldrei, Foucard& Son Ltd., Brookfield Drive, Liverpool, 9.~~~GREEN’S PURE FILTER PAPERSfor all kinds of filtrationWrite for descriptive catalogue 43/6.65J. BARCHAM GREEN LTD., HAYLE MILL,MAIDSTONE. KENTIJNIVERSITY OF BRISTOLDepartmcnt of Inorganic ChemistryM.Sc. COURSE Ilr; ANALYTICAL CHEMISTRY(With Special Reference to Instrumental Methods)Thc above course will be held ncxt scssion, and applicationsare invited from persons who hold, or expect to be awarded,an appropriate Honours degree or equivalent qualification.The Science Research Council has accepted the above courseas suitable for the tenure of its Advanced Course Studentships.This is a one-ycar course starting October Ist, 1966, andis based on the following topics:-Analytical spectroscopy, techniques of separation, clectro-analytical chemistry, and instrumental methods in general,including mass spectrorrietry and nuclear magnetic resonance.The aim of the course is to develop a research approach toAnalytical Chemistry and Instrumentation, and investigationsof certain problcnis will form a major part of the course.Applications should be sent to the Registrar, The Univer-sity, Senate House, Bristol, 2.CHEMISTArising out of internal promotion a vacancy exists in ourtechnical laboratory.The range of work is extremely variedand preference will be given to candidates with an interestin spectroscopy. Minimum qualification required is H.N.C.Chemistry. Starting salary will be in the range L900 toLll00 p.a.Please write for application forni to:The Personnel Manager,SMITHS INDUSTRIES LIMITED,K.L.G. Works,Putney Vale,London, S.W.1S.MINISTRY OF DEFENCE(ARMY DEPARTMENT)CENTRAL PATHOLOGY LABORATORYMALAYSIAEXPERIhlENTAL OFFICER (minimum age 26) requiredin the Central Pathology Laboratory, Tcrendak, Malaysia.DUTIES: Control of routine chemical pathology in thebiochemistry department, including quality control and theassessment and introduction of new techniques; assistancein the training of technicians in this subject.The appoint-ment will be for a period of 3 years initially and free familypassages will be available.QUALIFICATIONS : Degree, H.N.C. or equivalent in bio-chemistry.SALARY : E1440-LI819. Foreign Service Allowance a tpresent tax-free arid currently a t the following rates:Single A445-[585; married @lo-L1305.Interviews will be held in London.APPLICATIONS to Ministry of Defence, CE2(f)(AD),Northumberland House, London, W.C.2.STAFFORDSHIRE COUNTY COUNCILCOUNTY CHEMICAL LABORATORYAPPOINTMENT OF SENIOR ASSISTANT ANALYSTS (2)Applications are invited for the above-mentioned appoint-ments in the County Chemical Laboratory, Stafford fromcandidates who must have either the Higher National Certifi-cate with 5 years subsequent experience, or an OrdinaryDegree or Honours degree in Chemistry.The salary will be on the Grouped Grade A.P.T.III/IV/“A”(L1090-Ll745 per annum), with the commencing salaryaccording to qualifications and experience.Applications, giving age, qualifications, experience andthe names and addresses of two referees, and stating whetheror not the applicant is related to any member or senior officialof the County Council, should be scnt to the County MedicalOfficer of Health, County Buildings, Stafford, by March7th, 1966.County Buildings,Stafford.T. H. EVANSClerk of the Cou& CouncilFebruary, 19661 THE ANALYSTELECTRICITY E c 0 9ELECTRICITY COUNCIL RESEARCH CENTREwR E S E A R C H r.. . . . . When introducing a major new Research Establishmentthe first question* usually asked is "What is its purpose?" Well,our purpose is quite clear, it is to apply scientific methods toproblems of improving the use of electricity and its distribution.The range to be covered here is large, touching as it does thepossible use of electricity in all energy conversion processes inthe Industrial, Agricultural and Domestic fields, together with thecontrol of electricity supplied.The resultant research programme will develop in two ways-one, by tackling specific problems arising from currentusage-for example the control of electric arc furnaces;the other from applying basic research ideas formulated atthe Research Centre or elsewhere to produce novel orimproved application and equipment.To encourage the development of a really comprehensive approachto this programme, it will be tackled by teams composed ofResearch Officers from many disciplines-and these teams willinter-link with Project teams who will have the task of ensuringthat their research results in commercially reliable processes andequipment.We are in the first few months of the Research Centre's existenceand welcome queries, applications or 'phone calls (Hooton 31 29)from those who feel that they can, and would like to, take part inthis exciting research programme.D.C. Page, (Quote Ref: A/1)Head of Personnel Services,The Electricity Council Research Centre,Capenhurst, Cheshire.'The second question is usually *'What salary will they be offering?"f-this willdepend on ability experience and age, up to f3,OOO (approximately).ELECTRICITYELECTRICITY COUNCIL RESEARCH CENTRE 5 "ECf w0R E S E A R C H rxixIxxiv THE ANALYST [February, 1966THE SOCIETY FOR ANALYTICAL CHEMISTRYannounces the publication of“ANALYTICAL ABSTRACTS”DECENNIAL INDEX1954 to 1963This cumulative Index for Volumes I to 10will be published in mid1966 in two parts-Author and Subject sectionsFurther details will be nvailnble shortlFebruary, 19661 THE ANALYST xxvTHE SOCIETY FOR ANALYTICAL CHEMISTRYFOUNDED 1874.INCORPORATED 1907.THE objects of the Society are to encourage, assist and extend the knowledge and study ofanalytical chemistry and of all questions relating to the analysis, nature and compositionof natural and manufactured materials by promoting lectures, demonstrations, discussionsand conferences and by publishing journals, reports and books.The Society includes members of the following classes :-(a) Ordinary Members whoare persons of not less than 21 years of age and who are or have been engaged in analytical,consulting or professional chemistry; (b) Junior Members who are persons between the agesof 18 and 27 years and who are or have been engaged in analytical, consulting or professionalchemistry or bona Jide full-time or part-time students of chemistry.Each candidate forelection must be proposed by three Ordinary Members of the Society.If the Council intheir discretion think fit, such sponsorship may be dispensed with in the case of a candidatenot residing in the United Kingdom. Every application is placed before the Council andthe Council have the power in their absolute discretion to elect candidates or to suspend orreject any application. Subject to the approval of Council, any Junior Member above theage of 21 may become an Ordinary Member if he so wishes. A member ceases to be a JuniorMember on the 31st day of December in the year in which he attains the age of 27 years.Junior Members may attend all meetings, but are not entitled to vote.The Entrance Fee for Ordinary Members is L1 1s. and the Annual Subscription is L4-Junior Members are not required to pay an Entrance Fee and their Annual Subscription isE l 1s.No entrance Fee is payable by a Junior Member on transferring to Ordinary Member-ship. The entrance Fee (where applicable) and first year’s Subscription must accompanythe completed Form of Application for Membership. Subscriptions are due on January 1stof each year.Scientific Meetings of the Society are usually held in October, November, December,February, April and May, in London, but from time to time meetings are arranged in otherparts of the country. Notices of all meetings are sent to members by post.All members of the Society have the privilege of using the Library of The ChemicalSociety. Full details about this facility can be obtained from the Librarian, The ChemicalSociety, Burlington House, Piccadilly, London, W.1.T h e Analyst, the Journal of the Society, which contains original and review papers,information about analytical methods and reviews of books, and has a world-wide distribu-tion, and Proceedings of the Societyfor Analytical Chemistry, in which are reported the day-to-day activities of the Society, are issued monthly to all Ordinary and Junior Members. Inaddition, all Ordinary Members receive Analytical Abstracts, providing a reliable index to theanalytical literature of the world.Forms of application for membership of the Society may be obtained from the Secretary,The Society for Analytical Chemistry, 14 Belgrave Square, London, S.W.l.LOCAL SECTIONS AND SUBJECT GROUPSTHE North of England, Scottish, Western and Midlands Sections were formed to promote theaims and interests of the Society among the members in those areas.Specialised Groups within the Society are concerned with the study of various branchesof analytical chemistry of specialised or topical interest. Groups dealing with such topics asMicrochemical Methods , Biological Methods , Thin-Layer Chromatography, Atomic-AbsorptionSpectroscopy, Thermal Analysis, Automatic Methods and a Special Techniques Group,covering very new developments and specialised physical methods, are at present active-andfurther Groups are formed from time to time as the need arises.Non-members of the Society may be non-voting members of a Group.The Sections and Groups hold their own meetings from time to time in different places.Application for There is no extra subscription for membership of a Section or Group.registration as a member should be made to the Secretary of the Society

ISSN:0003-2654    

DOI:10.1039/AN96691BP037

出版商:RSC    

年代:1966

数据来源: RSC

摘要:

