摘要:

THE ANALYSTTHE ANALYTICAL JOURNAL OF THE CHEMICAL SOCIETYEDITORIAL ADVISORY BOARD"Chairman: J. M. Ottaway (Glasgow, U.K.)R. Belcher (Birmingham, lU.K.)L. J. Bellamy, C.B.E. (Waltham Abbey, U.K.)L. S. Birks (U.S.A.)E. Bishop (Exeter, U.K.)L. R. P. Butler (South Africa)"H. J. Cluley (Wembley, U.K.)E. A. M. F. Dahmen (The Netherlands)A. C. Docherty (Bi//ingham, U.K.)D. Dyrssen (Sweden)"P. Gray (Leeds, U.K.)J. Hoste (Belgium)H. M. N. H. Irving (South Africa)M. T. Kelley (U.S.A.)W. Kemula (Poland)G. W. C. Milner (Harwell, U.K.)G. H. Morrison (U.S.A.)"J. H. Knox (Edinburgh, L!K)H. W. Nurnberg (West Germany)"G. E. Penketh (Wilton, U.K.)E. Pungor (Hungary)D. I. Rees (London, U.K.)"R. Sawyer (London, U.K.)P. H. Scholes (Middiesbrough, U.K.)"W.H. C. Shaw (Greenford, U.K.)S. Siggia (U.S.A.)"D. Simpson (Thorpe-Ie-Soken, U.K.)A. A. Smales, O.B.E. (Thornaby, U.K.)"A. Townshend (Birmingham, U.K.)A. Walsh (Australia)T. S. West (Aberdeen, U.K.)"J. Whitehead (Stockton-on- Tees,A. L. Wilson (Medmenham, U.K.)P. Zuman (U.S.A.)U.K.)"Members of the Board serving on The Analyst Publications CommitteeREGIONAL ADVISORY EDITORSDr. J . Aggett, Department of Chemistry, University of Auckland, Private Bag, Auckland, NEW ZEALAND.Professor G. Ghersini, Laboratori USE, Casella Postale 3986,20100 Milano, ITALY.Professor L. Gierst. Universit6 Libre de Bruxelles, Facult6 des Sciences, Avenue F.-D. Roosevelt 50,Professor R . Herrmann, Abteilung fur Med. Physik., 63 Giessen, Schlangenzahl 14, W.GERMANY.Professor W. A. E. McBryde, Faculty of Science, University of Waterloo, Waterloo, Ontario, CANADA.Dr. W . Wayne Meinke, KMS Fusion Inc., 3941 Research Park Drive, P.O. Box 1567, Ann Arbor,Dr. I . Rubeska, Geological Survey crf Czechoslovakia, Kostelni 26, Praha 7, CZECHOSLOVAKIA.Professor J . Rhiicka, Chemistry Department A, Technical University of Denmark, 2800 Lyngby,Professor K. Saito, Department of Chemistry, Tohoku University, Sendai, JAPAN.Dr. A. Strasheim, National Physical Research Laboratory, P.O. Box 395, Pretoria, SOUTH AFRICA.Bruxel les, BELGIUM.Mich. 481 06, U.S.A.DENMARK.Published by The Chemical SocietyEditorial: The Director of Publications, The Chemical Society, Burlington House,London, W1 V OBN. Telephone 01 -734 9864. Telex No. 268001Advertisements: Advertisement Department, The Chemical Society, Burlington House, Piccadilly,London, W1 V OBN. Telephone 01 -734 9864Subscriptions (non-members) : The Chemical Society, Distribution Centre, Blackhorse Road,Letchworth, Herts., SG6 1 HNVolume 104 No 1239 June 1979((3 The Chemical Society 197

ISSN:0003-2654    

DOI:10.1039/AN97904FX021

出版商:RSC    

年代:1979

数据来源: RSC

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ANALAO 104 (1 239) 481 -592 (1 979)ISSN 0003-2654June 197948149150551 6525531538545552560566568572576580582584THE ANALYSTTHE ANALYTICAL JOURNAL OF THE CHEMICAL SOCIETYCONTENTSRelevance of the Approximately Hyperbolic Relationship Between Fluorescenceand Concentration t o the Determination of Quantum Efficiencies-NigelGains and Alan P. DawsonDetermination o f Cadmium in Blood and Urine by Flame Atomic-fluorescenceSpectrometry-R. G. Michel, M. L. Hall, J. M. Ottaway and G. S. FellEffect of Stray Light in Monochrornators on Detection Limits o f Flame Atomic-fluorescence Spectrometric Measurements-R. G. Michel, M. L. Hall, s. A. K.Rowland, J. Sneddon, J. M. Ottaway and G. S. FellSequential Multi-element Analysis of Small Fragments of Glass b y Atomic-emission Spectrometry Using an Inductively Coupled RadiofrequencyArgon Plasma Source-T.Catterick and D. A. HickmanSpectrophotometric Determination of Trace Amounts of Free Cyanide inPrussian Blue-G. J. Willekens and A. Van Den BulckePolarography o f Green S-F. E. PowellDetermination of Nitrogenous Gases Evolved from Soils in Closed Systems-Rapid Method for Determining Fluoride in Vegetation Using an lon-selectiveAutomated Catalytic Method for the Routine Determination of MolybdenumDetermination of the Prostaglandin F2cI Content o f Pharmaceutical Prepara-C. J. Smith and P. M. ChalkElectrode-Alberto Enrique Villain Plant Materials-B. F. Quin and P. H. Woodstions with Triangle Programmed Bromimetric Titration in FlowingSolutions-Zs.Feher, G. Nagy, K. T6th, E. Pungor and A. T6thSHORT PAPERSDetermination of Phenindione Using Organic Brominating Agents-A. Abou Ouf,M. I . Walash, M. Rizk and F. BelalApplication of Difference Spectrophotometry t o the Determination o fDipyrone-M. Abdel-Hady Elsayed, H. Abdine and M. E. Abdel-HamidSpectrophotometric Determination o f Cobalt(l1) with 2.2’-Pyridil Bis(2-quinolyl-hydrazone)-H. Kulshreshtha, R. B. Singh and R. P. SinghDetermination of Osmium(VIII) Alone or in Binary Mixtures with Some GroupVlll Cations by Potentiometric Titration o f lodide-H. Khalifa, N. T. AbdelGhani and M. S. RizkCOMMUNICATIONS *Gas Chromatographic - Mass Spectrometric Analysis o f Polyethylene BottlePacked Intravenous Solutions Contaminated with N-Ethylaniline from theRubber Part of the Two-component Closure-G. A. Ulsaker and G. TeienLuminescence Characteristics of Tubocurarine Chloride-Ernest P. Gibson andJames H. TurnbullBook ReviewsSummaries of Papers in this lssue-Pages iv, vi, vii, x, xii, xivPrinted by Heffers Printers Ltd Cambridge EnglandEntered as Second Class at New York, USA, Post Offic

ISSN:0003-2654    

DOI:10.1039/AN97904BX023

出版商:RSC    

年代:1979

数据来源: RSC

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iV SUMMARIES OF PAPERS IN THIS ISSUESum ma ries of Papers in thisJane, 1979IssueRelevance of the Approximately Hyperbolic Relationship BetweenFluorescence and Concentration to the Determination ofQuantum EfficienciesAn absorptivity-related constant and a quantum efficiency-related constantcan be derived, by a simple graphical procedure, from data obtained in astandard fluorimeter. The quantum efficiency of an unknown fluorophorecan be determined by comparison of its quantum efficiency-related constantwith that of a fluorophore of known quantum efficiency. The absorptivitycan similarly be determined using the absorptivity-related constant. Thismethod relies on the approximately hyperbolic relationship that existsbetween light absorbed and chromophsre concentration.The approximationmay be derived from the Beer - Lambert equation and the limits of its validityhave been tested using theoretical and1 experimental data.Keywords ; Beer - Lambert equation ; fluoyescewe eficiemiesNIGEL GAINSDepartment of Biology, University of York, York, YO1 5DD.and ALAN P. DAWSONSchool of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ.Anaiyst, 1979, 104, 481-490.Determination of Cadmium .in Blood and Urine by FlameAtomic-fluorescence SpectrometryThe development is described of an atomic-fluorescence method for thedetermination of cadmium in blood and urine. The method involves onlythe direct aspiration of acidified urine or diluted and acidified blood into theflame. Calibration is achieved simply by using acidified aqueous standardsand by the application of a pre-determined correction factor to account forchanges in the uptake rate. The sensitivity, accuracy and precision arecomparable to those given by most techniques that are currently in use €orthe determination of cadmium in biological materials.The simplicity ofthe method permits rapid analyses of large numbers of samples (more than25 samples per hour) and is particularly useful for the survey of populationsof people exposed to cadmium. The instrumentation used employs a two-source background correction system. This is essential for maximumaccuracy and allows automatic correction for the scatter, which is the primarycause of inaccuracies in the atomic-fluorescence spectrometric determinationof cadmium.Keywovds ; Flame atowtic-fluwescence spectrometvy ; cadmium determination ;blood analysis ; wine analysisR.G. MICHEL, M. L. HALL and 6. M, OTTAWAYDepartment of Pure and Applied Chemistry, University of Strathclyde, CathedralStreet, Glasgow, GI IXL.and G. S . FELLDepartment of Clinical Biochemistry, Royal Infirmary, Glasgow, G4 OSF.Analyst, 1979, 104, 491-504Vi SUMMARIES OF PAPERS I N THIS ISSUEEffect of Stray Light in Monocliromators on Detection Limits ofJune, 1979Flame Atomic-fluorescence Spectrometric MeasurementsQuantitative results are described that demonstrate that the use of a doublemonochromator to reduce stray light originating from strong thermal emissionin the flame gives significant reductions in noise on the background of thefluorescence measurement.This leads to worthwhile improvements indetection limit for all elements with analytically useful resonance lines a twavelengths shorter than approxima,tely 250 nm. The degree of improve-ment depends upon whether water or a real sample is being aspirated. Forthe determination of cadmium in uri:ne the detection limit is improved by afactor of three and for the determination of selenium in water the improve-ment is a factor of 5-6. Scatter of excitation source radiation is also shownto have a small but significant effect on detection limits when using electrode-less discharge lamps as source. Scatter is more serious in the air - hydrogenflame than the air - acetylene flame.Keywords : Flame atomic-fluorescence spectrometry ; stray light ; double mono-chromatorR.G. MICHEL, M. L. HALL, S. A. K. ROWLAND, J. SNEDDON and J. M.OTTAWAYDepartment of Pure and Applied Chemistry, University of Strathclyde, CathedralStreet, Glasgow, G1 1XL.and G. S. FELLDepartment of Clinical Biochemistry, Royal Infirmary, Glasgow, G4 OSF.Analyst, 1979, 104, 505-515.Sequential Multi-element Analysis of Small Fragments of Glassby Atomic-emission Spectrometry Using an Inductively CoupledRadiofrequency Argon Plasma SourceA method is described for the quantitative multi-element analysis of smallfragments (200-500 pg) of glass using an inductively coupled radiofrequencyargon plasma source. The glass samples are digested with a mixture ofhydrofluoric and hydrochloric acids and chromium is added as an internalstandard.An ultrasonic nebuliser is used in order to reduce to a minimumthe volume of solution required for eaxh analysis. A single monochromatorand detection system is employed, and the wavelength regions of interestare examined sequentially by means of a specially constructed control unit.The results for aluminium, barium, iron, magnesium and manganese showthat the analysis of glass fragments in the range 200-500 pg can be achievedwith coefficients of variation of approximately 10%. Standard glasses wereanalysed to assess the accuracy of the method.Keywords : Glass analysis ; acid digestion ; control unit for automatic sequentialselection of wavelength regions ; iwductively coupled radiofrequency argonplasma; forensic analysisT.CATTERICK and D. A. HICKMANThe Metropolitan Police Forensic Science Laboratory, 109 Lambeth Road, London,SE1 7LP.Analyst, 1979, 104, 516-524June, 1979 SUMMARIES OF PAPERS I N THIS ISSUESpectrophotometric Determination of Trace Amounts of Free Cyanidein Prussian BlueviiTrace amounts of free cyanide in Prussian blue are hydrolysed into hydro-cyanic acid. The latter is captured by a lithium picrate solution containedin a test-tube, which is placed in the reaction vessel. The colour changedue to the resulting lithium isopurpurate is measured spectrophotometricallyat 500nm. This method can detect cyanide down to a level of 2.5 pg in100 mg of Prussian blue and is accurate and reproducible.Keywords : Cyanide determination ; insoluble and colloidal Prussian blue ;spectrophotovnetvyG.J. WILLEKENS and A. VAN DEN BULCKEInstituut voor Hygiene en Epidemiologie, Departement Farmatoxicologie, AfdelingFarmacopee- en Standaardenonderzoek, Juliette Wytsmanstraat 14, 1050 Brussels,Belgium .Analyst, 1979, 104, 525-530.Polarography of Green SThe food dye Green S, 4-[4-dimethylammoniocyclohexa-2,5-dienylidene-(4-dimethylaminophenyl)methyl]-3-hydroxynaphthalene-2,7-disulphonic acid,monosodium salt, is reduced a t the dropping-mercury electrode from 50%ethanolic solutions with the total consumption of two electrons. Polarogramsfollow theoretical predictions in the pH range 2.7-8.75. The reductionmechanism involves two electron transfer steps that are sufficiently differenti-ated at higher pH for separate waves to appear.Keywords : Green S ; food dye ; polavographyF.E. POWELLDepartment of Science and Food Technology, Grimsby College of Technology, NunsCorner, Grimsby, South Humberside, DN34 5BQ.Analyst, 1979, 104, 531-537.Determination of Nitrogenous Gases Evolved from Soils inClosed SystemsA simple method is described for determining nitrogen oxide and nitrogendioxide, evolved from soils, in closed systems. These gases are absorbed byan acidic solution of potassium permanganate, and the resulting nitrate isdetermined by a steam distillation method. Excess of permanganate isreduced with iron(I1) sulphate and neutralised with sodium hydroxidesolution. Ammonium in solution is removed by distillation with magnesiumoxide, and nitrate is determined by distillation after reduction to ammoniumby Devarda's alloy.Nitrogen and dinitrogen oxide evolved from soils are measured using gaschromatography on a single 0.61-m column of molecular sieve 5A, tempera-ture programmed to 250 "C at 39 "C min-1, after an initial period of 1 mina t 35 "C. A complete analysis requires 19.5 min, and 2 pg of nitrogen canbe determined quantitatively for each gas.Keywords : A cidic permanganate ; gas chromatography ; nitrogenous gases ;steam distillation ; soilsC. J. SMITH and P. M. CHALKSchool of Agriculture and Forestry, University of Melbourne, Parkville, Victoria3052, Australia.Analyst, 1979, 104, 538-544

ISSN:0003-2654    

DOI:10.1039/AN97904FP037

出版商:RSC    

年代:1979

数据来源: RSC

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X SUMMARIES OF PAPERS IN THIS ISSUERapid Method for Determining Fluoride in Vegetation Using anIon-selective ElectrodeFluoride was extracted from dried vegetation by stirring with 0.1 N perchloricacid for 20 min at 20 "C. The flu0rid.e content was determined in this extract(pH 1) using the method of standard additions, thus eliminating the need tode-complex fluoride prior to analysis. The presence of up to 2.0% of silicon,0.06y0 of iron, 0.1% of aluminium, 0.7% of magnesium and 1.2% of calciumdid not result in any interferences and recoveries of 98-102y0 were obtained.The fluoride contents of standard saymples determined by this method werehighly correlated (Y = 0.999) with those obtained by reference methods overthe range 4-2000 p g g-l of fluoride in the dry matter.Keywords : Fluoride determination ; fluoride ion-selective electrode ; jluorideJune, 1979in vegetation ; perchloric acid extractionALBERT0 ENRIQUE VILLAEnvironmental Research Department, ALUAR Aluminio Argentino SAIC, 9120Puerto Madryn, Chubut, Argentina.Analyst, 1979, 104, 545-551.Automated Catalytic Method for the Routine Determinationof Molybdenum in Plant MaterialMolybdenum is determined by its catalytic effect on the liberation of iodinefrom iodide by hydrogen peroxide.'The detection limit (twice the standarddeviation of the blank) is 0.01 p.p.m. in plant material, using a 0.25-g sample.Interference from iron is eliminated by preventing any reduction to iron(I1)before complexation with fluoride. High concentrations of salts, othermetals and phosphate do not interfere, and agreement with establishedroutine methods is very good.Keywords Molybdenum determination ; plant material analysis ; automatedcatalytic analysisB.F. QUIN and P. H. WOODSWinchmore Irrigation Research Station, Ministry of Agriculture and Fisheries,Private Bag, Ashburton, New Zealand.Analyst, 1979, 104, 552-559.Determination of the Prostaglandin F2, Content of PharmaceuticalPreparations with Triangle Programmed BromimetricTitration in Flowing SolutionsA survey of the different methods for prostaglandin analysis is given. Theuse of bromine as a reagent for the accurate determination of prostaglandinFz, is indicated from its chemical structure. Several reasons, however, hinderthe use of classical bromimetry.In this paper the application of a new analytical method, the so-calledtriangle programmed titration technique, is described for prostaglandinanalysis. This method permits the simple and effective use of bromine as areagent by performing the titration in a continuous-flow system.The reagentis generated coulometrically during the titration.Methods are described for the determination of the prostaglandin F,,content of different pharmaceutical preparations.Keywords : Prostaglandin F2, determination ; coulometry ; biamperometvy ;triangle programmed bromimetric titration ; flowthrough analysis2s. FEHER, G. NAGY, K. TOTH and E. PUNGORInstitute for General and Analytical Chemistry, Technical University, Budapest,Hungary.and A.TOTHChinoin Pharmaceutical and Chemical Works, Budapest, Hungary.Analyst, 1979, 104, 560-565xii SUMMARIES OF PAPERS I N THIS ISSUEDetermination of Phenindione Using Organic Brominating AgentsJune, 1979Short PaperKeywords : Phenindione determination ; bromination ; titrimetryA. ABOU OUF, M. I. WALASH, M. KIZK and F. BELALFaculty of Pharmacy, Mansoura University, Mansoura, Egypt.Analyst, 1979, 104, 566-568.Application of Difference Spectrophotometry to the Determinationof DipyroneShov,t PaperKeywords : Dipyrone determination ; difference spectrophotometryM. ABDEL-HADY ELSAYED, H. ABDINE and M. E. ABDEL-HAMIDDepartment of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Uni-versity of Alexandria, Alexandria, Egypt.Analyst, 1979, 104, 568-572.Spectrophotometric Determination of Cobalt(I1) with2,2'- Pyridil Bis(2 quinolylhydrazone)Short PaperKeywords 2,2'-Pyridil bis-2(quinoi~lhydrazone) reagent; cobalt determina-tion ; alloy analysis ; spectrophotometryH. KULSHRESHTHA, R.B. SINGH and R. P. SINGHDepartment of Chemistry, University of Delhi, Delhi-110007, India.Analyst, 1979, 104, 572-575.Determination of Osmium(VII1) Alone or in Binary Mixtures withSome Group VIII Cations by Pstentiometric Titration of IodideShort PaperKeywords : Osmium( V I I I ) determination ; Potentiometric titration ; silverelectrodeH. KHALIFA, N. T. ABDEL GHANI and M. S. RIZKFaculty of Science, Cairo University, Giza, Cairo, Egypt.Analyst, 1979, 104, 576-579.Gas Chromatographic - Mass Spectrometric Analysis ofPolyethylene Bottle Packed Intravenous Solutions Contaminatedwith N-Ethylaniline from the Rubber Part of the Two-componentClosureCommunicationKeywords ; N-Ethylaniline migration ; rubber disc ; polyethylene plastics ;intravenous solutions ; gas chromatography - mass spectrometryG. A. ULSAKER and G. TEIENNational Centre for Medicinal Products Control, Sven Oftedalsvei 8, Oslo 9, Norway.Analyst, 1979, 104, 580-582xiv SUMMARIES OF PAPERS I N THIS ISSUELuminescence Characteristics of Tubocurarine ChlorideCommunicationKeywovds : Tubocurarine chlovide ; l:wnzinescence characteristicsERNEST B. GIBSON and JAMES €1. TURNBULLJune, 197

