摘要:

THE ANALYSTTHE ANALYTICAL JOURNAL OF THE CHEMICAL SOCIETYEDITORIAL ADVISORY BOARD"Chairman: H. J. Cluley (Wembley)"L. S. Bark (Salford)R. Belcher (Birmingham)L. J. Bellamy, C.B.E. (Waltham Abbey)L. S. Birks (U.S.A.)E. Bishop (Exeter)L. R. P. Butler (South Africa)E. A. M. F. Dahmen (The Netherlands)A. C. Docherty (Billingham)D. Dyrssen (Sweden)J. Hoste (Belgium)H. M. N. H. Irving (Leeds)M. T. Kelley (U.S.A.)W. Kemula (Poland)*G. F. Kirkbright (London)G. W. C. Milner (Harwell)"W. T. Elwell (Birmingham)"J. A. Huntor (Edinburgh)G. H. Morrison (U.S.A.)H. W. Nurnberg (West Germany)'J. M. Ottaway (Glasgow)"G. E. Penketh (Billingham)"T. B. Pierce (Harwell)E. Pungor (Hungary)D. I. Rees (London)"R. Sawyer (London)P. H. Scholes (Sheffield)"W.H. C. Shaw (Greenford)S. Siggia (U.S.A.)A. A. Smales, O.B.E. (Harwell)"A. Townshend (Birmingham)A. Walsh (Australia)T. S. West (Aberdeen)A. L. Wilson (Medmenharn)P. Zuman (U.S.A.)*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 CISE, Casella Postale 3986, 201 00 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 29, W. GERMANY.Professor W. A. E. McBryde, Dean of Faculty of Science, University of Waterloo, Waterloo, Ontario,Dr.W. Wayne Meinke, KMS Fusion Inc., 3941 Research Park Drive, P.O. Box 1567, Ann Arbor,Dr. 1. Rubeika, Geological Survey of Czechoslovakia, Kostelni 26, Praha 7, CZECHOSLOVAKIA.Dr. J . RGFicka, Chemistry Department A, Technical University of Denmark, 2800 Lyngby, DENMARK.Professor K. Saito, Department of Chemistry, Tohoku University, Sendai, JAPAN.Dr. A. Strasheim, National Physical Research Laboratory, P.O. Box 395, Pretoria, SOUTH AFRICAB ruxelles, BE LG I U M.CANADA.Mich. 481 06, U.S.A.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 103 No 1230 September 1978@ The Chemical Society 197