FEBRUARY, 1966 THE ANALYST Vol. 91, No. 1079 Some Sensitive and Selective Reactions in Inorganic Spectroscopic Analysis* BY T. S. WEST (Chemistry Department, Imperial College, London, S. W.7) THIS paper reviews some recent work in inorganic trace analysis by the author and his colleagues within the area of spectroscopic analysis. The work is concerned with analytical applications of the phenomena of absorption and emission of light by atoms or molecules in solution or flame media. The subject matter is, therefore, conveniently subdivided under four main headings, uiz.- Absorption Spectrophotometry in Solution (Absorptiometry). Absorption Spectrophotometry in Flames (Atomic-absorption Spectroscopy). Emission Spectrophotometry in Solution (Spectrofluorimetry) . Emission Spectrophotometry in Flames (Atomic-fluorescence Spectroscopy).In this paper, the philosophical aspects of each topic are stressed in relation to the achievement of maximum sensitivity and selectivity of determination, and examples are quoted from hitherto unpublished or recently published work. The main theme running through this work is the analytical application of chelation or co-ordination reactions. ABSORPTION TECHNIQUES SPECTROPHOTOMETRY- There are two main avenues worthy of exploration in search of selectivity of reaction for this technique. These are the design of highly selective reagents, and the use of mask- ing agents to achieve selectivity without recourse to separation. Most attempts to obtain selectivity have been directed towards the introduction of steric hindrance into reagent moleculcs close to the chelating centre.Some of the most commonly cited examples of selectivity do not, however, stand up to closer examination. For example, Merritt and Walker’s1 8-hydroxyquinaldine reagent is said to be unreactive towards aluminium, whilst retaining its activity towards other cations that react with 8-hydroxyquinoline. This is certainly true as far as non-precipitation of aluminium is concerned and the absence of any visible reaction] but we have found that, when attempts are made to extract other ions away from aluminium at pH 5.8 or above, significant amounts of aluminium are also extracted. The presence of the extracted complex may also be demonstrated in the chloroform extract by virtue of its fluorescence.2 Similarly, we have found that, although the literature describes neo-cuproine as a specific reagent for copper(I), it is only so within the context of spectrophoto- metry; many other ions, e.g., cadmium, cobalt, nickel and zinc, extract as colourless com- plexes.3 These examples illustrate that many selective reactions are only conditionally so, and that it is not always possible to transpose selectivity of reaction from one technique to another.* Presented at a meeting of the Society on Wednesday, October 6th, 1965. 6970 WEST: SOME SENSITIVE AND SELECTIVE [Analyst, Vol. 91 The principle of the clathrate or inclusion compound is now a familiar one. We have attempted to use this principle to construct a chelate cage molecule into which only ions of a certain size would fit. For example, the compound cycZo-tris-7-( 1-azo-8-hydroxynaphtha- lene-3,6-disulphonic acid) or Calcichrome, (I), at pH 12 reacts only with calcium (0.9 L&) and (1) not with strontium (1.1 A) or barium (1.3 A).4 Other ions that would normally be expected to fit into the cage are insoluble at pH 12 or are bound anionically in unreactive forms.This chromogenic reaction is, therefore, conditionally selective for calcium, and may be used to determine this ion spectrophotometrically in the presence of several thousandfold excesses of barium and strontium.6 The value of the molecular extinction coefficient is 7600, which compares with about 10,000 for murexide. The colour is, however, very stable and the selectivity is much greater. The compound Acid alizarin black SN, (11), resembles an opened out Calcichrome molecule and also possesses a good reactivity towards calcium, but its reaction with thorium, uranium and vanadium is of more interest.6 In the presence of masking agents such as cyanide, salicylate and 1,2-dihydroxybenzene-5-sulphonic acid at pH 4 to 4.2, thorium forms a 1 to 2 complex with Acid alizarin black SN,7 and gives a molecular extinction coefficient of 28,000 with only uranium and vanadium interfering.It is possible to determine all three metals sequentially by adding selective masking agents. Thus an excess of acetate masks thorium and uranium, whilst an excess of nitriloacetic acid masks thorium and vanadium. Both vanadium(v) and (IV) form complexes, of which the latter offers the better sensitivity.The molecular extinction coefficients for uranium and vanadium(1v) are 17,000 and 22,000. It occurred to us, however, that a better analytical sensitivity for thorium would be obtained by furnishing this molecule with additional hydroxyl groups so that it could form a 1 to 1 complex.6 This was done in the molecule (111), which behaves as expected and forms a (111) 1 to 1 complex with an extinction coefficient of 50,000 for thorium, 25,000 for uranium(v1) and 41,000 for vanadium(1v). This range of sensitivities compares favourably with the most sensitive reagents for these metals, e.g., morin ( E h 41,000) for thorium, dibenzoyl- meth;me (E fi 18,000) for uranium and diphenylbenzidene (E 23,000) for vanadium.February, 19661 REACTIONS IN INORGANIC SPECTROSCOPIC ANALYSIS 71 Some of the reagents that react with a great number of cations and can, therefore, be used as mass masking agents, have some gaps in their coverage.Thus EDTA may be said to be selectively unreactive towards silver, niobium, antimony and a very few other metals. We have found that the reagent bromopyrogallol red forms a particularly intense gelatin- sensitised colour reaction with niobium in a tartrate medium at pH 5 ~ 8 . ~ The 3 to 1 bromo- pyrogallol red - niobium complex has a molecular extinction coefficient of 60,000, which compares favourably with the standard thiocyanate method for niobium ( E = 35,000). In the presence of EDTA a 2 to 1 complex is formed, but the value of E falls to only 53,000. In this medium only tungsten, molybdenum, titanium and antimony interfere, and when a large excess of tartrate is added, the molecular extinction coefficient falls to only 47,500 whilst these ions no longer interfere.This sensitive and reproducible reagent system can be modified to allow the determination of niobium in steel down to 0.001 per cent. without recourse to the usual hydrolytic separation process for ni~bium.~ The reagent may also be applied to the determination of antimony(m), with which bromopyrogallol red forms a 1 to 1 complex in the presence of EDTA, cyanide and fluoride ions to give a molecular extinction coefficient of 39,000. The procedure appears to be superior in many respects to the standard Rhodamine B method ( E = 34,000) and is easier to operate.1° The sensitivity of absorbance measurements in solution is limited by two factors, both of which suggest that there is a lower limit of detection of about lo-* M for any complex in aqueous solution.The first factor is the inability of even a good spectrophotometer to measure to less than 0.001 absorbance units. Absorbance is defined as the logarithmic ratio of the intensity of the incident radiation (Io) to the intensity of the transmitted radiation (It). For trace amounts I , fi It and log I = 0. The second limiting factor is that molecules have a limited capacity to absorb light. Before light is absorbed, an electronic transition must occur within the molecule. The probability of such a transition is limited by several con- siderations that cannot be discussed in this paper, but any molecule can be regarded as having a light-capture cross-section. One method of increasing this is to spread a mesh of closely packed 7r orbitals in the molecule to capture photons and secure a transition.Braudell has discussed this extensively, and has shown that the maximum molecular extinction coefficient for any organic molecule is likely to be of the order of 100,000. It is, of course, possible to synthesise complex organic ligands possessing extensively conjugated-bond systems, but the task is a difficult one and the capability of such molecules to react efficiently with a metal cation is frequently rather small because of steric effects. For this reason we have paid very serious attention to the idea of utilising the formation of ternary complexes in which the cation reacts, not with one ligand species only, but rather with two.In this way it is possible for a much more complex absorbing organic envelope to be put around an ion than is normally possible, and consequently the sensitivity of such ternary systems is likely to be considerably superior. What is perhaps even more important is that the selectivity of ternary-complex formation is likely to be much superior to that of binary-complex formation. If we have a series of divalent metals, lM, 2rul, 3M, of similar chemical habit, they are likely to react to form complexes with a ligand, H2L of thenature of lML, 2ML, 3ML, etc., or lML2,-, 2MLi-, etc. As a generalisation, it is often permissible to say that the absorption characteristics of most metal complexes of a reagent tend to resemble closely the next higher ionisation stage of the ligand molecule considered in its reactive form as an acid.Hence, if a reagent reacts in the form HL-, its metal complexes frequently tend to have absorption spectra closely resembling that of L2-. Consequently, most metals form similarly coloured complexes so that selectivity is low. However, when two ligands are involved, e.g., H,L and H2R, the chances of duplication of ternary complexes of the nature M.L.R are much smaller, and this makes for selectivity of reaction. There are two chief routes to the formation of ternary complexes. In the first, before a metal ion can form a ternary complex of analytical utility, the situation must arise that one ligand does not fully satisfy all the co-ordinative requirements of the ion, so that the second ligand species can still react, i.e., neither ligand alone must form a co-ordination- saturated complex with the ion.Another alternative is that the first ligand, on entering the co-ordination sphere of the cation, fully satisfies it, but does so in a purely dative fashion, so that this primary complex ion still bears its over-all positive charge of the original central ion, and is free to ion-associate with a second ligand of suitable anionic charge to form a ternary complex.72 WEST: SOME SENSITIVE AND SELECTIVE [AnaZyst, Vol. 91 An example of the first type is the extremely useful ternary complex formed between alizarin complexan, cerium(II1) (or lanthanum) and fluoride ion.12J3,14,15716 This provides a sensitive and virtually specific method for the determination of the fluoride ion, which is probably the first instance recorded of the development of a positive colour reaction for the fluoride ion.The molecular extinction coefficient of this complex is of the order of 30,000, and we are currently developing similarly selective ternary complex systems for fluoride ions which yield molecular extinction coefficients of the order of 90,000.17 The second type of complex is well illustrated by a method we have recently described for the determination of the silver ion. A ternary complex formed between silver, 1,lO-phenan- throline and bromopyrogallol red, (AgPhen,) ,BPR, yields an extinction coefficient of 51,000, and in the presence of EDTA, fluoride and peroxide, only gold(II1) interferes.18 The stability of the colour system is vastly superior to that of silver - dithizone, and it is more sensitive (E -rr 30,000 for silver - dithizone).More recently we have developed3 this principle for the determination of the metals shown in Table I. The values quoted for the extinction coefficients TABLE I TENTATIVE SENSITIVITIES OF TERNARY ION-ASSOCIATION SYSTEMS Ion determined Cadmium Cobalt Copper (11) Manganese Nickel Lead Zinc Molecular absorptivity 92,000 (Ethyl acetate) 92,000 (Ethyl acetate) 75,000 (Ethyl acetate) 65,000 (Ethyl acetate) 50,000 (Chloroform) 70,000 (Nitrobenzene) 95,000 (Ethyl acetate) Molecular absorptivity (dithizone) 85,000 59,000 45,000 32,000 34,000 72,000 94,000 of these complexes extracted into chloroform, ethyl acetate or nitrobenzene, are tentative, and are capable of improvement by optimisation of conditions.In this series tetra-iodotetra- chlorofluorescein (Rose Bengal extra, C.I. 45440) is used as the counter ion for the central metal - phenanthrolinium cation. The mechanism of the colour reaction is of considerable interest, but this factor cannot be discussed in this paper. It should be added, however, that inter-element selectivity for the ions shown in the table can readily be achieved and we have so far devised specific procedures for copper and lead within the group itself.3 Another principle that appeared attractive in order to overcome the “sensitivity barrier” in spectrophotometry is the application of an amplification procedure. The Leipert amplification of iodine is, of course, well known.It results in a 6-fold yield of iodine per original iodide ion subjected to analysis. We have recently applied this principle to the determination of phosphorus.lg The phosphate ion is converted to phosphomolybdate, in which 12 molybdate ions are associated with each phosphate ion, and the complex is extracted away from the excess of molybdate and any other heteropoly acids formed from antimony, arsenic, germanium and silicon by means of butanol- chloroform. The extract is then put in contact with a pH 9 buffer, which re-extracts the phosphate ions and the associated 12 molybdate ions into the buffer. In this medium these are no longer chemically combined, so that the molybdate ions can now be made to react with the sensitive reagent, 4-chloro- 2-aminobenzenethiol, to give an easily measured complex.This amplification procedure results in an effective molecular extinction coefficient of 360,000 for phosphate ion, so that solutions as dilute as 0.008 p.p.m. of phosphorus may easily be determined. By contrast, the standard molybdenum-blue procedures have values of about 27,000. A similar, but considerably less sensitive and selective, procedure for phosphate has recently been described by Umland and Wunsch.20 ATOMIC-ABSORPTION SPECTROSCOPY- Atomic-absorption spectroscopy is, of course, concerned with the light-absorption charac- teristics of atoms as opposed to molecules, and solution and cuvette are replaced by a steady- state flame of suitable physical characteristics, to maintain a population of free ground-state atoms.A conventional monochromated light source is not acceptable for this technique because of the narrow profile of absorption bands due to free atoms. Consequently, sources such as hollow-cathode lamps, capable of emitting even narrower bands, must be used. The technique is more sensitive than flame (thermal emission) photometry because, for the majority of elements in the majority of flames, the overwhelming bulk of free atoms remainFebruary, 19661 REACTIONS IN INORGANIC SPECTROSCOPIC ANALYSIS 73 in the non-emitting ground state. It is also a more selective technique than flame photometry because it is very free from inter-element interference. It is not very dependent on flame temperature, whereas flame photometry exhibits an exponential dependence. However, like solution absorptiometry, it follows the same physical laws, and has a maximum sensitivity which is controlled by log Io/It fi 0 for traces (a signal that is independent of amplifier gain) and by the limiting laws of the probability of an electronic transition.Most instruments for atomic-absorption analysis of traces are, therefore, operated under conditions of maximum sensitivity. Devices such as long flames, heated tubes, multiple traverse of flames, are being examined on the one hand, and other substrates such as plasma jets and sheathed flames on the other, to obtain higher atomic populations for such “difficult” metals as niobium, molybdenum and titanium. Since selectivity of reaction is an inherent property of atomic-absorption spectroscopy, we have turned our attention principally to the use of organic complexing reactions as a means of increasing sensitivity.This can be achieved by virtue of the fact that most metals can be persuaded to partition into water-immiscible solvents as metal chelate complexes. After the solution has been sprayed into the flame, the, droplets evaporate to solid particles, which then dissociate to free atoms. When a solvent such as an ester or ketone is sprayed, the efficiency of aspiration increases greatly. Thus the throughput ratio of ethyl methyl ketone to water, in terms of ml per minute of liquid fed into the flame is about 4.19 When the ratio of absorbance signals of a metal, extracted into an equal volume of this solvent, is compared with that of an equal concentration of the metal in aqueous solution, a 4-fold enhancement of signal is generally obtained. Other advantageous factors inherent in the spraying of solutions of metal chelates are that the droplets evaporate more quickly and are smaller, and that the solids so formed are more volatile and the majority dissociate exothermally. In addition, ions that would formerly have been associated with anions such as phosphate (which are relatively difficult to break down) are no longer chemically bound to them.The relative importance of all these factors will vary from system to system, but undoubtedly one of the chief advantages is the improvement in rate of aspiration into the flame. Sensitivities can yet again be increased by control of phase ratios.Thus for an apparatus that gives a calibration-curve range of 1 to 10 p.p.m. of silver in aqueous solution (absorbance from about 0.04 to about 0.3), a calibration curve of 1 to 5 p.p.m. (absorbance from about 0.1 to about 0.5) is obtained after extraction into an equal phase volume of hexone, whilst extraction from a large volume of aqueous solution into a smaller volume of the same solvent in a simple separating funnel gives a calibration curve of 0.01 to 0.10 p.p.m. of silver in the original aqueous solution (absorbance from about 0.03 to 0.25).21 In some instances organo-metallic complexes break down too easily in a flame, e.g., tetraethyl-lead, so that calibration curves can only be constructed against standards prepared from tetraethyl-lead and measured at the lowest possible point in the flame.22 In other instances, e.g., the formation of a co-ordination complex of platinum with the thiocyanate ion or dithizone, can lead to the formation of stable complexes that do not break down to atomic species in the flame.23 Both these results were obtained with air - propane flames; variation of flame composition could alter these observations substantially.In other instances, such as the determination of copper in niobium and tantalum,24 the use of solvent extraction permits the separation and concentration of traces of copper from preponderant amounts of matrix material, which might otherwise lock up the ion being determined in a refractory oxide in the flame, or which might block up burner heads and other orifices.Within certain limits the type of extractant used is immaterial, because the specificity of the atomic-absorption technique circumvents interference from other co-extracted trace impurities. EMISSION TECHNIQUES Because of the inherent limitations of absorbance techniques with respect to lower limits of detection and determination, the author and his colleagues have been concerned with investigating simiIar techniques that might be suitable for adaption to analytical pro- cedures, with detection or determination limits several orders of magnitude beyond those of absorptiometric procedures. The answer in respect of both solution and flame techniques appeared to lie in the development of emission techniques in both media. There are three principal emission phenomena observed in solution.These are fluorescence, phosphorescence and chemi-luminescence. This paper is concerned only with the first74 WEST : SOME SENSITIVE AND SELECTIVE [Analyst, Vol. 91 mentioned. Phosphorescence in solution is rare, and may indeed be considered as a special type of long-lived fluorescence induced by the existence of triplet-excited electronic states. Chemi-luminescence is also rather rare in solution, and arises chiefly as a result of free-radical reactions involving the luminescent species. The stability of luminescent species is very small, so that the technique does not readily lend itself to direct analytical manipulation.26 The chief emission phenomena in flame media are fluorescence and thermal emission, although flame luminescence is also not unknown.The technique of flame photometry, which is based on thermal excitation, is well known, and chemi-luminescence in flames is relatively rare. Accordingly, this paper is once more concerned only with fluorescence phenomena in flames. SPECTROFLUORIMETRY- The fundamental aspects of analytical spectrofluorimetry in solution have recently been described elsewhere by the author,26 as have the basic requirements with respect to instru- mentation of the te~hnique,~’ and will not, therefore, be described here. A molecule in solution absorbs light $7 reacting with the photons that are in contact with it for about second (ie., the period of oscillation of the electromagnetic wave). The molecule will only absorb light that is keyed to the energy difference between its ground state and an excited electronic state (ie., the absorption is quantised).The excited-state molecule is unstable and will tend to get rid of its surplus energy. Those stable substances that we normally think of as coloured shed their energy by various intermolecular processes that may be called radiationless transfers and which amount to radiation of the energy as heat. Some absorbing species, however, have abnormally stable excited states, so that these can hold on to their acquired energy for up to lo-* second. During this relatively long period, part of the absorbed energy will be degraded by intramolecular vibrations that occur within 10-l2 second until the molecule reaches its lowest vibrational level. These excited- state molecules then have a very high probability of releasing their surplus energy radiatively as a single photon corresponding to the difference between the energy levels of the lowest vibrational state of the excited level and one of the vibrational states of the ground state, i.e., some of the absorbed light is re-emitted as light of a longer wavelength.The emitted light is characteristic of the molecule and invariably bears a mirror-image relationship to the longest-wavelength absorption band of the molecule. For dilute solutions that absorb only a small fraction of the exciting radiation, I,, the basic equation which relates the intensity of fluorescent light, F , to the concentration of the fluorescing species, C, may be simply stated as- where K is a proportionality constant made up of the quantum efficiency of the system, 4, the molecular extinction coefficient at the wavelength of excitation, E , and the absorbing pathlength in solution, 1. Comparison of this basic equation with that for absorbance, viz.- reveals that here no logarithmic dependence on a ratio of intensities is involved.Secondly, the law includes an I,, term so that the greater the intensity of the exciting radiation, the greater the analytical signal, F . Yet again, since F normally represents the output from a photomultiplier tube, it can be electronically amplified within reasonable limits imposed by the “noise” of the detector and the circuit. Furthermore, two sets of spectra, the excitation and emission spectra, become available for detection and determination.This also is in marked contrast to the availability of analytical information from absorbance measurements. Unquestionably, the technique is a far more sensitive one than absorption spectro- photometry, and it is surprising that analytical chemists have paid so little attention to it. The lack of suitable commercial equipment may be partly responsible, but there is also a dearth of reagents for inorganic analysis. The last-mentioned factor can only be set right by analytical chemists themselves. F = K . I , . C A = log Io/It = E . L . C We are currently investigating spectrofluorimetry from two angles- (a) the development of a range of spectrofluorimetric reagents for a wide range of inorganic ions; and (b) the tracing of relationships between spectrofluorimetric activity and reagent structure.February, 19661 REACTIONS IN INORGANIC SPECTROSCOPIC ANALYSIS 75 Generally, in absorption work, the more complicated and massive the molecule, the greater its molecular extinction coefficient.The converse, happily, appears to be true in spectrofluorimetric analysis. For example, we have found it possible to determine traces of thallium(1) down to 0.01 p.p.m. in a medium simply made 3.3 M with respect to hydrochloric acid and 0-8 M with respect to potassium chloride.28 Irradiation of this solution at 250 mp produces blue fluorescence emission peaking at 430 mp. Out of 42 cations and 11 anions examined, only relatively large amounts of lead, copper(II), tin and cerium(m) produced a fluorescence.Many of the other ions interfered by precipitating or by inner-filter effects, but all these except for gold, bismuth, platinum and antimony, were eliminated by a simple separation process. The thallium(1) was oxidised to thallium(m) by hydrogen peroxide and extracted from 1.5 M hydrochloric acid by diethyl ether. It was then back-titrated into an aqueous phase and concomitantly reduced to thallium(1) by aqueous sulphur dioxide, after which the latter was boiled out, the acidity adjusted with hydrochloric acid and measurements made as already described. Similarly, we have applied the reagent 2-hydroxy-3-naphthoic acid to the spectrofluori- metric determination of beryllium down to 0-0002 p.p.m. (20 nanograms) by using the calcium salt of CDTA as the masking agent29 at pH 5.8 in an acetate buffer.Only those ions interfere that cannot be held in solution as soluble salts under these conditions, i e . , bismuth, cerium(m), chromium, iron, tin, titanium and thorium. Similarly, the reagent salicylidene-o-aminophenol may be used to determine aluminium down to 27 nanograms.30 When the procedure is used in combination with a diethyldithio- carbamate extraction procedure, out of 46 cations examined, only chromium(11r) , scandium and thorium showed interference. We have similarly developed methods for the deter- mination of scandium with salicylaldehyde semicarba~one,~~ for molybdenum and tungsten with carminic gallium and aluminium with salicylidene-o-aminophenol,33 and are developing procedures for others such as gold, copper and phosphorus.There are few manipulative difficulties in spectrofluorimetry, and we have encountered no serious problems from quenching effects due to oxygen or from steep temperature gradients on calibration curves. Indeed, there is no real reason why this technique should not be developed as extensively as absorption spectrophotometry. Even with ordinary instru- mentation it appears to be capable of yielding limits of determination three orders of magnitude lower. ATOMIC-FLUORESCENCE SPECTROSCOPY- It is a logical sequence to proceed from emission spectrophotometry in solution to emission spectrophotometry in flames. Thermal excitation of atoms in flames is an inefficient process. Except for a very few metals, even in the hottest flames the majority of atoms present in the flame plasma remain in the ground state, and the small number of atoms in the emitting excited state varies exponentially with changes in flame temperature. The emission is also very subject to inter-element effects, so that extensive use has to be made of radiation buffers and similar devices.However, atoms in the ground state can readily be persuaded to fluoresce by irradiating them with light of the correct wavelength required to produce an electronic excitation. Usually this light corresponds to that used for measurements of atomic absorbance. The atomic state in a gaseous phase exhibits the phenomenon of resonance re-radiation, i.e, the excited atoms fluoresce light of the same wavelength as they have absorbed, but with diminished intensity owing to the quantum efficiency of the process.Normal fluorescence of longer wavelengths may also be observed in some instances, although it is a good deal less common than in (molecular) fluorescence in solution. The basic principles of this technique are so much a blend of the previously discussed techniques of spectrofluorimetry in solution and atomic absorbance in flames that little need be said of them. As before, the analytical fluorescence signal, F , may be expressed as- F = K . I o . C . Again, there is a linear signal that may be multiplied or augmented as necessary by control of I,,, and also the possibility of electronic amplification exists where it does not in atomic absorbance.76 WEST: SOME SENSITIVE AND SELECTIVE [Analyst, Vol. 91 The experimental arrangements of atomic-absorption spectroscopy and of atomic- fluorescence spectroscopy may best be expressed diagrammatically- I Absorbance = Log 2 I T Signal = I F IF = K.1, [Atom,] In some respects the instrumental demands of atomic-fluorescence spectroscopy are simpler than those of atomic-absorption spectroscopy.Incident light of a very narrow spectral profile is required for atomic-absorbance measurements because of the narrow band- width of the absorption bands. This is best obtained from a hollow-cathode lamp, with the cathode made from the metal concerned or one of its alloys. On the other hand, for atomic- fluorescence spectroscopy, much cheaper and more readily obtained non-reversed sources such as spectral-discharge lamps may be used. The freedom from interference, characteristic of atomic-absorption spectroscopy, should be almost as forthcoming in atomic fluorescence so that isotopic analysis should be possible, and the sensitivity of the method may well be several thousand times that of atomic absorption. Some factors such as self-absorption and scattering, which are of little or no account in atomic absorption, may produce additional problems in this newer technique, and others, such as quenching, which are not observed in absorption, require to be considered.Scattering may not be eliminated by light modulation in the same way as thermal emission is eliminated in atomic absorption, but the avoidance of total-consumption burners and the use of the fine sprays that are obtained when organic solvents are used, would greatly minimise such problems.Thermal emission in the flame may be rendered harmless by modulating the source and a.c. amplification. In preliminary experiments we have readily been able to detect cadmium down to M solutions, and obtain measurable responses with as little as 0-2 nanograms of cadmium ion.34 Using the 2288 A line, which promotes the 5 ‘ ~ ’ ~ --f 5’$1 transition, and measuring the resonance emission obtained with solutions of cadmium extracted into ethyl acetate as the [CdIZ-] complex from an acid solution, we have obtained results accurate to within + 1 per cent. at the level, and have found that cadmium suffers no interference from 100-fold amounts of NH,, Ag, Al, As, Au, Ba, Be, Bi, Ca, Ce, Co, Cr, Cu, Fe, Ga, Hg, In, K, La, Li, Mg, Mn, Mo, Na, Ni, Pb, Sb, Sc, Se, Sn, Sr, Te, Th, T1, U, V, W, Y, Zn, Zr or from acetate, borate, bromide, citrate, chloride, perchlorate, cyanide, fluoride, oxalate, phosphate, silicate, sulphite, sulphate or tartrate.When the following complexing agents were present : peroxide, EDTA, 1 ,lo-phenanthroline or reducing agents such as ascorbic acid or hydroxyl- ammonium chloride, interference was observed only from the phenanthroline. This was because of the formation of a precipitate. When sufficient acid was added to dissolve it, no interference was encountered. These results were obtained with rather crude and inefficient apparatus, and are capable of considerable improvement by modifications to obtain greater efficiency of irradiation and light collection. Undoubtedly, this is a technique that will prove to be invaluable for inorganic trace analysis in the nanogram range in the near future.Practically the only papers to have appeared on analytical aspects of this technique are those by Winefordne9~36,3~ ~ 3 8 and his co-workers. The author wishes to express his indebtedness to students cited in the references, and in particular to his colleagues, R. M. Dagnall and G. F. Kirkbright. Sincere thanks are also given to many industrial firms who have contributed apparatus and grants in aid of research, and especially to the Science Research Council, without whose support none of the work on atomic-absorption spectroscopy or spectrofluorimetry would have been possible.February, 19661 REACTIONS I N INORGANIC SPECTROSCOPIC ANALYSIS REFERENCES 77 1.2. 3. 4. 6. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 1 7 . 1s. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. Merritt, L. L., and Walker, J. K., I n d . Engng Chem., Analyt. Edn, 1944, 16, 387. Dagnall, R. M., West, T. S., and Young, P., Analyst, 1965, 90, 1066. Bailey, B., Dagnall, R. M., and West, T. S., Talanta, in the press. Close, R. A4., and West, T. S., Ibid., 1960, 5, 221. Herrero Lancina, M., and West, T. S., Analyt. Chem., 1963, 35, 2131. Kusakul, P., and West, T. S., in preparation. Belcher, R., Ramakrishna, T. V., and West, T. S., Talanta, 1965, 12, 681. Ramakrishna, T. \-., and West, T. S., in preparation. Christopher, D. H., and West, T. S., TaZanta, in the press. Uraude, E. A,, in Braude, E. A., and Nachod, F. C., Editors, “Determination of Organic Structures Belcher, R., Leonard, M. h., and West, T. S., Talanta, 1959, 2, 92. , , Analytica Chim. Acta, 1965, 32, 301. -- by Physical Methods,” Academic Press Inc., N.Y., 1955, L-olume I, p. 131 et seq. , , J.. Chem. Soc., 1958, 2390. --- 9 , , Ibzd., 1959, 3577. Leonard, M. A,, and )Vest, T. S., Ibid., 1960, 4477. Belcher, R., and West, T. S., Talanta, 1961, 8, 853 and 863. Cabello-Tomas, L., and West, T. S., in preparation. Dagnall, R. M., and West, T. S., Talanta, 1964, 11, 1533. Djurkin, V., Kirkbright, G. F., and West, T. S., Analyst, 1966, 91, 89. Umland, F., and Wiinsch, G., Z. analyt. Chem., 1965, 213, 186. Belcher, R., Dagnall, R. M., and West, T. S., TaEanta, 1964, 11, 1257. Dagnall, R. M., and West, T. S., Ibid., 1964, 11, 1553. Cabrera, A. M., and West, T. S., unpublished work. Kirkbright, G. F., Peters, M. I<., and West, T. S., unpublished work. Howard, P., and Wcst, T. S., unpublished work. West, T. S., Lab. Pract., 1965, 922. Kirkbright, G. F., West, T. S., and Woodward, C., Talanta, 1965, 12, 677. Dagnall, R. M., Smith, R., and West, T. S., Talanta, in the press. Kirkbright, G. F., West, T. S., and Woodward, C., ‘4naZyst, 1966, 91, 23. - ~ - , in ereparation. Dagiall, R. M. Smith, R., and West, T. S., Chem. & Ind., 1965, 1499. Dagnall, R. M., West, T. S., and Young, P., Talanta, in the press. Winefordner, J . D., and Vickers, T. J.. Analyt. Chem., 1964, 36, 161. Winefordner, J. D., and Staab, R. A., Ibid., 1964, 36, 165. -___ , Ibid., 1964, 36, 1369. Mankeld, J. M., Winefordner, J. D., and Veillon, C., Ibid., 1965, 37, 1061. ~-~ -, Ibid., 1965, 1030. , I , Analyt. Chem., 1965, 37, 137. ~ - - Received September loth, 1965