ISSN:0003-2654    

DOI:10.1039/AN97904BP043

出版商:RSC    

年代:1979

数据来源: RSC

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JUNE 1979 The Analyst Vol. 104 No, 1239 Relevance of the Approximately Hyperbolic Relationship Between Fluorescence and Concentration to the Determination of Quantum Efficiencies Nigel Gains* Department of Biology, University of York, York, YO1 5DD and Alan P. Dawson School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ An absorptivity-related constant and a quantum efficiency-related constant can be derived, by a simple graphical procedure, from data obtained in a standard fluorimeter. The quantum efficiency of an unknown fluorophore can be determined by comparison of its quantum efficiency-related constant with that of a fluorophore of known quantum efficiency. The absorptivity can similarly be determined using the absorptivity-related constant. This method relies on the approximately hyperbolic relationship that exists between light absorbed and chromophore concentration.The approximation may be derived from the Beer - Lambert equation and the limits of its validity have been tested using theoretical and experimental data. Keywords : Beer - Lambert equation ; fluorescence ej5ciencies The measurement of quantum efficiencies is most accurate when determined with optically dilute solutions with absorbances between 0.01 and 0.05.1-4 The measurement of low absorbances in a spectrophotometer is prone to inaccuracies. Further, as the spectral purity of the exciting light in the fluorimeter may not be the same as that in the spectrophotometer the effective absorbance in each may be differents** In a recent papel-4 a method was described for determining the absorbance of a solution by measuring the fluorescent intensities at two points along the absorbance path, thus overcoming the necessity for a separate measurement of absorbance in a spectrophotometer. This method would require some modification for right-angle fluorimeters and cannot be used with front-faced fluorimeters.However, it is also possible to determine an absorptivity-related constant and a quantum efficiency-related constant for a fluorophore in a standard, unmodified fluorimeter. The procedure depends on the approximately hyperbolic relationship between fluorescence and fluorophore concentration. A graph of fluorescence against fluorescence x [fluorophorel-1 approximates to a straight line and can be extrapolated to give intercept values at both zero and infinite fluorophore concentration.The derivation of this approximately hyperbolic relationship and the limits of its validity are shown. The determination, and potential accuracy, of absorptivity and quantum efficiency using this method are discussed. The work described in this paper was carried out as a control for some biological experi- ments. Its potential relevance to the determination of quantum efficiencies was realised only after reading a recent paper.* * Present address : Eidgenossische Technische Hochschule, Laboratorium fur Biochemie, ETH-Zentrum, 481 CH-8092 Zurich, Switzerland.482 GAINS AND DAWSON : FLUORESCENCE - CONCENTRATION RELATIONSHIP Analyst, VoZ. 104 Theoretical Derivation of an Approximately Hyperbolic Relationship Between Light Absorbed (Io - I ) , or Fluorescence, and Fluorophore Concentration The Beer - Lambert equation may be rearranged to give where I , is the intensity of the incident light, I is the intensity of the unabsorbed light, E is the absorptivity, c is the concentration and d is the path length. This can be expanded to give ( 2 ~ 3 ~ c d ) ( 2 .3 ~ ~ 4 ~ 2! 3! 1 - (2.3~cd) + -- - - For small values of ~ c d the cube term and above may be ignored and the following approxi- mation can be made : Also, for small values of Ecd the approximation can be made that 1 - 2.3~cd I--- 2 2.3~cd 1 + 7 This gives 2I02.3ecd 2 + 2.3ccd I o - I = or by rearrangement 2 ( I , - I ) . . .. .. * * ( 2 ) I, - I = 21,) - -~ 2.3~cd The first of these approximations is valid t:o within +4% provided that a d is less than 0.17, and the second approximation to within -4% provided that Ecd is less than 0.2.Details of the errors produced when these approximations are combined are given in a later section. The hyperbolic function described by equation (2) approximately describes the relation- ship between light absorbed and concentration in fluorimeters that have the optical arrange- ments shown in Fig. l(a), (b) and (c). Equations have been derived that describe the absorption of light (Ito - 1') in the path length, d, (where d2 = d - dl), monitored in right- angle fluorimeters with the optical arrangement shown in Fig. l ( d ) . 5 They are formally similar to the following: Equation (3) may be approximated in a similar fashion to equation (1) to give ;!(Ifo - 2 .3 ~ ~ * * (4) This equation, like equation ( 2 ) , describes a hyperbolic relationship between light absorbed (Ito - I f ) and concentration. In fact, equation (2) is a special example of equation ( 4 )June, 1979 AND THE DETERMINATION OF QUANTUM EFFICIENCIES 483 with d equal to d,; it is therefore not considered separately below. (2d - d,) approximates to 2d then equation (4) approximates to If d , is small so that If, as it is ideally, fluorescence is directly proportional to the amount of light absorbed then fluorescence should bear an approximately hyperbolic relationship to concentration in all the fluorimeter designs shown in Fig. 1. Equation (4) may be rewritten as where F is the fluorescence intensity, Y is a geometrical factor andais the quantum efficiency.If light absorbed, or fluorescence, is plotted against light absorbed (or fluorescence) x [fluorophorel-l then, to the limit to which the approximations in equations ( 2 ) , (4) and (5) hold, a straight line should result. As c tends to infinity I t , - I', or F , tends to a maximum value Imax., or Fmax., such that As c tends to zero (Ito - I')/c, or F/c, tends to a limiting value, such that Dividing the second intercept [equation (7)] by the first [equation (6)] gives a constant, K c d , such that 2 . 3 ~ ( 2 d - d,) 2 .. .. .. (8) .. Ked = Determination of Quantum Efficiencies The intercept on the fluorescence axis in a graph of fluorescence against fluorescence x [fluorophorel-1 is a t infinite concentration and infinite absorbance and is therefore indepen- dent of them [equation (S)] and of the spectral purity of the exciting light.It is dependent on the geometrical factor, the intensity of the illuminating light, a function of the two optical path lengths, 2d2/(2d - d,), and the quantum efficiency. Therefore, the direct comparison of this intercept value with that of a standard fluorophore with a known quantum efficiency will give the quantum efficiency of the unknown fluorophore: The minimisation of the errors arising from the mathematical assumptions in this procedure are discussedbelow. The procedure may also be subject to at least one non-mathematical error arising from overlap of the excitation and emission spectra. This may, as in other procedures, be minimised by using dilute ~olutions.l-~ Determination of Absorptivity The absorptivity of an unknown fluorophore can be found from the ratio of its Ked value [equation (S)] to that of a standard fluorophore of known absorptivity using the following equation : Kc- - 'unknown Kedatandard 'Btandard This procedure is equivalent to deriving a value of (2d - d,) from equation (S), using a fluorophore of known quantum efficiency and absorptivity, and then substituting this value484 GAINS AND DAWSON : FLUORESCENCE - CONCENTRATION RELATIONSHIP Analyst, VoZ.104 into the same equation to derive the absorptivity, from the KEd value, of an unknown fluoro- phore. Alternatively, the absorptivity can be calculated directly from equation (8) i f , for a right-angle fluorimeter [as in Fig.l ( b ) and (41, the emitted light analysed by the photo- multiplier is assumed to be sampled evenly about the centre of the cuvette. In this instance the term (2d - d,) is equivalent to the path length of the fluorimeter cell (d, in Fig. 1). As Ked is found by extrapolation to zero absorbance, it may be assumed that the level of illumination is the same throughout the fluorimeter cell. The second assumption, that the photomultiplier optics are symmetrical about the centre of the cuvette, depends not only on the accuracy to which the fluorimeter is built but also that to which the cell is made. The path length, found using this assumption, can be compared with the effective path length measured as described above. The value of the absorptivity determined using these pro- cedures will depend on the spectral purity of the exciting light.Materials and Methods Fluorescence was measured in two fluorimeters with different optical arrangements. One was a front-faced fluorimeter [Fig. l(a)] constructed at the University of East Anglia. The light source was a 40-W quartz iodide lamp. An image of the lamp element was focused through a Wratten 18A filter on to the cuvette so that it occupied the whole of the front face. The maximum intensity of the exciting light was at 380 nm. The emitted light was analysed by a Bausch and Lomb monochromator (No. 33-86-02) at 480nm, with a band width of 10 nm. The optical path length was 15 mm. The other fluorimeter was a Perkin- Elmer MPF3 spectrofluorimeter with an optical arrangement similar to that in Fig.l ( d ) . The exciting light was at 380 nm, and the emitted light was analysed at 480 nm, each with a band width of 4 mm. A 10-mm square cuvette was used. In both fluorimeters the contents of the cuvette were stirred continuously and maintained at 30 "C. .:. ?..D . r l .. ... 0 Light source a Photomultiplier (dl Q .. .. (c) Q .. . . .. .. .. . . Fig. 1. Four possible arrange.ments for a fluorimeter : d, = length of solution preceding the part monitored by the photomultiplier ; d , = monitored path length; d = total optical path length; and d , path length of fluorimeter cell. Absorbance was measured with a Zeiss spectrophotometer at 380nm using a cuvette with a 10-mm path length. The data were fitted by means of a least-squares fit to equations (9), (lo), (11) or (12).Each squared value, that is the square of the differences between the computer-generated values and the experimental value, was weighted by multiplying it by the reciprocal of the square of the experimental value from which it was derived. This is a fit based on the squares of the relative differences, as opposed to the squares of the absolute differences. This form of weighting is most suited to data that either have an insignificant error or have a similar relative error. It is also necessary to use this or a similar weighting if it is desired to fit a straight line through all the points of a chosen part of a curve rather than mainly through the higher values (Table I), or, as in Fig. 2 and Table 11, to fit a hyperbolic curve through data generated from the Beer - Lambert equation.June, 1979 AND THE DETERMINATION OF QUANTUM EFFICIENCIES 485 Magnesium 8-anilinonaphthalene-l-sulphonate (ANS) was obtained from Eastman Kodak Co., Rochester, N.Y., U.S.A. Triton X-100 and Tris were obtained from BDH Chemicals, Poole, Dorset.All other reagents were of analytical-reagent grade. Fluorescence and light absorbance are measured in arbitrary units. These arbitrary units are constant for the spectrophotometer and for both of the fluorimeters, but are different for each of them. TABLE I ERRORS INVOLVED IN THE ASSUMPTION T ~ A T THE RELATIONSHIP BETWEEN LIGHT ABSORBED OR FLUORESCENCE AND CONCENTRATION IS LINEAR For front-faced optics [or for right-angle optics as in Fig. 1 (b) and (c) where d = d,] values of a d were generated by substituting all the integral values of I , - I, up to the value shown in the left-hand column, into the Beer - Lambert equation using a value of 100 for I,.These values of acd were fitted, using a least- squares fit, to equation (9) where z should approximate to 2.303. For right-angle optics [as in Fig. l(d) where d > d2] the generated values of Ecd were substituted with their corresponding values of ( I , - I ) and with a value of acd2 equal to acd/100 into equation (3). These values of acd were fitted to the equation (I’,, - 1’) = I,zacd2. The maximum errors between the values of I , - I substituted into the Beer- Lambert equation and the corresponding values derived from the equation to which they were fitted are given.A . It should be noted that acd and not acd3 has been used in these calculations. For front-faced optics, and for right-angle optics where d2/d = 0.01- Light absorbed, 0 3 4 5 6 7 8 9 10 y) acd 0.0000 0.0132 0.017 7 0.022 3 0.026 9 0.031 5 0.036 2 0.041 0 0.0458 Front-faced optics r A \ Maximum difference, z % 2.303 2.279 +0.51, -0.51 2.274 +0.77, -0.76 2.268 +1.03, -1.02 2.262 +1.30, -1.28 2.256 +1.57, -1.54 2.250 +1.84, -1.80 2.244 +2.11, -2.06 2.237 +2.39, -2.33 B. For right-angle opics and for various values of d2/d- d2ld 0.001 0.01 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Light absorbed, 6 6 5 5 5 6 6 6 7 7 8 9 % €Cd 0.022 3 0.022 3 0.022 3 0.022 3 0.022 3 0.026 9 0.026 9 0.0269 0.03 1 5 0.031 5 0.036 2 0.041 0 Right-angle optics f L I Maximum difference, z % 2.303 2.256 +1.03, -1.00 2.245 +1.56, -1.51 2.233 +2.09, -2.01 2.221 +2.64, -2.52 2.209 43.12, -3.03 2.197 +3.76, -3.54 2.185 +4.34, -4.05 2.173 +4.93, -4.56 Maximum difference, % +2.09, -2.01 +2.09, -2.01 f2.00, -1.92 f1.89, -1.82 +1.78, -1.72 +2.11, -2.03 f1.97, -1.91 +1.84, -1.78 +2.06, -2.00 f1.90, -1.84 f2.03, -1.98 f2.13, -2.06 Results Computer-generated Model Showing the Approximately Hyperbolic Relationship Between Light Absorbed (10 - I ) and ecd For a front-faced optical arrangement Theoretical values of a d were obtained by substituting integral values of I into the Beer - Lambert equation using a value of 100 for I,.Values of (I, - I ) , up to lo%, with their corresponding values of ecd were fitted to the equation1s2 (I, - I ) = I,+cd .... .. * (9) where z is an operational constant. Values of (I, - I ) , up to 70%, with their corresponding value of Ecd were fitted to the equation486 GAINS AND DAWSON : FLUORESCENCE - CONCENTRATION RELATIONSHIP Analyst, VoZ. 104 (K.,:? € C d ) * * .. . . (10) where KEcd is the absorbance that reduces I,,,. to half. and the hyperbolic assumptions are shown in Tables I and 11. The errors involved in the linear CONSTANTS FOUND FROM AND ERRORS INVOLVED IN THE ASSUMPTION THAT THE RELATIONSHIP BETWEEN LIGHT ABSORBIED AND CONCENTRATION IS HYPERBOLIC Values of ecd were generated as described in Table I. The generated data were fitted, using a least-squares fit, to equation (10). Also given are the maximum errors between the values of I, - I substituted into the Beer - Lambert equation and the corresponding values derived from the least-squares fit to equation (lo), and a corrected value, I’max., for I,,,.(where I’max. = I,,,. x 0.8686 x KECd-l). It should be noted that ecd and not ecd, has been used in these calculations. A . For front-faced optics- The resulting values of I,,,. and KEcd are given. Light absorbed, 0 3 4 5 10 20 30 40 50 60 70 % ECd 0.0000 0.013 2 0.0177 0.022 3 0.045 8 0.096 9 0.1549 0.221 9 0.301 0 0.397 9 0.522 9 I,,,. 200.0 198.7 f 0.2 198.3 f 0.2 198.0 f 0.2 196.3 f 0.3 192.7 & 0.4 188.9 f 0.5 185.0 & 0.6 180.7 & 0.7 175.9 f 0.8 170.8 f 0.9 Kecd Maximum difference, % 0.868 6 0.8627 f 0.0009 0.861 3 f 0.0009 0.8598 f 0.0010 Values less than 0.01 0.85f!2 f 0.0014 -0.01, +0.01, -0.01 0.8362 f 0.0019 -0.05, +0.03, -0.06 0.8192 f 0.0024 -0.14, $0.08, -0.15 0.8007 f 0.0030 -0.28, +0.16, -0.32 0.7804 & 0.0035 -0.49, +0.27, -0.58 0.7576 f 0.0041 -0.77, +0.47, -0.96 0.7321 f 0.0048 -1.20, +0.69, -1.62 I’max.200.0 200.1 200.0 200.0 200.0 200.2 200.3 200.7 201.1 201.7 202.6 B. For right-angle optics, when d,/d = 0.01- Light absorbed, % rcd I m a x . K E e d Maximum difference, yo I’max. 0 0.000Q 100.0 0.4343 200.0 10 0.0458 94.9 0.4 0.411.9 5 0.0021 -0.07, $0.04, -0.07 200.1 20 0.0969 89.8 f 0.6 0.3801 f 0.0029 -0.34, +0.21, -0.37 201.0 30 0.1549 84.5 f 0.7 0.3628 f 0.0038 -0.88, +0.53, -1.00 202.3 40 0.2119 78.9 & 0.8 0.3358 & 0.0046 -1.77, +1.08, -2.16 204.1 C . For right-angle optics and for various values of d2/d- d2ld 0.001 0.01 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Light absorbed, 39 39 40 42 44 46 48 51 55 59 65 74 % cicd 0.215 0.215 0.222 0.237 0.252 0.268 0.284 0.310 0.347 0.387 0.456 0.585 Maximum difference, yo -1.66, +l.Ol, -2.02 -1.66, +1.01, -2.02 -1.61, +0.98, -1.97 -1.64, +1.00, -2.02 -1.64, +1.00, -2.04 -1.61, 1-0.98, -2.02 -1.55, $0.95, -1.97 -1.54, +0.94, -1.99 -1.55, $0.95, -2.05 -1.48, +0.91, -2.00 -1.45, +0.90, -2.01 -1.40, +0.86, -2.00 The computer-generated data were plotted in the form of light absorbed (Io - I ) against light absorbed x ( ~ c d ) - l (see Fig.2, upper curve). The line drawn is a computer fit of the data, for values of lo - I up to 70%, using equation (10). Smaller maximum values of lo - I were also used. From the computer analysis of these data, shown in Table 11, it can be seen that as the maximum values of I0 - I are decreased, the value of I,,,.more nearly approximates to the value of 210 [see equation ( 2 ) ] and Krcd more nearly approxi-June, 1979 AND THE DETERMINATION OF QUANTUM EFFICIENCIES 487 mates to 0.8686. The relative differences between the values of I , - I derived from the values of I substituted into the Beer - Lambert equation and those values of I , - I derived by substituting the corresponding values of Ecd into the hyperbolic function, derived from the least-squares fit, do not exceed -&2y0 (see Table 11). 201 \ 0 100 200 Light absorbed x (absorbance)-’/ arbitrary units I-’ pmol cm Fig. 2. Plot of light absorbed (I’, - 1’) against light absorbed x (absorbance) -l. The data were generated as described in Table 11.The upper curve (both and 0) is for a value of d,/d = 1.0, as in front- faced and some right-angle fluorimeters [see Fig. l(b) and (G)]. The line drawn through this curve is a computer fit to equation (10) using values of 1’, - I’ of up to 70% (e), assuming a hyperbolic function between I’o - I’ and absorbance. The lower curves in descending order are for values of d,/d equal to 0.9, 0.8, 0.6, 0.4 and 0.01. For a right-angle optical arrangement Theoretical values of Ecd were obtained by substituting integral values of I into the Beer - Lambert equation using a value of 100 for I,. Values of ccdl and a d Z were obtained by multiplying a d by dl/d and d,/d, respectively, where d = d, + d , [see Fig. 1 (a)]. These values of and ccd, were then substituted into equation (3) to obtain values of (16 - 1’).For comparative purposes these values were multiplied by d/d, before fitting them, with their corresponding values of ccd, to equation (10). The computer-generated data are plotted in Fig. 2 (lower curves) in the form of light absorbed (I’, - I’)d/d, against (Ito - I’)d/d, x (ccd)-l. A computer analysis of some of these data is given in Table 11. It shows the upper limits of (16 - I’)d/d, and ccd, to which the hyperbolic approximation holds, to within &2%, for various values of d,/d from 1.0 to 0.01. 6 Relationship Between Light Absorbed or Fluorescence and the Concentration of Dissolved Magnesium 8-Anilinonaphthalene- 1 - sulphonate The absorption of light by ANS in 0.005 M Tris - HCl (pH 7.6) and in 5% (m/V) Triton X-100 obeys the Beer- Lambert relationship, in that graphs of absorbance against con- centration give straight lines, Fig.3 (a). If the same data are plotted in the form of light488 GAINS AND DAWSON : FLUORESCENCE - CONCENTRATION RELATIONSHIP Analyst, VoZ. 104 absorbed (I, - I ) against (I, - I ) x [ANSIw1 the graphs appear to be linear within the experimental error and up to a value of 70% far I , - I [see Fig. (3)]. The data in Fig. 3 (b) were fitted to the following equation : The relative difference between any of the experimental and the computer-generated data was less than 2.2% (see legend to Fig. 3). Values of I,,,, were found to be 169.8 in 5% (m/V) Triton X-100 and 175.7 in 0.005 M Tris - HC1 (pH 7.6). These values of I,,,.are lower than the value of 200 for ZI, [see equation (2:)]. However, it can be seen from Fig. 2 that the magnitude of I,,,, will depend on the maximum value used of the light absorbed. These experimental values of I,,,. compare favourably with the theoretical values shown in Table 11. From Fig. 3 (b) and from the analysis shown in the legend to Fig. 3 it can be seen that in practice the amount of light absorbed. (I, - I ) bears an approximately hyperbolic relationship to the concentration, provided thilt I,,,. is not fixed at a value of 21,. 180 I 0 20 40 60 80 100 [ANSI /pM 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Light absorbed x [ANSI-‘/arbitrary units x pM-’ Fig. 3. Relationship between light absorbed and concentration of magnesium S-anilinonaphthalene- l-sulphonate (ANS) .The same data are plotted in .two ways : (a) absorbance against concentration and ( b ) light absorbed (I, - I) against light absorbed x (concentration)-l. For both (a) and (b) ANS was dissolved in 0.006 M Tris - HC1 (pH 7.6) (O), or in 6% ( m / Q Triton X-100 (0). The data in (b) were fitted to equation (11). The following were found in Tris - HC1, with results obtained in Triton X-100 given in parentheses: the maximum difference between the experimental and generated data are -2.2%, +0.9%, -1.1% (-1.8%, +0.8%, -1.3%), I,,,. is 175.7 -+ 2.8 (169.8 f 2.0), K , is 188.1 & 3.7 p~ (126.5 & 2.0 p ~ ) , KEcd is 0.7543 (0.7295) ; the maximum value of I,, - I used was 60% (70%!. (The data are averages of ten separate titrations.) Similarly, a plot of fluorescence against fluorescence x [ANSI-l for the fluorophore dissolved in 5% (m/V) Triton X-100 is, over a limited concentration range, apparently a straight line.The front-faced fluorimeter was used for the former and the Perkin-Elmer MPF3 spectrofluorimeter for the latter. All the data in Fig. 4 (a) and some of the data in Fig. 4 (b) (closed circles) have been fitted to the following equation : This is shown in Fig. 4 (a) and (b).June, 1979 AND THE DETERMINATION OF QUANTUM EFFICIENCIES 489 F = Fm,,. ("> . . .. .. . . (12) K , + c Values of Fm,,, and K , are given in the legend to Fig. 4. 160- (6) 60- 0 - 0 - 0 40 20 - I I I I I I 1 1 1 1 I ~ 0 0.2 0.4 0.6 0.8 1.0 1.2 Fluorescence x [ANSI-'/arbitrary units x pM-' Fig. 4. Plots of fluorescence against fluorescence x [ANSI-l: (a) using a front-faced fluorimeter, (b) using a right-angle fluorimeter (both 0 and 0).For both, the fluorophore was dissolved in 5% m/ V Triton X-100. All the data in (a) and some of the data in (b) (a) were fitted by means of a least-squares fit t o equation (12). The lines drawn represent these fits. In both, the errors between the experimental and generated data were within f1.4y0, for (a) a 15-mm cell was used and K, is 78.2 f 1.7 PM and for (b) a 10-mm cell was used and K, is 119.1 1.6 p ~ . (The data are averages of three separate titrations.) Discussion For front-faced fluorimeters the assumption that fluorescence is directly proportional to fluorophore concentration is valid to within 52% provided that 8% or less of the exciting light is absorbed (Table I A).Similarly, the assumption that fluorescence bears a hyperbolic relationship to fluorophore concentration is valid to within -+2% provided that 70% or less of the exciting light is absorbed (Table I1 A). The errors involved in these assumptions should also be the same for the right-angle optical arrangements shown in Fig. 1 (b) and (c). For most commercial right-angle fluorimeters the optical arrangement is similar to that in Fig. 1 (d). In this instance the upper limit to which the linear and hyperbolic assumptions are valid to within &2y0 depends on the ratio d,:d [see Fig. 1 (d) and Table I B]. When this ratio is 0.1 or less the linear assumption is valid provided that 4y0, or less, of the light is absorbed and the hyperbolic assumption is valid provided that 39% or less of the exciting light is absorbed.As the ratio d,:d approaches unity the upper limits tend towards 8% and 74y0, respectively (Tables I B and I1 C). One possible criticism of the data in Figs. 3 and 4 is that the maximum fluorescence value may be caused by the saturation of ANS binding sites on the Triton X-100 micelles. How- ever, even at the highest fluorophore concentrations used there is an approximately &fold excess of Triton X-100 micelles over ANS ions. Although the results were similar the data could not be used to determine values of F,,,. and K , as the Beer - Lambert relationship was not followed. A slight upward curvature was found in graphs of absorbance against ANS concentration. Ethanol was also used as a solvent.490 GAINS AND DAWSON The accuracy to which the quantum efficiency and absorptivity can be determined depends on the error of the intercept values derived from the graph of fluorescence against fluorescence x [flu~rophorel-~.These values are dependent on the concentration range used (Table 11). For front-faced fluorimeters the error between I,,,. and the model value of 210 and between K,, and its maximum value of 0.8686 is about 2y0.per 0.05 absorbance. For right-angle fluorimeters the errors are about 5% at small slit widths (where d > d,) and will decrease towards 2% as d, approaches its maximum value of d. The theoretical errors in deriving I,,,. and K,, have been calculated using equal increments of (I’o - I’); the use of equal increments of Ed will slightly alter these errors.However, for both I,,,. and K,, the theoretical errors will be self-cancelling if the titrations of fluorescence against concentration of the unknown and standard fluorophores are made at the same, or reasonably similar, absorbance values. This can be seen from the values of I’max. (where I‘max. = Imax, x 0.8686 x shown in Table 11. If the fluorescence titration data for the standard and unknown fluorophores are such that the absorbance values of each bear a fixed relationship to one another then the value of I,,,. x K,,--l will be insensitive to the different absorbance ranges used, and the quantum efficiency may be calculated from the following equation : The errors in I,,,. and K,, are in the same direction and are very nearly equal. However, in this instance the absorptivity must be measured in a spectrophotometer or by the method described by Britten et aL4 Similarly, if the experimental data are such that the error on Imax. is unacceptably large, but that on the intercept of the fluorescence x [fluoro- phorel-l axis is acceptable, then the quantum efficiency may be calculated from equation (7) if the absorptivities are known. The data in this paper suggest that it is tlneoretically possible, by using the procedures discussed above, to determine the quantum efficiency to an accuracy of 2% or less. Whether or not these procedures are of any practical value can only be independently assessed by those who, unlike us, regularly use the already established methods for measuring quantum efficiencies. We thank Dr. M. J. Selwyn for helping us with the approximations that transform equation (1) into equation (2), P. D. Bolton for compiling the computer program and the University of York Computer Advisory Service for helping us to modify it to our needs. N.G. gratefully acknowledges the MRC for a Research studentship, the SRC for a Post- doctoral Fellowship, and the Centre National! pour la Recherche Scientifique for financial support whilst writing the paper. References 1 . 2. 3. 4. 5. Parker, C . A., and Rees, W. T., Analyst, 1960, 85, 587. Parker, C . A., “Photoluminescence of Solutions,” Elsevier, Amsterdam, 1968. Demas, J. N., and Crosby, G. A., J . Phys. Chem., 1971, 75, 991. Britten, A., Archer-Hall, J.. and Lockwood, G., Analyst, 1978, 103, 928. Brand, L., and Whitholt, W., Meth. Enzym., 1967, 11, 776. Received November 29th, 1978 Accepted January 16th, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790400481