ISSN:0003-2654    

DOI:10.1039/AN97803FX033

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

ANALAO 103 (I 230) 897-1 008 (1 978)I SS N 0003-2654September 1978THE ANALYSTTHE ANALYTICAL JOURNAL OF THE CHEMICAL SOCIETYCONTENTS897 A Highly Sensitive Flow-through Phototransducer f o r Unsegmented Continuous-flowAnalysis Demonstrating High-speed Spectrophotometry a t the Parts per 109Level and a New Method o f Refractometric Determinations-D. Betteridge, E. LmDagless, B. Fields and N. F. GravesDetermination of Lead in Irons and Steels by Atomic-absorption Spectrophotometryw i t h the Introduction o f Solid Samples into an Induction Furnace-David GodfreyAndrews, Abdullah M. Aziz-Alrahman and James €3. Headridge91 6 Measurement o f Silver i n Blood by Atomic-absorption Spectrophotometry Using theMicro-cup Technique-Catherine Howlett and Andrew Taylor921 Determination of Copper i n Plasma Ultrafiltrate by Atomic-absorption Spectro-metry Using Carbon Furnace Atomisation-H.Kamel, J. Teape, D. H. Brown, J. M.Ottaway and W. E. SmithTechnique f o r the Determination o f Fluorescence Quantum Efficiencies: a MethodAvoiding Direct Measurement o f Absorbance-Adam Britten, John Archer-Hall andGeoffrey Lockwood909928937 Some Fluorescent Derivatives of the Drug Phenelzine--5. Caddy and A. H. Stead950 Polarographic Determination o f Folic Acid in Tablets Containing Iron(l1) Sulphate-L. R6zaiiski955 Extraction and Determination of Polyoxyethylene Alkyf Ether Non-ionic Surfactantsi n Water a t Trace Levels-L. Favretto, B. Stancher and F. Tunis963 Cyclohexane-1.3-dione Bisthiosemicarbazone Monohydrochloride as a Spectro-photometric Reagent for the Rapid Determination o f Chlorate i n PerchloricAcid Medium-M.RomBn Ceba, J. A. MuAoz Leyva and J. J. Berzas NevadoAssessment o f Mixing Efficiency Using the Oxidation of Iodide by Hydrogen Peroxide-T. J. N. Carter and B. R. Stanbridge968REPORT BY THE ANALYTICAL METHODS COMMITTEEDetermination o f Fish Content of Coated Fish Products 973SHORT PAPERS979 Determination of the Cyclic Trimer of Formaldehyde by Gas - Liquid Chromato-graphy-V. B. Kapoor, S. K. Chopra and S. C. Vishnoi982 Determination of Minimum Detectable Amounts of Atmospheric Particulates byX-ray Diffraotometry-C. Plowman985 Techniques for the Determination of Mercury i n Silicon-containing OrganomercurialsUsing Atomic-absorption Spectrophotometry-D.T. Burns, F. Glockling, V. B.Mahale and W. J. Swindall990993994998Determination o f the Resin Content of Carbon Fibre - Resin Composites-H. SwiftExtended Application of Folin - Ciocalteu Reagent i n the Determination o f Drugs-Calculated Equivalence Volume in the Iron( II) - Cerium( IV) Titration-W. DeschachtLow-level Determination of Hydrazine i n Boiler Feed Water w i t h an UnsegmentedG. R. Rao, G. Kanjilal and K. R. MohanHigh-speed Continuous-flow System-W. D. Basson and J. F. Van Staden1002 Book ReviewsSummaries of Papers in this Issue-Pages iv, vi, vii, x, xii, xivPrinted by Heffers Printers Ltd Cambridge EnglandEntered as Second Class at New York, USA, Post OfficJOURNALS BOOKSMONOGRAPHSOrders for all publicationsformerly published by the Societyfor Analytical Chemistry shouldbe sent direct or through abookseller toTHE CHEMICAL SOCIETY,Distribution Centre,Blackhorse Road, Letchworth,Herts., SG6 1 HNSelected Annual Reviewsof the Analytical SciencesVolume 4CONTENTS'Advances in Voltammetric Techniques, byB.Fleet and R. D. Jee'High-frequency Electrodeless PlasmaSpectrometry,' by B. L. SharpPp. vi + 73 f 9.50ISBN 0 85990 204 8CS Members' price f4.00Orders should be sent direct, with remittance, orthrough your usual bookseller to-THE CHEMICAL SOCIETYDistribution Centre,Blackhorse Road, Letchworth,Herts. SG6 IHN, EnglandCS Members must write direct to the above addressenclosing the appropriate remittance.NOTICE TO SUBSCRIBERS(other than Members of the Society)Subscriptions for The Analyst, Analytical Abstracts and Proceedings shouldbe sent to:The Chemical Society, Distribution Centre,Blackhorse Road, Letchworth, Herts., SG6 1 H NRates for 1978The Analyst, Analytical Abstracts and Proceedings (including indexes) :* .. . f99.00Proceedings . . .. . . . . . . . . . . . . . . €105.00(a) The Analyst, Analytical Abstracts and Proceedings . . . .(b) The Analyst, Analytical Abstracts printed on one side of the paper, andThe Analyst and Analytical Abstracts without Proceedings (including indexes) :The Analyst, and Analytical Abstracts printed on one side of the paper(c) The Analyst, and Analytical Abstracts . . .. .. . . . . . . f87.00(d) . . €93.00(Subscriptions are NOT accepted for The Analyst and/or for Proceedings alone)Analytical Abstracts only (two volumes per year, including indexes):(e) Analytical Abstracts . . .. .. .. .. .. .. . . €67.00(f) Analytical Abstracts printed on one side of the paper . . .. .. . . f73.0