ISSN:0003-2654    

DOI:10.1039/AN9669100069

出版商:RSC    

年代:1966

数据来源: RSC

摘要:

78 MUNRO AND FLECK: RECENT DEVELOPMENTS IN THE jlifzaiyst, i’o1. 91 Recent Developments in the Measurement of Nucleic Acids in Biological Materials A Supplementary Review* B Y H. N. MUKRO AND A. FLECK? (Department of Biochemistry, The University of Glasgow) SUMMARY OF CONTENTS Introduction Preparation of tissue samples for nucleic acid determinations Precautions during removal of tissues Extraction of acid-soluble compounds Extraction of lipids 1. Methods of determining nucleic acids in the tissue residue The procedure of Schmidt and Thannhauser The use of alkaline hydrolysis to separate RNA from DNA The RNA fraction of the alkaline digest The DN-4 fraction of the alkaline digest Recommendations for the use of the Schmidt - Thannhauser method 2. The Schneider procedure 3.4. Other procedures The procedure of Ogur and Rosen General recommendations for nucleic acid determination. IN 1961, Hutchison and Munrol reviewed the literature on methods of determining nucleic acids in biological materials. This article is an account of developments that have taken place since 1961 in the field of nucleic acid analysis. For convenience, the main topics will be dealt with in the same order as that used in the earlier review. Under each heading the conclusions reached in the 1961 review will be summarised, and subsequent developments will then be discussed. A comprehensive survey of nucleic acid analysis based on the literature covered by both reviews is to be published elsewhere.2 Since 1961, most investigators have relied on modifications of the procedure of Schmidt and Thannhauser3 as the method of choice for determining the nucleic acid content of tissues.Consequently, in this survey of recent developments we shall devote more space to this method than to the other two main procedures, namely the Schneider method4 and the method of Ogur and R o ~ e n . ~ Nucleic acids contain 3 distinct components ( a ) purine and pyrimidine bases, ( b ) ribose or deoxyribose and (c) phosphorus. The principal methods of determining nucleic acids have therefore been based on the strong ultraviolet absorption of the bases, or on specific colour reactions for the pentoses, or on the determination of phosphorus. Procedures depend- ing on ultraviolet absorption or on phosphorus determination are clearly common to both RNA and DNA and therefore demand preliminary separation of the two nucleic acids.This is the objective in the original Schmidt - Thannhauser3 method in which phosphorus deter- mination is used, and in the Ogur - Rosen5 procedure which depends on ultraviolet-absorption measurement of the separated nucleic acids. On the other hand, in the Schneider method4 RNA and DNA are measured on the same tissue extract by specific colour reactions for ribose and deoxyribose, respectively. Before these specific methods for nucleic acid determination are used, certain procedures have first to be applied to the tissue. Usually this preliminary treatment involves the removal of small molecules (e.g., free nucleotides) and if necessary, lipids, since these may interfere with later chemical determinations. This initial treatment is followed by extraction of the * Reprints of this paper will be available shortly.For details sce Summaries in advertisement pages. t Present address : Departmcnt of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass.February, 19661 MEASUREMENT OF KUCLEIC ACIDS IK BIOLOGICAL MATERIALS 79 nucleic acids from the tissue residue and their subsequent determination. Consequently, the main methods for nucleic acid determination can be considered as having 3 stages- (a) Preliminary preparation of the tissue samples for nucleic acid determinations. This generally involves removal of small molecules and sometimes lipids; this stage is common to all three main procedures for nucleic acid measurement.(b) A procedure for the extraction and, if necessary, separation of the nucleic acids. In the Schmidt - Thannhauser method, hydrolysis of RNA in alkali is used to extract it from the residue and separate it from DNA, which resists attack by alkali and can therefore still be precipitated with acid. In the Schneider procedure, both nucleic acids are extracted from the tissue simultaneously with hot acid. In the Ogur - Rosen method, the RNR is extracted with cold perchloric acid and the DNA is then obtained by applying hot perchloric acid to the tissue. Application of a specific procedure for determining the nucleic acid present in the extract. (c) THE PREPARATION OF TISSUE SAMPLES FOR NUCLEIC ACID DETERMINATIONS PRECAUTIONS DURING REMOVAL OF TISSUES- It was pointed out in the earlier review1 that considerable losses of nucleic acids can occur through enzymic action if a tissue is not rapidly cooled on removal and kept chilled during the preliminary stages of the analytical procedure.In order to avoiasuch losses, it has long been the practice to excise and homogenise the tissue at 0" C. In most instances, rapid excision, homogenisation in cold water and addition of a cold protein precipitant have been found to be adequate. However, some workers have considered it necessary to take additional precautions for special purposes. If the free nucleotide pool is to be examined, Saukkonen6 recommends freezing the tissue instantly in liquid nitrogen, a procedure for which he gives full details and references. This method was used by Logan, Mannell and Rossiter' for determining nucleic acids in nervous tissue, as the time required for dissecting out this tissue can be quite lengthy.Despite this, no such special precautions were recorded in some recent investigations into the concentrations of nucleic acids in the b ~ a i n . ~ ~ ~ In a review of nucleic acid determination in plants, Markhamlo suggested that the tissue should be plunged into boiling ethanol in order to inactivate nucleases. Several authors studying plant material1l9l2 9 1 3 and ova14 have recently reported that this procedure is satis- factory, and in unpublished studies we have found the method to be readily applicable to rat liver. Rapid dissolution of tissue with inactivation of enzymes has also been achieved by immersing the sample in detergent solutions, such as deoxycholate in a solution of urea16 and 1 per cent.Triton X 100.16 If the tissue is not immediately analysed, the investigator usually freezes the sample. The conditions of storage can sometimes be critical. May and Grenelll' reported large losses of RNA from rat brain stored in a deep-freeze cabinet, but Smithls found.no change in liver RNA content under these conditions. However, Curriel9 has demonstrated that the xanthine content of rat liver increases during storage at -13" C, suggesting that some enzyme activity continues even at this temperature. I t is therefore essential that the analyst should ensure, by comparison of results obtained from both fresh and stored tissue samples, that the conditions of storage are adequate to prevent serious losses of nucleic acids.EXTRACTION OF ACID-SOLUBLE COMPOUNDS- Three groups of tissue components of low molecular weight may interfere with the estimation of tissue nucleic acids : ( a ) free nucleotides ; (b) carbohydrates ; (c) inorganic phos- phate and organic phosphorus compounds of low molecular weight. For example, it has been calculated2 that failure to remove adenine nucleotides would result in a 10 per cent. over-estimation of the RNA content of rat liver. The usual procedure for removal of such compounds of low molecular weight is by precipitation with cold acids, usually trichloroacetic acid or perchloric acid. Various concentrations of acid and conditions of extraction have been used; these were discussed, in detail, in the earlier review.1 In general, maximal removal of acid-soluble phosphorus has been used as the criterion of satisfactory extraction of small molecular weight compounds.Recently, Hallinan, Fleck and Munro20 have used a modified Schmidt - Thannhauser method of nucleic acid determination in order to ascertain the optimal conditions of extraction of low molecular weight contaminants with trichloroacetic acid and80 MUNRO AND FLECK: RECENT DEVELOPMENTS IN THE [Analyst, lTOl. 91 perchloric acid. After homogenisation of liver samples in cold water, cold acid was added to give various concentrations of acid, and the tissue residue was then washed twice with the same concentration of acid. They found that various concentrations of trichloroacetic acid, from 5 per cent.to 20 per cent., all gave maximal recovery of RNA. With perchloric acid, the same recovery of RNA was also obtained after precipitation with cold 0.2 N acid, but with concentrations above 0-3 N, the recovery of RNA fell progressively. This latter observation is in agreement with the findings of Ogur and R o ~ e n . ~ It is consequently important not to use high concentrations of perchloric acid to remove small molecules. In unpublished experi- ments we have shown that the use of 0.2 N perchloric acid at 0" C also results in maximal precipitation of DNA and protein from rat liver. The general use of this concentration of perchloric acid in the initial stages of nucleic acid determination can therefore be recom- mended, especially since perchloric acid has the advantage over trichloroacetic acid that it does not absorb ultrcviolet light and thus does not interfere with subsequent nucleic acid deter- minations involving ultraviolet absorption.However, it should be noted that the standard conditions of extraction with 0.2 N perchloric acid or with 10 per cent. trichloroacetic acid have been found to give rise to some difficulties with certain tissues. This has been reported by two authors2lyz2 studying yeast, and detailed discussion of difficulties in the use of cold acid to extract plant tissues is provided by Smillie and K r ~ t k o w . ~ ~ Finamore and Volkin24 reported earlier that half of the RNA isolated with phenol from amphibian eggs is soluble in 0.5 N perchloric acid, but this material has since been shownz5 to consist of small oligo- nucleotides." The claim by Levy and Lynt26 that some RNA in HeLa cells remains unpre- cipitated by 1.0 N perchloric acid may merely imply that the expected degree of breakdown of RNA at this concentration of perchloric acid had occurred in their experiments.The technique of cold-acid precipitation for removal of small molecules can be applied in two ways. We prefer to begin by homogenising the tissue in cold water and then adding cold acid to samples of the homogenate. The alternative approach is to homogenise the tissue directly in the cold-acid precipitant, but this often leads to clogging of the homogeniser with precipitate, specially in the Potter - Elvehjem type of homogeniser, and, in addition, sampling of the homogenate becomes difficult.A specially designed homogeniser for rapid tissue disintegration and simultaneous addition of acid has been recently de~cribed.~' EXTRACTION OF LIPIDS- The original procedures of Schmidt and Thannhauser3 and of Schneider4 include a stage at which tissue lipids are extracted, in order to remove the phosphorus present in the tissue in the form of phospholipids. As most modern methods of nucleic acid determination do not depend on phosphorus determination, this step is no longer obligatory, but is never- theless still extensively used. A considerable variety of lipid-extraction procedures have been used in the past. They are discussed in detail in the earlier review,l in which a standard extraction procedure was offered involving ethanol, then ethanol - chloroform, ethanol - ether and finally ether.The question of adverse effects of lipid solvents on recoveries of nucleic acids was raised by Venkataraman and Lowe,28~29 who treated liver samples with 5 per cent. trichloroacetic acid to remove small molecules and then extracted the lipids with cold 95 per cent. ethanol. Under these conditions, they observed losses of up to 30 per cent. of the tissue RNA. In our earlier review,l it was pointed out that several other authors4~30~31~32 had reported that no losses occurred when lipid solvents were used to extract tissues precipitated with cold trichloroacetic acid, and it was concluded that the findings of Vcnkataraman and Lowe did not have general application to nucleic acid determination. However, we subsequently noted that all the investigators who observed no loss of RNA had used 10 per cent.trichloroacetic acid, whereas Venkataraman and Lowe had used 5 per cent. trichloroacetic acid. Accordingly, Hallinan, Fleck and Munro20 re-investigated the situation and found that the concentration of cold acid applied initially to the tissue was critical in determining the extent of loss of RNA into ethanol and other lipid solvents. They showed that about 40 per cent. of rat-liver RNA was extracted when ethanol was applied to liver samples after precipitation with 5 per cent. trichloroacetic acid, whereas after treatment with 10 per cent. trichloroacetic acid the loss is only about 10 per cent., and after using 15 per cent. trichloroacetic acid it fell to 5 per cent.Hallinan, Fleck and MunroZO also demonstrated that a similar effect occurred when tissues precipitated with perchloric acid were subsequently extracted with ethanol. After precipitation with 0-2 N perchloric acid, ethanol extracted about 25 per cent. of the RNAFebruary, 19661 MEASUREMENT OF NUCLEIC ACIDS IN BIOLOGICAL MATERIALS 81 from liver samples, whereas after treatment with 0.7 N perchloric acid, the loss into ethanol was small. However, when perchloric acid is used, degradation of RNA by the cold perchloric acid becomes significant at concentrations above 0.3 N, so that the reduced loss into lipid solvents resulting from higher acid concentrations is offset by degradation at the stage of cold-acid precipitation. As pointed out in the earlier review, Marko and Butler33 observed that DNA was often degraded if the tissue samples were treated with cold trichloroacetic acid and then extracted with hot lipid solvents, owing to the retention of acid by the lipid solvents.This effect could be prevented by buffering the first lipid solvent with potassium acetate. Steele, Okamura and BuschN have shown that this procedure can be successfully used to prevent loss of RNA into lipid solvents after treatment of the tissue with cold acid. They recommend using ethanol buffered with 2 per cent. sodium acetate as the first lipid solvent after precipitation with perchloric acid, and using ethanol containing 10 per cent. potassium acetate as first lipid solvent after precipitation with trichloroacetic acid. I t has been found by our colleagues that 1 per cent.potassium acetate is effective in preventing losses of RNA into ethanol from samples of adrenal gland35 and kidney (Halliburton, unpublished results) after treatment of these tissues with cold 0.2 N perchloric acid. Investigators who choose to use acetate buffers in this way should ensure that they are using them under optimal conditions, since these have not yet been fully defined. I t would therefore be desirable for the analyst to check recoveries of RNA following the addition of various concentrations of acetate buffer to ethanol. As pointed out earlier, the use of lipid solvents is usually not obligatory, and the analyst should therefore consider using procedures such as described by Fleck and M ~ n r o ~ ~ and Fleck and Begg37 in which lipid extraction is not part of the technique.However, if it is thought desirable to remove lipids, as with plant nuclei38 which contain an ultraviolet-absorbing lipid component, there are two possible procedures. First, the investigator can use cold acid to extract small molecules and then use ethanol buffered with acetate for the initial step in the removal of lipids. Alternatively, he can carry out lipid extraction as his first step, with subsequent cold-acid treatment. The latter approach was first described by Ogur and Rosen5 and has since been used by several other investigators, mainly working with plants, whose findings were reviewed in the earlier artic1e.l The use of lipid solvents as the initial step can be combined with inactivation of enzymes by first immersing the tissue in boiling ethanol ; this procedure has been adopted in several recent investigations.ll J2 J3 914 METHODS OF DETERMINING NUCLEIC ACIDS IN THE TISSUE RESIDUE 1.THE PROCEDURE OF SCHMIDT AND THANNHAUSER THE USE OF ALKALINE HYDROLYSIS TO SEPARATE RNA FROM DNA- In this method, the tissue residue is digested in alkali, which hydrolyses the RKA to products that are no longer precipitable on acidification. The DNA resists attack by alkali and is consequently precipitated when the digest is acidified. As pointed out in the earlier review,l a considerable variety of conditions of alkaline hydrolysis has been used. The conclusions reached at that time remain valid, namely- (a) Incubation in 0.3 N alkali for 1 hour at 37" C is adequate to extract all the RNA from mammalian tissues in an acid-soluble form, although these conditions do not degrade the RNA quantitatively to mononucleotides. ( b ) More prolonged alkaline incubation and the use of stronger alkali have the dis- advantages of rendering acid-soluble increasing amounts of tissue protein and, with N alkali, leading to de-amination of cytidylic acid. These effects can interfere seriously with subsequent measurement of RNA by ultraviolet absorption.(c) There is good reason to believe that DNA is not degraded under the usual conditions of alkaline incubation. Consequently, we recommend a 1-hour period of digestion in N potassium hydroxide at 37" C for mammalian tissues. IJnder these circumstances the RNA can be determined by measuring the ultraviolet absorption of the acid-soluble fraction obtained on acidifying the dige~t.369~7 For plant tissues, two 1-hour periods39 and a 3-hour period38 of digestion in alkali have been found necessary to extract all the RNA.82 MUNRO AND FLECK: RECENT DEVELOPMENTS IN THE [AWZjJl'st, VOl.91 THE RNA FRACTION OF THE ALKALINE DIGEST- The original Schmidt - Thannhauser determination depended on measurement of RNA as phosphorus. However, since only 80 per cent. or less of the phosphorus in the RNA fraction can be accounted for by ribonucleotides,l this method of assessing RNA content is not generally used. RNA has frequently been measured by ribose determination, usually by the orcinol reaction. The conditions for this determination have been fully discussed in the earlier review.l New colorimetric reactions for pentoses are occasionally described, such as those with dimethyl- phenol and chloro-m-creso140 and with benzofurane derivative^.^^ The first two reactions have been used as a basis of methods for RNA determination, but the last has not so far been used €or this purpose and might warrant further exploration.As pointed out in the earlier review,l many carbohydrates can interfere with colorimetric reactions for ribose. With animal tissues, the error is usually not serious, but particularly with plant tissues and yeast, the use of these reactions to determine the RNA in the untreated acid-soluble fraction of the digest is commonly subject to gross errors. Smillie and K r ~ t k o w ~ ~ and de Deken- Grenson and de D ~ k e n ~ ~ recommend using ion-exchange resins to separate the ribonucleotides from orcinol-reacting contaminants.Subsequent workers43 have also found this procedure t o be advantageous. The characteristic intense ultraviolet absorption of RNA has been widely used for determining ribonucleotides in the acid-soluble fraction of the alkaline digest, It is, however, subject to error when appreciable amounts of protein-degradation products are released by prolonged alkaline digestion. The error due to the ultraviolet absorption of peptides present in the RNA fraction can be dealt with in three ways. First, the period of digestion can be limited to 1 hour in 0.3 N potassium hydroxide at 37" C as recommended earlier. These conditions of digestion have generally been found to cause minimal solubilisation of tissue protein.36 There are, however, some exceptions. With the thyroid gland, even this short period of digestion results in significant peptide contamination of the RNA fraction.44 Barker and Hollin~head~~ found an appreciable contamination with peptide material when they digested plant tissue with alkali for two periods of 1 hour; this may have arisen through their use of acid ethanol to precipitate the DNA-protein fraction, since some tissue protein is soluble in this s0lvent.~5 Alternatively, one can correct the observed ultraviolet absorption of the RNA fraction for the presence of peptide material in one of two ways.First, the protein can be deter- mined directly46 and a suitable adjustment made in the observed ultraviolet absorption of the acid-soluble fraction.36 939 Secondly, measurements of ultraviolet absorption can be made at two wavelengths, one of which is that of maximum absorption of RNA, namely 260 mp.It has been common practice to select a second wavelength around 280 mp, since this is in the region of maximum absorption of protein. Tsanev and T~4arkov~~ explored this approach extensively and describe a procedure claimed to correct for the extensive protein contamina- tion of the RNA fraction that occurred after 18 hours of alkaline digestion. El~ewherel,~~ we have shown that this procedure results in considerable errors owing to the difficulty of standardising the protein correction. Fleck and Regg37 have recently shown that 232 mp, the absorption minimum in the RNA spectrum, is a sensitive point at which to determine the presence and amount of protein contamination. These auth0rs,~7 as well as Tsanev and M a ~ k o v , ~ ~ Fleck and R1unr0~~ and Warburg and Christian,48 provide a discussion of the principles and correct application of two-wavelength procedures in the analysis of mixtures. I n order to use such methods, the ultraviolet-absorption characteristics of the tissue RXA must be used, not that of another species of RNA, such as yeast RXA.47 Thirdly, the ultraviolet absorption due to protein can be eliminated on separating the ribonucleotides from contaminant peptides by passage through an ion-exchange resin, as recommended by Smillie and K r ~ t k o w ~ ~ and by de Deken-Grenson and de Deken.42 Several recent investigators measuring RNA in plant tissues have found this method to give reliable ~ a l u e ~ .~ ~ ~ ~ ~ ~ ~ ~ The peptides have also been removed with charcoal,51 but with plant-issue digests this procedure has been found unsatisfactory (Holdgate and Goodwin ; private com- munication). Finally, the nucleotides can be separated from the contaminating peptides by ele~trophoresis5~ or possibly by paper ~hromatography.