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, June, 1979, Vol. 104, pp. 491-504 491 Determination of Cadmium in Blood and Urine by Flame Atomic-fluorescence Spectrometry R. G. Michel, M. L. Hall and J. M. Ottaway Department of Pure and Applied Chemistry, University of Strathclyde, Cathedral Street, Glasgow, G1 1XL and G. S. Fell Department of Clinical Biochemistry, Royal Infirmary, Glasgow, G4 OSF The development is described of an atomic-fluorescence method for the determination of cadmium in blood and urine. The method involves only the direct aspiration of acidified urine or diluted and acidified blood into the flame. Calibration is achieved simply by using acidified aqueous standards and by the application of a pre-determined correction factor to account for changes in the uptake rate. The sensitivity, accuracy and precision are comparable to those given by most techniques that are currently in use for the determination of cadmium in biological materials. The simplicity of the method permits rapid analyses of large numbers of samples (more than 25 samples per hour) and is particularly useful for the survey of populations of people exposed to cadmium.The instrumentation used employs a two- source background correction system. This is essential for maximum accuracy and allows automatic correction for the scatter, which is the primary cause of inaccuracies in the atomic-fluorescence spectrometric determination of cadmium. Keywords : Flame atomic-fluorescence spectrometry ; cadmium determination ; blood analysis ; urine analysis The most commonly used method for the determination of cadmium in biological samples is currently flame atomic-absorption spectrometry.Other methods that have been used or that are under development include spectrophotometric methods involving extraction with diphenylthiocarbazone (dithizone),lS2 neutron a~tivation,~,~ atomic-absorption spectrometry using the Delves cup5s6 or tantalum boat7s8 for blood cadmium, atomic-absorption spectro- metry with electrothermal atomiser~~-~~ and electrochemical methods.12-14 Pulido et al.15 have used a long path length absorption cell to increase the sensitivity of the determination of cadmium in urine and serum by direct aspiration into a flame. Smith et aZ.16 used an electrically heated ceramic tube through which the sample, after dithizone extraction of blood, was aspirated into a hydrogen flame.The relative merits of some of these techniques have been discussed by Friberg et aZ.,17 Pierce et aZ.l8 and O’Laughlin et al.19 Most methods have the required sensitivity and specificity , and inter-laboratory s t u d i e ~ l ~ - ~ ~ are beginning to give indications of the accuracy and precision of these methods. Almost all of the reported procedures employ some form of chemical pre-treatment of the sample in order to remove interferences from the biological matrix and to concentrate the cadmium in the final solution for analysis. Examples in the literature include neutron- activation analysis4 and atomic-absorption s p e ~ t r o m e t r y , ~ ~ ~ ~ 7 - 1 ~ ~ ~ ~ - ~ ~ where it is necessary to employ acid digestion or dry ashing, often followed by chelation and extraction steps.An alternative is to use ion-exchange techniques20s25 to separate cadmium. Electrochemical methodsl2,l4 also usually involve destruction of the biological matrix by wet digestion or low-temperature ashing before analysis by the various versions of anodic-stripping voltam- metry. Preliminary results from this l a b ~ r a t o r y ~ ~ - ~ ~ have shown that flame atomic-fluorescence spectrometry has potential as a simple and rapid method for determinations of cadmium in blood and urine. This speed and simplicity are attributable to the high sensitivity of the atomic-fluorescence technique and the minimum sample pre-treatment required. In this paper we report the use of more sophisticated atomic-fluorescence instrumentation.Cadmium in urine is determined by direct aspiration into a separated air - acetylene flame and cadmium in blood by 1 + 4 dilution and then direct aspiration. Our previous r e s u l t s ~ ~ ~ ~ demonstrated492 MICHEL et al. : DETERMINATION OF CADMIUM IN BLOOD AND Analyst, VOZ. 104 less than satisfactory accuracy for the atomic-fluorescence determination of cadmium in urine. However, the improved instrumentation includes a two-source background correction facility, which ensures high accuracy when determining low (pg 1-l) levels of cadmium, AI f r Mechanical- chopper J I Experiment a1 P.M. tube Instrumentation The choice of components for atomic-fluorescence instrumentation has been reviewed recently by Winefordner31 and there are also a number of other review papers available329 that discuss atomic-fluorescence spectrometry.A schematic diagram of the instrumentation used here is shown in Fig. 1 and a list of components in Table I. Cadmium atomic fluorescence was excited using a microwave-excited electrodeless discharge lamp (EDL) that was the subject of two previous p~blications.3~~~~ I I Double Separated monochromator Fig. 1. Instrumentation. Choice of jlame Cadmium atomic-fluorescence signals have been shown36 to be greater in hydrogen-based flames than in acetylene-based flames. However, we found that a number of disadvantages arose from our use of hydrogen flames. Salt deposits on the burner head appeared much more rapidly when using the air - hydrogen flame rather than an air - acetylene flame.This caused unacceptable instability while aspirating urine and blood samples with their high solids contents. Further, despite the low background and slightly larger cadmium fluorescence signals in the hydrogen flame, the sensitivities obtained with acetylene and hydrogen flames were comparable when aspirating biological samples.37 This was a result of the greater scatter of excitation-source radiation by unvapourised sample particles in the hydrogen flame. This erroneous signal could be background corrected. However, noise levels associated with the scatter signal degraded the detection limits more in hydrogen than in acetylene flames.37 The flame background noise at 228.8 nm, the cadmium analytical wavelength, was five times smaller in the separated air - acetylene flame than in the same unseparated flame. This was a result of a reduction in the total flame background, upon separation, by a factor of 25-30.Separated flames have been discussed in more detail by various authors.3839 The nitrogen-separated air - acetylene flame supported on a circular capillary burner was therefore used throughout this work. It was noted that the reduction in noise upon flame separation was equivalent to the square root of the reduction in flame background. This indicated that the noise on the flame background was shot noise at this wavelength.June, 1979 URINE BY FLAME ATOMIC-FLUORESCENCE SPECTROMETRY TABLE I INSTRUMENTATION AND OPERATING CONDITIONS 493 Component Double monochromator .. Manufacturer Spex Industries, Metuchen, N.J., USA Operating conditions Spectral band pass 0.6 nm, grating 1200 grooves mm-l, blaze 300 nm, wavelength 228.8 nm Model No. 1672 Photomultiplier . . .. .. 9789 QB EMI, Electron Tube Division, Hayes, Middx. Photomultiplier housing . . PRI 400RF Products for Research, Danvers, Mass., USA EM1 Photomultiplier power supply Photon counting system . . Synchronous sampler . . .. PM 28A 6C1 5C21 High voltage, 1100 V Count time 1 or 10 s. Ortec Brookdeal, Bracknell, Berks. Synchronous sampler used for demodulation Pre-mix flame system . . .. Perkin-Elmer Nitrogen-separated air - acetylene flame operated under stoicheiometric conditions. Signals observed 30 mm above burner head Capillary burner and flame separator .. .. .. Chopper .. .. .. .. Broida Q-wave cavity . . ..Laboratory constructed Laboratory constructed Electromedical Supplies, Wan tage Modulation frequency 300 Hz 210L Rlk I11 Microwave generator . . .. (EDL) . . .. .. .. facility . . .. .. .. Electrodeless discharge lamp EDL temperature control Electromedical Supplies Laboratory constructed References 34 and 35 Laboratory constructed. (Fan and heater elec- tronics-surplus components) EM1 - Varian Ltd., Middx. Temperature of heater con- trolled by 0-240-V Variac v1x-300uv High-pressure xenon arc . . See text Xenon arc housing . . .. Xenon arc power supply . . Lenses . . .. .. .. R300-2 P5300-1 EM1 - Varian EM1 - Varian Thermal Syndicate Ltd., Tyne and Wear Ealing Beck Ltd., Watford Focal length 50 mm, diameter 50 mm Mirror . . .. .. .. Focal length 75 mm, diameter 75 mm Optical benches and optical mounts .. .. .. Mechanite Ealing Beck Choice of monochromator The monochromator chosen for the instrument was an f/4 double monochromator (Table I ) . A double monochromator was used in order to prevent stray light, resulting from wavelengths other than the analytical wavelength, from reaching the exit slit. By reducing the stray light the associated noise was reduced and the signal to noise ratio of the measure- ment improved. The magnitude of this improvement was a factor of three in the detection494 Analyst, VOl. 104 limit for cadmium in urine at 228.8 nm. At wavelengths other than 228.8 nm the magni- tude of the improvement in noise level varied. Stray light from the flame when aspirating biological samples is a result of the sum of the flame background emission and the emission from the biological matrix itself.This emission is both atomic, i.e., line, and molecular, i.e., broad band in character. The stray light problem in flame atomic-fluorescence spectro- metry is discussed in detail el~ewhere.~’ MICHEL et al.: DETERMINATION OF CADMIUM I N BLOOD AND Two-source background correction The background correction instrumentation that was used is shown in Fig. 2 and was based on that described by Rains et aL40 The mechanical chopper modulated the signal at a frequency of 300 Hz. The principle of operation of this atomic-fluorescence scatter correction system is identical with that discussed by Rains et al. and is also similar to the two- source systems used in atomic absorption.*l At omic-fluorescence signals were excited using a cadmium microwave-excited electrodeless discharge lamp (EDL) and the scatter was simulated by using a high-pressure xenon arc.The EDL and xenon arc scatter signals were balanced using a 2% aluminium solution in a similar fashion to that described by Rains et al. However, the intensity of the xenon arc was varied by using a series of metal gauze discs of different densities placed directly in front of the arc lamp. An alternative could be a series of neutral density filters. Each gauze disc was placed around the circum- ference of a wheel that could be rotated in front of the lamp. Fine control of the lamp intensity was obtained by using the power control on the xenon arc power supply. This method allows variation of intensity without changing the size of the image of the xenon arc in the flame.To match the EDL image and the xenon arc image at the flame the optical system was aligned to ensure that the centres OE each image were coincident and that both images were taller than the 10-mm slit height of the monochromator, wider than the flame and in the same horizontal plane as the slit. This alignment is simple to carry out and the different geometry of the two sources is not a problem if the above conditions are fulfilled. This procedure ensures detection of both scatter signals over the same region and depth of the flame and hence will give an accurate scatter correction. Electrodeless Front view \ Plane mirrors discharge lamp Mechanical chopper ,\~:qpl Xenon arc \ ’ ? 2 I I wheel Measurement ; : ; \\\ \* Fig.2. Chopper for background correction. Electronic components A photon counting system was used to monitor signals from The photon counter included a phase-sensitive detector driven the photomultiplier tube. by a photodiode-derivedJune, 19 79 URINE BY FLAME ATOMIC-FLUORESCENCE SPECTROMETRY 495 reference signal from the mechanical chopper. To reduce the cost of an atomic-fluorescence instrument it is feasible to use a conventional analogue lock-in amplifier instead of the photon counting system without any significant loss in performance or essential facilities. The background count (photomultiplier dark count plus stray light) of the instrument with the flame and sources off was typically 8 counts s-l whereas flame background was typically 130 counts s-l with a 0.5-nm spectral band pass at 228.8 nm.Scatter signals were of the same order of magnitude as the flame background (130-230 counts s-l). The noise at the detection limit was assumed to be the quadratic sum of the noise on the flame background and the noise on the scatter signal. This was measured by aspirating a blood, urine or aqueous solution and observing the total signal in the background channel of the phase-sensitive detector. When the continuum source is operating and the system balanced to correct for scatter this total background represents the sum of the flame and scatter backgrounds. The square root of this number was the noise figure used for calculating detection limits. This method of defining and measuring the detection limit is essentially the same as that used by Johnson et ~ 1 .~ ~ and the same assumption was made that white (shot) noise was dominant. This assumption was verified by measurements of the variation of noise with (a) flame background, (b) slit width, (c) analytical signal and ( d ) count time. The results confirmed that the limiting noise followed the square root relationship with each variable. Optical components optical benches with conventional mounting components. chopper had facilities for vertical and horizontal translations. that shown in Fig. 1, placed around the lens in front of the monochromator. mirror provides the normal double pass of source radiation through the flame. A count time of 1 s was used for routine analyses. All lenses, the mirror, mechanical chopper and light sources were mounted on triangular The burner and mechanical The only light baffle was The spherical Reagents content.Glassware was acidiwashed and rinsed with de-ionised water before use. Cadmium stock solution A stock solution of 2000 pg ml-l of cadmium in 0.04 M hydrochloric acid was prepared by dissolving a known amount of spectrographically pure cadmium in 10 ml of cadmium-free 11 M hydrochloric acid. Standard solutions of cadmium over the range 0.001-0.1 pg ml-1 were prepared daily in 0 . 0 4 ~ hydrochloric acid. Solutions of metals for the interference study were prepared from the AnalaR-grade chlorides. The 2% aluminium solution for balancing the scatter correction system was prepared from spectrographically pure aluminium wire dissolved in AnalaR-grade hydrochloric acid.BZood Standard disposable syringes and needles were used to take blood by venepuncture (5 ml). The blood was kept in plastic sample tubes containing anticoagulant (potassium EDTA or lithium heparin). This collection procedure was shown to be free from cadmium contamina- tion on this and on previous o c c a ~ i o n s . ~ ~ ~ ~ ~ No significant changes in cadmium concentration occurred when venous blood collected in this way was stored for more than 1 month either at 4 to 10 "C or at -10 to -20 "C. Development work on calibration was carried out using pooled blood from outdated blood bank samples. Urine Twenty-four hour urine samples were collected in plastic bottles, containing thymol as a bacteriostatic agent.Concentrated (1 1 M) hydrochloric acid was added dropwise to 25-ml portions to give a final concentration of 0.04 M hydrochloric acid. This adjustment required about 3 drops, or 0.3 ml of 11 M hydrochloric acid. The acidified portions showed no deterioration in cadmium content when stored in their 25-ml plastic specimen tubes at 4-10 "C for up to 4 weeks. All reagents were of the highest purity available and each batch was checked for cadmium High-purity de-ionised water30 was used for the preparation of all solutions.496 Blood sample preparation Blood (2ml) was taken from the thawed samples after ensuring thorough mixing and added to a clean 10-ml centrifuge tube. The sample was diluted to 10 ml with 2 ml of 0.2 M hydrochloric acid, 2 ml of 2.5% Triton X.(to ensure complete haemolysis) and 4 ml of de-ionised water. The diluted blood was then centrifuged (30s; 3000revmin-1) to remove cellular debris and aspirated directly into the flame. The 10-ml sample was sufficient for duplicate analyses. MICHEL et Ul. : DETERMINATION OF CADMIUM I N BLOOD AND Analyst, V d . 104 Urine sample preparation If acid had not been added at the collection stage it was added shortly before analysis. Otherwise no further sample preparation was necessary. The undiluted urine was then aspirated directly into the flame. Results and Discussion Cadmium Calibration Standards It was possible to use aqueous cadmium solutions to construct calibration graphs for the analysis of both blood and urine solutions prepared as described above. However, it was found necessary to acidify samples and standards with hydrochloric acid to a level above 0 .0 3 ~ . Fig. 3 shows that the commonly occurring enhancing effect of hydrochloric acid on trace-metal atomic absorption and fluorescence in flames was observed and that in aqueous solutions this effect became constant at acid concentrations greater than 0.01 M. However, when the same experiment was carried out in urine the enhancing effect did not stabilise until above 0.03 M hydrochloric acid. 450 250 I I I I I I , 0.01 0.03 0.05 Hydrochloric acid concentration/M Fig. 3. Effect of hydrochloric acid on cadmium fluorescence. A, Aqueous stan- dards, 4 pg 1-1 of cadmium; B, 4 pg 1-1 of cadmium in urine. The addition of hydrochloric acid was advantageous for the usual purposes of stabilising the concentration of metal ions in solution and for taking advantage of the enhancing effect of the acid.With aqueous calibration graphs no change in linear range was observed. The results of standard additions of cadmium to urine with and without the presence of 0.04 M hydrochloric acid show that without acid linearity extends to 100 pg l-l, whereas with acid linearity is the same as for aqueous standards and extends to 2000 pug 1-1 (Fig. 4). Further, the calibration graph with acid added was identical with the calibration graph prepared using aqueous standards 0.04 M in hydrochloric acid (Fig. 4), except that there remained a constant 4% depression of the cadmium signals in urine relative to aqueous solutions. This depression corresponded with a 4% reduction in the rate of uptake of urine into the spray chamber. A correction for this and a similar correction for blood is discussed below but this correction has been applied and incorporated into the results shown in Fig.4. The standard additions calibration graph obtained for the diluted blood samples prepared as described above was identical with graphs prepared using aqueous standards 0.04 M in hydrochloric acid (Fig. 4) except that there remained the constant (19%) depression of the cadmium signals in blood This enhancement also has an important effect on calibration graphs.Jzcne, 1979 URINE BY FLAME ATOMIC-FLUORESCENCE SPECTROMETRY 497 relative to aqueous solutions. This corresponded to a 19% reduction in rate of uptake caused by the high viscosity of diluted blood.The blood calibration graph was linear up to 2000 pg 1-1 of cadmium. 