ISSN:0003-2654    

DOI:10.1039/AN97803BX035

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

iv SUMMARIES OF PAPERS I N THIS ISSUE SeptelPz bey, 19 78Summaries of Papers in this IssueA Highly Sensitive Flow- through Phototransducer for UnsegmentedContinuous-flow Analysis Demonstrating High-speed Spectrophoto-metry at the Parts per 109 Level and a New Method ofRefractometric DeterminationsA simple photometric detector is described which, because of the highstability of the light source, permits determinations of metal ions a t theparts per lo9 level with 4-(2-pyridylazo)resorcinol as the spectrophotometricreagent. By virtue of the design of the transducer it also functions as arefractometer capable of determinations of solutions of organic and inorganiccompounds down to a lower limit of approximately 0.01% m/m. The theoryof this function is discussed.A gallium phosphide light-emitting diode and a silicon phototransistor actas light source and sensor, respectively.The output current from the photo-transistor is converted into voltage by the current t o voltage converterdescribed.The transducer is designed as a flow-through cell which, when used inconjunction with standard unsegmented continuous-flow apparatus, iscapable of sampling rates of up to 300 per hour with a relative standarddeviation of the result of 1.5%. At slower flow-rates, with a sampling rateof 160 per hour, the relative standard deviation is less than 1%.Keywords : Flow injection analysis ; $ow cell; photometric detector ; ye fracto-metry; light-emitting diode - photodiodeD. BETTERIDGE, E. L. DAGLESS, B. FIELDS and N.F. GRAVESDepartment of Chemistry, University College of Swansea, Swansea, SA2 8PP.Analyst, 1978, 103, 897-908.Determination of Lead in Irons and Steels by Atomic-absorptionSpectrophotometry with the Introduction of Solid Samples into anInduction FurnaceAtomic-absorption spectrophotometry with an induction furnace has beenused for the determination of 0.1-100 p g g - l of lead in 1-12-mg samples ofirons and steels dropped into the furnace. Calibration graphs of peakabsorbance vevsus mass of lead have been constructed by using standardsteels.Samples of alloys can be added to the furnace a t 4-5-min intervals.Information is presented on the calibration graphs and on the accuracy,precision and limits of detection of the method for 44 irons and steels.Withsteels containing more than 1 pg g-l of lead relative standard deviations areusually d 10%. The limit of detection for lead is ~ 0 . 0 5 pg g-1 when usingthis method.Keywords : Lead determination ; iron analysis ; steel analysis; atounic-absorption spectroplzotonzetry ; induction furnaceDAVID GODFREY ANDREWS, ABDULLAH M. AZIZ-ALRAHMAN andJAMES B. HEADRIDGEDepartment of Chemistry, The University, Sheffield, S3 7HF.Analyst, 1978, 103, 909-915September, 1978 THE ANALYST VFOR SALEAFBS(sulphonated alizarin fluorine blue)an improved reagent for the positiveabsorptiometric determination of fluoride,general metal reagent and complexometricindicator, is available in quantities up to1 g from Mr. A. Robinson, ChemistryDepartment, Queen’s University, KeirBuilding, Belfast BT9 5AG, at a price ofS1.50 for 0.2 g.See Analyst, 99, p. 645;100, p. 275; 102, p. 340 and in press,probably October 1978.NewEU RO - STA N DAR Dnow availableE.S.877-1Furnace Dust (LD Converter)Certified for the following elements :Fe, Si, Ca, Mg, Al, Ti, Mn, P, S, Na,K, F, V, Cr, Ni, C, Zn, Pb, Cu and AsFull details obtainable from :Bureau of Analysed SamplesLtd.Newham Hall, Newby,Middlesbrough, Cleveland TS8 9EATelephone: Middlesbrough 31 721 6ADVERTISERS PLEASE NOTEAll advertisements forTHE ANALYSTshould from now on be addressed to our ownAdvertisement Department,The Chemical Society,Burlington House,Piccadilly, London WlV OBNTel: 01-734 