~~ THE DNA FRACTION OF THE ALKALINE DIGEST- Difficulties have frequently been encountered in determining the DNA contained in the precipitate formed on acidifying the alkaline digest. The DNA can be determined from itsFebruary, 19661 MEASUREMENT OF NUCLEIC ACIDS IN BIOLOGICAL MATERIALS 83 phosphorus content by deoxyribose assay, by its ultraviolet absorption or by some miscel- laneous methods.As pointed out in the earlier review,l determination of the total phosphorus content of the precipitate has usually given acceptable values for the DNA content of the tissue, provided that phospholipids have first been removed. However, with nervous tissue, even defatting leaves phospho-inositides which increase the phosphorus content of the DNA Measurement of DNA by phosphorus determination has the advantage that the whole pre- cipitate can be digested and used for the determination. Other methods of measuring DNA, such as colorimetric determinations of deoxypentose or ultraviolet-absorption measurements, first involve either dissolving the precipitate in alkali or extracting the DNA by means of hot acid or enzymes.The DNA-protein precipitate can usually be rapidly and completely dissolved in 0.3 N potassium hydroxide at room temperature or at 37" C. This solution will clearly also contain the tissue protein which can interfere with DSA determination, especially by ultraviolet absorption. Consequently, many investigators extract the DNA with hot acid, usually either with 5 per cent. trichloroacetic acid for 15 minutes at 90" C, or with 0.5 N perchloric acid for 10 minutes at 80" C.l However, as pointed out in the earlier review, these extraction procedures may either be insufficient to remove all the DNA, or, if too vigorous, can lead to destruction of deoxypentose and to extraction of significant amounts of protein degradation products by the hot acid. This is the dilemma also inherent in Schneider's method for extracting nucleic acids from tissues with hot trichloroacetic acid or perchloric acid, which will be discussed later.Few investigators have studied in detail the optimum conditions for extraction of DNA with hot acid. Recently, Threlfall (private communication) obtained maximal recoveries of deoxyribose from liver samples by heating in N perchloric acid at 70" C, but the optimum temperature for kidney samples was found to be 65" C. At 85" C, destruction of deoxyribose was serious, indicating the importance of carefully controlled conditions. Wannemacher et report a study in which they extracted tissue samples with 0.5 N perchloric acid at various temperatures and examined the recoveries of DNA by measurement as deoxyribose, as phosphorus and by ultraviolet absorption.Optimal recovery was obtained after heating for 45 minutes at 96" C. However, as will be discussed in more detail under the heading of the Schneider procedure, Lprvtrup and R W S ~ have demonstrated that the apparent maximal extraction is, in reality, a balance between incomplete extraction and de&-uction of DNA and therefore does not imply full recovery of DNA. A few investigators have attempted to extract DNA from tissue residues by using deoxyribon~clease~~ or a combination of deoxyribonuclease and phosphodiestera~e.~~ The fraction is first incubated with the enzymes and then the protein is removed by precipitation with acid, the products of DKA hydrolysis being left in solution. This procedure can obviously be applied to the DNA-containing fraction of the Schmidt - Thannhauser procedure.How- ever, in a few preliminary studies on rat-liver samples we have not found conditions under which adequate extraction of DXA can be attained; a similar failure to extract all the DNA enzymically from chloroplasts has been recorded by Kirk.58 The DNA contained in the fraction dissolved in alkali or in the extract has frequently been measured by reactions for deoxypentose. In the earlier review, a wide variety of procedures for determining deoxypentose was discussed. In practice, only the Burton59 modification of the diphenylamine method and the indole method of Ceriotti60 are extensively used nowadays. Croft and Lubran6l describe a form of diphenylamine reaction based on Burton's procedure, but having greater sensitivity and not subject to interference by sialic acid.Giles and ;Llyers62 have also improved the sensitivity of the Burton method by 70 per cent. and have reduced the absorption of the reagent blank to one-third after an investigation of the optimal proportions of reagents. Schmid, Schmid and B r ~ d i e ~ ~ have explored optima! conditions for the indole reaction. It was originally claimed by CeriottiG0 that his form of the indole procedure was subject to interference only by arabinose, which is not likely to be present in the DNA fraction of the Schmidt - Thannhauser method. However, glycoproteins and sialic acidel have recently been found to cause erroneously high values by the Ceriotti method. Samples of thyroid glandM have been shown to give an unsatisfactory absorption spectrum for the coloured complex formed in the Ceriotti procedure; this is presumably due to the carbohydrate contained in thyroglobulin.As to determination of DNA by ultraviolet absorption, the solution of the DNA-con- taining precipitate in alkali includes such a large amount of protein that it is not practicable84 MCNRO -4ND FLECK: RECEXT DEVELOPMENTS IN THE [Amdyst, vol. 91 to measure the DNA with adequate accuracy, even when a correctly designed two-wavelength method is applied. Consequently, many authors have extracted the DNA with hot trichloro- acetic acid or perchloric acid, as described in the earlier review.l The problem involved in this approach is to secure adequate extraction without also solubilising some of the tissue protein that absorbs ultraviolet light.In consequence, Scott, Fraccastoro and TaftG4 describe conditions that are a compromise between these opposing effects. Wannemacher, Banks and W ~ n n e r ~ ~ used 0-5 N perchloric acid to extract the DXA and compared the ultraviolet absorp- tion at 265 and 290mp in order to detect the presence of peptide. At temperatures of extraction up to 96" C, the ratio of 265 to 290 was constant, but at higher temperatures a fall in the ratio was interpreted as being due to peptide contamination. Trace amounts of protein were detected at lower temperatures by the Lowry method.46 A more sensitive method of detecting contamination by the products of protein degradation would be to examine the ultraviolet spectrum of the extract in the region of the absorption minimum of pure DNA (around 233 mp), as suggested for RNA by Fleck and Begg.37 A correctly designed two-wavelength procedure should provide an accurate procedure for correcting the hot acid extract for peptide contaminants, provided that appropriate standards for tissue DSA and contaminant protein can be prepared.65 This approach was taken by Santen and Agranoffg when measuring brain D1JA4; however, their use of salmon-sperm DNA as a standard may have led to erroneous values.In the earlier review1 we referred to two sensitive fluorimetric methods for measuring DSA4.6C 9 G 7 Although no further methods for Auorimetric measurement of DXA have since been described, new fluorescent reactions have been reported for adenine,68 and after treatment of RNA and DNA with berberineC9; these might become the basis of quantitative procedures.The high sensitivity of fluorescent methods has been utilised recently by workers studying the DlVA content of ova and e m b r y o ~ . l ~ 9 ~ ~ DSA can also be determined as nucleotides or bases, as discussed in the previous review.1 Recent advances in the chromatography of these products, including notably the use of thin-layer chromatography6 and of gas chrornat~graphy,~~ increase the potential usefulness and sensitivity of this approach. Isotope-dilution methods for measuring DNA, mentioned in the earlier review, appear not to have been used during the past few years. A review of the measurement of DNA by microbiological methods was provided recently by Lervtrup and Roos.71 RECOMMENDATIONS FOR THE USE OF THE SCHMIDT - THANNHAUSER METHOD- In view of the studies of Hallinan, Fleck and Munro,20 the general application of lipid solvents in the preliminary treatment of the tissue is no longer considered desirable.Further, we now prefer to use cold 0.2 N perchloric acid rather than 10 per cent. trichloroacetic acid for the initial inactivation of enzymes and precipitation of the nucleic acids, especially when the RNA content of the tissue is to be measured by ultraviolet absorption. The digestion of the samples in 0.3 N potassium hydroxide for 1 hour a t 37" C is satisfactory for mammalian tissues. These various recommendations and modifications are incorporated in the Schmidt - Thannhauser procedure described by Fleck and 111unr0~~ and by Fleck and Begg.37 Our final procedure is set out below in a form suitable for the analysis of rat-liver samples.The tissue is homogenised in 19 volumes of ice-cold de-ionised water, and 5 ml (zz 250 mg wet weight of tissue) are transferred by pipette into a 15-ml centrifuge tube. To this are added 2-5 ml Qf ice-cold 0.6 N perchloric acid. After thorough mixing and standing for 10 minutes in ice, the tube is centrifuged, the supernatant (acid-soluble) fraction is dis- carded and the precipitate is then washed twice with ice-cold 0.2 N perchloric acid. Excess of acid is finally drained off by inverting the tube briefly on to filter-paper. After incubation for 1 hour at 37" C (air-oven or water-bath), the digest is cooled in ice, and the protein and DNA are precipitated by adding 2-5 ml of 1-2 N perchloric acid.After 10 minutes' standing in ice, the precipitate is separated centrifugally and washed twice with 0-2 N perchloric acid. The supernatant fluid from the first centrifugation and the washings are combined, 10 ml of 0-6 N perchloric acid are added, and the solution is made up to 100 ml with de-ionised water to give a final solution of 0.1 N perchloric acid. The ultraviolet absorp- tion of this solution at 260 mp (and 232 mp also, if the absence of protein is to be checked) is then measured. An extinction of 1.000 at 260 mp is given by a concentration of 32 pg of The recommendations made in the earlier review1 remain valid in general. To the tissue residue 4 ml of 0.3 N potassium hydroxide are added and mixed.February, 19661 MEASUREMENT OF NUCLEIC ACIDS I N BIOLOGICAL MATERIALS 85 RNA per ml for rat liver.The original figure of 35 pg of RNA per ml given by Fleck and Munro36 is wrong because of an error in calculation. The precipitate containing the DNA is dissolved in 5 ml of 0.3 N potassium hydroxide, if necessary by warming to 37" C for a few minutes, and a further 12 ml of 0.3 N potassium hydroxide are added. The solution is then made up to 50 ml with water to give a solution of DNA in 0.1 N potassium hydroxide; 2-ml samples of this solution are taken for DNA estimation by the Ceriotti indole procedure60 or by Burton's modification59 of the diphenyl- amine method. Analysts using the latter procedure should consult the papers by Croft and Lubran61 and by Giles and Myers62 for further improvements in this method.The procedure described above can be used for analysing samples containing 0.7 to 3.5 mg of RNA and 0.2 to 1 mg of DNA. It can readily be scaled down to measure one-tenth or less of these amounts by reducing the volumes in which the RNA and DNA fractions are made up. 2. THE SCHNEIDER PROCEDURE In the original Schneider procedure,* the nucleic acids are extracted from the tissue residue after the removal of small molecules and lipids by heating in 5 per cent. trichloroacetic acid at 90" C for 15 minutes. The RNA is then measured in the extract by the orcinol method and the DNA by the diphenylamine reaction. Since the original description of this method was published, extraction of the nucleic acids has also been commonly carried out with hot perchloric acid.As pointed out in the earlier review,l both acids have been used at a wide variety of concentrations, temperatures and duration of extraction by investigators who did not appreciate that the conditions of acid extraction were critical. Hutchison, Downie and Munro30 showed that for liver samples extracted with perchloric acid at 70" C, increasing the concentration of the acid resulted in excessive amounts of orcinol-reacting material appearing in the extract. M'ith DNA, two opposing effects were observed. At low concentrations of perchloric acid the DNA was incompletely extracted, but at higher concentrations, especially when the temperature was raised to 90" C, there was destruction of deoxyribose.No condi- tions of hot-acid extraction were found that gave full recovery of the DNA of the tissue, as compared with the values obtained for the Schmidt - Thannhauser method. These findings have been confirmed and amplified by Lovtrup and R o o s ~ ~ ~ ~ ~ ~ ~ ~ in a series of careful studies of the kinetics of DNA extraction and deoxyribose destruction by hot 0.5 N perchloric acid at various temperatures. On the basis of these kinetic studies, they offer a method of deter- mining the tissue DNA, after treatment at 90" C for 1 hour in 0.5 N perchloric acid. The deoxyribose value obtained for the extract is corrected to give 100 per cent. recovery by calculations derived from the kinetic studies. They found that the correction equations differed for each tissue studied, and in consequence no single set of universally applicable conditions can be recommended.Therefore, the use of the Lovtrup - Roos correction method has to be worked out for each tissue independently and would be tedious for ordinary manual determination. However, since the Schneider method has now been made the basis of an automated analytical method for measuring the RNA and DNA in bacteria,74 the incorporation of the correction for deoxyribose loss can be readily carried out as part of the analytical procedure. Automated analysis may thus restore the attractiveness of the Schneider pro- cedure, in spite of the inherent errors in extraction. This does not, however, guarantee the exclusion of errors in measurement of RNA and LISA due to solubilisation by hot acid of tissue constituents that give the reactions for ribose and deoxyribose.The earlier review1 contains numerous examples of erroneous values for KNA and DNA by sugar determination due to this cause. Consequently, the analyst who is embarking on the use of sugar reactions for automated analysis with a new tissue should first examine the spectrum of the coloured product obtained with the tissue extract and compare it with the corresponding spectrum for pure ribose or deoxyribose. 3. THE PROCEDURE OF OGUR AND ROSEN Ogur and Rosen5 described a procedure in which the RNA is extracted with cold per- chloric acid; DKA is subsequently extracted with hot perchloric acid. The ultraviolet absorption of each extract is then used as a measure of the RNA and DNA contents, respec- tively, of the tissue.In the previous review,l numerous reports of incomplete separation of RNA from DNA by this procedure were quoted. During the last few years the method86 MUNRO AND FLECK: RECENT DEVELOPMENTS I N THE [Andyst, Vol. 91 has been used on several occasions, on some of which the authors report unsatisfactory r e ~ ~ l t ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Consequently, we see no reason to modify the conclusion reached in the earlier review that this method is inadequate for the accurate determination of nucleic acids in biological materials. 4. OTHER PROCEDURES In the earlier review,l several miscellaneous methods for nucleic acid determination were discussed. There is little to add to the comments made there. Claims have been made that extraction of DNA from tissues by the phenol procedure can be made quantitati~e.~89~~ Samis, Wulff and FalzoneS0 explored methods of recovering nucleic acids quantitatively after phenol extraction.They report the effects of ethanol concentration on precipitation of nucleic acids and conclude that it is difficult, with this method, to obtain satisfactory quantitative recoveries. Accordingly, they investigated the use of indium salts and were able to obtain conditions under which both nucleic acids were completely precipitated. The precipitate was then treated with alkali to dissolve the RNA and DNA, the indium being discarded as the insoluble hydroxide. The nucleic acids could then be separated by incubation in the alkaline medium, as in the Schmidt - Thannhauser procedure. This approach may be worth further exploration.In less frequently used methods for determining the extracted nucleic acids, recent advances in thin-layer chromatography6 and in gas chr~matography~~ may find applications to quantitative measurements of bases, nucleosides or nucleotides of RNA and DNA. The application of microbiological assays to measurement of nucleic acids has been reviewed by Lprvtrup and R o o s . ~ ~ GENERAL RECOMMENDATIONS FOR NUCLEIC ACID DETERMINATION It was concluded in the earlier review1 that of the three major methods of nucleic acid estimation, the Schmidt - Thannhauser procedure is least subject to analytical error. This conclusion has been strengthened by a study of the recent literature, and we offer in an earlier section of this review an outline of a procedure found satisfactory for many animal tissues.However, investigators continue to report new and unsuspected sources of error in the standard methods of analysis, as for nervous t i ~ s u e , ~ > 8 y 9 thyroid gland,44 gastric mucosa61 and For plant tissues, considerable analytical errors have been encountered. Smillie and K r ~ t k o v ~ ~ made an extensive study of analytical methods for measuring nucleic acids in plant tissues and concluded that the Schmidt - Thannhauser procedure gave the most satisfactory results, provided that contaminants are removed from the RNA fraction by passing it through an ion-exchange resin. Subsequent authors studying nucleic acids39 lg2 ~8~ from plant tissue have confirmed the importance of resin treatment in order to obtain valid results for RNA content by phosphorus or orcinol determination or ultraviolet absorption.Barker and H~llinshead~~ used a short period of incubation in alkali in order to minimise contamina- tion of the RNA fraction with protein. Nevertheless, they found significant amounts of protein in this fraction; they were able to correct for the ultraviolet-absorption error due to this material on determining the amount of protein present by the Lowry method46 and then applying a correction factor to the ultraviolet absorption. In this way they obtained results that were in good agreement with those obtained by treating the RNA fraction with an ion-exchange resin. I t should be pointed out that the unusually large amount of protein made acid-soluble by the short period of alkaline digestion may have occurred because they used acid ethanol to precipitate the Dh’A and protein at the end of digestion; it has been shown that acid ethanol extracts a considerable amount of tissue proteing5 that would subsequently appear in the RNA fraction.From these reports of difficulties in the application of standard nucleic acid procedures to various tissues, it is apparent that no single method can be expected to be universally applicable. Consequently, the analyst making a new study of the nucleic acid content of a tissue should be on his guard against sources of error, and should provide evidence that his method of choice has been rigorously tested. REFERENCES 1. 2. Hutchinson, W-.C., and Munro, H. N., Analyst, 1961, 86, 768; 1962, 87, 303. Munro, H. N., and Fleck, A., in Glick, D., Editor, “Methods of Biochemical Analysis,” Interscience Publishers, a division of John Wilcy & Sons Inc., New York, London and Sydney, 1966, Volume XIV, in the press.February, 19661 MEASUREMENT OF NUCLEIC ACIDS IN BIOLOGICAL MATERIALS 87 3. 4. 5. 6. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 60. 51. c 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. Schmidt, G., and Thannhauser, S. J., J . Biol. Chem., 1945, 161, 83. Schneider, W. C., Ibid., 1945, 161, 293. Ogur, M., and Rosen, G., Archs. Biochem., 1950, 25, 262.Saukkonen, J. J., Chromat. Rev., 1964, 6, 53. Logan, J . E., Mannell, W. A., and Rossiter, R. J., Biochem. J., 1952, 51, 470. Zamenhof, S., Bursztyn, H., Rich, K., and Zamenhof, P. J., J . Neurochem., 1964, 11, 505. Santen, R. J., and Agranoff, B. W., Biochim. Biophys. Acta, 1963, 72, 251. Markham, R., in Paech, K., and Tracy, M. V., Editors, “Moderne Methoden der Pflanzenanalyse,” Springer-Verlag, Berlin, 1955, Volume IV, p. 246. Stange, L., Kirk, M., Bennett, E. L., and Calvin, M., Biochim. Biophys. Acta, 1962, 61, 681. Giles, K. W., and Myers, A., Ibid., 1964, 87, 460. Icessler, B., and Engelberg, N., Ibid., 1962, 55, 70. Baltus, E., and Brachct, J., Ibid., 1962, 61, 167. Kovacs, E., Archs. Biochem. Biophys., 1958, 76, 546. Ledoux, L., Galand, P,, and Huart, R., Biochim.Biophys. Acta, 1962, 55, 97. May, L., and Grenell, R. G., Proc. SOC. Exp. Biol. illed., 1959, 102, 235. Smith, 0. K., Yale J . Biol. filed., 1953, 26, 126. Currie, R., Nature, 1965, 205, 1212. Hallinan, T., Fleck, A., and Munro, H. N., Biochim. Biophys. Acta, 1963, 68, 131. Kuroima, Y., and Hashirnoto, N., Bull. Agric. Chem. SOC. Japan, 1960, 24, 547. Katchman, B. J., and Fetty, W. G., J . Bact., 1955, 69, 607. Smillie, R. M., and Krotkov, G., Can. J . Bot., 1960, 38, 31. Finamore, F. J., and Volkin, E., J . Biol. Chem., 1961, 236, 443. Finamore, F. J., Ibid., 1964, 239, 1882. Levy, H. B., and Lynt, R. K., Biochim. Biophys. Acta, 1963, 72, 529. Nekhorocheff, J., and Cantan, B., Bull. SOC. Chim. Biol., 1964, 46, 805. Venkataraman, P.R., and Lowe, C. U., Biochem. J . , 1959, 72, 430. Venkataraman, P. R., Biochim. Biophys. Acta, 1960, 39, 352. Hutchison, W. C., Downie, E. D., and Munro, H. N., Ibid., 1962, 55, 561. Greenbaum, A. L., and Slater, T. F., Biochem. J . , 1957, 66, 155. Cooper, W. D., and Loring, H. S., J . Biol. Chem., 1957, 228, 813. Marko, A. M., and Butler, G. C., Ibid., 1951, 190, 165. Steele, W. J., Okamura, N., and Busch, H., Biochim. Biophys. Acta, 1964, 87, 490. Imric, R. C., Ramaiah, T. R., Antoni, F., and Hutchison, W. C., J . Endocr., 1965, 32, 303. Fleck, A., and Munro, H. N., Biochim. Biophys. ,4cta, 1962, 55, 571. Fleck, A., and Begg, D. J., Ibid., 1965, 108, 333. McLeish, J., Proc. Roy. Soc., B, 1963, 158, 261. Barker, G. R., and Hollinshead, J. A., Biochem. J., 1964, 93, 78.Staron, T., Xuong, N. D., Allard, C., and Chambre, M. M., Hebd. Sdanc. Acad. Sci., 1962,254, 3048. Mikulaszek, E., Merkel, M., Osowiecki, H., and Zawadowski, T., Bull. Acad. Polon. Sci., B, 1963, de Deken-Grenson, M., and de Deken, K. H., Biochim. Biophys. Acta, 1959, 31, 195. Young, E. G., Can. J . Bot., 1964, 42, 1471. Begg, D. J., McGirr, E. M., and Munro, H. N., Endocrinology, 1965, 76, 171. Munro, H. N., and Downie, E. D., Archs. Biochem. Biophys., 1964, 106, 516. Lowry, 0. H., Kosebrough, N. J., Farr, A. L., and Randall, R. J., J . Biol. Chem., 1951, 193, 265. Tsanev, R., and Markov, G. G., Biochim. Biophys. Acta, 1960, 42, 442. Warburg, O., and Christian, W., Biochem. Z., 1942, 310, 384. Rottger, B., and Fritz, H. G., Biochim. Biophys. Acta, 1962, 61, 621. Marcus, A., and Feeley, J., Ibid., 1962, 61, 830. Kitagawa, T., Schmidt, G., and Thannhauser, S. J., “Abstracts of papers presented a t A.C.S. National Meeting, ” Division of Biological Chemistry, American Chemical Society, Washington, D.C., 1962, Abstract 701. 11, 565. Davidson, J. N., and Smellie, R. M. S., Biochem. J . , 1952, 52, 594 and 599. Gerlach, E., Dreisbach, R. H., and Deuticke, R., J . Chromat., 1965, 18, 81. Wannemacher, R. W., jun., Banks, W. L., jun., and Wunner, W. H., Analyt. Biochem., 1965,11,320. Lavtrup, S., and Roos, K., Biochim. Biophys. Acta, 1961, 53, 1. Haggis, A. J., Devl. Biol., 1964, 10, 358. Iwamura, T., Biochim. Biophys. .4cta, 1962, 61, 472. Kirk, J . T. O., Ibid., 1963, 76, 417. Burton, K., Biochem. J., 1966, 62, 315. Ceriotti, G., J. Biol. Chem., 1955, 214, 59. Croft, D. N., and Lubran, M., Biochem. J . , 1966, 95, 612. Giles, K. W., and Myers, A., Nature, 1965, 206, 93. Schmid, P., Schmid, C., and Brodie, D. C., J . Biol. Chem., 1963, 238, 1068. Scott, J. F., Fraccastoro, A. P., and Taft, E. B., J . Histochem. Cytochem., 1956, 4, 1. Fleck, A,, Ph.D. Thesis, University of Glasgow, 1963. Kissane, J. M., and Robins, E., J . Biol. Chem., 1958, 233, 184. Roberts, D., and Friedkin, M., Ibid., 1958, 233, 483. Estabrook, R. W., and Maitra, P. K., Analyt. Biochem., 1962, 3, 360. Yamagishi, H., .J. Cell Biol., 1962, 15, 589. Hancock, R. I,., and Coleman, D. L., Analyt. Biochem., 1966, 10, 365. Lavtrup, S., and Roos, K., Acta Biochim. Pol., 1963, 10, 73. -- , Biochim. Biophys. Acta, 1063, 68, 425.88 73. 74. 75. 76. 77. 78. 79. 80. 81. MUNRO AND FLECK [Analyst, Vol. 91 Lavtrup, S., Actu Biochim. Pol., 1962, 9, 411. Gerke, J. R., Watson, R W., and Umbreit, W. W., “Continuous Analysis of Microbial Nucleic Acids and Protein with an AutoA4nalyzer Instrumental System,” Paper presented a t the Technicon 25th Anniversary International Symposium, “Automation in Analytical Chemistry,” London, 1964. Brawerman, G., Pogo, A. O., and Chargaff, E., Biochim. Biofihys. Ada, 1962, 55, 326. Ambellan, E., Ibid., 1964, 80, 8. Ambellan, E., and Webster, G., Ibid., 1963, 68, 119. Colter, J . S., Brown, R. A., and Ellem, K. A. O., Ibid., 1962, 55, 31. Lyttleton, J . W., and Petersen, G. B., Ibid., 1964, 80, 391. Samis, H. V., Wulff, V. J., and Falzone, J. A., Ibid., 1964, 91, 223. Holdgate, D. P., and Goodwin, T. W., Phytochem. hTewsl., 1965, 4, 831. Received July 2211d, 1965