1 I I I I I I I I I 0 1 10 100 1000 Cadmium concentration/pg I-’ Fig. 4. Effect of hydrochloric acid on cadmium in urine calibra- A, Spiked urine, 0.04 M acid (identical with aqueous calibra- tion. tion, 0.04 M acid) ; B, spiked urine, no acid. Correction for Changes in Uptake Rate The ratio of the rate of uptake of aqueous standards to the rate of uptake of urine or blood was used as the arithmetical correction factor to be applied to analytical measurements of cadmium atomic fluorescence. These ratios were defined as follows: Uptake rate of water (U,) Uptake rate of blood ( U b ) = Blood correction factor u , / u b was typically in the range 1.2-1.3 and Uw/Uu was in the range 1.03-1.07.Twenty consecutive measurements obtained each week for 6 weeks gave an average weekly ratio of 1.23 for blood (120 different samples of normal blood) and 1.04 for urine (120 different samples of normal urine) with a standard deviation in these ratios of 3% for blood and 2% for urine. This predictable and long-term precise performance of measurements of u w / u b and U,/Uu allowed the use of a correction factor rather than a more time-consuming standard addition to each sample or the addition of glycerol to the standards.43 It is possible that widely differing concentrations of haemoglobin in blood could change these rates by more than the above standard deviations indicate. The range of haemoglobin values over which the blood correction factor of 1.23 is valid was not determined.If massive deviations in haemoglobin values, e.g., for anaemic people, do change the rates of uptake significantly such changes could be accounted for by prior knowledge of particular samples and the application of specific rate measurements or the use of the standard additions technique. The procedure used to obtain and apply the correction factor was simple. The rates were obtained by using a 10-ml measuring cylinder and stop-watch to measure the time taken for the spray chamber to consume a fixed volume of liquid. A measurement of rates of uptake to obtain the correction factor was made in duplicate before each batch of cadmium analyses in blood and urine. The signals obtained throughout the batch were then multi- plied by the appropriate ratio to obtain the figures that could be used to deduce the cadmium concentrations from the aqueous calibration graphs.The correlation between the cali- bration graph obtained using aqueous standards 0.04 M in hydrochloric acid and the graph obtained by standard additions incorporating the above correction factors was excellent for498 MICHEL d.: DETERMINATION OF CADMIUM IN BLOOD AND Analyst, Vd. 104 both blood (correlation coefficient 1.02) and urine (correlation coefficient 0.99). To compute these correlation coefficients 11 concentrations were chosen to cover the full linear range of each calibration graph. Repeated measurements over a period of several months have demonstrated the continued reliability of this method of correction.Correction for Scatter of Source Radiation The procedure for scatter correction was aut.omatic once the scatter signals caused by each source had been equalised in the following manner. When the EDL was operating under optimum conditions of temperature and microwave a 2% solution of aluminium was aspirated into the separated air - acetylene flame. This solution gave a large signal caused by the scatter of EDL radiation off aluminium salt particles in the flame. The radiation from the xenon arc, irradiating the flame 180" out of phase with the EDL and scattered by the aluminium particles gave a similar large signal that was subtracted by the phase-sensitive detector from the EDL scatter signal. The intensity of the xenon arc was adjusted until zero signal was obtained at the output of the photon counter. After this balance had been achieved the aspiration of the 2*& aluminium solution could be discontinued.The instrument would then correct automatically for scatter off blood and urine matrix particles in the flame. With the instrumentation described here the 2% aluminium solution scattered the EDL radiation to give a signal of, typically, 9000 counts s-l. Diluted blood and undiluted urine gave scatter signals in the range 130-230 counts s-l. The flame itself gave a scatter signal of about 25 counts s-l, i.e., after scatter correction the signal was zero to within the shot noise of the flame background while aspirating cie-ionised water. This observation verified that scatter off the flame gases and unvapourised water droplets does take place but its magnitude is small and equivalent to that of a cadmium fluorescence signal close to the detection limit.Two alternative solutions, sodium chloride and calcium hydrogen orthophosphate, were investigated to determine whether or not they gave the same results as the 2% aluminium solution. They gave similar results, i.e., the scatter correction for blood and urine turned out to be the same, as did the magnitude of the scakter signal while aspirating the concentrated balancing solution (approximately 9000 counts s-l) . The use of calcium orthophosphate was thought inadvisable in view of the possibility of PO molecular fluorescence at 228.8 nm.44 However, no PO fluorescence was observed, probably because the xenon arc was used at low power for background correction purposes.It was necessary to ensure that the EDL was stable before attempting to use the back- ground correction system because any instability caused an imbalance in the two source intensities. A 0.5-h warm-up time was found to be necessary in order to achieve sufficient stability and maximum fluorescence. Once this was obtained balance checks with 2% aluminium solutions demonstrated that the instrument was in balance and therefore sufficiently stable for 2-3 h operation. Rebalancing after this period took less than 1 min and was not therefore a serious problem. More precise temperature control is currently being developed to reduce the time required to stabilise the EDL and to maintain its output constant. This is because the stability of the fluorescence and scatter signals depends primarily on the operating temperature of the EDL.During operation the temperature has a tendency to increase slowly (approximately 2-3 "C h-l). This increase causes the total line width to increase owing to self-absorption and self-reversal and hence an increase in scatter and a simultaneous decrease in fluorescence are observed. A more precise temperature control system would prevent this temperature increase, stabilise both fluorescence and scatter and maintain the balance between the two sources. 'The magnitude of this effect is of the order of 6% in 2 h. No problems were encountered with the stability of the xenon arc. Interferences The effects of the inorganic ions Mg2+, Ca2+, Fez+, Na+ and K+ as chlorides and C1-, NO3-, PO4% and S042- as acids on the atomic fluorescence of cadmium were investigated for the two flames, air - hydrogen and nitrogen-separated air - acetylene.The ions were studied in the concentration range 10-5000 pg ml-l.June, 19 79 URINE BY FLAME ATOMIC-FLUORESCENCE SPECTROMETRY 499 Two methods were used to confirm whether or not an apparent enhancement interference was caused by scatter. Firstly, the signals from blank solutions, containing only the inter- fering ion and no cadmium, were subtracted from the observed signals from interference test solutions. Secondly, this result was checked by using the background correction facility, which automatically subtracts scatter and reveals the presence of other types of interference. The second method was the only one that could be used when looking at the interferences affecting cadmium in urine directly rather than cadmium in synthetic aqueous solutions.In the nitrogen-separated air - acetylene flame, without hydrochloric acid present, all ions enhanced the 4 pg 1-1 cadmium atomic-fluorescence signals. Hydrochloric acid itself gave the greatest enhancement of about lOOyo (Fig. 3). The remaining ions caused enhancements that gradually increased from low to high concentrations of interfering ion with an average maximum of 50% enhancement. For the ions NO3-, PO4-, Cl-, Na+ and K+ scatter of source radiation was negligible (less than 5% of the total signal) at all concentrations of interfering ion (e.g., Na+, Fig. 5). Slight scatter was evident at the 5000 pg ml-l levels of Fe2+ and SO,2- (e.g., Fez+, Fig.6). Scatter was serious for Ca2+ and Mg2+ at all concentra- tions and especially at high concentrations (e.g., Mg2+, Fig. 7). $ 1 I , I 0 I 0 100 1000 Concentration of sodium in aqueous soIution/pg m1-l Fig. 6 . Effect of sodium on cadmium fluorescence. A, 4 p g 1-1 of cadmium, 0.04 M hydrochloric acid; B, 4 pg 1-l of cadmium, no acid. In 0.04 M hydrochloric acid solution the cadmium fluorescence was enhanced by the acid as demonstrated in Figs. 3 and 4. None of the other ions investigated caused any change in this signal although Ca2+ and Mg2+ continued to cause large scatter signals (e.g., Mg2+, 0 10 100 1000 Concentration of iron in aqueous soIution/pg mI-’ Fig. 6. Effect of iron on cadmium fluorescence.0, .=, 4 pg 1-1 of cadmium in 0.04 M hydrochloric acid; 0, 0, 4 pg 1-1 cadmium, no acid; 0, =, no correction for scatter; 0, 0, scatter corrected.500 MICHEL et al.: DETERMINATION 0 1 7 CADMIUM IN BLOOD AND Analyst, VoZ. 104 I 0 10 100 1000 Concentration of magnesium in aqueous soIution/pg mI-’ Fig. 7. Effect of magnesium on cadmium fluorescence. 0,. ., 4 pg 1-1 of cadmium in 0.04 M hydrochloric acid; 0, 0, 4 pg 1-l in cadmium, no acid; 0, a, no correction for scatter; 0, 0, scatter corrected. Fig. 7), SO,” and Fe2+ scatter was significant only at high concentrations of interferent (e.g., Fe2+, Fig. 6) and little scatter (less than 5:/0) was observed for the remaining ions (e.g., Na+, Fig. 5). It appears, therefore, that the presence of the metal chlorides and inorganic acids causes various enhancement interferences.Hydrochloric acid releases cadmium from the effects of the other ions and enables it to be determined in hydrochloric acid medium without interference. In urine the releasing effect is not completely effective until the hydrochloric acid concentration is above 0.03 M i B shown in Fig. 3. Moreover, it was found that orthophosphoric, sulphuric and nitric acid!; behave in a similar way. Urine acidified with one of these acids could probably be anal.ysed successfully using aqueous calibration standards containing the appropriate acid. This was not investigated further because hydrochloric acid provided the best analytical sensitivity, i.e., it caused the greatest enhance- ment of the cadmium signal in urine.The presence of 0.04 M hydrochloric acid in the diluted blood samples also appeared to have a releasing effect on cadmium in blood. This effect was demonstrated by the coincidence of the calibration graph (corrected for rate of uptake) obtained by standard additions to blood with the calibration graph obtained using aqueous standards which were 0 . 0 4 ~ in hydrochloric acid. Although the air - hydrogen flame was not used extensively, the ions K+, Na+, Ca2+, C1- and P0,3- were investigated in order to compare the behaviour of this flame with that of the acetylene flame. Similar results were obtained except that interferences in unacidified solutions were greater in magnitude with a maximum enhancement of 100%. Hydrochloric acid had a similar releasing effect to that found in the separated air - acetylene flame.Scatter was high for calcium solutions but little scatter (less than 5%) was observed for the remaining ions. Calcium scatter signals in the hydrogen flames were always three to five times greaterJune, 1979 URINE BY FLAME ATOMIC-FLUORESCENCE SPECTROMETRY 501 than the scatter signals in the separated air - acetylene flame; this was an example of the inadvisability of using the air - hydrogen flame for heavy matrices. A large scatter signal carries a noise component that degrades precision and detection limits. Results for Blood and Urine Analyses Table I1 summarises the results for the determination of cadmium in the blood and urine of a number of different groups of people. Blood and urine was taken from samples sub- mitted for analyses as part of health checks by Employment Medical Advisors.The samples that constituted the reference populations were obtained from hospital patients who were known to be unexposed to cadmium. The exposed groups of workers were coppersmiths engaged in brazing operations and men engaged in the shipbreaking industry. All these industrial workers showed increases in blood and urine cadmium relative to the reference population indicating evidence of recent exposure to cadmium. However, statistical comparison of results would require details as to the smoking habits of each of the populations studied as cigarette smoking can cause moderate elevations of blood cadmium. Samples analysed by standard additions gave agreement with samples analysed as described above, using aqueous standards.The average level of blood cadmium in the reference population, 3.1 pg 1-1 (27.6 nmoll-I), was similar to the control value of 35.6 nmol 1-1 reported by Cernik and Sayers9 and to other published values17 for non-exposed subjects. The mode (most frequently occurring value) of the reference population was lower than the average or mean value and indicated a non-normal distribution in the population. The average level of urine cadmium in the reference population, 0.5 pgl-1, was close to published valued7 and again the mode was lower than the mean. It was possible to measure the magnitude of the scatter signals associated with the atomic fluorescence signals by analysing blood and urine with and without use of the two-source background correction.For a batch of 54 blood samples from cadmium-exposed workers it was found that the average magnitude of the scatter signal was equivalent to 5.7 pg 1-1 of cadmium in the original undiluted blood with a standard deviation of 3.3 pg 1-1 (1.14 pg 1-1 and 0.66 pg l-l, as measured by the instrument, for diluted blood). Similarly, 25 samples of urine gave an average of 2.0 pg 1-1 and a standard deviation of 1.3 pg 1-l. To verify that the two-source system gave an accurate correction for scatter the alternative correction procedure described by Haarsma et aZ.45 was also applied. The Haarsma method is not rapid or automatic, but it is useful for verification purposes. It is based on the principle that the different dependence of EDL and fluorescence intensity on temperature permits a correction for scattering to be based on measurements at two EDL temperatures.Results using the Haarsma correction were in good agreement with the two-source method. TABLE I1 SUMMARY OF RESULTS OF BLOOD AND URINE ANALYSES Number in sample . . .. .. Standard deviation/pg 1-1 . . .. Rangelpg 1-1 . . .. .. .. Class interval of modelpg 1-1 Mean cadmium concentration/pg 1-1 .. - Reference population 100 3.1 1.5 1.1-6.4 1.4-1.7 Blood Copper- smiths* 20 10.4 4.8 3.3-20 6-6.9 - * Blood and urine samples were not from the same workers. 1 Ship- breakers 20 7.3 1.9 4-11.8 6-6.9 Urine w Reference Copper- population smiths* 0.5 7.2 0.4 6.8 20 20 0.2-1.5 2-26.4 0.2-0.29 2-2.9 It was previously reported from this l a b o r a t ~ r y ~ , ~ ~ that the level of urine cadmium in a reference population was 7 & 2 pg 1-1, which was higher than recent published values and higher than some results obtained on the same urine samples during inter-laboratory com- parisons.The value reported here, 0.5 pg 1-1, is in closer agreement with published values and demonstrates the improvement brought about by using the atomic-fluorescence instru- ment described above. The figures for the magnitude of the scatter correction show that502 MICHEL et d. : DETERMINATION OF CADMIUM I N BLOOD AND Analyst, 'vd. 104 the increased accuracy can be attributed primarily to the use of the scatter correction facility with a contribution resulting from the use of the separated air - acetylene flame rather than an air - hydrogen flame.The air - hydrogen flame tends to give scatter signals a factor of two to three times greater than air - acetylene when aspirating blood and urine solutions. An at omic-absorption spectrometric met hod with electrothermal at omisation developed in this laboratoryll was compared with the flame atomic-fluorescence method. The results of the analysis of the urine of a group of 30 exposed workers using both methods gave a correlation coefficient between those two sets of results that showed good agreement (Table 111). Further comparisons with more diverse methods of cadmium analysis are required to characterise fully the accuracy of the atomic-fluorescence method relative to other techniques. TABLE I11 DETERMINATION OF CADMIUM IN URINE USING TWO TECHNIQUES Flame atomic-fluorescence spectrometry (AFS) and atomic-absorption spectrometry using an electrothermal atomiser'l (AAS) .Urine samples taken from industrial workers some: of whom had possible exposure to cadmium in the workplace. Samples 26-29 were from four of our students. Sample number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Cadmium contentlpg 1-1 - AFS AAS 0.3 0.5 0.5 0.5 0.4 0.5 0.2 0.2 0.6 0.6 0.4 0.6 3.1 2.6 1.8 1.4 0.8 0.3 1.6 1.0 11.0 10.0 4.6 4.4 15.0 14.0 2.1 2.2 2.6 2.5 Sample number 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Cadmium contentlpg 1-1 -7 AFS AAS 7.9 8.8 1.5 1.3 1.9 1.5 0.6 0.2 8.4 8.5 0.3 0.7 0.2 0.4 3.6 3.8 3.6 3.2 0.3 0.4 0.3 0.2 0.2 0.2 0.3 0.2 0.3 0.9 .. . . 2.6 2.5 .. . . 3.6 3.6 .. .. 0.2-15 Mean cadmium concentration/ pg 1-l Standard deviationlhg 1-1 .. .. . . - Rangelpg 1-1 .. .. .. Correlation coefficient between the results of each technique . . .. .. .. .. .. 0.994 p <0.05 a t 95% confidence Regression equation .. .. .. . . A=mF + c (A = AAS, F = AFS) Gradient of regression equation . . .. . . 0.954 Intercept of regression equationlpg 1-1 . . . . 0.021 Some analytical figures of merit for typical analyses are shown in Table IV. The detection limit for blood is five times worse than that of urine because of the dilution required for blood. The noise on the scatter signal accounts for the difference in detection limit between aqueous standards and biological samples. Analyses of 15 aliquots each of a pooled blood and a pooled urine spiked with 20 pg 1-1 and 4pg1-1 of cadmium, respectively, were carried out to obtain the relative standard deviation within batch. The spiked, pooled blood and urine were then stored for about 3 months and an aliquot was analysed about once a week during routine analyses to obtain the between-batch figure (Table IV).The precisions and recoveries obtained were those normally expected of flame techniques. Conclusions The atomic-fluorescence method that has been described involves only direct aspiration of acidified urine or diluted and acidified blood into the flame. Calibration is achieved simply by using acidified aqueous standards and by the application of a pre-determined correction factor to account for changes in the uptake rate.June, 1979 URINE BY FLAME ATOMIC-FLUORESCENCE SPECTROMETRY 503 TABLE IV ANALYTICAL PERFORMANCE FOR THE DETERMINATION OF CADMIUM Precision as relative standard deviation (rsd)- Blood spiked with 20 pg 1-’ Urine spiked with 4 pg I-’ of Cd of Cd c A \ A Rsd, % Recovery, % Rsd, yo Recovery, %’ Within-batch (15 aliquots) ..2.8 101 2.0 101 Between-batch (9 aliquots) .. 9.5 103 10.0 102 Detection limitsipg l-l*- Aqueous Count timeis standards Blood Urine 1 0.2 1.4 0.3 10 0.07 0.5 0.1 * Spectral band pass was 0.5 nm. The sensitivity of the method is comparable to that of most techniques that are currently in use for the determination of cadmium in biological samples. The accuracy and precision are also satisfactory when compared with other techniques reported in the literature. Inter- laboratory studies need to be carried out to define the accuracy more completely. However, it is clear that background scatter correction is essential for accurate cadmium determinations when using flame atomic-fluorescence spectroscopy at the pg 1-1 level.This probably holds true for most elements, especially those with analytical lines in the ultraviolet region. The instrument described has a dynamic range for background correction that is more than adequate for all matrices. This is because of the use of the high-power second source. Moreover, the background correction will work throughout the visible and ultraviolet regions because the output of the xenon arc is maintained through them. The simplicity of the method permits rapid analyses of large numbers of samples (more than 25 samples h-l) and is particularly useful for the surveys of industrial workers currently being carried out in our laboratories.The instrumentation described has potential for the determination of other elements, although success depends upon the availability of suitable excitation sources. For the determinations requiring greater sensitivity electrodeless discharge lamps are often suitable and efforts are being made in our laboratory to develop well controlled methods for the preparation of these lamps.34~~5 In addition, it can be seen, from Fig. 1, that if the EDL is not switched on the xenon arc is positioned suitably to excite atomic fluorescence. 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M., Solomon, A., and Wakshal, E., Analyt. Chem., 1975, 47, Valenta, P., Rutzel, H., Nurnberg, H. W., and Stoeppler, M., 2. Analyt. Chem., 1977, 285, 25. Pulido, P., Fuwa, K., and Vallee, B. L., Analyt. Biochem., 1966, 14, 393. Smith, T. J., Temple, A. R., and Reading, J. C., Clin. Toxicol., 1976, 9, 75. Friberg, L., Piscator, M., Nordberg, G., and Kjellstrom, T., “Cadmium in the Environment,” Second Edition, CRC Press, Cleveland, Ohio, 1974. Pierce, J. O., O’Laughlin, J. W., and Hemphill, D. D., in “Edited Proceedings of the First Inter- national Cadmium Conference, San Francisco, 31st January-2nd February, 1977,” Metal Bulletin Ltd., London, p.88. O’Laughlin, J. W., Hemphill, D. D., and Pierce, J. O., “Analytical Methodology for Cadmium in Biological Matter-A Critical Review December 1976,” International Lead Zinc Research Organi- zation, Inc., New York. Lauwerys, R., Buchet, J . P., Roels, H., Berlin, A., and Smeets, J., Clin. Chem., 1975, 21, 551. Kjellstrom, T., Tsuchiya, K., Tompkins, E., Takabatake, E., Lind, B., and Linnman, L., “Pro- ceedings of an International Symposium on Environmental Health, Paris, 1974,” Commission of the European Communities, EUR 5360, 1974, 4, 2197. Berman, E., Atom. Absorption Newsl., 1967, 6, 57. Westerlund-Helmerson, U., Atom. Absorption h’ewsl., 1970, 9, 133. Evans, W. H., Read, J. I., and Lucas, B. E., Analyst, 1978, 103, 680. Vens, M. D., and Lauwerys, R., Arch. Mal. Prof. Mkd. Trav., 1972, 33, 97. Hough, D. C., and Ottaway, J. M., Proc. SOC. Analyt. Chem., 1974, 11, 223. Scott, R., Mills, E. A., Fell, G. S., Hussein, F. E. R., Yates, A. J., Paterson, P. J., McKirdy, A., Ottaway, J . M., Fitzgerald-Finch, 0. P., Lamont, A., and Roxburgh, S., Lancet, 1976, 396. Fell, G. S., Hough, D. C., Hussein, F. E. R., i%nd Ottaway, J. M., Proc. Analyt. Div. Chem. Soc., 1976, 13, 271. Fell, G. S., Ottaway, J. M., Hussein, F. E. R., Michel, R. G., and Hall, M. L., in Brown, S. S., Editor, “Clinical Chemistry and Chemical Toxicology of Metals,” Elsevier North Holland, Amsterdam, 1977, p. 367. Fell, G. S., Ottaway, J . M., and Hussein, F. E. R., BY. J . Ind. Med., 1977, 34, 106. Winefordner, J. D., J . Chem. Educ., 1978, 55, 72. Browner, R. F., Analyst, 1974, 99, 617. West, T. S., Analyst, 1974, 99, 886. Michel, R. G,, Coleman, J., and Winefordner, J. D., Spectrochim. Acta, 1978, 33B, 195. Michel, R. G., Ottaway, J. M., Sneddon, J., and Fell, G. S., Analyst, 1978, 103, 1204. Bratzel, M. P., Dagnall, R. M., and Winefordner, J. D., Analyt. Chem., 1969, 41, 713 and 1527. Michel, R. G., Hall, M. L., Rowland, S. A. K., Sneddon, J., Ottaway, J. M., and Fell, G. S., Analyst, Aldous, K. M., Browner, R. F., Dagnall, R. M., and West, T. S., Analyt. Chem., 1970, 42, 939. Kirkbright, G. F., and Sargent, M., “Atomic Absorption and Fluorescence Spectroscopy,” Academic Rains, T. C., Epstein, M. S., and Menis, O., Analyt. Chem., 1974, 46, 207. Zander, A. T., Int. Lab., Jan/Feb, 1977, 15. Johnson, D. J., Plankey, F. W., and Winefordner, J . D., Analyt. Chem., 1975, 47, 1739. Butrimovitz, G. P., and Purdy, W. C., Analytica Chim. Acta, 1977, 94, 63. Haraguchi, H., Fowler, W. K., Johnson, D. J., and Winefordner, J. D., Spectrochim. Acta, 1976, Haarsma, J . P. S., Vlogtman, J., and Agterdenbos, J., Spectrochim. Acta, 1976, 31B, 129. 534. 1979, 104, 505. Press, New York, London, 1974. 32A, 1539. Received September 2.5512, 1978 Accepted November Zlst, 1978