9864Please send all space orders, copy, enquiries etc.to this addressv1 SUMMARIES OF PAPERS I N THIS ISSUE September, 1978Measurement of Silver in Blood by Atomic-absorptionSpectrophotometry Using the Micro- cup TechniqueA rapid and precise method for the measurement of silver in blood by atomic-absorption spectrophotometry using the micro-cup (Delves) technique isdescribed. The limit of detection is 2.7 ng ml-l and the calibration graphis linear up to a concentration of 400 ng ml-I. In 30 subjects industriallyexposed to fine silver dust the silver concentration in their blood was lessthan 2.7 ng ml-l, so that the principal use of the method will be in detectingcases of excessive exposure t o this metal.Keywords Blood analysis ; silver determination ; micro-cup atomic-absorptionspectrophotometry ; battery plate manufactuveCATHERINE HOWLETT and ANDREW TAYLORSouth West Thames Regional Heavy Metals Reference Laboratory, Department ofBiochemistry, University of Surrey, Guildford, Surrey, GU2 5XH.Analyst, 1978, 103, 916-920.Determination of Copper in Plasma Ultrafiltrate byAtomic- absorption Spectrometry Using CarbonFurnace AtomisationA method for the direct determination of the level of copper in plasma ultra-filtrate using atomic-absorption spectrometry with carbon furnace atomisationis reported. The relative standard deviation and the detection limit expressedas 26 were 3.6% (50 pl of 0.05 pg ml-l copper solution) and 0.0036 pg ml-l,respectively. Interferences from the salt matrix, the use of backgroundcorrection and the problems of contamination caused by the low level ofcopper are discussed. The method has been applied to the determination of thelevel of copper in the plasma ultrafiltrate of patients with rheumatoidarthritis.Keywords ; Copper detevmination ; plasma ultrafiltrate ; atomic-absorptionspectrometry ; carbon furnace atomisationH.KAMEL, J. TEAPE, D. H. BROWN, J. M. OTTAWAY and W. E. SMITHDepartment of Pure and Applied Chemistry, University of Strathclyde, CathedralStreet, Glasgow, G1 1XL.Analyst, 1978, 103, 921-927September, 1978 SUMMARIES OF PAPERS IN THIS ISSUETechnique for the Determination of Fluorescence QuantumEfficiencies : a Method Avoiding Direct Measurement ofAbsorbanceA method is described for the determination of the absorbance of a fluorescentsolution by recording the fluorescence intensities at two points along theexcitation path.An expression is developed that relates the ratio of thesetwo fluorescence intensities to the absorbance of the solution. Thereby boththe fluorescence and the absorbance of a solution can be obtained by recourseonly to a spectrophotofluorimeter. This approach is extended to the deter-mination of fluorescence quantum efficiences and an expression is presentedthat relates the fluorescence intensities at two points along the excitationpath to the quantum efficiency of the fluorescent material. Quantumefficiences can therefore be determined without the need for an accurateseparate measurement of the absorbances of solutions of standard andsample materials. A cuvette with a nitrogen deoxygenation facility isdescribed for use in these and other related determinations.viiKeywords Fluorescence eficiency ; absorbance ; nitvogen deoxygenationADAM BRITTENDepartment of Pharmacy, University of Aston in Birmingham, Gosta Green,Birmingham, B4 7ET.JOHN ARCHER-HALLDepartment of Physics, University of Rston in Birmingham, Gosta Green,Birmingham, B4 7ET.and GEOFFREY LOCKWOODDepartment of Chemistry, University of Sheffield, Sheffield, S10 2TN.Analyst, 1978, 103, 928-936.Some Fluorescent Derivatives of the Drug PhenelzinePyridazine, phthalazinone and some substituted phthalazinone derivativeshave been prepared from the drug phenelzine. The application of thesefluorescent compounds to the analysis of the drug