ISSN:0003-2654    

DOI:10.1039/AN9669100078

出版商:RSC    

年代:1966

数据来源: RSC

摘要:

February, 19661 DJURKIN, KIRKBRIGHT AND WEST 89 A Sensitive and Selective Spectrophotometric Procedure for the Determination of Phosphorus BY V. DJURKIK,* G. F. KIRKBRIGHT AND T. S. WEST (Chemistry Department, Imperial College, Logadon, S. W . 7) Phosphorus as phosphate is determined by an amplification procedure in which the heteropoly acid H,PO,(MoO,),, is formed and extracted away from excess of molybdate reagent. The 12 molybdate ions associated with the phosphate are then determined spectrophotometrically a t 7 10 m p as the green molybdenum(vr ) complex with 2-amino-4-chlorobenzenethiol in chloro- form. Depending on the procedure used, the effective molar absorptivity for phosphorus is 96,900 or 359,000. The proposed procedure is therefore much more sensitive than previously described methods for phosphorus.Amounts of phosphorus down to 0.2 pg (0.008 p.p.m.) may be determined. Large excesses of silicon, germanium, arsenic or antimony do not interfere. A simple masking procedure obviates any interference from up to a 30-fold excess of tungsten(v1). A VOLUMINOUS literature exists on methods for the spectrophotometric determination of phosphorus based on measurement of the absorbance of phosphomolybdic acid or its reduction product (molybdenum blue) in aqueous or organic media. These methods have recently been reviewed by Riemann and Beukenkampl and Wadelin and Mellox2 The selectivity of the methods may be improved by careful choice of reducing agent, acidity and reagent c~ncentration,~ or by selection of a suitable solvent mixture for extraction of the molybdenum blue or unreduced heteropoly acid into an organic solvent.2 The sensitivity of spectrophotometric methods based on molybdenum blue, phospho- molybdic acid and phosphovanadomolybdate is not high, and depends inter alia on the reaction conditions and whether the absorbing species has been first extracted into an organic solvent.The most sensitive methods, however, appear to be those based on the formation of molybdenum blue in aqueous solution after reduction of phosphomolybdic acid with l-amino-2-naphthol-4-sulphonic acid or hydrazine sulphate ( ~ 8 2 0 ~ ~ = 26,600 and csgornlr = 26,800, respectively) . 4 9 5 In the procedure described in this paper, phosphate is converted into phosphomolybdic acid, H,P0,(Mo03),2, and extracted into an organic solvent.I t is then broken down by alkali and the 12 molybdate ions associated with each original phosphate ion are determined by their spectrophotometric reaction with 2-amino-4-chlorobenzenethiol hydrochloride. By virtue of this amplification procedure an effective molar absorptivity of €720 = 96,900 is attained. The extraction procedure used to separate phosphomolybdic acid from unreacted sodium or ammonium molybdate is that described by Wadelin and Mellon.2 The molybdate is determined spectrophotometrically as its green complex with 2-amino-4-chlorbenzenethiol hydrochloride in chloroform by the method of Kirkbright and k'oe.'j EXPERIMENTAL APPARATUS- Beckman model DB rtcordiizg s$ectro$hofometer, with 1-cm glass cells. Vibron pH meter, model 39A (Electronic Instruments Ltd., Richmond, Surrey, England).REAGENTS- 2-Amin.0-4-chloro beizzenetlziol hydrochloride solution-Prepare a 4 per cent. solution of 2-amino-4-chlorobenzenethiol hydrochloride (Eastman Kodak Catalogue No. 3279) in 95 per cent. ethanol. Molybdate solzttion-Dissolve 7.5 g of sodium molybdate dihydrate or 5.348 g of am- monium molybdate tetrahydrate, analytical-reagent grade, in 200 ml of distilled water, add 100 ml of hydrochloric acid, and dilute the solution to 500 ml with distilled water. Standard phos$horus solution-Dissolve 0.1098 g of analytical-reagent grade potassium dihydrogen phosphate (KH2P0,) in distilled water and dilute to 1 litre. This solution contains * Present address : Faculty of Mathematics and Science, Unil-ersity of Sarajevo, Yugoslavia. Prepare the reagent solution freshly every 2 days.90 [Analyst, Vol. 91 25 pg of phosphorus per ml.Dilute this stock solution as required to 1 pg of phosphorus per ml. Extractant-Use the mixed solvent proposed by Wadelin and Mellon2 (a mixture of analytical-reagent grade chloroform and butanol, 4 + 1 by volume), Rufler solution, pH 2-2-Prepare Clark and Lubs’ buffer solution, pH 2.2, by mixing 33.5 ml of 0.2 M hydrochloric acid and 250 ml of 0.2 M potassium chloride solution and diluting to 1 litre with distilled water. Diverse ions-Use analytical-reagent grade salts to prepare solutions of various ions. Make up the solutions to contain 0.1 g of the ion per litre of solution. Tiron-Make a 0.6 per cent. solution of 1,2-dihydroxybenzene-3,5-disulphonic acid disodium salt (Reagent grade, B.D.H., Poole, Dorset).DJURKIN, KIRKBRIGHT AND WEST: A SENSITIVE AND SELECTIVE CALIBRATION GRAPH FOR PHOSPHORUS- Transfer 1.0, 2.0, 3-0, 4.0 and 5-0-ml aliquots of standard phosphorus solution (1 pg of phosphorus per ml) to 50-ml separating funnels, add 10ml of molybdate reagent solution t o the contents of each funnel, and dilute to 25 ml with distilled water. Add 10 ml of butanol- chloroform mixture and shake the funnels for 2 minutes. When the phases have completely separated, transfer each non-aqueous phase into a clean, dry 50-ml separating funnel, and extract the aqueous phase with a second 10-ml portion of butanol - chloroform mixture for 2 minutes. Discard the aqueous phase, combine the butanol - chloroform extracts, and wash them by shaking with 10ml of 2~ hydrochloric acid for 1 minute.Discard the aqueous wash solution, add 10 ml of 2 N aqueous ammonia solution, and shake the mixture for 1 minute. Transfer the ammoniacal aqueous solutions to 50-ml beakers and wash the contents of the funnels with 8 ml of 2 N hydrochloric acid and a few millilitres of distilled water. Combine the washings with the appropriate ammoniacal extracts, adjust the pH of each solution t o 2.2 with 2 N hydrochloric acid and transfer the solutions to 100-ml calibrated flasks. Add 3 ml of buffer solution, pH 2-2, to each and dilute to volume with distilled water. Transfer 25-ml aliquots of these solutions to 50-ml separating funnels, add 0.25 ml of 4 per cent. 2-amino-4-chlorobenzenethiol reagent solution, mix and allow to stand for 15 minutes.Add 10.0 ml of chloroform and shake the funnels vigorously for 1 minute. Allow the phases to separate, filter the chloroform extract through Whatman KO. 1 filter-paper into 1-cm glass cuvettes, and measure the absorbance of each solution immediately at 710 mp against a reagent blank, similarly prepared. The conditions, the volume of solution, pH, time of shaking and so on, should be carefully controlled to attain good reproducibility. Test solutions of phosphate should be approxi- mately neutral. DISCUSSION The optimum conditions for the solvent extraction of phosphomolybdic acid2 and for the spectrophotometric determination of molybdatc6 by the method used have already been established, and were therefore not studied here.An investigation was made, however, of the effect of several important factors on the practical procedure. EXTRACTION OF PHOSPHOMOLYBDIC ACID- I t was found necessary to make two successive extractions with 10-ml portions of the butanol - chloroform mixture to achieve quantitative extraction of the phosphomolybdic acid irom 2-2 N hydrochloric acid at the concentrations studied. Two minutes’ shaking time with each aliquot of solvent mixture was found sufficient. Experiments were made, in the absence of phosphate, to determine whether any molybdate reagent was extracted into the organic solvent with the phosphomolybdic acid. I t was found that in the initial experiments a considerable amount of molybdenum was recovered on back-extraction, owing to incomplete separation of the solvent mixture from the molybdate solution.The organic phase was, therefore, separated and washed with 2 N hydrochloric acid to remove any excess of molybdate solution before the back-extraction with ammonia. This effectively removed any molybdate mechanically transferred with the organic phase, and only a negligible amount of molybdenum was found when a blank containing no phosphate was taken through the extraction and spectrophotometric procedures.February, 19661 SPECTROPHOTOMETRIC PROCEDURE FOR PHOSPHORUS 91 BACK-EXTRACTION- Experiments were conducted in which the organic phase containing the phospho- molybdic acid was shaken with successive 10-ml aliquots of 2 N ammonia solution, The molybdenum content of each aliquot was then determined spectrophotometrically a t pH 2-22.It was found that equilibration for 1 minute with a single 10-ml portion of 2 N ammonia solution effected the destruction of the phosphomolybdic acid and gave quantitative recovery of the molybdenum from the organic phase. A small volume of 2 N hydrochloric acid was used as wash-liquid to achieve quantitative transference of the ammoniacal back-extract from the separating funnel and to facilitate the pH adjustment. CALIBRATION GRAPH AND OPTIMUM CONCENTRATION RANGE- When the recommended procedure was used, the calibration graph for phosphorus was rectilinear over the range 0-04 to 0-16 p.p.m. of phosphorus in the original solution, i.e., 1 to 4 pg (see Fig. 1). 0 Phosphorus, p.p.m. Fig. 1. Calibration graphs for phosphorus deter- mination.Curve A, recommended procedure for 1 to 5 pg of phosphorus (25-ml aliquot of final 100ml of solution taken) ; curve B, procedure for 0.2 to 1.25 pg of phosphorus (all 100 ml of final solution taken) It is possible to increase the sensitivity of the procedure further by taking the whole back-extract for the molybdenum determination instead of only one quarter of it as described above. A calibration graph that was rectilinear over the range 0.008 to 0.05 p p m . of phos- phorus (0.2 to 1 pg) could thus be attained (curve R), The absorbance at these concentrations was 0.123 and 0.580, respectively. In this way the theoretical 4-fold increase in sensitivity was virtually attained by determining the total molybdenum recovered. The precision in this procedure was not as high as in the recommended procedure.The less sensitive procedure has the advantage that, if it is desired, up to four molybdenum determinations may be performed on each back-extract to act as a check on the spectrophotometric procedure. SENSITIVITY- For the recommended procedure, in which one quarter of the molybdenum recovered is determined, the effective molar absorptivity, based on 1 gram-atom of phosphorus deter- mined, is €710 = 96,900. In the same manner, when the whole of the back-extract is taken, the molar absorptivity for phosphorus is €710 = 359,000. In the original procedure for the determination of molybdenum with 2-amino-4-chlorobenzenethiol hydrochlorideG the molar92 DJURKIN, KIRKBRIGHT AXD WEST: A SENSITIVE AND SELECTIVE [Akdyst, Vol.91 absorptivity for molybdenum was reported to be ~ 7 1 0 = 36,000, and our re-determination of this value gave €710 = 32,000. Hence, the molar absorptivities obtained in the phosphorus determination, when account is taken of the slightly lower sensitivity of the spectrophoto- metric procedure when the molybdenum-2-amino-4-chlorobenzenethiol complex is developed in a large volume of aqueous solution (100 ml), agree reasonably well with the association of 12 molybdenum atoms with one phosphorus atom in phosphomolybdic acid. PRECISION- During the development of the recommended procedure and study of the interferences, a series of 25 absorbance values for the replicate analysis of an original solution containing 0.16 p.p.m. (4pg) of phosphorus was obtained.The average absorbance was 0-500 and the standard deviation was 0.024 or 4.8 per cent. EFFECT OF DIVERSE IONS- The effect of a selected group of elements on the determination of phosphorus by the recommended method has been investigated. The elements studied were those known to form the central atom of a heteropoly acid in which molybdate is the co-ordinated group (arsenic, germanium and silicon) and the important related elements antimony and tungsten. As shown in Table I, phosphorus can be determined in the presence of a 100-fold excess of TABLE I EFFECT OF FOREIGN IONS ON DETERMINATION OF 0.16 P.P.M. (4pg) OF PHOSPHORUS BY THE RECOMMENDED PROCEDUREI Added Excess by weight Ion as over phosphorus - - - 50 100 Sb (11 I) SbCl, 50 100 Si(1v) Na,Si03 50 100 As(v) KH,AsO, 50 100 Ge(1v) NazGeO3 Na,WO, 10* 20* 30* 50* Y V I ) Absorbance a t 710 m p 0-490 0.490 0.470 0.495 0.500 0.488 0.500 0.490 0.515 0.490 0.490 0.490 0.455 Error, per cent.0 -4.1 + 1.0 + 2.0 - 0.4 + 2.0 0 + 6.1 0 0 0 - 7.14 L * In the presence of 3 mg of tiron. germanium, antimony, silicon or arsenic. The interference of tungsten can be minimised by addition of excess of tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) to the original solution before e~traction.~ With the quantity of tiron taken, a 30-fold excess of tungsten causes no interference, and the presence of a 50-fold excess causes -7.1 per cent. error in absorbance in the determination of 4pg of phosphorus. A greater excess of tiron cannot be taken, as formation of its molybdenum complex then significantly depletes the amount of molybdate available for formation of phosphomolybdic acid.Results for the determination of phosphorus in mixtures containing moderate excesses of the diverse ions studied are shown in Table 11. TABLE I1 DETERMINATION OF 0.16 P.P.M. OF PHOSPHORUS IN SYNTHETIC SOLUTION OF DIVERSE IONS p, As@), Sb(rII), Ge(Iv), Si(Iv), W(VI), Tiron, Absorbance* Error, P.lg Pg PLg Pg Pg PLg mg a t 710 mp per cent. 4.0 0 0 0 0 0 0 0.508 - 4-0 80 80 80 80 40 3 0.491 - 3.35 4.0 120 120 120 - 80 3 0.515 + 1.4 4.0 120 120 120 - - - 0.504 - 0.8 4.0 - - - 80 80 3 0.511 + 0.6 * Values represent mean of three determinations.February, 19661 SPECTROPHOTOMETRIC PROCEDURE FOR PHOSPHORUS 93 Further work on the development of alternative amplification procedures for phosphorus is in progress and will be reported at a later date. After this manuscript had been prepared for publication, F. Umland and G. Wiinsch8 described a similar, but less sensitive and selective amplification procedure based on the use of thiocyanate. Mie are grateful to UNESCO for the award of a Fellowship to one of us (V.D.). REFERENCES 1. Riemann, W., IIT, and Beukenkamp, J., in Kolthoff, I. M., and Elving, P. J., Editors, “Treatise on Analytical Chemistry,” Part 11, Volume 5, Interscience Publishers Inc., New York and London, 1961. 2. 3. 4. 5. 6. 7. 8. Umland, F., and Wunsch, G., 2. anaZyt. Chem., 1965, 213, 186. Wadelin, C., and Mcllon, M. G., ilnalyt. Chern., 1953, 25, 1668. Levines, H., Sowe, J . J., and Grimaldi, F. S., Ibid., 1955, 27, 258. Griswold, B. L., Humoller, F. L., and McIntyre, A. R., Ibid., 1951, 23, 192. Boltz, D. F., and Mellon, M. G., l b i d . , 1947, 19, 873. Kirkbright, G. I?., and Yoe, J. H., Talanta, 1964, 11, 415. Kusakul, P., and West, T. S., Analytica Chim. Acta, 1965, 32, 301. Received September 9th, 1965