ISSN:0003-2654    

DOI:10.1039/AN9790400491

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, June, 1979, Vol. 104, fie. 505-515 505 Effect of Stray Light in Monochromators on Detection Limits of Flame Atomic-fluorescence Spectrometric Measurements R. G. Michel, M. L. Hail, S. A. K. Rowland, J. Sneddon and J. M. Ottaway Department of Pure and Applied Chemistry, University of Strathclyde, Cathedral Street, Glasgow, G1 1XL and G. S. Fell Department of Clinical Biochemistry, Royal Injirmary, Glasgow, G4 OSF Quantitative results are described that demonstrate that the use of a double monochromator to reduce stray light originating from strong thermal emission in the flame gives significant reductions in noise on the background of the fluorescence measurement. This leads to worthwhile improvements in detection limit for all elements with analytically useful resonance lines at wavelengths shorter than approximately 250 nm.The degree of improve- ment depends upon whether water or a real sample is being aspirated. For the determination of cadmium in urine the detection limit is improved by a factor of three and for the determination of selenium in water the improve- ment is a factor of 5-6. Scatter of excitation source radiation is also shown to have a small but significant effect on detection limits when using electrode- less discharge lamps as source. Scatter is more serious in the air - hydrogen flame than the air - acetylene flame. Keywords Flame atomic-fluorescence spectrometry ; stray light ; double monochromator The presence and effects of stray light in monochromators have been described for atomic emission,l atomic2 and molecular-3 absorption and Rarnan4s5 spectroscopy.Larson et aZ.1 have discussed the effect of stray radiation on background levels and hence on accuracy in atomic-emission spectroscopy. They also discussed the causes of stray radiation in mono- chromators and cited publications on the grating imperfections which are the primary cause.6 Here, the one quoted by Goode and Crouch2 is suitable, i.e., the stray radiation is expressed as the ratio of the spectrometer’s response to radiation within the monochromator’s band pass to its response to radiation outside the band pass. The most effective method of reducing stray radiation to negligible levels is to use a double monochromator. This method is almost universally used in Raman spectroscopy and has been suggested as a solution for atomic-emission spectroscopy1 and atomic-fluorescence spectroscopy.7~8 Barnett and Kahn7 found that high concentrations of sodium appeared to interfere with the flame atomic fluorescence of iron.They concluded that the intense sodium emission in the flame resulted in stray light in the monochromator. The intensity of the stray light was such that it saturated the electronics and caused an apparent interference. Haarsma et aZ.8 found that the noise associated with the flame background at the cadmium resonance line (228.8nm) could be reduced by use of a double monochromator. It was clear therefore that stray light originated from the flame background itself as well as from intense sodium emission in the flame. Flame atomic-fluorescence spectrometry has potential as a rapid means for the determina- tion of low levels of toxic metals in blood and urine.g This type of sample contains essential elements such as sodium, magnesium and calcium, which emit strongly in flames and which can cause stray light in monochromators.Stray light originating from the flame does not affect the accuracy of atomic-fluorescence measurements because modulation of the excitation source allows the fluorescence signal to be distinguished from all other signals originating in the flame. However, the noise associated with the stray light signal does degrade precision and detection limits. This can be contrasted with the modulated excitation source radiation, which, when scattered by incompletely dissociated matrix particles present in the flame, A number of definitions of stray radiation have been used in the past.506 MICHEL et nl.: EFFECT OF STRAY LIGHT IN MONOCHROMATORS Analyst, VoZ. 104 does affect the accuracy of the measurement. Scatter signals can be corrected9 but cannot be prevented from reaching the detector if excitation and fluorescence take place at the same wavelength (resonance fluorescence). Noise on scatter signals therefore also degrades detection limits. An attractive means of avoiding scatter noise is to use direct line fluorescence10 or similar transitions where excitation and fluorescence occur at different wavelengths. With microwave-excited electrodeless discharge lamp (EDL) excitation the use of direct line fluorescence often leads to an unacceptable loss in sensitivity, which is not usually balanced by the reduction in scatter noise.For some elements laser excitation does provide adequate sensitivity for direct line fluorescence measurements.11 In summary, it can be seen that, with modulated EDL excitation, accuracy can be ensured by using a background correction system but it is not practical to reduce the noise associated with the scatter signal. However, it is possible to improve precision and detection limits by minimising the contribution of stray light to background noise. Quantitative results are described here that demonstrate that the use of a double mono- chromator to reduce stray light gives significant reductions in the noise on the background of the fluorescence measurement. This leads to worthwhile improvements in detection limit for all elements with analytically useful resonance lines at wavelengths shorter than approximately 250 nm.The double monochromator is particularly useful for the reduction of stray light originating from strong thermal emission in flames caused by the aspiration of biological samples such as blood and urine. Experimental Instrumentation A double monochromator is two similar or identical monochromators which are arranged in tandem and which disperse the incident radiation twice. The second of the two mono- chromators is therefore able to re-disperse the stray light that is present at the exit slit of the first monochromator with the result that there is a negligible amount of stray light remaining at the exit slit of the second monochromator.As a consequence of the double dispersion the spectral band pass of the double monochromator is normally half the spectral band pass of the equivalent single monochromator if the same slit width is used on both. The same band pass can be achieved by doubling the slit width on the double monochromator. The exit slit of the first monochromator and entrance slit of the second monochromator of the tandem are normally physically the same slit (middle slit) in a true double mono- chromator. There are twice the usual number of mirrors and two gratings in a double monochromator, and therefore some light losses are expected relative to the equivalent single monochromator. The instrumentation that was used here has been more fully described in a previous publicati~n.~ Microwave-excited electrodeless discharge lamps were used as excitation sources.Cadmium lamps were made in the laboratory using the method published else- ~here.129~3 Selenium lamps were prepared using the same method but optimised specially for this element.14 The instrument included a two-source background correction system that employed a 300-W high-pressure xenon arc as the second source. All flames were supported on a circular capillary burner with facilities for flame separation. In order to determine the effect of using a double monochromator, it was necessary to compare all measurements with those obtained. using an equivalent single monochromator. The two monochromators chosen were those manufactured by Spex Industries Inc. (Metuchen, N.J., USA). These were the Doublemate double monochromator and Minimate single monochromator. The Minimate is in every respect identical with half of the Doublemate and hence clear comparisons could be made. The detailed specifications of these mono- chromators, taken from the manufacturer’s literature, are shown in Table I. Fluorescence, scatter, flame background and stray light signals were all measured using a photon counter in the digital lock-in mode (the Ortec-Brookdeal 5 C1/5C21 photon counting system was used routinely with a 1 s count time). Simultaneous measurements of flame background on the double monochromator and the single monochromator were made by placing them on opposite sides of the same flame andJune, 1979 ON DETECTION LIMITS OF FLAME AFS MEASUREMENTS 507 taking the signals from the two photomultiplier tubes to the two separate channels on the photon counter.The photomultiplier tubes used were as near identical as possible and both EM1 9789QB tubes were operated at 1100 V. TABLE I SPECIFICATIONS OF MONOCHROMATORS Manufacturer, Spex Industries, Inc., Metuchen, N. J , , USA. Monochromator Focal length . . .. . . .. Aperture . . .. .. .. .. Grating . . .. . . . . .. Blaze . . .. .. . . .. Dispersion .. . . . . .. Resolution .. . . . . .. Near stray light (slits 5 nm band pass, within 3.0 band widths) . . .. Far stray light (> 10 band widths) Configuration . . .. .. .. Slits .. .. .. .. .. Band pass .. .. .. .. . . 1670 Minimate single monochromator 220 mm 1200 grooves mm-1 300 nm 4 nm mm-l 1 nm f14 1672 Dou blemate double monochromator 220 mm fI4 (2) 1 200 grooves mm-1 300 nm 2 nm mm-1 0.5 nm 10-s 10-4 5 x 10-4 10-9 In-line Czerny - Turner Double in-line Czerny - Turner Five pairs 20 nm high, 0.25, 0.5, 1.25, 2.5 and 5 mm 1-20 nm 0.5-10 nm Reagents High-purity de-ionised water acidified to 0.04 M with hydrochloric acid was used for bIank measurements and is referred to as water throughout this paper.Urine samples were obtained as discussed elsewhere9 and were normally from persons exposed to cadmium in their workplace. Urine was also acidified to 0 . 0 4 ~ with hydro- chloric acid. The acid was of AnalaR grade. New batches of acid were checked for cadmium and selenium contents. Undiluted rather than diluted urine was aspirated into the flame. Results and Discussion When using a single monochromator (SM) it is not possible to distinguish easily between true flame background at a particular wavelength and the stray light that is a result of flame background at wavelengths removed from the monochromator wavelength setting.However, it can be assumed that the double monochromator (DM) can discriminate against stray light. Simultaneous measurements of flame background using an SM and a DM then reveal differences that are due to stray light alone. Variation of Flame Background and Stray Light with Wavelength Fig. 1 shows the variation in magnitude of the flame background with wavelength for a nitrogen-separated air-acetylene flame when measured on the DM and the SM. The difference between the two spectra shows that stray light was a significant component of the flame background at short wavelengths.De-ionised water acidified to 0.04 M in hydro- chloric acid was aspirated into the flame to obtain Fig. 1. In Fig. 2 urine was aspirated instead of water and again the stray light difference between the double and single mono- chromators can be seen. The results in Fig. 3 are taken from Figs. 1 and 2 and are the flame background as seen by the double monochromator, L e . , stray light was not present. Accordingly, the difference in background between water and urine being aspirated was a real change in flame background. At wavelengths longer than 300 nm this change leads to increased noise and degraded detection limits when aspirating urine rather than water. However, there was no shift in flame background below 300 nm.This result indicates that the only increase in noise to be expected upon aspiration of urine is caused by scatter of excitation source radiation if a double monochromator is used and elements with resonance lines below 300 nm are being determined.508 MICHEL et d. : EFFECT OF STRAY LIGHT IN MONOCHROMATORS Analyst, VOl. 104 300 400 500 600 - u 200 Wavelength/nm Fig. 1. Flame background with water. Flame background was measured a t 10-nm intervals while aspirating water into a stoicheio- metric nitrogen-separated air - acetylene flame. Spectral band pass on both monochromators was 1 nm. 0, Single monochromator; and @, double monochromator. On this and subsequent figures, Na, K and Ca indicate the position of atomic lines and OH the band spectrum of this radical, which are superimposed on the flame background .1 200 360 400 500 600 Wavelengthhm Fig. 2. Flame background with urine. Flame background was measured a t 10-nm intervals while aspirating urine into the separated air - acetylene flame. Spectral band pass on both monochromators was 1 nm. 0, Single monochromator; and @, double monochromator. Improvement in Flame Noise Levels by Use of the Double Monochromator It was possible to demonstrate the improvement in flame background noise levels by using the results in Figs. 1 and 2. It was assumed that the noise on the flame background was white or shot noise and therefore the noise could be calculated by taking the square root of the flame background measured in counts per second.Prior measurements of various parameterss had indicated that this assumption was correct a t 228.8nm when using both monochromators. Shot noise was probably also dominant throughout the low background region of the flame. Fig. 4 shows the noise improvement with wavelength. The noise improvement factor is the ratio of the noise as measured in the single monochromator to the noise on the double monochromator, where noise is measured as the square root of the flame backgrounds taken from Figs. 1 and 2 at each wavelength plotted. Improvements in the shot noise as a result of using the double monochromator are the smallest improvements that could be expected from the large reductions in flame background that were observed. 200 300 400 500 600 Wavelengthhm Fig.3. Flame background water and urine. Flame background was measured a t 10-nm intervals while aspirating either urine or water into a stoicheiometnc :nitrogen-separated air - acetylene flame. Detection using the double monochromator with a spectral band pass of 1 nm. 0, Water; and a, urine.June, 1979 ON DETECTION LIMITS OF FLAME AFS MEASUREMENTS 509 If proportional noise15 had made important contributions to flame background noise it would have decreased linearly with the flame background. Therefore, the improvements in proportional noise were the largest that could have been expected. From Fig. 4 it can be seen that the improvement in shot noise is much greater for urine than for water. This was a result of the intense emission from the flame when aspirating urine and which causes greater stray light levels.The improvement was only significant, ie., greater than a factor of 2, at wavelengths shorter than 250 nm and varied from a factor of 25 at 200nm to a factor of 2 at 250nm for urine. When aspirating water the corre- sponding figures were 8 at 200 nm and 2 at 225 nm. From Fig. 3 it is clear that the major portion of the stray urine emission from the flame probably originates from increases in flame background over a broad band of wavelengths above 300 nm, together with contributions from sodium, potassium and calcium atomic line emission. b c, m + c, I! E 3 P .- .- 0 z Wavelengthhrn Fig. 4. Noise improvement, single to double mono- chromator. Ratio of noise in single monochromator to noise in double monochromator using results from Figs.1 and 2 (see text). Stoicheiometric nitrogen-separated air - acetylene flame. Spectral band pass 1 nm. 0, Water; and 0, urine. Improvement in Flame Background Noise at Particular Wavelengths Atomic fluorescence and flame background measurements were made at the selenium and cadmium resonance lines at 204.0 and 228.8 nm, respectively. Slit width and hence spectral band pass were varied in order to determine differences in flame background more accurately. Measurements of flame background at only one spectral band pass (Fig. 4) are biased by relative differences in slit width and other physical parameters between the two mono- chromat ors. Figs. 5 and 6 show the variation of flame background with spectral band pass for the two elements and for both monochromators while aspirating urine or water. As predicted in Fig.3 negligible background shift was observed with the DM in changing from water to urine. However, large shifts caused by stray light were observed when using the SM. In Fig. 5 the background at 228.8 nm (cadmium) while aspirating urine was 17 times greater in the SM than in the DM, which indicated a reduction in flame noise of 1/17 or 4.1 when using the double monochromator. When aspirating water the background was 3.8 times greater in the SM than the DM, i.e., a reduction in flame noise of 1.9 when using the double monochromator. Fig. 6 shows similar results for selenium, which indicate a 1/562 or 24 times reduction in flame noise when using the double monochromator and aspirating urine.A 1/31 or 5.6 reduction in flame noise was obtained when aspirating water, Shifts in flame background caused by scatter of excitation source (EDL) radiation have been corrected in Figs. 5 and 6. The magnitudes of the scatter signals are discussed in relation to detection limits in a later section.510 MICHEL et aZ. : EFFECT OF STRAY LIGHT IN MONOCHROMATORS Analyst, VoZ. 104 Spectral band pass/nm rn + C : Y lo4- 2 l o 3 - D 3 m Y n Fig. 5. Background versus spectral band pass for cadmium. Flame back- ground measured at the cadmium resonance line (228.8 nm) using both single and double monochromators. Separated air - acetylene flame. Ratios of flame backgrounds in single mono- chromators to double monochromators are 17 for urine and 3.8 for water. A, Single monochromator with urine; B, single monochromator with water ; and C, double monochromator for urine and for water.Slope of each line = 2. Spectra I band pass/nm Fig. 6. Background vevsus spectral band pass for selenium. Flame back- ground measured at the selenium line (204.0 nm) using both single and double monochromators. Separated air - acet- ylene flame. Ratios of flame back- grounds in single monochromators to double monochromators are 562 for urine and 31 for water. A, Single monochromator with urine; B, single monochromator with water; and C, double monochromator with urine and with water. Light Losses in the Double Monochromatar The cadmium fluorescence signals obtained simultaneously with the background measure- ments of Fig. 4 are shown in Fig. 7. These results show that at the same spectral band pass the fluorescence signals were the same in both monochromators.If there had been no light losses as a result of using the DM rather than the SM then the signals obtained in the DM would have been twice those obtained in the SM. This is because at the same spectral band pass the slit width of the DM is twice the slit width of the SM. As the signals turned out to be equal in both monochromators then light losses were close to 50%. Light losses did not affect detection limits because at the same spectral band pass there were no effective light losses between the two monochromators. There was an approximately 6% difference in sensitivity between the two photomultiplier tubes attached to the two monochromators. A correction for this difference has been incorporated into Figs.1-3 and 7. Scatter of line source radiation, when detected, is effectively similar in nature to the atomic fluorescence signal. One consequence was that when urine was aspirated into the flame scatter signals were of the same magnitude in both monochromators at the same spectral band pass. The effect of the scatter signals on detection limits, however, was different in the two monochromators because of the differences in the flame background. Effect of the Double Monochromator on Detection Limit At the detection limit the noise on the fluorescence signal approaches in magnitude the noise on the background.16 Therefore, the decreases in flame noise brought about by using the double monochromator should improve detection limits in proportion, if flame noise is the limiting noise.For example, the reduction in flame noise by a factor of 4.1, which was obtained for the aspiration of urine (cadmium, Fig. 5) should improve detection limits by aJune, 1979 ON DETECTION LIMITS OF FLAME AFS MEASUREMENTS Io4 [- 51 1 I " 0.5 1.0 2.0 5.0 10.0 Spectral band p a d n rn Fig. 7. Cadmium fluorescence vey- sus spectral band pass. Cadmium fluorescence signals from 4 pg 1-1 of cadmium in water (228.8 nm). Stoi- cheiometric separated air - acetylene flame measured using both single (0) and double (0) monochromators. factor of 4.1.8,However, the scatter of excitation source radiation had a significant effect on detection limits for determinations of both cadmium and selenium in urine.Table I1 shows some typical figures for the magnitude of flame background and scatter at a l-nm spectral band pass on both DM and SM. The scatter signals given are average figures TABLE I1 EFFECT OF FLAME BACKGROUND AND SCATTER ON DETECTION LIMITS USING THE SINGLE OR DOUBLE MONOCHROMATORS Element Parameter Cadmium (228.8 nm) . . Double monochromator, DMS Single monochromator, SMS Improvement factors, SM/DM- in background in noise in detection limit Selenium (204.0 nm) . . Double monochromator, DM Single monochromator, SM Improvement factors, SM/DM- in background in noise in detection limit Flame back- ground*/ Scatter/ counts s-l counts s-1 500 500 50 460 1900 8500 50 460 - 3.8 17 - 1.95 4.1 - - 40 40 4 30 1240 22480 4 NDY - 31 562 - 5.6 24 - - Detection limitt/ PLg 1-1 0.2 0.26 0.37 0.81 - - - 1.9 3.1 160 210 890 3800 - - 5.6 19 * Flame background for stoicheiometric separated air - acetylene flame.Flame background increases for leaner or richer fuel conditions and affects detection limits appreciably. t Detection limit for signal to noise ratio of 2 where noise is expressed as the square root of the total background (flame background plus scatter). : Urine samples were those of workers exposed to cadmium in their work place. Scatter signals of persons unexposed to cadmium tend to be lower by about half. 5 Spectral band pass 1 nm for both monochromators. 7 ND = not detectable.512 MICHEL et al. : EFFECT OF STRAY LIGHT IN MONOCHROMATORS Analyst, Vol. 104 which varied from urine to urineg and which were measured using the two-source background correction facility which was incorporated into the instr~ment.~ Detection limits (signal to noise ratio = 2) in Table I1 were estimated by measuring signals close to the detection limit and calculating noise by taking the square root of the total background, i.e., flame back- ground plus scatter signal.It can be seen in Table I1 that the improvement in detection limit in going from the SM to the DM was not as great as the improvement in flame background noise. This was a result of the addition of the scatter noise into the total noise. The scatter noise was signifi- cant only for urine. The aspiration of water caused little scatter off water droplets and flame gases as shown in Table 11. Despite the scatter noise component the double mono- chromator improved detection limits for cadmium in urine by a factor of 3 and for selenium in urine by a factor of 19.As a result, the determination of cadmium in urine became feasible, because although the concentration of cadmium in the urine of persons unexposed to cadmium is close to 0.2 pg l-l, it was possible to determine cadmium in the urine of persons exposed to cadmium (cadmium present at concentrations above 2 pg 1-l) by direct aspiration of un- diluted urine into the flame.9 It was not possible to determine selenium in this way because this element is present at levels below 1 pug 1-l. Selenium is discussed in this paper simply to demonstrate the utility of the double monochromator for the analysis of real samples, with urine taken as a convenient example.It is apparent from these results that the considerable lowering of the flame background by discrimination against stray light in the DM caused the scatter noise to have an approxi- mately equal effect, with flame noise, on the total noise and hence on the detection limit. In contrast, when using the SM the scatter noise was negligible relative to flame noise, which was then the limiting noise at the detection limit. With the double monochromator scatter was important relative to the flame background for both cadmium at 228.8 nm and selenium at 204.0 nm. It appears, therefore, that scatter will probably be important for all elements with resonance lines at wavelengths shorter than 250 nm where flame background is low and where the corresponding EDLs tend to perform well in terms of atomic-fluorescence detection limits.For elements with resonance lines at I 200 300 400 500 600 Wavelength/nrn Fig. 8. Background in three flaines with water. Using the double monochromator, flame backgrounds were measured at 10-nm intervals while aspirating water into either a stoicheiometric separated air - acetylene flame (0) or the hydrogen flames operated under fuel-lean conditions ; 0, air - hydrogen flame ; and 0, separated air - hydrogen flame. (Optimised for cadmium atomic fluorescence measurements.) Spectral band pass 1 nm.Jane, 1979 ON DETECTION LIMITS OF FLAME AFS MEASUREMENTS 513 wavelengths longer than 250 nm, where the flame background is high and not improved by the DM, scatter will probably not determine the detection limit even with EDLs of high radiant output. This is primarily because cadmium EDLs are normally superior to EDLs of other metals and therefore scatter signals caused by a cadmium EDL are the largest likely to be experienced.Furthermore, scatter of ultraviolet radiation is known to be of greater magnitude than scatter of longer wavelength radiation. If continuum or line excitation sources of greater intensity than EDLs are used then scatter noise will amost always domi- nate. This effect has been discussed elsewhere for laser excitation.17 Choice of Flame The variation in flame background with wavelength for three flames was obtained when using the double monochromator. These backgrounds are shown in Fig. 8 (aspiration of water) and Fig. 9 (aspiration of urine).As would normally be expected the nitrogen- separated air - hydrogen flame had the smallest background at all wavelengths. The separated air - acetylene flame had a background that was lower than the unseparated air - hydrogen flame when aspirating water (Fig. 8) but higher than the same flame when aspirating urine. It appeared, therefore, that the low background, and hence low noise, separated air - hydrogen flame would give the best detection limits when the fluorescence signals are similar in all flames. However, the hydrogen flames have a low temperature and probably do not dissociate matrix particles as efficiently as the air - acetylene flame, which leads then t o increased scatter of excitation source radiation. 200 300 400 500 600 Wavelength/nm Fig.9. Background in three flames with urine. Using the double monochromator flame backgrounds were measured a t 10-nm intervals while aspirating urine into the same flames as in Fig. 8. Spectral band pass 1 nm. 0, Separated air - acetylene flame; 0, air - hydrogen flame; and 0, separated air - hydrogen flame. The increased scatter in hydrogen flames was demonstrated by the results shown in Table I11 for which total background, i e . , flame background plus scatter, was recorded in all three flames at the wavelength of the cadmium resonance line (228.8nm). The detection limits that resulted from these backgrounds are also shown in Table 111. There was little scatter when aspirating water and therefore the separated air - hydrogen flame gave the best detec- tion limit.When urine was aspirated the detection limits deteriorated more in hydrogen flames than in the acetylene flame as a result of the noise on the larger scatter signals in the hydrogen flames. The nitrogen-separated air - acetylene flame is therefore the best choice when analysing urine and will also be a good choice for most other matrices.514 MICHEL et al. : EFFECT OF STRAY LIGHT I N MONOCHROMATORS Analyst, VOl. 10 By changing the fuel flow-rate it was possible to improve cadmium atomic-fluorescence signals and hence detection limits in the hydrogen flames by up to a factor of two. How- ever, the flame was then lean and the burner became less resistant to the formation of salt deposits in the orifices of the burner head. These deposits rapidly caused instability of the fluorescence signals.The cadmium fluorescence signal changed little with changes in fuel conditions in the air - acetylene flame. The optimum signal to noise ratio was obtained in the stoicheiometric flame, where the flame background was at a minimum. TABLE: I11 RELATIVE DETECTION LIMITS OF CA:DMIUM BY ATOMIC-FLUORESCENCE SPECTROSCOPY I N THREE FLAMES Detection limits defined as in Table I1 and the text. Signals in all three flames were approximately the same. Fuel conditions as described in caption to Fig. 8 and in the text. Flame background (228.8 nm*)/ counts s-l Detection limitlpg 1-1 A 3 Separated - 3 d (Separated Separated Sample air - C,H, Air - H, air - H, air - C,H, Air - C,H, air - H, Water . . .. . . 660 1250 70 0.2 0.36 0.09 Urine .. .. . . 960 1950 990 0.26 0.45 0.35 * Double monochromator, spectral band pass 1 nm. Conclusions The use of a double monochromator to reduce stray light originating from strong thermal emission in the flame gives significant improvements in detection limits for all elements with resonance lines at wavelengths shorter than approximately 250 nm. The degree of improve- ment depends upon whether water or a real sarnple such as urine is being aspirated. When water is aspirated the flame background at all wavelengths causes stray light, which is reduced by using the double monochromator. The improvements obtained with urine are much greater than for water because urine in the flame causes intense emission over a broad band of wavelengths, as well as line emission from sodium, potassium and calcium, which all contribute to stray light at analytical wavelengths shorter than 250 nm.It is possible to estimate the performance required of a monochromator in discriminating against stray light by considering the total amount of light passing through the entrance slit. With urine in the flame and thej/4 double monochromator this is approximately 108 counts s-1. This figure was obtained by integrating the flame background over all wavelengths using the data in Fig. 2. A double monochromator with a far stray light specification of is therefore probably more than adequate to reduce stray light to insignificant levels. A normal ruled grating single monochromator has a typical specification of 10-4 and a holographic grating single monochromator has a typical specification of The latter will clearly reduce stray light to lower levels but will not be as effective as the double monochromator.With a double monochromator scatter of (EDL) excitation source radiation has a small but significant effect on detection limits at wavelengths shorter than 250 nm. With a single monochromator scatter is not important because the flame background, which includes the stray light component, is high and the noise on the scatter signal is small in comparison. However, the same inaccuracies caused by the scatter signal will occur when using either monochromator and, to compensate, a scatter correction must be applied. Scatter is more serious in the air - hydrogen flame than in the air - acetylene flame. This degrades detection limits more in air - hydrogen flames than in the air - acetylene flame and favours the latter for routine use.The authors acknowledge the support of the Scottish Home and Health Department for the purchase of the major items of equipment used in this project and for the award of a Postdoctoral Fellowship (to support R.G.M.). We also thank the Eastern District, GreaterJune, 19 79 ON DETECTION LIMITS OF FLAME AFS MEASUREMENTS 515 Glasgow Health Board, for a maintenance grant in support of J.S. and the HSE, Employ- ment Medical Advisory Service, for a maintenance grant in support of M.L.H. Practical and equipment support of this work has also been given by Pye Unicam Ltd. and this is p a t ef ully acknowledged. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. References Larson, G. F., Fassel, V. A., Winge, R. K., and Kniseley, R. N., Appl. Spectrosc., 1976, 30, 384. Goode, S. R., and Crouch, S. R., Analyt. Chem., 1974, 46, 181. Slavin, W., Analyt. Chem., 1963, 35, 561. Stamm, R. F., and Salzman, C. F., Jr., J . Opt. SOC. Am., 1953, 43, 126. Leite, R. C. C., and Porto, S. P. S., J . Opt. SOC. Am., 1964, 54, 981. Sharpe, M. R., and Irish, D., Optica Acta, 1978, 25, 861. Barnett, W. B., and Kahn, H. L., Analyt. Chem., 1972, 44, 935. Haarsma, J. P. S., Vlogtman, J., and Agterdenbos, J., Spectrochim. Acta, 1976, 31B, 129. Michel, R. G., Hall, M. L., Ottaway, J. M., and Fell, G. S., Analyst, 1979, 104, 491. Kirkbright, G. F., and Sargent, M., “Atomic Absorption and Fluorescence Spectroscopy,” Weeks, S. J., Haraguchi, H., and Winefordner, J. D., Analyt. Chem., 1978, 50, 360. Michel, R. G., Coleman, J., and Winefordner, J . D., Spectrochim. Acta, 1978, 33B, 196. Michel, R. G., Ottaway, J. M., Sneddon, J., and Fell, G. S., Analyst, 1978, 103, 1204. Michel, R. G., Ottaway, J. M., Sneddon, J., and Fell, G. S., Analyst, in the press. Chester, T. L., and Winefornder, J. D., Analyt. Chem., 1977, 49, 119. Winefordner, J. D., Schulman, S. G., and O’Haver, T. C., “Luminescence Spectrometry in Analytical Green, R. B., Travis, J. C., and Keller, R. A., Analyt. Chem., 1976, 48, 1954. Academic Press, New York, London, 1974. Chemistry,” Wiley-Interscience, New York, 1973, p. 149. Received September 26th, 1978 Accepted November 21st, 1978