has been explored.Asensitive and specific test for phenelzine in urine has been developed.Keywords : Phenelzine determination ; Juovescence ; spot testB. CADDY and A. H. STEADForensic Science Unit, Department of Pharmaceutical Chemistry, University ofStrathclyde, Glasgow, G1 1XW.Analyst, 1978, 103, 937-949.Polarographic Determination of Folic Acid in TabletsContaining Iron(I1) SulphateA polarographic method has been developed for the determination of folkacid in tablets containing a large excess of iron(I1) sulphate. A preparedextract of the sample is treated with lactate -phosphate buffer and theprecipitate removed by filtration. The pH of the iron-free filtrate is adjustedwith ascorbic acid to 6.8-7.5 and the folk acid determined polarographically.Keywords Folic acid determination ; iron(II) sulphate-containing tablets ;p o lar ograp hyL.RO~ANSKIResearch Laboratory, Pharmaceutical Factory “Polfa,” Poznafi, Poland.Analyst, 1978, 103, 950-954September, 1978 ...Vlll THE ANALYSTPublication Mid-SeptemberA Handbook of Decomposition Methodsin Analytical Chemistryby Rudolf Bock. Translated, updated and extended by Iain L. MarrA highly practical handbook, in a previously underpublished field, for the analyst faced withan ever-increasing range of new materials.both inorganic and organic solids are consideredthere is critical comment on the suitability and efficacy of methods in different situationsadvice is given on the choice of containers and vesselsover 3000 literature references are included -a valuable, time-saving list30 pp of the appendix comprise a list of the most commonly used dissolution proceduresContents: Introduction. Dissolution without chemical reaction.Decomposition basedon supply of energy. Dissolution and opening-out by chemical reaction, but without changein oxidation state. Oxidising procedures. Decomposition procedures involving reduction.Appendices and bibliography.229 x 152 mm 430 pp 56 line diagrams cased SBN 7002 0269 2 cE20.00(North American rights held by Halsted Press)Please order through your booksellerDescriptive leaflet from the publishers :International Textbook Company(a member of the Blackie Publishing Group)Bishopbriggs, Glasgow 664 2NZ, ScotlandPractical Research in South Africain one of the most modernlaboratories of its kindAnglo American Corporation provides comprehensivemanagement and technical services to its operatingDivisions (Gold and Uranium, Coal, Diamond and BaseMinerals), and to its many subsidiaries and associatecompanies throughout South Africa and other parts ofthe world.The Johannesburg based Research Laboratories provideservices to the Corporation’s prospecting activities aswell as its metallurgical operations including processdevelopment and experimental investigations on plants.RESEARCH TECH NOLOGISTSGraduates and diplomates in chemical-, metallurgical-and minerals processing engineering and allied fields,with or without experience, in plant or laboratory work,are invited to fill vacant as well as newly created posts.ANALYTICAL CHEMISTSChemistry graduates and holders of the H NC or H N D inanalytical chemistry with several years analyticalexperience are required for the analytical laboratorieswhich are equipped with sophisticated moderninstruments.An attractive package includes -% guaranteed annualbonus of 10% annual salary earned * up to 30 days leave. * medical and life insurance + pension scheme.Free air passagesfor incumbents and immediate familywill also be provided i n addition to generous relocation/resettlement allowances.In the first instance, applicants should send full personaland career detailsto Mr.N. R . Coulson, Anglo CharterInternational Services Limited, (Appointments Division).Ref. TA, 40 Holborn Viaduct, London ECI P 1 AJ.Anglo American Corporation SOUTH LiMtTEoh