ISSN:0003-2654    

DOI:10.1039/AN9669100089

出版商:RSC    

年代:1966

数据来源: RSC

摘要:

94 ABBOTT, BUNTING AND THOMSON: THE DETERMINATION OF RESIDUES [AfldySt, VOl. 91 The Determination of Residues of Dimethoate with Multi-band Chromatoplates BY D. C. ABBOTT, MRS. J. A. BUNTING AND J. THOMSON (Ministry of Technology, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, S.E.1) A method is proposed for the determination of dimethoate residues in vegetable material. Tripartite multi-band plates are prepared and used to separate the dimethoate from co-extracted materials ; spot-area measurement is used t o give a quantitative determination of the amount of pesticide. CONSIDERABLE interest has been shown1 l2 l3 3* in the thin-layer chromatographic separation of standard organophosphorus pesticides. However, little use of these procedures has been reported for the determination of residues on crop samples.A molybdenum-blue colorimetric method has been described5 for the determination of dimethoate and its oxygen analogue after they have been separated on a silica-gel chromatoplate developed with acetone + chloro- form, (3 -t 1). This chromatographic technique gives a degree of identification that is lacking in most other colorimetric meth0ds,~9~7* but the operations required are cumbersome and time-consuming. Recent work by Kovacsg has illustrated the uses of thin-layer chromato- graphy as a semi-quantitative process applied to extracts of kale, lettuce, strawberries, apples and carrots, co-extractives causing little interference with those organophosphorus pesticides studied. The use of similar techniques as clean-up and identification stages before gas- chromatographic determination and infrared examination has proved invaluable,1° but the apparatus required is expensive and considerable time is required for the satisfactory completion of the exercise.EXPERIMENTAL The use of spot-area measurement as a means of determining pesticide residues on thin- layer chromatograms offers an inexpensive and rapid method of combining identification with accuracy. Graphs of the square root of the area of the spot against the logarithm of the weight of material contained have been shown to be linearll; such a system has already been used successfully for the determination of some triazine herbicides in soil and water.12 Where clean extracts are readily obtained, similar procedures may be used for many organophosphorus-pesticide residues on vegetable tissue,l0 silica-gel chromatoplates being developed with hexane + acetone, (9 + 1). Dimethoate [dimethyl S-(N-methylcarbamoyl- methyl)phosphorothiolothionate] is not amenable to this treatment since, under these con- ditions, it remains at the origin together with a proportion of co-extracted interfering materials; its presence is thereby masked and determination is not possible.If the polarity of the mobile solvent is increased, higher RF values are obtained, as shown in Table I, but only at the cost of similarly increasing the mobility of the residual co-extractives with resultant streaking and malformation of the dimethoate spot. TABLE I RF VALUES OF DIMETHOATE I N VARIOUS SYSTEMS Mobile solvent Nitromethane + carbon tetrachloride, 1 + 1 .. .. Nitromethane + dichloromethane, 1 + 1 .. .. Light petroleum 40" to 60" + 2-butoxy-ethanol, 1 + 1 . . Light petroleum 40" to 60" + ethyl methyl ketone, 1 + 1 Butanol + benzene, 1 + 1.. . . * . .. ., Butanol + toluene, 1 + 1 . . . . . . . . . . Chloroform + acetone, 9 + 1 . . .. . . .. Chloroform + acetone, 9+ 1 . . . . . . . . Chloroform + acetone, H+ 1 . . . . . . . . Chloroform + acetone, 9 + 1 . . .. . . . . Chromatoplate RF value Silica gel 0.17 Silica gel 0.72 Silica gel 0.36 Silica gel 0.48 Silica gel 0.55 Silica gel 0.39 Alumina 0.90 0.52 0.35 Silica gel 0.21 Kieselguhr + silica gel, 1 + 1 Multi-band plate as in methodFebruary, 19661 OF DIMETHOATE WITH MULTI-BAND CHROMATOPLATES 95 The use of a tripartite multi-band ~hromatoplatel~ has been found effective in over- coming most of the difficulties experienced with this compound and a method is now proposed for the determination of dimethoate in vegetable tissue.The layers of adsorbent comprising the chromatoplate have been so chosen and arranged that the pesticide is caught quantitatively on a narrow band of silica gel, while migrating interfering materials move away rapidly across a band of inactive kieselguhr; chloroform + acetone, (9 + l), has been chosen as mobile solvent for this purpose. Other commonly used organophosphorus pesticides such as parathion, malathion, chlorthion, fenchlorphos, phorate, diazinon, azinphos-methyl and carbophenothion do not interfere as they migrate close to the solvent front; elution of the adsorbent in this area with dichloromethane yields a clean extract suitable for examination for the presence of other compounds on a silica-gel chromatoplate. Schradan, dichlorvos and dimethoate oxygen analogue remain on the lower kieselguhr + silica-gel band.Spots are rendered visible by means of a Brilliant green + bromine treatment which is sensitive to 0.1 pg of dimethoate. Although the spots are slightly distorted in shape, a linear relationship is observed between the square root of the area and the logarithm of the weight, as is illustrated in Fig. 1, over the range 0.2 to 1Opg. The extraction procedure used is a modification of that previously advocated for gas - liquid chromatographic determinations.14 'I- 1 5 1 7 1 9 I 0 0 3 0 5 0 7 0 9 Log (weight, pg) Fig. 1 .Quantitative determination of dimethoate METHOD APPARATUS- Multi-band spreader-Insert two close-fitting spacers into the body of a thin-layer spreader as described by Abbott and Thomson.12 The Desaga (Camlab Ltd, Cambridge) and Shandon (Shandon Scientific Co. Ltd, London) spreaders are suitable for this purpose Cork spacers covered with aluminium foil are suitable. Carrier $lates-20 x 20-cm glass plates. Chronzatographic tank--22 x 21 x 9.5 cm internal measurements. Chronzatographic spray-The laboratory Spray Gun (Shandon Scientific Co. Ltd, London) Top-drive macerator. Air-oven-Set at 120" C, suitable for drying and activating thin-layer chromatoplates. Use analytical-reagent grade materials whenever possible. Ethyl methyl ketone.Hexane-Kedistilled, boiling-point 68" to 70" C. Acetone. Dichloromethane. Chloroform. Sodium suZphate-Granular anhydrous material. Sodium sulphate solution, 5 per cent. w/v, aqueous. is suitable. REAGENTS-96 ABBOTT, BUNTING AND THOMSON : THE DETERMINATIOK OF RESIDUES it 5 per cent. v/w of water and mix the solid thoroughly in a closed vessel. [Analyst, Vol. 91 Aluminium oxide-Heat aluminium oxide at 800” C for 4 hours, cool, carefully add to Silica-gel G-For thin-layer chromatography. Kieselguhr G-For thin-layer chromatography. Brilliant green soldon-A 0.5 per cent. solution of Brilliant green (C.I. 42040) in acetone. Bromine. Standard dimethoate solutions-Prepare a stock solution containing 2 pg per ml of dimethoate in acetone. PROCEDURE- Preparation of multi-band chromato$lates-Position the two spacers within the body of the spreader at distances of 5 cm and 8 cm from one end.Simultaneously prepare the layer-mixes described in Table 11, shake them for l$ minutes and pour each mix into the appropriate compartment before layering 5 carrier plates, each 20 x 20 cm, in the usual way. Activate the 250-p thick layers by heating at 120” C for at least 1 hour. Dilute with acetone as required. TABLE I1 COMPONENTS OF MULTI-BAND CHROMATOPLATE Weight of Volume of Compartment Width, Material material, water, cm g m! 1 5 Kieselguhr + silica gel, 1 + 1 11.0 22 2 3 Silica gel 6.5 13 3 12 Kieselguhr 25 30 Extraction and clean-Ufi-Macerate 50 g of sample with three successive portions each of 75 ml of a mixture of ethyl methyl ketone + hexane, (3 $- a), and separate the solvent extracts by filtration.Reduce the combined extracts to a volume of about 5 ml and transfer the solution with 20 ml of hexane into a separating funnel containing 500ml of sodium sulphate solution. Shake the funnel well, discard the hexane layer and wash the aqueous solution with 25ml of hexane. Extract with three 25-nil portions of dichloromethane an6 dry by passage through a short column of anhydrous sodium sulphate, then reduce the volume of the combined extracts to 1 ml. Transfer the solution to a column of aluminium oxide (8 cm x 1.8 cm i.d.) and elute with 200 ml of dichloromethane. (With samples con- taining little wax or oil this step may be omitted.) Reduce the solution to dryness, take up the residue in 40 p1 of acetone and spot 20 pl on to the mixed kieselguhr + silica-gel band about 2 cm from the lower edge of the chromatoplate. Apply to the same plate a series of spots of standard dimethoate solutions, each in 2Opl of acetone, covering the range 0.2 to 10pg.DeveZopment and visualisation of the chrornato9Zate-Develop by ascending chromato- graphy with chloroform + acetone, (9 + 1) for 45 minutes; in this time the solvent front will travel about 12 cm. Spray the developed plate with Brilliant green solution and then place it immediately in an atmosphere o f bromine. After 30 seconds, remove the plate and allow the bromine to evaporate; dimethoate spots are shown as yellow areas with blue “haloes” at about the centre of the silica-gel band. Fig. 2 shows a typical range of standards so obtained.Quantitative rneasurement-Circumscribe the well defined spots carefully and measure their area by any suitable method. It may be more convenient to measure the area of the spot on a direct copy15 of the developed plate than on the layer itself. Plot a graph relating the square root of the area of the standard spots to the corresponding logarithm of the weight of dimethoate. By reference to this linear graph determine the amount of dimethoate in the sample spot. RESULTS Dimethoate was added to a variety of samples to give concentrations varying from 0.025 to 1.0 p.p.m. Recoveries obtained are listed in Table I11 and these are close to 100 per cent., only celery giving low yields of 80 per cent. All blanks carried out on untreated samples were negative.February, 19661 OF DIMETHOATE WITH MULTI-BAND CHROMATOPLATES 97 t I Kieseiguhr +Silica gel ( 1 + 1 ) I * * * * * * * * 0 2 5 0 5 I 2 4 6 8 1018 Fig.2. Multi-band chromatoplate veloped in chloroform + acetone, (9 + de- 1) TABLE I11 RECOVERY OF DIMETHOATE ADDED TO VARIOUS SAMPLES Sample Weight of sample, Dimethoate added, Dimethoate recovered, g p.p.m. p.p.m. 50 0.5 0.52 50 0.2 0-18 50 0.1 0.09 Apple . . .. .. J I Brussels sprouts . . Celery . . .. .. 50 50 I 50 50 i 50 0.5 0.2 0.1 0.2 0.1 0.54 0.22 0.10 0-16 0.08 Sugar beet . . .. 50 0.2 0.18 Strawberries . . .. Water . . .. .. 50 50 200 200 0.2 0.1 0.05 0.025 0.20 0.09 0.045 0-024 Permission to publish this paper has been given by the Government Chemist, Ministry of Technology. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. REFERENCES IValker, K. C., and Beroza, M., .J. Ass. Off. -4grir. Chent., 1963, 46, 250. Baumler, J., and Kippstein, S., Helv. Chiwi. Acta, 1061, 44, 1162. Bunyan, P. J., Analyst, 1964, 89, 615. Stanley, C. W., J . Chramat., 1964, 16, 467. Steller, \V. A., and Curry, A. N., J . Ass. Off. Agric. Chem., 1964, 47, 645. Heath, D. F., Cleugh, J . , Other, 1. K. H., and Park, P. O., J . Agric. Fd Chem., 1956, 4, 230. Chilwell, E. D., and Beecham, P. T., J . Sci. Fd Agric., 1960, 11, 400. Laws, E. Q., and Webley, D. J., Analyst, 1961, 86, 249. Kovacs, M. F., J . Ass. On. Agric. Chem., 1964, 47, 1097. Abbott, D. C., Crosby, N. T., and Thomson, J., in Shallis, P. W., Editor, “Proceedings of the Purdy, S. J., and Truter, E. V., Analyst, 1962, 87, 802. Abbott, D. C., Bunting, J . A., and Thomson, J., Ibid., 1965, 90, 356. Abbott, D. C., and Thomson, J., Chem. G. Ind., 1965, 310. Egan, H., Hammond, E. W., and Thomson, J., Awalyst, 1964, 89, 175. Abbott, D. C., Egan, H., and Thomson, J., J. Chronzat., 1964, 16, 481. SAC Conference, Nottingham, 1965,” W. Heffer & Sons Ltd., Cambridge, 1965, 121. Received June loth, 1966

ISSN:0003-2654    

DOI:10.1039/AN9669100094

出版商:RSC    

年代:1966

数据来源: RSC

摘要:

98 DALZIEL AND THOMPSON SOLVENT-EXTRACTION AXD ABSORPTIOMETRIC [ A ? Z d J ' S f , VOl. 91 The Solvent-extraction and Absorptiometric Determination of Iron with 2-Mercaptopyridine-l-oxide BY J. A. W. DALZIEL AND M. THOMPSON* (Department of Chemistry, Chelsea College of Science and Technology, Manresa Road, London, S. W.3) Iron(1r) and iron(II1) can be completely extracted from M sulphuric acid into a 0.02 M solution of 2-mercaptopyridine- 1-oxide and determined absorp- tiometrically at 550 mp. The only significant interference is from copper(I1); the sensitivity of the method is 0-02 pg cm-2. COLORJMETRIC reagents for iron are numerous,l but relatively few are in common use. Those that depend on reaction with iron(1rr) are generally subject to interference from anions such as fluoride and phosphate.Some methods, viz., the thiocyanate and the thioglycollate (mercaptoacetate) methods, have the additional disadvantage of unstable reactions that cause colour fading. Methods that depend on the reaction of reagents such as 2,2'-dipyridyl, 1,lO-phenanthroline and related compounds with iron(I1) are generally preferred because of their good sensitivity. The reagent 2-mercaptopyridine-l-oxide, subsequently referred to as thione, has already been used for the gravimetric determination of iron.2 This paper reports an extension of the use of thione in the determination of smaller amounts of iron by a spectrophotometric method, and one reagent can now be used for determining iron over a wide range of concentrations. In weakly acidic solutions both iron(r1) and iron(II1) are precipitated as the black iron(II1) complex (C,H,NOS),Fe. This complex can be extracted into chloroform, from relatively strong acidic solutions of iron, to give a violet solution that forms the basis of the method.EXPERIMENTAL THE VISIBLE SPECTRA OF SOME THIONE COMPLEXES- Thione complexes of chromium(m), manganese(II), iron(III), cobalt(n), nickel(II), copper(I1) and zinc(I1) were prepared by precipitation from homogeneous solution by the hydrolysis of S-2-pyridyl thiuronium bromide 1-oxide (Thiurone) in the presence of the appropriate metal ion, at a suitable pH. The visible spectra were recorded, on a Unicam SP500 spectrophotometer, of solutions of known concentration of each neutral inner-complex in analytical-reagent grade chloroform. The spectra are reproduced in Fig.1. Their most notable feature is the exceptional absorption of the iron complex compared with the other complexes. Analytically this is important as it imparts an inherent selectivity to the colorimetric method for iron. The molecular extinction coefficient at 550 mp is about 3400, which is greater than the extinction coefficient of the other complexes by a factor of more than ten, e.g., for the copper(rr) complex at 550 mp the value is only about 150. The strong absorption of the iron(II1) complex has been attributed to charge-transfer rather than to d - d transition^.^ THE EXTRACTION OF IRON AND OTHER METALS- The extraction of iron(II1) from aqueous sulphuric acid into a 0.02 M solution of thione in chloroform was studied ; it was found that iron was extracted quantitatively from solutions with acidities varying between 5 M and 0.005 M.Of the other metals studied only copper was extracted over the whole of this range. Nickel was partly extracted from solutions weaker than O ~ M , and cobalt from solutions weaker than 0.01 M in sulphuric acid. Chrom- ium(m) was not extracted at all, even after prolonged mixing of the phases. A molar solution of sulphuric acid was selected for the composition of the aqueous phase in the recommended method, as this concentration would prevent the extraction of nickel and cobalt. Iron can also be extracted with equal efficiency from 2 N solutions of hydrochloric acid, nitric acid (free from nitrous acid), acetic acid and even phosphoric acid and hydrofluoric acid, * Present address : London Transport Research Laboratories, 566 Chiswick High Road, London, W.4.February, 19661 DETERMINATION OF IRON WITH 2-MERCAPTOPYRIDINE-1-OXIDE 99 Wavelength, mp Fig.1. Visible spectra of 1 + 2 complexes of cobalt(II), copper(II), nickel(II), zinc(I1) and 1 + 3 complexes of iron(II1) and chromium(II1) with thione METHOD REAGENT- Thione solution, 0.02 M, in analytical-reagent grade chloroform. Dissolve 10 g of Thiurone, S-2-pyridylthiuronium bromide-1-oxide (Hopkin and Williams Ltd.), in 40 ml of M sodium hydroxide and boil the solution for about 2 minutes to complete the hydrolysis. Acidify the solution, cool it rapidly with continuous agitation, and filter off, wash and dry the freshly prepared thione. Dissolve 2-5 g of thione in 1 litre of chloroform. The preparation should be carried out with as little exposure to light as possible, and the chloroform solution should be stored in a brown bottle. PROCED u RE- Prepare a solution of iron in M sulphuric acid by a method appropriate for the sample; the iron can be in the (11) or (111) oxidation state. Transfer a 25-ml portion, containing up to 0.4 mg of iron, into a 100-ml separating funnel.Add to the solution with a pipette, 25 ml of the thione solution, and mix the phases by shaking the funnel for 3 minutes. Allow the phases to separate, and run off some of the chloroform through a plug of cotton-wool in the stem of the funnel into a 1-cm spectrophotometer cell. Measure the optical density at a wavelength of 550mp, with some thione solution extracted with pure M sulphuric acid as reference solution.CALIBRATION- A calibration graph was prepared by the extraction of various known amounts of iron(m), up to about 0.4 mg, by the recommended procedure. The best straight line through twelve points was calculated and found to be of the form : optical density = 0.002 + 2.06 x mg of iron extracted. This is proof of adherence to Beer’s law within the experimental error. The sensitivity of the method as described is thus 0.02 pg cm-2. REPRODUCIBILITY OF THE METHOD The reproducibility of the method was investigated by performing replicate extractions on constant amounts of iron, to give a mean optical density of about 0.6. The estimate of100 DALZIEL AND THOMPSON : SOLVENT-EXTRACTION AND ABSORPTIOMETRIC [l!%a&Sif, VOl.91 the standard deviation of the observed optical densities was found to be 1.3 per cent. of the mean. The estimate of the standard deviation due to the instrument alone was 0.9 per cent., giving (by subtraction of the variances) the reproducibility of the extraction as 1.0 per cent. It has been previously noted that thione is decomposed by light,2 and therefore the solution of thione in chloroform must be stored away from light. However, the solution is stable for at least 1 week if stored in a brown bottle, and exposure to light during normal extraction manipulations causes no measurable change in the solution. Chloroform solutions containing extracted iron stored in the open laboratory, but not exposed to direct sunlight, showed no change in their optical densities during several days.Exposure to direct sunlight causes rapid fading of the iron colour, presumably due to the continuous photo-decomposition of the small equilibrium concentration of free thione. INTERFERENCES Interference with the method was tested by performing the extraction of a fixed amount (0-3mg) of iron(m) by the recommended procedure, in the presence of known amounts of foreign substances. The optical density of the resulting solution was compared with that of an extract of an aliquot of pure iron solution. If the difference amounted to more than 2 per cent., the experiment was repeated with a smaller proportion of the interfering substance. The proportion of interfering substance causing an error of 2 per cent.was regarded as being a tolerable ratio, and is the figure quoted in Table I. TABLE I INTERFERENCE FROM VARIOUS SUBSTANCES IN THE DETERMINATION OF 0.3mg OF IRON COLOUR STABILITY Interfering substance Mg2+ . . Ti4+ . . Cr3+ . . Mn2+ . . co2+ . . Ni2+ . . cu2+ . . Zn2+ . . Cd2+ . . Hg2+ . . Sn2+ . . A13+ . . Ce4+ . . Th4+ . . Zr02+ . . NH30H+ NO,- . . NO,- . . c1- . . H,PO,- . . F- . . Aceiic acid Thiourea v0,- . . uo22+ . . . . . . .. .. .. .. . . .. .. . . . . . . . . .. . . . . .. . . . . .. . . . . .. . . . . . . .. .. .. . . . I .. .. .. . . .. .. .. . . . . . . . . .. . . .. . . .. .. . . . . Amount added, mg 6.0 6-0 1-5 6.0 6.0 6.0 6.0 6.0 6-0 6.0 6.0 6.0 6.0 6.0 6-0 6.0 6.0 12-0 300 30 150 450 900 300 600 Iron found, per cent. added 100 103 100 99 99 100 100 98 97 95 101 100 99 100 100 100 100 97 100 100 100 100 100 * * Tolerable ratio of other ion > 20 13 >5 PO 40 > 20 > 20 20 13 8 40 > 20 40 > 20 > 20 > 20 > 40 670 0.2 - > 500 > 1500 > 3000 > 1000 > 2000 * Denotes that gross interference occurs.It can be seen that the determination of iron is free from interference of a wide range of substances. The only important exception is copper, which will cause gross interference if it is not separated prior to the determination of iron. Attempts to “mask” the copper by the addition of 2 per cent. of thiourea to the aqueous phase were unsuccessful. DISCUSSION The colorimetric method of determination is inherently more selective for iron than the gravimetric method that we have previously reported.2 This is because of the relatively large molecular extinction coefficient of the iron(II1) complex and the high acidities at whichFebruary, 19661 DETERMINATION OF IRON WITH 2-MERCAPTOPYRIDINE-1-0~1~~ 101 it is extracted.The latter is due to the combination of the stability of the complex with its favourable partition coefficient from the aqueous phase into chloroform. The method can be used to determine both iron@) and iron(rrr), i.e., total iron, and is exceptional among methods of this kind in being unaffected by high concentrations of fluoride and phosphate. The colour is stable, provided that chloroform solutions of the complex are not left in strong sunlight. The method appears to have two disadvantages in comparison with those that use 1,lO-phenanthroline with iron(I1). These are the lower sensitivity of the thione method, although this is, to some extent, compensated by the possibility of concentration of the iron complex in the solvent-extraction stage, and the gross interference of copper which necessitates its prior separation from the iron. We are indebted to London Transport Board for the provision of study leave for one of us (M.T.). REFERENCES 1. 2. 3. Sandell, E. B., “Colorimetric Determination of Traces of Metals,” Interscience Publishers Inc., Dalziel, J . A. W., and Thompson, M., Analyst, 1964, 89, 707. Robinson, M. A . , J . Inorg. Nucl. Chem., 1964, 26, 1277. New York, 1959, pp. 522-554. Received July 23rd, 1965