ISSN:0003-2654    

DOI:10.1039/AN9790400505

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

516 Analyst, June, 1979, VoL. 104, pp. 516-524 Sequential Multi-element Analysis of Small Fragments of Glass by Atomic-emission Spectrometry Using an Inductively Coupled Radiofrequency Argon Plasma Source T. Catterick and D. A. Hickman The Metropolitan Police Forensic Science Laboratory, 109 Lambeth Road, London, SE 1 7LP A method is described for the quantitative multi-element analysis of small fragments (200-500 pg) of glass using an inductively coupled radiofrequency argon plasma source. The glass samples are digested with a mixture of hydrofluoric and hydrochloric acids and chromium is added as an internal standard. An ultrasonic nebuliser is used in order to reduce to a minimum the volume of solution required for each analysis. A single monochromator and detection system is employed, and the wavelength regions of interest are examined sequentially by means of a specially constructed control unit.The results for aluminium, barium, iron, magnesium and manganese show that the analysis of glass fragments in the range 200-500 pg can be achieved with coefficients of variation of approximately 10%. Standard glasses were analysed to assess the accuracy of the method. Keywords ; Glass analysis ; acid digestion ; control unit for automatic sequential selection of wavelength regions ; inductively coupled radiofrequency argon filasma ; forensic analysis Glass is a commonly encountered material in forensic science, but until recently much of the evidential value has been based on the measurement of physical properties such as refractive index and density.Several worker~l-~ have shown that chemical analysis of a glass sample will increase the evidential value and may well enable the sample to be classified as sheet, container, tableware, headlamp, etc., glass. Although the measurement of a large number of variables (such as trace-element concentrations) should give rise to the best classification,* this must be related to such factlors as the analytical technique employed and the operator time involved. The preferred situation would be to classify an unknown glass sample by measuring the concentrations of a relatively small number of trace elements. Previous work in this lab0ratory~9~9~ has shown that aluminium, barium, iron, manganese and magnesium are useful elements for classifying glass samples.Alternative analytical techniquesls2 have indicated that other elements, such as antimony, arsenic and potassium, are also useful for classification purposes, but again the determination of these elements must be viewed in relation to the capabilities of the analytical instrumentation available. A number of techniques have in fact been employed for the chemical analysis of glass: neutron- activation analysis,lv7 d.c. arc - atomic-emission spectr~graphy,~~~ atomic-absorption spectro- metry,596 spark-source mass spectrometry2 and X-ray fluorescence spectrometry.9 This paper describes an inductively coupled argon plasma (ICP) - atomic-emission spectro- metric procedure, which is a logical progression from the d.c. arc - atomic-emission3 and atomic-absorption6s6 spectroscopic methods previously reported from this laboratory.The emission spectrographic method, which involved grinding the glass fragments in a graphite matrix, was very demanding on the operator and was also prone to airborne contamination. It was also a fairly lengthy procedure, incorpclrating densitometry of the photographically recorded spectra. Atomic-absorption spectromletry is inherently a single-element technique, and the limited linear range for each element would mean that the measurement of several elements in a dissolved glass sample would be time consuming and might require dilutions of the sample. Electrothermal atomisation might be necessary for the determination of low concentrations of some elements, and this would increase further the analysis time.The inductively coupled argon plasma - atomic-emission method takes advantage of the large linear working ranges characteristic of plasma sources to reduce sample preparation to aCATTERICK AND HICKMAN 617 minimum. The sequential wavelength selection control unit employed enables a single monochromator and photomultiplier tube to be used for signal detection, and thus reduces instrumental costs. It also provides a more flexible system than a direct-read spectrometer. A dissolved glass sample can be analysed for five elements in under 4 min. Control unit Experimental Apparatus components and operating conditions are listed in Table I. A schematic diagram of the instrumental system is shown in Fig. 1, and details of the Argon 1 nebuliser U'trasonic I Fig.1. Schematic diagram of experimental system. Sequential analysis control unit The control unit was designed especially for this work, and operates by controlling auto- matically a stepping motor coupled to the wavelength drive of the monochromator, the synchronous motor of the monochromator and the detection and recording system. Up to TABLE I INSTRUMENTATION AND OPERATING CONDITIONS Plasma power supply . . Plasma torch . . .. Argon flow-rates . . Nebuliser . . .. Optics .. .. Spectrometer . . .. Wavelength selection . . Read-out . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. Radyne, Model R50P, 27 MHz. Output power variable, 0-5 kW, normally operated a t 2.5 kW. Work coil, 3 turns 4 x 4 mm cross-section copper tubing. De-mountable torch; coolant gas tube, fused silica, 28 f 0.6 mm 0.d.; plasma gas tube, fused silica, 23 f 0.5 mm 0.d.; sample gas tube, borosilicate glass, 7 mm 0.d.; jet, 1.5mm i.d.A PTFE tube, 3.5 mm o.d., 3.0 mm i.d., is positioned inside the sample gas tube and is connected to the nebuliser. Coolant, 20.0; plasma, 0.0; sample, 0.55 1 min-l. Ultrasonic nebuliser, based on a published design,1° using a Siemens Sonostat 633 ultrasonic supply, of maximum output 12 W. Plasma is imaged on to the entrance slit of the spectrometer (a distance of 550 mm away) with an optical arrangement of two fused silica lenses. Entrance slit: width 0.02 mm, height 4-18 mm. Rank Hilger Monospek 1000; grating of 1200 lines mm-l blazed at 300 nm, reciprocal linear dispersion 0.82 nm mm-l. The control unit for sequentially selecting and scanning across wavelength regions is described in the text.Signal from photomultiplier tube [RCA, Type IP28 (selected)] , a t a potential of 600V (Brandenberg power supply, Model 475R), is amplified by the internal operational amplifier of the control unit and monitored on a potentiometric chart recorder (Servoscribe, Model RE 54 1.20). Sample uptake rate, 0.2 ml min-l.518 CATTERICK AND HICKMAN : SEQUENTIAL MULTI-ELEMENT ANALYSIS OF Analyst, VOl. 104 eight wavelength regions can be examined using the control unit, and for each wavelength region one of four possible amplifier gain settings can be selected. The stepping motor performs a rapid slewing (278 nm min-l) between each wavelength region. The response of the photomultiplier tube at the exit slit of the monochromator, suitably amplified by the operational amplifier in the control unit, is only monitored during the scan of the synchronous motor and the signal is fed to a chart recorder. The advantage of this system of scanning across a wavelength region is that the background levels on either side of the atomic-emission line are automatically recorded, and thus the need to carry out discrete measurements of background and background plus signal is avoided.Entering data for sequential analysis into the control unit The selected analytical atomic-emission lines are arranged in increasing order of wave- length, together with a reference “Start” position chosen at a convenient value just below the shortest wavelength. The stepping motor for altering the monochromator wavelength drive requires 96 pulses to complete one revolution, corresponding to a 2.5-nm wavelength region. It is thus straightforward to calculate the number of pulses required to advance the monochromator wavelength position from the chosen “Start” position to the selected wavelength regions.In practice, these numbers are then converted into equivalent binary numbers. The binary numbers associated with each of the selected wavelengths are entered sequentially in order of increasing wavelength into the control unit, using a set of 16 toggle switches. The 16-bit number chosen for each wavelength region is stored in a random access memory device (RAM, Sygnetics 82509). The position of each entry into the memory is determined by incrementing the setting of a multi-pole PROGRAM SWITCH.Each 16-bit binary number contains two items of information : (a) The first 14 bits represent the number of pulses required by the stepping motor for slewing the monochromator wavelength drive from the selected “Start” position to a position adjacent to the chosen analytical wavelength. In practice the maximum range from the “Start” position is about 425 nm. (b) The remaining 2 bits represent the amplifier gain selected from one of four possible settings. Logic of the control unit Automatic wavelength drive control. After the necessary combination of up to nine 16-bit binary numbers has been entered into the memory, the following sequence of events is initiated by pressing the START button. The COMPARATOR and COUNTER are cleared and the MEMORY ADDRESS CONTROL enters the second binary number from the MEMORY STORE into the MEMORY BUFFER (the Jirst binary number being the “Start” value).The first 14 bits of this 16-bit binary number are com- pared with the value in the COUNTER. While the COMPARATOR registers a “not equal to” state between the MEMORY BUFFER and the COUNTER, pulses are sent simultaneously from the INTERNAL CLOCK to the STEPPING MOTOR CONTROL and to the COUNTER. When the value in the COUNTER equals the 14-bit binary number in the MEMORY BUFFER the COM- PARATOR sends out an “equal to” signal. This “equal to” signal is sent to the MEMORY ADDRESS CONTROL, the STEPPING MOTOR CONTROL and the SYNCHRONOUS MOTOR CONTROL. This has the effect of stopping the pulses, which were both incrementing the value in the COUNTER, and also activating the stepping motor; the “equal to” signal results in the initiation of the time-controlled scans of the !synchronous motor.The switching of both motors is achieved via their relevant control units operating the appropriate relays (see Fig. 2). The SYNCHRONOUS MOTOR CONTROL uses pulses from the INTERNAL CLOCK to give a synchronous motor scan of fixed duration (about 9 s). At the end of the first scan the scan direction is reversed and a second scan is made across the same wavelength region. A counter in the SYNCHRONOUS MOTOR CONTROL registers when both scans have been completed. A signal is then generated, which is sent to the MEMORY ADDRESS CONTROL. This unit then updates the MEMORY BUFFER with the next 16-bit binary number and the above sequence of events is repeated for each binary number in the sequential program.The COUNTER This is shown schematically in Fig. 2.June, 1979 SMALL FRAGMENTS OF GLASS BY ATOMIC-EMISSION SPECTROMETRY 2 + Bits e 14 Bits -b Memory store (9 x 16-bit Program address control I I I I B e-. 1 Comparator w 4 UP 1 I Down Start L r t I Internal clock - - - Pu Ise generator v I L r Synch. motor Synch. : Relay - +- control motor Drive shaft to grating l-r-7 Step ping 519 r Stepping motor motor : Relay : control Fig. 2. Automatic wavelength drive control. keeps a running tally of how far the stepping motor has advanced from the original “Start” position. Thus, with each subsequent larger binary number (associated with the analytical regions of interest programmed in increasing order of wavelength), the number of pulses necessary to advance from the “old” to the “new” wavelength position is always available within the control unit.At the completion of each synchronous scan sequence the MEMORY ADDRESS CONTROL compares the position allocated in the MEMORY STORE of the current binary number in the MEMORY BUFFER with the position of the multi-pole PROGRAM SWITCH, This switch is left set at the position of the final entry. If the positions of the current binary number in the memory and the multi-pole switch are equal, the MEMORY ADDRESS CONTROL recognises that the sequential analysis program has been completed. A series of signals is then generated from the MEMORY ADDRESS CONTROL and the following actions result : (a) the COMPARATOR is cleared; ( b ) the Jirst 16-bit number in the program (i.e., the “Start” value) is entered into the MEMORY BUFFER; (c) the STEPPING MOTOR CONTROL reverses the direction of the stepping motor; ( d ) the COUNTER is set to count down as it receives pulses via the MEMORY ADDRESS The stepping motor then returns the monochromator to the selected “Start” position, and the control unit is now ready to repeat the programmed sequence of events. Automatic detection and recording system.In addition to the wavelength drive control system, the control unit contains a complementary system for controlling the detection and recording of the analytical signal. This is shown schematically in Fig. 3. CONTROL.520 CATTERICK AND HICKMAN : SEQUENTIAL MULTI-ELEMENT ANALYSIS OF Analyst, Vol.104 2-Bit binary Output from value from comparator memory buffer i output from PM T su p p I y to chart drive Chart recorder Fig. 3. Automatic detection and recording system. The output from the photomultiplier tube (PMT) detector is only switched through to the operational amplifier during the synchronous motor scans. This is achieved by activating a switch (SWITCH 1) with the “equal to” signal from the COMPARATOR. The same signal activates the chart paper drive of the recorder (SWITCH 2). The third part of the detection system that is automatically controlled is the gain selected by the AMPLIFIER CONTROL. This employs the remaining 2 bits of the 16-bit word. in the MEMORY BUFFER that is not involved with the wavelength drive.According to the value of these 2-bit binary numbers one of four pairs of resistors (Rl-R4 in Fig. 3) is selected. The linkage of the resistors to the operational amplifier gives the gain selected as appropriate for each particular wavelength region. The combination of the functions described above provides the necessary controls for the programmed sequential examination of wavelength regions and for selectively recording the signal from the photomultiplier tube during each of the synchronous motor scans. Ultrasonic fiebuliser As the method was designed to accommodate small sample sizes (200-500pg), it was necessary to keep to a minimum the corresponlding volume of dissolved sample, in order to retain reasonable concentrations of the trace elements.The requirement of effecting sequential analysis on a small sample volume led to the selection of an ultrasonic nebuliser, with the relatively low uptake rate of 0.2 ml min-1. The construction of the nebuliser was based on a design by Hoare et aZ.,lO with the modification of adding a PTFE sample transfer tube; this tube was extended to form an inner lining to the borosilicate glass sample injection tube of the plasma torch. The PTFE tube ended just short of the jet on the borosilicate tube. Although this meant that the jet was uinprotected from attack by hydrofluoric acid, in practice no visible signs of etching have been observed. No problems have been encountered with the trace-element concentrations in blank acid solutions run through the system.The problem of the presence of hydrofluoric acid could have been overcome by complexing the excess of hydrofluoric acid prior to analysis; the use of boric acid for this purpose has been described in the literature.1l This procedure was not adopted as it was felt that any additional steps in the sample digestion procedure would increase the risk of contamination and would also increase the sarnlple preparation time. Materials and Reagents Polystyrene tubes (5 ml, Sterilin) were emp1o:yed for the acid digestion of the samples and as containers for the working standard solutions. The tubes were pre-washed with an aqueous solution of hydrofluoric and hydrochloric acids (2HF + HC1 + 9H,O), were rinsedJune, 1979 SMALL FRAGMENTS OF GLASS BY ATOMIC-EMISSION SPECTROMETRY 521 with doubly distilled water and ethanol and dried in an oven at 60 “C.All acids used were of Aristar quality (BDH Chemicals), and elemental standard solutions were commercially available 1 000 pg ml-l solutions (BDH Chemicals or Hopkin & Williams). Procedure Sample preparation The glass fragments (200-500 pg) were cleaned by soaking in concentrated nitric acid for 30min, followed by a triple rinse with doubly distilled water. A final rinse with ethanol preceded drying at 60 “C. Glass samples were weighed on a microbalance (Perkin-Elmer, Model AD-2) before being transferred into the pre-washed 5-ml tubes. A 0.5-ml volume of a mixed acid solution (HF + 2HC1) was added to each sample,6 and these were then agitated for 30 min in an ultrasonic bath (Megason, Model 60-1, Schuco Scientific Ltd.).To the resulting glass solutions were added 1.5 ml of doubly distilled water and 0.5 ml of a 2.0 pg ml-l solution of chromium (as an internal standard) to give a total volume of 2.5 ml. Blanks were prepared in a similar manner. Standards tion ranges are listed in Table 11. samples, using equivalent amounts of the mixed acids and the chromium internal standard. Four multi-element standard solutions were used to calibrate each run, and their concentra- The standards were prepared in a similar manner to the TABLE I1 ANALYTICAL WAVELENGTHS, CONCENTRATION RANGES AND DETECTION LIMITS Element “Start” position Manganese . . Iron . . .. Chromium . . Magnesium A Magnesium B Aluminium . . Barium . . Ionisation Wavelength/ state nm ..249.80 . . I1 257.61 . . 11 259.94 . . I1 267.71 .. 277.98 . . I 285.21 .. I 396.15 . . I1 455.40 Concentration range of standards/ pg ml-1 - 0.007 5-0.045 0.05-0.30 (0.40) 1.0-6.0 1.0-2.0 0.25-1.50 0.007 5-0.045 Concentration Detection limit 0.5 mg of glass glass range for for 0.5 mg of - - 0-225 p.p.m. 10 p.p.m. Internal standard 0-0.15% o.oo5~0 0-3.0y0 0.2% o-1.0yo 0.05% 0-0.75% 0.05% 0-225 p.p.m. 5 p.p.m. Analysis The samples, still in the original 5-ml tubes used for digestion, are connected in turn to the ultrasonic nebulising system. The resulting aerosol is transferred into the plasma by a carrier flow of “sample” argon. After a delay of 25 s to allow equilibrium conditions to become established, and with the monochromator set to the “Start” wavelength, the pro- grammed sequence of scans is initiated at the control unit.The seven analytical wavelengths monitored are listed in Table 11, together with the “Start” wavelength position. Two magnesium emission lines are studied in order to cover the large range of magnesium concentra- tions found in glass samples. The control unit activates the stepping motor, which stops at the first programmed value, about 0.4 nm in front of the emission line. The stepping motor is electrically disengaged by the control unit and the synchronous motor of the mono- chromator is activated. This scans forward slowly (5.0 nm min-1) across a 0.8-nm spectral region. The direction of the synchronous motor is then reversed, and a second traverse across the spectral region is made, resulting in a duplicate record of each analytical feature.When the scan starting position is reached the stepping motor is re-engaged and moves the wavelength drive forward to the second programmed value. The procedure is repeated to cover all seven analytical wavelengths ; the stepping motor then returns the wavelength drive to the original “Start” position. During the scans of the synchronous motor the out- put of the photomultiplier tube, suitably amplified at one of the four gain settings selected in the initial programming, is fed to a chart recorder. The control unit holds the signal to the operational amplifier at earth potential except during the synchronous motor scans.522 CATTERICK AND HICKMAN : SEQUENTIAL MULTI-ELEMENT ANALYSIS OF Analyst, ‘cl‘ol.104 Similarly, the chart-paper drive of the recorder is only activated during the synchronous motor scan periods. Fig. 4 shows a typical chart recorder output from the analysis of a fragment of sheet glass. F C 1‘ il Fig. 4. Chart-recorder trace from the analysis of a 340-pg fragment of sheet glass (refractive index, 1.517 1). Emission lines: A, manganese 257.6nm; B, iron 259.9nm; C, chromium 267.7 nm; D, magnesium 278.0 nm; E, magnesium 285.2 nm; F, aluminium 396.1 nm; and G, barium 455.4 nm. The total analysis time for the programmed sequence of seven wavelength regions is 3 min 30 s; the control unit provides the option of scanning four times (instead of twice) over the emission lines, and using this option the analysis takes 5 min 40 s. A sample of distilled water is nebulised into the plasma in between each sample analysis, during the time when the wavelength drive returns from the highest wavelength to the “Start” position.The peak heights of the blanks, standards and samples are fed into a simple computer program together with the elemental concentrations of the standards and the masses of the glass samples. For each analysis the ratios of the peak heights to the chromium peak height are calculated. Using the data for the blanks and standards a line of best fit is constructed for each element and the ratioed sample peaks are compared with this. A final listing is generated of the samples and their corresportding concentrations of the trace elements measured. Listed in Table I1 are the working concentration ranges in glass (for 500-pg samples) for the elements determined by this method.Also listed are the corresponding detection limits, but these are not