ISSN:0003-2654    

DOI:10.1039/AN97803FP081

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

September, 1978 THE ANALYST ixPHOTOMETRIC DETERMINATION OF TRACES OF METALS4th Ed.: General Aspectsby E. 6. Sandell, University of Minnesota,and H. Onishi, University of Tsukuba, JapanThis edition examines the determination of metal traces by absorptiometry and fluorimetry. It also includesother phases of trace analysis, such as sampling, losses of metals and their minimisation, sources ofcontamination and means of minimising them, and separation methods. Special attention is given toliquid-liquid extractions and ion-exchange separations. (Chemical Analysis Series.)0471 030945 approx. 1669 pages In Press approx. €32.40/$58.35HANDBOOK OF CHEMICAL MICROSCOPY 2nd Ed. VoI. 2:Chemical Methods and Inorganic Qualitative Analysisby E. M. Chamot and C. W. Mason, Cornell UniversityA reprinted edition of this classic, published in 1940 and brought back into print by popular demand.0471 04122 X approx.483 pages In Press approx. fl9.40/$34.95PHYSICOCHEMICAL APPLICATIONS OF GASCHROMATOGRAPHYby R. J . Laub, University College of Swansea,and R. L. Pecsok. University of HawaiiAn up-to-date, critical review of recent advances covering all aspects in a simplified, user-oriented way.The author gives a brief historical review, and compares the technique with classical static methods forobtaining thermodynamic data, emphasising gas chromatography's accuracy and reproducibility. Thepotential for investigating kinetic phenomena and the properties of pure substances are examined; acomprehensive bibliography is included.0471 51 838 7 approx.336 pages In Press approx. €1 4.80/$27.05ANALYSIS INSTRUMENTATION Vol. 15edited by J . F. Combs, Monsanto Company, F. D. Martin, Dow Chemical USA,and W. H. Wagner, Union CarbideProceedings of the 23rd Annual ISA Analysis Instrumentation Symposium May 17-1 9, 1977 Charleston,West Virginia.The symposium theme, "Industrial Innovation, through Advancements in Process Analysis" emphasisesthe importance of providing a focal point for the exchange of knowledge and experience in all aspects ofanalytical instrumentation.087664361 6 153 pages December 1977 f7.80/$13.20 (paper only)Published by Instrument Society of America and distributed by John Wiley & Sons LtdX SUMMARIES OF PAPERS I N T H I S I S S U EExtraction and Determination of Polyoxyethylene Alkyl EtherNon-ionic Surfactants in Water at Trace LevelsPolyoxyethylene non-ionic surfactants of the type RO(CH,CH,O) EH (whereR is an n-alkyl group and n is the number-average degree of polymerisation)are extracted and concentrated from water at trace levels with 1,Z-dichloro-ethane and then determined by a spectrophotometric procedure.Their charac-teristic polydispersity is checked by gas chromatography.The proposed method, which has been studied in detail on polyoxyethylenedodecyl ethers, is free from interferences from anionic surfactants and theinterference of cationic surfactants is also reduced about 15-fold. Thedetermination of non-ionic surfactants in polluted sea waters is consideredas a tentative application to a complex matrix.Septenzbev, 1978Keywords : Polyoxyethylene alkyl ether non-ionic surfactant determination ;water analysis ; spectrophotometryL. FAVRETTO, B.STANCHER and F. TUNISIstituto di Merceologia, UniversitL di Trieste, 34100 Trieste, Italy.Analyst, 1978, 103, 955-962.Cyclohexane - 1,3 - dione Bisthiosemicarbazone Monohydrochlorideas a Spectrophotometric Reagent for the Rapid Determination ofChlorate in Perchloric Acid MediumCyclohexane- 1,S-dione bisthiosemicarbazone monohydrochloride producescoloured solutions with chlorate ions in perchloric acid medium, in thepresence of chloride ions. The yellow colour obtained (molar absorptivity1.7 x lo4 1 mol-l cm-l a t a wavelength of 402 nm) has been used for thespectrophotometric determination of trace amounts of chlorate.Thereaction is of considerable interest, as a t present few useful colour reactionsare known for the chlorate ion. An important advantage of using thisreagent is the stability of the yellow coloured solution obtained.Keywords : Cyclohexane- 1,S-dione bisthiosemicarbazone monohydrochloride ;spectrophotometry ; chlorate determination ; perchlovic acid mediumM. ROMAN CEBA, J. A. MUG02 LEYVA and J. J. BERZAS NEVADODepartment of Analytical Chemistry, Universidad de Extremadudura, Badajoz,Spain.Analyst, 1978, 103, 963-967.Assessment of Mixing Efficiency Using the Oxidation of Iodideby Hydrogen PeroxideA method of assessing mixing efficiency in spectrophotometric cells is described.The rate at which iodide is oxidised by hydrogen peroxide, indicated by thetime taken to consume a finite amount of thiosulphate, was found to bedependent on mixing efficiency in a system in which force of injection wasthe sole mixing mode.In the system studied it was shown that mixing toa total volume of 1 ml by injecting less than 0.5 ml is inadequate.Keywords : Mixing eficiency ; iodide oxidation ; hydrogen peroxide ; reactionrateT. J. N. CARTER and B. R. STANBRIDGEWolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston,Birmingham, B15 2TH.Analyst, 1978, 103, 968-972September, 1978 THE ANALYST xir 4 Analvtical Chemist dRank Xerox, one of