ISSN:0003-2654    

DOI:10.1039/AN9669100098

出版商:RSC    

年代:1966

数据来源: RSC

摘要:

102 HALL, GRAY AND FLYNN: THE USE OF TITAN YELLOW FOR THE [Analyst, Vol. 91 Observations on the Use of Titan Yellow for the Determination of Magnesium with Special Reference to Soil Extracts BY R. J. HALL, G. A. GRAY AND L. R. FLY” (Ministry of Agriculture, Fisheries and Food, National Agricultural Advisory Service, Kenton Bar, Newcastle upon Tyne) Examination by paper and thin-layer chromatography of several prepara- tions of titan yellow from different manufacturers has shown them to be complex mixtures. By using a titan yellow selected chromatographically, it has been possible to improve the stability and sensitivity of the coloured complex produced with magnesium. Studies have been made of the effects of alkalis, interfering ions and protective colloids, and the use of gelatin with a lithium hydroxide - glycine buffer is proposed for the determination of magnesium in soil extracts.Results have been compared with those obtained by atomic-absorption spectroscopy and show close correlation. THE acceptance of titan yellow (the sodium salt of methylbenzothiazole (1,3)-4,4’-diazoamino- benzene-(2,2’)-disulphonic acid) as the reagent for the absorptiometric determination of small amounts of magnesium is far from universal. Despite the claims made in its favour, many workers have reported inconsistent results, and difficulties were experienced in this laboratory with the published methods for plants and soi1s.l y 2 v3 The advantages of atomic-absorption spectroscopy are to some extent offset by the fact that it requires expensive equipment, which may not be available in some laboratories.It was felt, therefore, that a re-assessment of the determination of magnesium with titan yellow should be undertaken with particular reference to its application to soils. Bradfield: in a study of the factors that affect the adsorption of titan yellow on to magnesium hydroxide, observed differences in the composition of four commercial prepara- tions, and noted the presence of a substance exhibiting an intense violet fluorescence that could be seen on paper chromatograms in ultraviolet light. Hall and F l ~ n n ~ ? ~ have now shown by thin-layer chromatography that laboratory grades of titan yellow can be extremely variable and complex mixtures of magnesium-reactive and non-reactive fractions, and may also contain a variety of fluorescent components.They have established that the main fraction reacting with magnesium has a mean RRF value of 0.32 when chromatographed on silica gel - alumina and, although all of the samples that they examined were heterogeneous, it was possible to select those in which the magnesium-reactive dye was predominant as suitable for analytical determinations. This paper describes observations that have been made on a variety of titan yellows from different sources and proposes a method for determining available magnesium in soil extracts by using a chromatographically selected dye in a buffered medium. The results correlated closely with those obtained by atomic-absorption spectroscopy. REAGENTS- Where possible all reagents should be of analytical-reagent grade.Preferably the water should be distilled from an all-glass apparatus and be free from carbon dioxide. Calcium chloride soil extractant, 0.025 N CaCl,-Dissolve 160 g of dried calcium chloride (analytical-reagent grade 70 per cent. CaCl,) in 2 litres of water. Standardise with a suitable solution of silver nitrate or by EDTA titration and adjust the concentration to N. Prepare a 0.025 N solution by appropriate dilution with water. This solution has a pH of 5.0 to 5-1. Prepare also a 0.1 N calcium chloride solution. Compensating solution-Solution A : Dissolve 0-2 g of hydroxylammonium chloride and 0.1 g of aluminium chloride (A1C1,.6H20) in 60 ml of water, add 40 ml of triethanolamine and make up to 100 ml with water. Solution B: Prepare a 0.2 N calcium chloride solution from the N solution.Mix equal quantities of A and B immediately before use. MATERIALS AND METHODSFebruary, 19661 DETERMINATION OF MAGNESIUM WITH REFERENCE TO SOIL EXTRACTS 103 Titan yellow solution-Dissolve 0.01 g of selected titan yellow in about 20 ml of water. Dissolve 0.1 g of gelatin (Difco or other source low in magnesium) in about 50 ml of water at 40" to 50" C. Mix the two solutions and make up to 100 ml with water. The final solution should be clear and may be kept for a few days at 2" C. The titan yellow may be stored as 0.1 per cent. w/v solution in water and suitably diluted to give 0.01 per cent. in 0.1 per cent. gelatin solution. The titan yellow must consist predominantly of magnesium-reactive material, and have been selected after chromatographic examination as described by Hall and F l ~ n n .~ , 6 Lithium hydroxide, N-Dissolve 6.94g of freshly cut lithium metal in water and make up to 1 litre. Store in a polythene bottle. Glycinne solution, 0.1 M-Dissolve 0.75 g of glycine in 100 ml of water. Store in a polythene bottle at 2" C. Bufer solution pH 12.5-Mix 10 ml of N lithium hydroxide with 10 ml of 0.1 M glycine solution and make up to 100 ml with water. Standard magnesium solution-Dissolve 0-8289 g of Specpure magnesium oxide (Johnson Matthey Ltd.) in 42 ml of N hydrochloric acid and &lute to 1 litre with water. This solution contains 500 pg of magnesium per ml. Dilute 5 ml of this solution to 250 ml to give a working standard of 10 pg of magnesium per ml.METHOD FOR THE DETERMINATION OF MAGNESIUM- Extract 5 g of air-dry soil (ground to pass 2-mm mesh) by shaking the soil for 2 hours with 50 ml of 0.025 N calcium chloride solution. Filter through a Whatman No. 2 filter-paper. With a pipette transfer 2 ml of the clear filtrate into a suitable tube of about 15 ml capacity and add, in the following order, 1.0ml of water, 1.0ml of compensating solution, 2 ml of titan yellow solution and 4 ml of the lithium hydroxide - glycine buffer. Mix after each addition. It may be necessary to use smaller volumes of soil extract and to make up to 2 ml with 0-025 N calcium chloride solution. Prepare a set of standards containing 0, 5, 10, 15, 20 and 25 pg of magnesium and add 0.5 ml of 0.1 N calcium chloride to each. Make the volumes up to 3.0 ml with water and proceed as for a soil extract.Measure the red colour at 530 mp or with an Ilford 624 filter. ATOMIC-ABSORPTION SPECTROSCOPY- of the design described by Box and Walsh.' same results. PAPER CHROMATOGRAPHY- For chromatography, 10-pl spots containing 25 pg of dye were applied to sheets of Whatman No. 4 filter-paper and developed for 16 hours at ambient temperatures (usually 17" to 22" C) by ascending solvent consisting of N ammonium hydroxide + butanol + methanol (20 + 40 + 40 v/v). THIN-LAYER CHROMATOGRAPHY- Standardise the solution against N hydrochloric acid. Measurements were made with a Hilger instrument, a Unicam SP9OOA and two others All four instruments gave essentially the Plates were prepared with the Desaga apparatus. Silica gel - alumina plates-A mixture was made of 6 g of silica gel (Stahl) and 12 g of alumina (Stahl) ; 20 ml of water and 6 ml of ethanol were mixed and added to the powder to form a paste, Then 10 ml of water were added and the whole mixed to give sufficient slurry to spread three 20 x 20-cm plates.The plates were allowed to air-dry and then heated at 100" C for 1 hour. The dye preparation (2-5 and 5.0 mg per ml in water) was applied in spots of 10 pl and 2 0 4 , allowed to dry, and the chromatograms were developed by ascending solvent for 4 to 5 hours with butanol that had been equilibrated by shaking with a 1.5 N solution of ammonium hydroxide and then discarding the aqueous phase. PREPARATION OF DYE FRACTIONS FROM THIN-LAYER CHROMATOGRAMS- After development, selected yellow dye fractions were carefully removed by loosening the material with a micro spatula, shaking it through a small funnel into test-tubes and extracting with 5ml of methanol to which ammonium hydroxide, sp.gr. 0.880, had been added at the rate of 0.2 ml per 100 ml.The methanol was removed by compressed air and replaced by 5ml of water.104 HALL, GRAY AND FLYNN: THE USE OF TITAN YELLOW FOR THE [Analyst, VOl. 91 EXPERIMENTAL RESULTS All experimental observations were carried out in duplicate or triplicate, and a Unicam SP600 spectrophotometer was used for measuring optical densities. SELECTION OF TITAN YELLOW- Fourteen different samples of titan yellow supplied by British Drug Houses Ltd., Hopkin & Williams Ltd. and Merck Chemicals Ltd. were examined spectrophotometrically and by filter-paper and thin-layer chromatography. SPECTROPHOTOMETRIC MEASUREMENTS- Table I shows the wide range of optical densities when the yellow intensity of the dye was measured at 405 mp, the peak in that part of the visible spectrum.The concentration was 2.5 mg in 100 ml of aqueous solution. TABLE I OPTICAL DENSITY MEASUREMENTS OF TITAN YELLOW Sample of Titan yellow. . . . A B C D E F G Sample of Titan yellow. . .. H I J K L M N Optical density at 405 mp . . 0.425 0.912 0-942 0-465 0.820 0.859 0.689 Optical density at 405 mp . . 0-325 0.490 0.859 0.689 0.949 0.707 0-509 There appears to be little relationship between concentrations of titan yellow and magnesium when used on a weight to volume basis. With certain outstanding exceptions closer correlation was obtained when the titan yellow was used at a standard optical density.In order to determine this characteristic, titan yellow solutions were prepared such that aliquots, when diluted to 2 ml, would have an optical density of 2.0 at 405 mp. This was done by calculating, and accurately measuring, volumes of the concentrated titan yellow solutions which, when diluted four times with 0.2 per cent. gelatin solution, gave optical densities of 0.500 at 405 mp. It had previously been established that Beer’s law was obeyed at this wavelength. Each diluted titan yellow solution was checked at 405mp and, where necessary, suitable adjustments were made to ensure the standard optical density of 0-500 for 8 ml. The same volumes of the concentrated titan yellow solutions were then taken for the reaction with 0 and 15 pg of magnesium under the conditions of the method for soil extracts.It can be seen from Table I1 that most of the titan yellows then gave somewhat similar measurements in their reactions with magnesium, but when correlated with weights there was no relationship at all. Reactions with some samples, particularly A, H, I and M, produced brownish colours rather than red. TABLE I1 REACTION BETWEEN MAGNESIUM AND TITAN YELLOW OF STANDARDISED OPTICAL DENSITY Titan yellow in test, tG A 248 13 109 C 113 D 221 E 122 F 240 G 133 Optical densities* a t 530 mp for- r no magnesium 0.037 0-052 0.049 0.057 0.049 0.051 0.047 15 CLg of magnesium 0.112 0.161 0-155 0.160 0.168 0.172 0.178 Titan yellow in test, pg H 321 I 209 J 120 K 150 L 110 M 139 N 206 Optical densities* at 530 mp for- 7- magnesium magnesium 0.033 0.088 0-038 0.123 0.047 0.185 0-057 0.156 0-059 0.164 0.049 0.167 0.05 1 0.171 no 15 P.lg Of * The figures for 15 pg of magnesium have been corrected by subtraction of the blank.CHROMATOGRAPHIC STUDIES- On filter-paper, 10 of the samples showed a major yellow component having a mean RF value of 0.32. Other yellow components were also seen as well as fluorescent substances. When chromatographed on thin-layer plates of silica gel - alumina the dyes separated into many fractions, several of which exhibited blue and green fluorescence when viewed inFig. 1. Separation of titan yellow on silica gel + alumina, developed with ascending butanol equilibrated with 1.5 N ammonium hydroxide : spotted with 20-4 volumes of solutions containing 5 mg per ml; photographed in ultraviolet light 0 0 i Blue, green and yellow fluorescent spots Mg2+ reactive __ Relatively unreactive w i t h blue fluorescent bands -1 I- 0 0 0 0 G E N M K H Fig.2. Key to Fig. 1 [To face page 105February, 19661 DETERMINATION OF MAGNESIUM WITH REFERENCE TO SOIL EXTRACTS 105 ultraviolet light (Figs. 1 and 2). There was, however, essentially the same order of separation of the main fractions as on filter-paper, and the same 10 samples of dye were seen to contain a major yellow fraction with a mean RF value of 0-32 (range 0.25 to 0.40). The difficulties in isolating large amounts of each fraction of dye have been described by Hall and Flynn,6 and it was not practicable to test each dye fraction of each sample.Sufficient amounts of the dye component, moving with an RF of 0.32, were isolated from one sample from each manu- facturer, and removed from the support by extraction into ammoniacal methanol. The volumes of these solutions were adjusted with water so that an optical density of 0.75 was obtained at 405mp. Reactions with various amounts of magnesium were then carried out under the conditions described for soil extracts except that the amounts of reagents were adjusted to give a total volume of 5ml. The results are shown in Table 111. TABLE I11 REACTION WITH MAGNESIUM OF THE MAIN TITAN YELLOW FRACTION FROM SAMPLES D, E ,4ND J Optical density at 530 mp from sample- Magnesium, f A \ D E J 0 0.021 0.017 0-018 2 0.046 0.046 0.046 4 0.119 0.115 0.121 6 0-168 0.176 0.176 8 0.191 0.197 0.198 10 0-204 0.206 0.207 Within experimental limits, the readings indicate that all three dye fractions were giving essentially the same reaction with magnesium per unit optical density at 405 mp.The lack of linearity was almost certainly due to taking insufficient titan yellow for the amount of mag- nesium present, but the readings were reasonably consistent throughout for the three samples. It was concluded that the fraction having a mean RF value of 0.32 was, therefore, primarily responsible for the reaction with magnesium. From these findings it was decided that those samples, in which this fraction was the major component, were probably suitable for the determination of magnesium and should be used for further studies. Tests with fractions of other RRF values showed either a reduced reaction or none at all.Most of the work described in this paper was carried out with titan yellow E. EFFECT OF COLLOIDS- Bradfield* examined the stabilising effect of 14 different colloids on the reaction between titan yellow and magnesium, and found that the greatest sensitivity with effective protection of the complex was achieved with a final concentration of 0.01 per cent. w/v poly(viny1 alcohol) and 10 per cent. v/v glycerol, and his recommendations were adopted for the determination of magnesium in soil extracts and plant-ash preparations. In this laboratory, however, poly- (vinyl alcohol) as the protective colloid was often unsatisfactory for soil extracts ; precipitates frequently formed making photometric measurements unreliable.Attempts to improve stability with agar and methyl cellulose were unsuccessful, but excellent results were obtained with the titan yellow prepared in a 0-1 per cent. aqueous solution of gelatin. Through the generous co-operation of Messrs. B. Young & Co., Ltd., London, S.E.l, some 16 different gelatins were tested and most were found to have the same protective action without inter- fering with the determination. In common with poly(viny1 alcohol), gelatin has an enhancing effect on the optical density of the magnesium - dye complex, but this was reasonably constant within the final concentration of 0.01 to 0.10 per cent. A final reaction concentration of 0.02 per cent. w/v was adopted for this work.Although glycerol used with starch or poly(viny1 alcohol) has been said to increase the sensitivity of the reaction, when used by itself the optical density due to 25 pg of magnesium was reduced from 0.322 (in 0.02 per cent. gelatin) to 0.186 (in 5 per cent. v/v glycerol)-some 42 per cent. Table IV compares the sensitivity of the reaction with titan yellow E in the presence of glycerol and gelatin, which in this case was used at a final concentration of 0.04 per cent.106 HALL, GRAY AND FLYNN: THE USE O F TITAK YELLOW FOR THE [Analyst, VOl. 91 TABLE IV EFFECT O F GELATIN -4KD GLYCEROL ON FORMATION OF MAGNESIUM - TITAN YELLOW COMPLEX Optical density" at 530 mp developed with lithium hydroxide - glycine pH 12.5 in presence of- r 1 Magnesium, gelatin - Pg water gelatint glycerol: glycerols 0 0.056 0.064 0.081 0.08 1 5 0-022 0.037 0.002 0-005 16 0.080 0.168 0.057 0.087 25 0.182 0.310 0.129 0.194 * The readings for 5, 15 and 25 pg of magnesium have been corrected by subtraction of the blank value.t Final concentration 0.04 per cent. w/v. Final concentration 10 per cent. v/v. Final concentration 0.04 per cent. w/v and 10 per cent. v/v. EFFECT OF ALKALI- I t has been well established that the reaction between magnesium and titan yellow requires a strongly alkaline medium, and sodium hydroxide appears to have been generally used at final concentrations from 0.5 to 1.0 N. No previous publications have been found sug- gesting buffering the reaction. Experiments with lithium, potassium and sodium hydroxides with and without glycine to form buffers showed a lithium hydroxide - glycine reagent to be superior for calcium chloride extracts of soil.Development of the complex was accelerated in the presence of the lithium reagents. Table F7 shows a comparison of the reaction with 5 and 25 pg of magnesium with three different titan yellows containing a low, a medium and a high level of the active fraction after development of the colour with solutions of different alkalis. The increased sensitivity that was attained by producing the colour with the lithium buffer is clearly evident, and particularly so at the 5 pg of magnesium level. The considerable increase in the optical density of the blank in those reactions with unbuffered alkalis masked the readings attributable to the magnesium - titan yellow complex and may well be due to a separate reaction of the alkali with the dye.Reactions with unbuffered sodium hydroxide were extremely variable. TABLE V REACTIONS OF TITA?: YELLOW WITH MAGNESIUM IN ALKALINE SOLUTIONS Lithium hydroxide - *Magnesium, glycine buffer 0 0.055 E (high) . . { 5 0.048 25 0.330 0 0.022 25 0.066 0 0.056 5 0.040 i 25 0.264 Titan yellow PLg at pH 12.5 H (low) ..I 5 0.028 M (medium) . . 