necessarily absolute values a s compromise experimental conditions were employed. The values represent the limits attainable under the conditions needed to cover the concentration ranges expected in glass samples. Results and Discussion Analysis of Standard Glasses (Accuracy Check) Six glasses were prepared3 by grinding together mixtures of Specpure (Johnson Matthey) oxides and carbonates and fusing them in platinum crucibles at 1400 “C. The results of three separate analyses of each glass are given in Table 111. The values in parentheses are the levels predicted from the masses of the compounds used in preparing the standard glass.June, 1979 SMALL FRAGMENTS OF GLASS BY ATOMIC-EMISSION SPECTROMETRY 523 TABLE I11 ANALYSIS OF STANDARD GLASSES Sample All % Ba, p.p.m.Fe, p.p.m. Mg, % Mn, p.p.m. A 0.16 (0.13) 23 (17) 1380 (1 890) 0.043 (0.052) 272 (303) B 0.20 (0.18) 50 (49) 1000 (1 220) 0.073 (0.096) 141 (170) D 0.40 (0.50) 95 (100) 380 (450) 1.49 (1.6) 169 (206) E 0.82 (0.81) 126 (134) 260 (290) 1.96 (2.2) 85 (82) F 0.64 (0.85) 576 (657) 90 (80) 3.99 (4.1) 28 (33) C 0.41 (0.51) 85 (80) 740 (870) 1.04 (1.1) 112 (119) Analysis of Sheet Glass (Precision Check) A typical commercially produced sheet glass (refractive index 1.517 1) was analysed a number of times in order to assess the over-all precision of the method. Table IV gives the results of a series of nine determinations made on one day, and a series of 24 determinations made over several weeks, with different operators and standard solutions. Also listed in Table IV are the results of analyses of the same sheet-glass sample by d.c.arc - atomic- emission ~pectrography~ and atomic-absorption spectrometry.6 This method gives better precision for replicate analyses of the sheet glass than it does for an equivalent number of analyses of one of the laboratory-produced standard glasses (coefficients of variation 4-10 yo compared with 8-25%). This is probably a consequence of inhomogeneity in the standard glass. TABLE IV ANALYSIS OF SHEET GLASS Method ICP - AES (within-day) ICP - AES (long-term) D.c. arc - AESB . . AASs . . .. .. Mean Standard deviation No. of determinations C.V., yo Mean Standard deviation No.of determinations C.V., yo Mean Standard deviation No. of determinations C.V., yo . . Mean Standard deviation No. of determinations C.V., yo A], % 0.51 0.049 9.6 9 0.51 0.063 12.2 24 0.45 0.05 11.1 11 Ba, p.p.m. 116 5.1 4.4 9 108 14 13.0 24 114 18 15.8 11 Fe, p.p.m. 560 38 6.7 9 611 86 14.1 24 576 75 13.2 11 5 70 20 10 3.5 Mg. % 1.79 0.069 3.9 9 1.73 0.15 8.5 24 1.70 0.17 10.0 11 2.03 0.041 2.0 10 Mn. p.p.m. 71 5.9 8.3 9 77 11 14.0 24 102 17 16.6 11 84 2.6 3.1 10 Conclusions A method has been described for the sequential quantitative determination of five elements in small samples of glass. The samples are dissolved in a mixture of hydrofluoric acid and hydrochloric acid at ambient temperature.6 Although the standard solutions are matched to the samples with respect to acid concentration, it was found that matching to the sodium, calcium and silica matrix present in a digested glass sample was unnecessary.This is in agreement with the conclusions of other workers12 with inductively coupled plasmas that chemical interferences are insignificant. An ultrasonic nebuliser with an acid-resistant transfer system enables the digested samples to be nebulised without undergoing neutralisa- tion or massive dilution. The use of an internal standard improves the precision of the method. The coefficient of variation for magnesium on replicate analyses of the sheet glass was 8.6% without the internal standard and 3.9% (see Table IV) with the internal standard. This finding is in agreement with that of Hoare and Mostyn,13 who used a very similar ultrasonic nebulising system.524 CATTERICK AND HICKMAN The method works well for samples in the range 200-500 pg, giving relative standard deviations of 4-10%.The variation in relative standard deviation reflects the demands of the analysis; magnesium occurs in glass at levels of several per cent. and is thus easier to determine than manganese, which is present at the 20-200 p.p.m. level. Iron determina- tions will lie somewhere between these two extremes, but may be subject to contamination. An advantage of the method is that it gives absolute levels of the trace elements, compared with methods such as spark-source mass spectrometry and X-ray fluorescence spectrometry, in which the results are normally expressed as ratios. For the purposes of data interpretation it is more satisfactory to use absolute trace-element values. A novel feature of the method is the wavelength-drive control unit, which enables a sample to be analysed for several elements sequentially, at a lower cost than a direct-read spectrometer. The unit also provides a greater degree of flexibility than a polychromator system as it can be re- programmed for a new set of elements in a few minutes. The authors thank Dr. A. G. Knapp and M:r. J. Russell for their help in the design and construction of the sequential analysis control unit. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. References Goode, G. C., Wood, G., Brooke, N., and Coleman, R. F., A.W.R.E. Re$., No. 024/71, 1971. German, B., and Scaplehorn, A. W., J. Forens. Sci. SOC., 1972, 12, 67. Blacklock, E. C., Rogers, A., Wall, C., and Wheals, B. B., Forens. Sci., 1976, 7, 121. Sneath, P. H. A., and Sokal, R. R., “Numerical Taxonomy,” Freeman, San Francisco, 1973, p. 5. Hughes, J. C., Catterick, T., and Southeard, G., Forens. Sci., 1976, 8, 217. Catterick, T., and Wall, C. D., Talanta, 1978, 25, 573. Sayre, E. V., and Smith, R. W., in Bishay, A., Editor, “Recent Advances in the Science and Tech- Harvey, C. E., J . Forens. Sci., 1968, 13, 269. Reeve, V., Mathiesen, J., and Fong, W., J. Forms. Sci., 1976, 21, 291. Hoare, H. C., Mostyn, R. A., and Newland, B. ‘r. N., Analytica Chim. Ada, 1968, 40, 181. Price, W. J., and Whiteside, P. J., Analyst, 197’7, 102, 664. Fassel, V. A., and Kniseley, R. N., Analyt. Chem., 1974, 46, lllOA and 1155A. Hoare, H. C., and Mostyn, R. A., Analyt. Chem., 1967, 39, 1153. nology of Materials,’’ Volume 3, Plenum Press, New York, 1974, pp. 47-70. Received December 18th, 1978 Accepted January 15th, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790400516

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, June, 1979, Vol. 104, pp. 525-530 525 Spectrophotometric Determination of Trace Amounts of Free Cyanide in Prussian Blue G. J. Willekens and A. Van Den Bulcke Instituut VOOY Hygiene en Epidemiologie, Departement Farmatoxicologie, A fdeling Farmampee- en Standaardenonderzoek, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium Trace amounts of free cyanide in Prussian blue are hydrolysed into hydro- cyanic acid. The latter is captured by a lithium picrate solution contained in a test-tube, which is placed in the reaction vessel. The colour change due to the resulting lithium isopurpurate is measured spectrophotometrically at 500 nm. This method can detect cyanide down to a level of 2.5 pg in 100 mg of Prussian blue and is accurate and reproducible. Keywords ; Cyanide determination ; insoluble and colloidal Prussian blue ; spectrop hotowetry Prussian blue is used as an antidote for metal intoxications such as those due to thalliumJ1s2 radioactive strontium3 and caesium.475 As the Prussian blue is given in daily doses of up to 20g6-* and generally over a 14-d period, the purity of Prussian blue is most important.Because of the extreme toxicity of cyanides their detection and determination in small amounts are of great importance. A great variety of methods are available for the identifica- tion of cyanides. Of these, spectrophotometry is the most convenient for determining small amounts. Prussian blue exists in either an insoluble or a colloidal form, the colloidal form being the so-called soluble formJg which is the most active antidote for thallium poisoning6910p11 owing to its small particle size (down to 0.03 pm).Because of the small particle size extraction of adulterants by solvents followed by filtration or centrifugation is impracticable. The free cyanides can, however, be converted into hydrocyanic acid, which diffuses as a gas to react with a suitable colorimetric reagent, either in solution or absorbed on a filter-paper. Distillation by heating an acidic mixture of the sample to give the hydrocyanic acid is not suitable for routine quantitative determinations as it is time consuming and requires complex apparatus. It may also give inaccurate results when small amounts of alkali metal hexacyanoferrate(I1) and hexacyanoferrate(II1) are present in the sample, as they break down into hydrocyanic acid.Hence, we focused our research on the detection and quantita- tive determination of the hydrocyanic acid evolved at 20 "C. From preliminary tests it appeared that a compromise between pH and time had to be found, which would result in a slow evolution of hydrocyanic acid by hydrolysis, with no addition of proton donors other than water. The choice of the reagent for detection was limited. Although procedures based on the Konig synthesis12-16 are generally considered satisfactory for determining small amounts of cyanide, the colour developed is not sufficiently stable. Alkaline picrates combine with hydrogen cyanide to give isopurpurates,l7 which are specific and stable colour reactions and the possibilities of using these reactions were examined.NO2 Picric acid Lithiu rn isopurpurate Attempts to use squares of filter-paper soaked with the reagent to react with the hydro- cyanic acid liberated from the test solution, by heating with sodium hydrogen carbonate526 WILLEKENS AND VAN DEN BULCI'E SPECTROPHOTOMETRIC AnaZyst, "ol. 104 and a slight excess of acid,l8 were unsuccessful as there was no significant change in the colour intensity with increase in the amount of hydrocyanic acid. Other tests such as the copper acetate - benzicline test,lS the test depending on the fonna- tion of Prussian blue,20 the copper sulphide test21 and the guaya resin test,22 although sensitive, proved to be unsatisfactory for the satme reason. The method finally developed used a simple gas-testing device (Fig.1). A small flat-bottomed test-tube of approximately 2-ml capacity containing lithium picrate solution was placed in a glass-stoppered flask, which contained the sample of Prussian blue to which a definite amount of water had been added. The flask was stoppered immediately, protected from the light and allowed to stand over- night at 20 O C , after which the absorbance of the solution within the test-tube was measured against a blank prepared under the same conditions. The procedure, described below, easily detected 2.5 pg of cyanide added to 100 mg of Prussian blue (Table I). Experimental Reagents Lithium picrate solution. Dissolve 0.25 g of lithium carbonate and 0.5 g of picric acid in 80 ml of boiling distilled water. Cyanide standard solution. Dissolve 100 mg of potassium cyanide in 1000 ml of distilled water and dilute 5 ml of this solution to 100 ml.Prussian blue test sample (colloidal and insolu:ble). Obtained from Hopkin & Williams, Chadwell Heath, Essex. Allow to cool aind dilute to 100 ml. This solution is to be used within 12 h. Standard Additions Procedure Weigh 100-mg samples of the Prussian blue under test into each of five glass-stoppered, wide-necked, 200-ml flasks (Fig. 1). Add to four of them, respectively, 0.5, 1, 2 and 3 ml of the cyanide standard solution and sufficient distilled water to produce 5ml. To the fifth flask, used as a blank, add 5 ml of water. S'hake the flasks gently. Accurately pipette 1 ml of the lithium picrate solution into five small, flat-bottomed test-tubes and introduce each of them into one of the conical flasks and stopper the flasks immediately.Allow the flasks to stand overnight, protected from light, at 20 "C. Lithium picra , Prussian blue and cyanide ,te Fig. 1. Gas-testing device. Add to the contents of the flat-bottomed test-tubes 2 ml of the lithium picrate solution, To construct the calibration graph, plot the absorbances veysus the corresponding amounts mix and measure the absorbance in 1-cm cells against the blank at 500 nm. of cyanide added to the Prussian blue solutions and draw the regression line. Results and Discussion Neither the insoluble nor the colloidal (soluble) form of Prussian blue can be totally removed by filtration. Filtration of the colloidal solution, even through a micro-crystalline filter-paper (Whatman No.42) yielded a pale blue filtrate. Therefore, distillation of theJune, 1979 DETERMINATION OF TRACE AMOUNTS OF FREE CYANIDE IN PRUSSIAN BLUE 527 hydrocyanic acid was needed and although this distillation required a 24-h period the effective work for one determination was only 30min. The reaction was carried out at 20 "C with protection from the light. The recovery of different amounts of cyanide added to 100 mg of insoluble and colloidal Prussian blue was evaluated spectrophotometrically at 500 nm (Fig. 2). 240 3 20 400 4 80 5 60 Wave le ngt h/nm Fig. 2. Ultraviolet absorption spectra in water : solid line, lithium picrate; and broken line, lithium isopurpurate. Plots of the absorption values veysus the corresponding amounts of cyanide gave straight lines with a significant correlation coefficient of 0.9999 for both the insoluble and colloidal forms.Only the graph for insoluble Prussian blue passed through the origin, which means that this Prussian blue released no cyanide at 20 "C. The colloidal form lost approximately 0.7 pg per 100 mg of Prussian blue. This cyanide release was calculated from the total absorbance measured for each mixture and the deviation of the calibration graph on the y-axis. Subtraction of the related values yielded the cyanide release (Table I). The confidence limits (95% confidence interval) of the mean cyanide recoveries are given by x (o/dT x t*), where o/d< is the standard error, o the standard deviation, rz the number of measurements and t* is 1.96. The recovery errors were between -3.4% and +0.9%.These values are listed in Table I and indicate the acceptable accuracy of the spectrophotometric method using lithium picrate as a reagent. The results are listed in Table I. The calibration graphs (Fig. 3) at different temperatures show good linearity except for the colloidal form at 50 "C. The recovery errors were between -4.4% and +4% at 30 "C and between -4.6% and +11.6% at 40 "C. Thus, change of temperature had no significant influence on the recovery of cyanide added to 100mg of Prussian blue. The increase in temperature , however, increased exponentially the cyanide release of Prussian blue itself. The insoluble form released no cyanide at 20 "C when no cyanide was added. At 30 "C the release was 1.36 pg, at 40 "C 3.35 pg and at 50 "C 9.63 pg.For the colloidal form this cyanide release increased to a considerable amount , which was three times higher than for the insoluble form. Values of 0.61, 2.89, 8.67 and 22.56 pg were found at the different temperatures, respectively. Addition of cyanide, on the other hand, also increased this cyanide release and the greater the cyanide addition the greater was the cyanide release of Prussian blue itself, especially The influence of temperature on the recovery of the added cyanide was determined.528 WILLEKENS AND VAN DEN BULCKE: SPECTROPHOTOMETRIC Analyst, VoZ. 104 TABLE. I RECOVERY OF CYANIDE ADDED TO PRUSSIAN BLUE AND INFLUENCE OF TEMPERATURE ON THAT RECOVERY AND THE RELEASE OF CYANIDE BY PRUSSIAN BLUE Form of TemDeraturel Prussian blue Cvanide "C 20* 30t 40t Colloidal Insoluble Colloidal Insoluble Colloidal Insoluble Colloidal aidedlpg 0.00 2.50 5.00 10.00 15.00 0.00 2.50 5.00 10.00 15.00 0.00 2.50 5.00 10.00 15.00 0.00 2.50 5.00 10.00 15.00 0.00 2.50 5.00 10.00 15.00 0.00 2.50 5.00 10.00 15.00 0.00 2.50 5.00 10.00 15.00 0.00 2.50 5.00 10.00 15.00 Cyanide recovered/ P*.g 0.00 2.49 4.83 10.02 15.05 0.00 2.43 4.97 10.09 14.96 0.05 2.60 4.86 9.93 15.08 0.09 2.39 4.88 10.22 14.91 0.12 2.59 4.94 9.54 15.31 -0.18: 2.79 4.77 10.24 14.86 - 0.093 2.54 5.63 8.89 15.52 2.92 8.48 10.58 13.38 - 2.863 Cyanide Confidence limits released by Recoverv.& Prussian blue/ % - 99.8 96.6 100.2 100.3 97.3 99.4 100.9 99.7 104.0 98.8 99.0 100.5 95.6 97.6 102.2 99.4 103.6 98.8 95.4 102.1 111.6 95.4 102.4 99.0 101.6 112.6 88.9 103.4 116.8 169.6 105.8 89.2 - - - - - - - .I, Maximum Minimum Pt4 0.00 0.09 0.06 0.00 0.61 0.63 0.69 0.78 0.77 1.36 1.29 1.29 1.32 1.34 2.89 2.97 3.05 3.17 3.36 3.35 3.58 3.79 4.23 4.76 8.67 8.69 8.75 8.74 9.77 9.63 9.75 9.89 10.04 10.35 22.56 23.38 24.12 24.40 24.73 - * Each result is the mean of ten determinations. t Each result is the mean of three determinations.No confidence limits were calculated. These results were determined by calculation of the regression lines. for colloidal Prussian blue. The cyanide release of the insoluble form increased to a lesser extent. The influence of temperature, light, reaction time, acid and mass of sample on the release of cyanide from Prussian blue in the absence of cyanide was also investigated. The results are listed in Tables I1 and 111.Under the influence of light, and especially sunlight, there was an uncontrollable cyanide release. After an exposure of 24 h of the reaction vessel to daylight we found a cyanide release of 35 pg pler 100 mg of colloidal Prussian blue. The insoluble form released 3.85 pg per 100 mg. Even in the dark at 20 "C, 100 mg of colloidal Prussian blue lost 0.72 pg of cyanide. Under the influence of 0.06 N hydrochloric acid. at 37 "C, 100 mg of inSoluble Prussian blue released no cyanide. The colloidal form, however, released 1.90 pg. This value is notJzwze, 1979 DETERMINATION OF TRACE AMOUNTS OF FREE CYANIDE IN PRUSSIAN BLUE 529 0.800 0.700 r- 0.600 a, 0.500 0 2 2 a & 0.400 0.300 0.200 0.100 0 5 10 15 20 Cyanide concentration/pg Fig.3. Calibration graphs for the detec- tion of cyanide added to a 100mg per 5 ml of Prussian blue solution at different temperatures : 0, insoluble state ; and A, colloidal state. similar to that reported by Kamerbeek,' who incubated colloidal Prussian blue for 4 h with gastric juice (pH 2) and 0.1 N hydrochloric acid in the presence of oxygen and could not detect any release of cyanide. The mass of Prussian blue did not influence the cyanide release. Conclusion The spectrophotometric determination of cyanide by use of a lithium picrate solution is a simple method, which is based on the distillation of hydrocyanic acid and is used in all instances where interfering substances may hamper direct detection of cyanide. The proposed method is a standard additions procedure and permits the simple detection of cyanide down to a level of 2.5 pg.TABLE I1 CYANIDE RELEASED BY COLLOIDAL PRUSSIAN BLUE IN WATER UNDER DIFFERENT CONDITIONS The results given are the amounts of cyanide released, in micrograms; each value is the mean of three determinations. Mass of Prussian bluelmg 7 Temperaturel'C Acid Time/h Daylight 50 20 - 24 + 8.0 0.3 24 20 1.4 30 - 24 1.4 37 0.06 N HCI 2 4.4 40 - 24 50 - 24 - 19.1 - - - - - 100 150 200 250 16.2 18.2 29.1 35.4 0.7 1.4 2.2 2.8 2.9 4.6 5.7 7.8 1.9 1.9 2.0 3.0 8.4 12.1 17.1 30.0 23.6 30.8 36.3 47.2530 WILLEKENS AND VAN DEN BULCKE TABLE I11 CYANIDE RELEASED BY INSOLUB:LE PRUSSIAN BLUE IN WATER UNDER DIFFERENT CONDITIONS The results given are the amounts of cyanide released, in micrograms; each value is the mean of three determinations. Mass of Prussian blue/mg I A Temperaturel’C Acid Time/h Daylight 50 100 150 200 20 - 24 + 2.6 3.9 4.3 4.2 20 - 24 - 0.05 0.09 0.05 0.09 24 - 0.6 1.0 1.4 1.5 30 37 0.06 N HCl 2 40 - 24 - 1.8 3.1 3.9 5.4 50 - 24 - 5.9 10.8 14.4 17.4 - - - - - - 7 250 4.3 0.07 2.0 1.4 7.8 18.2 The authors thank Prof.Dr. Apr. van Peteghem (Rijksuniversiteit Gent) for literature communications and Mr. Legrand (I.H.E., Brussels) for statistical analysis. They are obliged to Mr. J. W. Lightbown of the National Institute for Biological Standards and Control, Hampstead, London, for assistance with the manuscript. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. References Dvorak, P., Naunyn-Schmiedebergs Arch. Ex*. Ptzth.Pharmak., 1971, 269, 48. Stevens, W., Van Peteghem, C., Heyndrickx, A, and Barbier, F., Int. J . Clin. Phavmac. Thev. Toxic., 1974, 10, 1. Borisov, V. P., I l k , L. A., Kendysh, M. M., Shomorokhova, T. N., Mikhailovich, S. M., and Seletskaya, L. I., Hlth Phys. Probl. Int. Contaiw. Proc. IRPA (Int. Rudiat. Pvot. Ass.), 2nd Eur. Congr. Radiat. Prot., 1972; Chem. Abstr., 1973, 79, 143311. Havlicek, F., Int. J. Appl. Radiat. Isot., 1968, 19, 487. Madshus, K., and Stromme, A., 2. Naturj., 1968, 236, 391. Kamerbeek, H. H., Rauws, A. G., Ten Ham, M., and Van Heyst, A. N. P., Acta Med. Scand., 1971, Kamerbeek, H. H., “Therapeutic Problems in Thallium Poisoning,” Proefschrift Utrecht, Gianotten, Rauws, A. G., Kamerbeek, H. H., and Ten Ham, Id., “Verslag 130e Wetenschappelijke Vergadering,” Pascal, P., “Trait6 de Chimie MinCrale,” Masson, Paris, 1933, p. 862. Dvorak, P., 2. Ges. Exp. Med., 1960, 151, 89. Dvorak, P., Arzneimittel-Forsch., 1970, 20, 1886. Konig, W., J. Prakt. Chem., 1904, 69, 105. Konig, W., 2. Angew. Chem., 1905, 70, 115. Aldridge, W. N., Analyst, 1944, 69, 262. Aldridge, W. N., Analyst, 1945, 70, 474. Epstein, J., Analyt. Chem., 1947, 19, 272. Auterhoff, H., and Boehme, K., Arch. Pharm., Bed., 1968, 301, 793. Schwapowalenko, A. M., Chem. ZentBl., 1930, 11, 588. Moir, J., Chem. News, Lond., 1910, 102, 17. Vichoever, A., and Johns, C. O., J . Am. Chem. SOG., 1915, 37, 601. Barnebey, 0. L., J . Am. Chem. Soc., 1914, 36, 10!)2. Prodanov, P., Izv. Khim. Inst. Bulg. Akad. Nauk, 1964, 10, 277. 189, 321. Tilburg, 1974. R.I.V., Utrecht, Bilthoven, 1973. Received September 12th, 1978 Accepted December 28th, 1978

ISSN:0003-2654    

DOI:10.1039/AN9790400525

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, June, 1979, Vol. 104, pp. 531-537 531 Polarography of Green S F. E. Powell Department of Science and Food Technology, Grimsby College of Technology, Hurnberside, DN34 5BQ N u n s Corner, Grimsby, South The food dye Green S, 4- [4-dimethylammoniocyclohexa-2,5-dienylidene- (4-dimethylaminophenyl)methyl]-3-hydroxynaphthalene-2,7-disulphonic acid, monosodium salt, is reduced a t the dropping-mercury electrode from 50% ethanolic solutions with the total consumption of two electrons. Polarograms follow theoretical predictions in the pH range 2.7-8.75. The reduction mechanism involves two electron transfer steps that are sufficiently differenti- ated at higher pH for separate waves to appear. Keywords : Green S ; food dye ; polarograplay Carbonium ions of the triarylmethane type are reducible at the dropping-mercury electrode (D.M.E.), and the parent species, triphenylmethane, has been studied in various media.For example, in liquid sulphur dioxide, the cation undergoes single-electron reduction to the radical, which dimerisesl : .. . . - - (1) #&+ + e + . . .. .. .. - * (2) 2$3C' -+ dimer .. In protonating solvents, a second reduction step is possible : Thus, in methanesulphonic acid2 and sulphuric acid3 media, this step is in competition with the dimerisation. Dyes based on this structure have also been examined polarographically.4-7 The methane carbon atom is the electroactive centre of these molecules also and reduction occurs through the steps described above. Variety of behaviour is afforded by the nature of the attached aryl groups.Strongly electron-attracting aryl groups lower the electron density of the electroactive centre, which is then readily reduced in a two-electron wave. However, two distinct single-electron waves are observed with strongly donating aryl groups. The mechanism is also sensitive to acid - base eq~ilibria.~ Within this framework, a study has been made of the dye Green S (C.I. 44090), the mono- sodium salt of 4-[4-dimethylammoniocyclohexa-2,5-dieny~dene-(4-~methylaminophenyl)- methyl]-3-hydroxynaphthalene-2,7-disulphonic acid. As the dye is a permitted food colour, the information so obtained should provide a rational basis for the design of polarographic procedures for its analytical determination in foodstuffs. Experimental For polarography, 50% aqueous ethanol solvent was used to eliminate possible adsorption effects.4 The ethanol was of analytical-reagent grade and was free from impurity waves.Conventional McIlvaine and Clark - Lubs buffer components were used and the solutions were made up to a consistent ionic strength of 0.15 by the addition of potassium chloride when necessarya8 Solutions also contained 0.01 yo of methylcellulose as maximum suppressor ; protein surfactants were avoided because of the possibility of binding with the dyestuff .g Solutions were filtered and well de-aerated before polarography. A water-jacketed electrochemical cell was employed, consisting of a working D.M.E., auxiliary electrode of area 19.6 cm2 and a saturated calomel electrode (S.C.E.) as reference. Polarograms were recorded with a Heath EUW-401 polarograph used in conjunction with a Servoscribe RE51 1 recorder and EIL 38B external millivoltmeter.Data for logarithmic A commercial sample of Green S was recrystallised from ethanol in low yield.532 POWELL : POLAROGRAPHY Analyst, Vol. 104 analysis of the waves were obtained manually. Corrections of the faradaic currents for residual current and mercury column heads (h) for the back pressure were made. Plateau currents were compared at the h value of 61.!)cm, where the mercury flow-rate, m, was 1.62 mg s-l. Drop times (t) were determined indirectly from the current versus time traces displayed on a Telequipment S51 B oscilloscope, direct visual observation being impossible because the dye absorbs strongly in the visible region at 635nm.In all solutions t was 2.9-3.0 s in the potential range of interest. All measurements were made at 25 & 0.2 "C. Results and Discussion The dye is reduced in a single wave up to pH 7.3, where wave splitting becomes discernible. At pH 8.75 the division has developed to an extent that distinct waves can be recognised. Representative polarograms are shown in Fig. 1. Fig. 1. Representative polarograms. A, pH 4.2, E , = -0.600 V, depolariser concentration c = 1.17 x C, pH 7.3, E , = -0.754V, c = 1.03 x m; and D, pH 8.75, E+ (first wave) = -0.'700 V, E , (second wave) = 1 0 - 3 ~ ; B, PH 5.7, E , = -omov, c = 1.16 x 1 0 - 3 ~ ; -0.932 v, G = 1.13 X M. Limiting Currents Well defined plateau regions were observed on polarograms of the dye in each of the buffer solutions.A linear dependence of the mean limiting current (iL) on depolariser concentra- tion (C) at six values between 0.2 and 1.2 mol EL-^ was established in each instance. These lines passed through the origin and their respective gradients ( i L / C ) are given in Table I. Further, iL was found to be directly proportion,al to h* when the mercury head was varied between 32 and 62 cm (Table I). These results show that limiting currents are diffusion con trolled. TABLE I EXPERIMENTAL RESULTS ir./C/ Mean deviation I l El4 PH w-4 m3 mol-1 of iLh-f,% WA m* mol-1 mg-P s* n v vs. S.C.E. 2.7 2.04 0.4 1.23 1.9 -0.495 3.7 1.98 1.1 1.20 1.9 -0.582 4.2 1.94 1.1 ~. ~ 4.6 2.00 1.7 5.7 2.06 0.9 6.3 2.02 0.4 7.3 2.20 0.9 7.95 2.06 1.1 8.75 1.10 0.8 (1st wave) (total wave) 2.18 1.2 * 1st wave.f 2nd wave, 1.17 1.21 1.24 1.22 1.33 1.24 0.66 1.32 1.8 -0.600 1.9 -0.617 1.9 -0.690 1.9 -0.724 2.1 -0.754 1.9 -0.706* - 0.8741 1.0 -0.700* 2.0 -0.932t E F / dEt/dPH/ 0: Vvs. S.C.E. mV 0.35 +o.oin - 0.37 -0.291 -87 0.37 -0.350 -36 0.37 -0.410 -42.5 - - - - - - - - 0.874 - 1.00 - 0 0.76 -0.932 -73 P 2.02 0.99 - 0.85 - - - - - 0 0.94June, 1979 OF GREEN S 533 The diffusion current constant ( I ) , viz., iLICmW, of the total wave was found to be essentially uniform over the pH range studied (Table I), indicating a common stoicheiometry for the reduction process. According to the IlkoviE equation, this term can be identified with the product 607nD*, where n is the number of electrons involved in the reduction process. The adoption of a diffusion coefficient (D) value of 1.14 x 10- cm2 s-l, as deter- mined for the related dye Brilliant Green in 50% ethanol by a porous diaphragm m e t h ~ d , ~ produces the n values shown in Table I.Evidently, the complete reduction is a two-electron process, but at pH 8.75 the reduction takes place in two single-electron steps, each of which is diffusion controlled. Attempts to extend the range to higher pH values resulted in colourless solutions, probably owing to the formation of the electroinactive carbinol.6 Experimental Wave Forms At pH 8.75 the difference between half-wave potentials (E,) of the split waves exceeds 200 mV, permitting separate logarithmic analysis (Fig. 2) ; from the slopes of these plots it appears that the first wave corresponds to a reversible single-electron transfer but that the second electron is taken up irreversibly in the subsequent wave with a transfer coefficient (a) of 0.76.Comparison of the E* values at pH 8.75 and pH 7.95 (Table I) shows that the second but not the first wave is pH dependent. The number of protons (9) involved in the rate-determining step of the more negative irreversible process evaluated fromlO suggests the uptake of a single proton (Table I). Fig. 2. at pH 8.75. and ( b ) , second wave slope 79 mV. Logarithmic analysis of waves (a), First wave slope 59 mV; Hence, the first wave can be ascribed to process (1) and the second to process (3). Dimerisa- tion of the free radical does not take place, as the difference between E, for the separate waves does not change with depolariser concentration.ll On decreasing the pH, the half-wave potential of the irreversible wave is displaced to more positive potentials, overtaking that of the reversible wave, resulting in a merger.The appropriate logarithmic analyses are shown in Figs. 3 and 5 and the data are given in Table I.534 POWELL : POLAROGRAPHY Analyst, Vol. 104 Theoretical Wave Forms Mizutani et aZ.12 have presented a theoretical treatment of the reduction scheme below for an expanding plane electrode : O + e + Z 2 + e + R (heterogeneous rate constant = k ) This treatment can be readily adapted to the proposed mechanism for Green S with the recognition that here k is a pseudo-first-order rate constant dependent on the hydrogen-ion concentration : k = k, [H-t]' . . .... .. * * (5) p protons being involved in the transition state of the rate-determining step and k , is the true heterogeneous rate constant. Provided that the half-wave potential of the irreversible step, E r , is sufficiently more positive than that of the first, reversible step, E r , then a fused wave results, with i.e., the logarithmic plot is linear. pH 4.6. taken as 0.4 in this pH range. This situation arises in the reduction of Green S up to From the experimental slopes (Fig. 3), the a value of the irreversible step can be Further, the composite E t can be written as12 -1.0 -0.5 0 0.5 1.0 1.5 I L -- I Fig. 3. Logarithmic analysis of experimental . . Log10 7- waves in pH range 2.7-6.3. Hence, E$' values can be calculated in this pH range, utilising the experimental ments and taking EY as -0.700 V, as established at pH 8.75.measure- Differentiating equation (7) with respect to pH and utilising equation (4) givesJune, 1979 OF GREEN S 536 from which $ can be calculated (Table I). Of course, identical results are obtained by direct application of equation (4) to the calculated E r values of the second step. At pH 4.6 and 4.2 a single proton is involved in the rate-determining step of the irreversible process and the over-all reduction mechanism proposed in alkaline solution remains applicable. However, at pH 3.7 and below, a second proton is consumed, presumably in the protonation of an amine group, whose basicity in the intermediate free radical is enhanced over that in the parent carbonium ion because of a reduction in the positive charge density.Mizutani et al. derived the current - potential relation of the consecutive electron transfer (ee) mechanism in closed forrn [equation (19) in their paper12]. This relationship was employed to explore the evolution of the waveforms with pH on a theoretical basis, utilising the following parameter values as guided by the experimental results: D = 10-6cm2s-1, t = 3s, a = 0.4, E r = -0.30 V, Er = -0.70V and a total mean limiting current of 2 PA. The experimental curve found at pH 3.7 could be reconstructed theoretically with close agreement with the observed E+ and logarithmic slope, by assuming k = 6.5 x cm s-l (Fig. 4). I I I I -1.0 -0.5 0 0.5 1 .O - . Log,o - Fig. 4. Logarithmic analysis of theoretical waves in pH range 2.7-6.3.A, pH = 2.7, EF = 0 V, slope = 42 mV; B, pH = 3.7, Eim = -0.3 V, slope = 43 mV; C, pH = 4.3, Ef" = -0.4 V, slope = 42 mV; D, pH = 5.65, Eim = -0.6 V; and E, pH = 6.3, EP = -0.7 V. Theoretical curves at other acidic pH values were generated by duplicate calculations modifying only the parameter E r in accordance with equation (4) and taking p = 2 between pH 2.7 and 3.7 and j5 = 1 elsewhere. This variation in Ep contains implicitly the necessary change in k with pH, as the parameters are related bylo dE$' 59 d loglok (mvl - = - x - .. dpH o! dpH .. .. (9) If required, the inferred k at each pH can be obtained from the basis value at pH 3.7 using equation (5). The self-consistent current - potential curves so obtained are plotted in logarithmic form at 10-mV intervals in Fig.4. On comparison with the experimental data536 POWELL POLAROGRAPHY Analyst, Vol. 104 in Fig. 3, good agreement is observed between slopes and operational E+ values at low pH. Also, the development of a two-segment logarithmic plot with increasing pH is reproduced. In alkaline solutions, the larger a value foundl indicates facilitation of the transfer of the second electron. Consequently, in the theoretilcal calculation at pH 7.3, a has been given the value 0.7, and E f extended to -0.80V in accordance with equation (4) with a in transition from 0.4 to 0.7. These changes again permit a close fit between the theoretical and experimental waveforms (Fig. 5 ) . -1.0 -0.5 0 0.5 1 .o Fig. 5. Logarithmic analysis of wave at pH 7.3. Open circles denote experimental results, full line represents Mizutani et al.relation, with parameter values as in text. Conclusion The following reduction scheme forms the foundation of a consistent theoretical interpreta- tion of the polarographic behaviour of Green S in the pH range of interest:June, 1979 OF GREEN S 537 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. References Elving, P. J., and Markowitz, J. M., J . Phys. Chem., 1961, 65, 686. Wawzonek, S., Berkey, R., and Thomson, D., J . Electrochem. Soc., 1956, 103, 513. Plesch, P. H., and SestAkova, I., J . Chem. SOC. (B), 1970, 87. Kaye, R. 0.. and Stonehill, H. I., J . Ckern. Soc., 1952, 3231. Ramaiah, N. A., and Katiyar, S. S., Curr. Sci., 1961, 30, 175. Kemula, W., and Axt-Zak, A., Roczn. Chem., 1962, 36, 737; 1963, 37, 113. Bengtsson, G., Acta Chem. Scand., 1966, 20, 1176; 1967, 21, 1138 and 2544; 1968, 22, 1241; 1969, Elving, P. J., Markowitz, J. M., and Rosenthal, I., Analyt. Chem., 1956, 28, 1179. Gurr, F., “Synthetic Dyes in Biology, Medicine and Chemistry,” Academic Press, New York, 1971, Elving, P. J., Pure Appl. Chem., 1963, 7 , 423. Pozdeeva, A. A., and Zhandov, S. I., PYOG. 3rd Int. Polarogr. Congr., Southampton, 1964, 2, 781. Mizutani, F., Sato, N., and Sekine, T., Denki Kagaku, 1978, 46, 247. 23, 435, 448 and 455. p. 404. Received December 7th, 1978 Accepted January 19th, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790400531

出版商:RSC    

年代:1979

数据来源: RSC