0.4 N lithium hydroxide 0.219 0.009 0.276 0.035 0.002 0.085 0.162 0.001 0.244 0.4 N potassium hydroxide 0.210 0.024 0.24 1 0.050 0.010 0.07s 0.141 0.030 0-234 0.4 N sodium hydroxide 0.21 1 0.0 18 0.218 0.038 0.006 0.066 0.125 0.006 0.245 * The readings a t 530 mp for 5 and 25 pg of Mg2+ have been corrected by subtraction of the blank value.With unbuffered potassium or sodium hydroxide at reaction concentrations of 0-1 to 0.6 N the colour development took as long as 90 minutes to reach completion, but full development of the red magnesium complex was virtually instantaneous with unbuffered 0.4 N lithium hydroxide, and within a few minutes when buffered. Although all the results reported here were obtained with a lithium hydroxide - glycine reagent, similar results were obtained with glycine buffers prepared with the same volumes of normal potassium or sodium hydroxide. From Table VI and Fig. 3 it can be seen that no red magnesium complex was formed until the final reaction mixture reached a pH of 11-45, and little change in the net optical density with 20 pg of magnesium took place between pH 12.2 and 1'7.5, although the optical The reactions were developed as described for soil extracts.February, 19661 DETERMINATIOK OF MAGNESIUM WITH REFERENCE TO SOIL EXTRACTS 107 density of the blank rose markedly with increasing pH and alkali content of the reaction mixture.Fig. 3. Effect of pH on the development of the magnesium - titan yellow complex in lithium hydroxide - glycine buffer TABLE VI EFFECT OF pH ON MAGNESIUM - TITAN YELLOW REACTION N lithium hydroxide in buffer, * ml 2.0 2.4 3.0 3.6 4.0 7.2 8.0 8.8 10.0 16.0 20.0 Buffer 11.85 12-0 12.1 12-15 12-25 12-4 12.45 12-5 12.5 12.5 12-5 PH Reaction 11.0 11.3 11.45 11.6 11.75 12-0 12.2 12.2 12-25 12.4 12-5 PH Optical densities a t 530 mp for- r 1 20 pf magnesium blank 0.108 0.196 0.233 0.276 0.297 0.306 0.316 0.333 0.340 No reaction 0.022 0.022 0.025 0.042 0.047 0.050 0.062 0.078 0-088 Optical density difference 0.086 0.172 0.208 0.234 0.250 0.256 0.254 0.255 0.252 * Volume of lithium hydroxide mixed with 10 ml of 0.1 M glycine solution and made up to 100 ml.It seemed convenient to adopt the buffer containing equal volumes of 0-02 M glycine solution and 0-2 N lithium hydroxide. Fig. 4 shows a typical calibration curve. 0 35 I Magnesium, pg Fig. 4. Calibration curve for the reaction between magnesium and titan yellow108 HALL, GRAY AND FLYNN: THE USE OF TITAN YELLOW FOR THE [Analyst, VOl. 91 INTERFERENCE BY OTHER IONS A great deal of conflicting evidence has been published on the effect of aluminium, calcium, iron, manganese and phosphate.Bradfield4 has reported comprehensively on this literature and concluded that the extent to which these ions interfere depends on the reaction conditions. Evidence obtained in this laboratory confirms Bradfield’s findings to some extent. The observations of Goffinets have also been confirmed, in that calcium ions cause an initial increase in colour between magnesium and titan yellow, rising to a maximum when 3 to 4mg are present and then decreasing. In Table VII these effects can be seen. It is of interest that, although the optical density of the magnesium - titan yellow complex increased with increasing calcium to a maximum value in the presence of 3 mg of calcium, that of the blank with the same amounts of calcium in fact decreased. The reactions were carried out as described in the method, except that solution R was omitted from the com- pensating reagent and various amounts of calcium were introduced separately.TABLE VII EFFECT OF CALCIUM ON THE MAGNESIUM - TITAN YELLOW REACTION Optical density* a t 530 mp with- 7 - 7 Calcium, no 5 tGof 15 Pg of 25 Clg of mg magnesium magnesium magnesium magnesium 0 0.098 0.045 0.138 0.188 0.5 0.097 0.048 0.186 0-290 1 0.085 0.046 0-175 0.338 2 0-065 0.041 0.179 0.349 3 0.054 0.042 0.189 0.355 4 0.039 0.034 0.163 0.311 6 0.029 0.028 0.158 0.302 10 0.027 0.019 0.155 0.283 * The values have been corrected by the subtraction of the blank. Under the conditions of the test, copper and iron were found to exert little effect. Copper tended to increase the optical-density values of the reaction, but even at the 100-pg level the influence was small.Iron slightly decreased the optical densities as the levels of both iron and magnesium increased. For the results in Table VIII the reactions involved 2 mg of calcium. TABLE VIII EFFECT OF COPPER AND IRON ON THE MAGNESIUM-TITAN YELLOW REACTION Optical densities a t 530 mp corrected for blank value r A I in absence in presence of copper in presence of iron A A Magnesium, of iron f \ r \ CLg or copper 10 pg 50 pg 100 CLg 10 50 t% 100M 0 0.065 0.050 0.053 0.074 0.050 0.048 0.039 5 0.04 1 0.042 0.033 0.056 0.038 0.035 0.035 15 0.179 0.179 0.181 0.203 0.188 0.183 0.171 25 0-349 0.340 0.340 0-352 0.346 0.346 0.342 No interference was encountered by the presence of 50 pg of phosphorus as phosphate ions in the soil extracts, but a turbidity was produced by 100 pg.These levels are not usually extracted by 0-025 N calcium chloride. Up to 10 pg of manganese per test had little effect on the formation of the magnesium - dye complex; there was a slight increase in the optical density measured with 25 pg of manganese(I1) ions. DETERMINATION OF MAGNESIUM IN SOIL EXTRACTS It is probable that the determination of magnesium by means of titan yellow has been applied more to soil analysis than to any other subject, and it was Mikkelsen and Totho who first described differences in titan yellow when using it in their soil-magnesium studies. This present investigation was prompted by difficulties in assessing accurately the available magnesium levels in calcium chloride extracts of soils that were suspected of being deficientFebruary, 19661 DETERMINATION OF MAGNESIUM WITH REFERENCE TO SOIL EXTRACTS 109 in the element.Frequently the values which were obtained by a method involving the use of strong unbuffered alkali indicated that the soils were not deficient in available magnesium, although crops grown in them exhibited symptoms of magnesium deficiency. Comparisons with determinations by atomic-absorption spectroscopy revealed striking differences in those soil extracts that were expected to be low in magnesium. Although there was a tendency for higher figures to be obtained in non-deficient soils by using titan yellow with unbuffered alkali than by using atomic-absorption spectroscopy, the differences were not so great as in those extracts from deficient soils.By developing the complex with buffered alkali, close correlation was obtained with atomic absorption values. A selection of results by the three methods is shown in Table IX. TABLE IX AVAILABLE MAGNESIUM LEVELS IN SOIL DETERMINED BY ATOMIC-ABSORPTION SPECTROSCOPY AND ABSORPTIOMETRY WITH TITAN YELLOW Magnesium, mg per 100 g of air-dry soil, by- atomic- buffered unbuffered absorption titan yellow titan yellow spectroscopy procedure procedure 14.2 14.3 18.6 14.4 14.3 16.8 15.2 15-0 17.5 14.8 15.0 18.6 13-0 12.9 16.6 80.3 81.6 104.5 5.9 5.4 7.1 112.8 113.3 152.5 I A \ I Magnesium, mg per 100 g of air-dry soil, by- atomic- buffered unbuffered absorption titan yellow titan yellow spectroscopy procedure procedure A \ 23.1 7.9 73.8 23.1 0.7 0.8 1-1 0.8 23.6 7.9 80.0 24.5 0.8 1.3 0.9 0.7 27-2 9.8 118.8 28.9 6-3 6-3 6.2 5.0 Statistical analysis of 127 comparisons between values obtained by atomic-absorption spectroscopy and the buffered titan yellow procedure described in this paper gave a corre- lation coefficient of 0.999.Not all soil extractions for magnesium determination are made with calcium chloride solutions; other reagents are sometimes used such as N ammonium nitrate or N ammonium acetate. The presence of large amounts of these reagents inhibits the development of the complex with titan yellow when buffers are used of the concentration proposed for calcium chloride extracts. The problem may be overcome by increasing the molarity of the buffers. Results of reactions of standard magnesium solutions in the presence of 2 ml of each of five common soil extractants are shown in Table X.The table also shows the results after the colour had been developed by glycine buffers of pH 12.5 prepared with lithium, potassium and sodium hydroxide. I t would seem that notwithstanding the proposed use of a lithium hydroxide - glycine buffer for calcium chloride extracts, one prepared with potassium hydroxide appears promising for other extractants. There was a consistently lower blank with increased sensitivity, but departures from Beer’s law tended to be greater than with a lithium buffer. DISCUSSION The observation that different batches of titan yellow varied in their reaction with mag- nesium was first reported by Mikkelsen and Toth8 nearly 20 years ago, and SchubertlO demon- strated that the ordinary commercial product was mainly the 7-sulphonate with a variable amount of the 5-sulphonate isomer always present. The observations reported here show that commercial preparations can be very complex indeed.All those examined contained more than one component that fluoresced strongly in ultraviolet light. Bradfield4 has suggested that one of these may be an excess of the sodium salt of dehydrothio-9-toluidine sulphonic acid formed by an incomplete coupling reaction with a diazotised solution of the same acid. On the thin-layer and paper chromatograms developed in this laboratory, most of the blue or violet fluorescent spots were colourless in daylight. Some of the titan yellows certainly did contain large amounts of yellow components that were strongly fluorescent and which were not magnesium-reactive.These substances tended to remain at the point of origin on the thin-layer plate or to move more slowly than the active fraction. From the investigations described in this paper it seems clear that the reaction between magnesium and the titan yellows available commercially must necessarily be non-specificTABLE X REACTIONS OF MAGNESIUM WITH TITAN YELLOW IN DIFFERENT SOIL EXTRACTANTS 0.025 N calcium chloride* 0.5 N acetic acidf N ammonium acetate? A f A \ 7 \ f \ A sodium CLg hydroxide hydroxide hydroxide hydroxide hydroxide hydroxide hydroxide hydroxide hydroxide 0 0.043 0.062 0.059 0.077 0.183 0.175 0.047 0.143 0.074 6 0-035 0.039 0.043 0-075 0.016 0.034 0.055 0.058 0-045 15 0.174 0.172 0.169 0.247 0.164 0.171 0.223 0.188 0,216 25 0-350 0.333 0.327 0.416 0.312 0.328 0.378 0-347 0-364 Magnesium, potassium lithium sodium potassium lithium sodium potassium lithium 0.5 N sodium acetate and 0.5 N acetic acid: N ammonium nitrate? f A A \ c \ Magnesium, potassium lithium sodium potassium lithium sodium Pg hydroxide hydroxide hydroxide hydroxide hydroxide hydroxide 0 0.052 0.095 0.115 0.032 0.090 0.054 5 0.060 0.067 0.056 0.042 0.044 0.074 15 0.223 0.212 0.217 0.204 0.170 0-237 25 0.373 0.360 0-375 0.352 0.341 0.422 * Buffers prepared with 10 ml of N solutions of the alkalis, as in method.t Buffers prepared with eight times the amount of alkali and glycine used after calcium chloride extraction. Buffers prepared with twice the amount of alkali and glycine used after calcium chloride extraction.w 0 z 2 M n bFebruary, 19661 DETERMINATION OF MAGNESIUM WITH REFERENCE TO SOIL EXTRACTS 11 1 owing to the heterogeneity of the dye. The reaction, however, can be applied usefully for quite accurate quantitative studies by selecting a dye that shows a high level of the mag- nesium-reactive component when examined by thin-layer or even paper chromatography. The reaction can also be made more specific by developing the red titan yellow - magnesium complex with a buffer at pH 12-5. This has two important advantages. The optical density of the blank is reduced to a level that is analytically acceptable and permits optical differences to be measured for less than 5 pg of magnesium in a reaction volume of 10 ml.When con- centrated alkali is used to develop the complex, a much more intense colour is produced with the blank, which renders the reaction quite insensitive for less than 5 pg of magnesium. I t is believed that independent reactions take place between the alkali and the different fractions of the titan yellow, and that these mask the true reaction of magnesium with the active portion of the dye. Because the complex was produced under conditions which, as far as can be ascertained, have not been previously described, it was decided to re-investigate the effects of some ions that have always been regarded as interfering with the reaction. Of several workers who have studied titan yellow, Bradfield1y294 has made some notable contributions and some of his recommendations formed the basis for the compensating solution to overcome the effects of copper, iron, manganese and zinc.In this work rather larger amounts of calcium were found to be required than Bradfield proposed, the optimum amount being 3 to 4mg of calcium for each test. Interference by copper, iron and manganese was negligible, and phos- phate ions had no effect below 50 pg for each test. Although poly(viny1 alcohol) has been widely accepted as a suitable protective colloid, it seemed to be of limited value for soil extracts, and after many attempts with other reagents efforts were directed to the use of gelatin. Of the few workers who have used gelatin, Carlesll appears to have been the first, and he found that 0.1 per cent. gave adequate protection. Experience in this laboratory showed that a final concentration of 0-02 per cent.of gelatin gave excellent protection of the magnesium complex. The complex from 5 to 25 pg of magnesium remained stable in the gelatin solution for up to 48 hours. Since this paper was submitted for publication our attention has been drawn to the presence of magnesium in manufactured gelatin, which has prompted us to examine all our samples by atomic-absorption spectroscopy. The magnesium levels were found to vary from 70 to 1980 p.p.m. of dry material. The “Difco” gelatins had the lowest levels of from 70 to 80 p.p.m., and one of these was used throughout this work. By using 2 ml of a 0.1 per cent. solution, a small amount of magnesium (0.14 pg) was therefore introduced into each reaction.The magnesium may be removed from the gelatin by precipitating the protein with 20 ml or more of a 1 per cent. solution with 5 ml of normal perchloric acid. The precipitated protein may then be re-constituted by re-dissolving it in water and neutralising the small amount of residual acid with N lithium hydroxide solution. Such a procedure can be expected to remove the calcium and phosphate that are also likely to be present as impurities in gelatin. The precipitated gelatin was found to exert the same protective effect on the magnesium complex. Of the other reagents only the calcium chloride solutions were found to contain a measurable amount of magnesium. This amounted to the addition of 0.12 pg of magnesium per test. These amounts do not seriously affect the accuracy of the determinations. The proposed method has been applied to neutral calcium chloride extracts of soil which, according to SchachtschabeP gives a more correct indication of the available magnesium, Comparisons with results by atomic-absorption spectroscopy have shown that the procedure recommended in this paper for using titan yellow produces close correlation, and that the use of strong unbuffered alkali may give erroneously high values in calcium chloride extracts of soils low in magnesium. The observations made during the course of this study have been applied to the deter- mination of magnesium in plant materials. It is hoped to report these findings in a separate communication. We are grateful to our colleagues in the N.A.A.S. laboratories at Cambridge and Reading and to Mr. H. T. Jobson, F.R.I.C., analyst at the School of Agriculture, University of Newcastle upon Tyne, for the determinations by atomic-absorption spectroscopy, to Detective Chief Inspector C. Watson of the Forensic Science Laboratory, Gosforth, Newcastle upon Tyne, for the photography, and to Miss Sheila Rutherford and Mrs. Carole Williams for skilful technical assistance .112 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. HALL, GRAY AND FLY" [Analyst, Vol. 91 REFERENCES Bradfield, E. G., Analyst, 1960, 85, 666. -, Ibid., 1961, 86, 269. Schachtschabel, P., 2. Pflanzenerahr. Dung Bodenk., 1954, 67, 9. Bradfield, E. G., Analytica Chim. Ada, 1962, 27, 262. Hall, R. J., and Flynn, L. R., Nature, 1965, in the press. Box, G. F., and Walsh, A., Spectrochim. A d a , 1960, 16, 255. Goffinet, A,, Bull. Inst. Agron. Gembloux, 1955, 23, 166. Mikkelsen, D. S., and Toth, S. J., J . Amer. SOC. Agron., 1947, 39, 165. Schubert, M., Justus Liebigs A n n l n Strs. Rech. Chem., 1947, 558, 10. Carles, J., Bull. SOC. Chim. Biol., 1957, 39, 445. -- , , J . Chromat., awaiting publication. Received August loth, 1965

ISSN:0003-2654    

DOI:10.1039/AN9669100102

出版商:RSC    

年代:1966

数据来源: RSC