the leading namesin communication and informationhandling systems requires an AnalyticalChemist to join a small team engagedin occupational hygiene and chemicalsafety studies at their Welwyn GardenCity location.The work will include the analysis ofa range of materials and samples forthe detection and qualification of sub-microgram quantities of contaminants.We look for an innovative approachto continuously push back the detectionlimits of the techniques involved, thisnecessitates a high level of practical abil-ity whichshouldincludeexperienceinIRand AAspectrometry, GLC and polaro-graphy.Additionally knowledge ofGPC and TLC would be an advantage.Male or female applicants should bequalified to degree level but M Chem Ais preferred.In addition to a highly competitivesalary we are offering 4 weeks’ holiday,generous sick pay and pension schemes,free life assurance, BUPA discountsRANI< XEROX-7 ENGINEERING GROUP-and discounts on a wide range ofgoods through a staff purchase scheme.Generous assistance with relocationwill be given where necessary, coveringreimbursement of all reasonable legaland estate agency fees, up to twoBuilding Society survey fees, plusbridging loan facilities if required.Financial assistance will also be givencovering travel and subsistence, etc.,during the period prior to movinginto a new home.Working conditions at Rank Zeroxare extremely attractive, with some ofthe finest equipment and facilitiesavailable anywhere.If you are interested, contact JimCollingham on Milton Keynes 316611,extension 260, for an application formand company information, or write tohim at Rank Xerox, EngineeringGroup, Linford Wood, Milton KeynesMK14 6LA.In the evenings and at weekends ananswering service is available onMilton Keynes 312870.CLASSIFIED ADVERTISEMENTST h e Rate f o r Classijed Advertisements i s E2.30 per single column centimetre(minimum E4.60).B o x Numbers are charged an extra 50p.Deadline f o r classijed copy i s 20th of the month preceding month of issue.All space orders, co$y instructions and enquiries should be addressed toT h e Advertisement Department,T h e Chemical Society, Buylington House,Piccndilly, London W I V oBN.Telephone 01-734 9864 Telex 26800x ii SUMMARIES OF PAPERS IN THIS ISSUEDetermination of Fish Content of Coated Fish ProductsSeptember, 1978Report prepared by the Fish Products Sub-committee.Keywords Coated fish products ; fish contentANALYTICAL METHODS COMMITTEEThe Chemical Society, Burlington House, London, WlV OBN.Analyst, 1978, 103, 973-978.Determination of the Cyclic Trimer of Formaldehyde byGas - Liquid ChromatographyShort PaperKeywords : 1,3,5-Trioxan determination ; gas chromatography ; polyoxy-ethylene sorbitan monooleate stationary phaseV.B. KAPOOR, S. K. CHOPRA and S. C. VISHNOIIndian Institute of Petroleum, Dehradun-248005, India.Analyst, 1978, 103, 979-982.Determination of Minimum Detectable Amounts of AtmosphericParticulates by X-ray DiffractometryShort PaperKeywords : A tvnospheric particulates ; X-ray diflraction ; limit of detectionC .PLOWMANCentral Electricity Generating Board, Scientific Services Department, BeckwithKnowle, Otley Road, Harrogate, Yorkshire, HG3 1PR.Analyst, 1978, 103, 982-985.Techniques for the Determination of Mercury in Silicon- containingOrganomercurials Using Atomic-absorption SpectrophotometryShort PaperKeywords : Mercury determination ; silicon-containing organomercurials ;atomic-absorption spectrophotometryD. T. BURNS, F, GLOCKLING, V. B. MAHALE and W. J. SWINDALLDepartment of Chemistry, Queen’s University of Belfast, Belfast, BTQ 5AG,N. Ireland.Analyst, 1978, 103, 985-989.Determination of the Resin Content of Carbon Fibre - ResinCompositesShort PaperKeywords : Carbon fibre - vesin composites ; resin content determinationH.SWIFTAnalytical Chemistry Branch, Chemistry Division, Atomic Weapons Research Estab-lishment, Aldermaston, Reading, RG7 4PR.Analyst, 1978, 103, 990-993x1v SUMMARIES OF PAPERS I N THIS ISSUEExtended Application of Folin - Ciocalteu Reagent in theDetermination of DrugsSeptcwzbcv, 1978Keywords : Folin - Ciocalteu reageiat ; drug detetmiPmtionG. R. RAO, G. KANJILAL and K. R. NOHANQu+ity Control Laboratories, Indian Drugs and Pharmaceuticals Ltd., Hyderabacl,India, PIN 500037.Analyst 1978, 103, 903-994.Calculated Equivalence Volume in the Ison(I1) - Cerium(1V)TitrationShOYt PaperKeywovds : Potentiovlzetvic titration ; oxidation - Yeduction titvation ; cnlcu?atedend -Po in tW. DESCNACHTFisheries Research Station, Aiikcrstraat 1, B-8400 (Mend, Belgium.Analyst, 1978, 103, 994-998.Low-level Determination of Hydrazine in Boiler Feed Water withan Unsegmented High- speed Continuous - flow SystemShovt PaperKeywovds : High-speed continuous-flow analysis ; unsegmented system ;lzydvazine detevmination ; boilev feed watevW. D. BASSON and J. F. VAN STADENDepartment of Inorganic and Analytical Chemistry, University of Pretoria, Pretoria,Republic of South Africa.Analyst, 1978, 103, 998-1001

ISSN:0003-2654    

DOI:10.1039/AN97803BP087

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

SEPTEMBER 1978 The Analyst Vol. 103 No. 1230 A Highly Sensitive Flow-through Phototransducer for Unsegmented Continuous-flow Analysis Demonstrating High-speed Spectrophotometry at the Parts per lo9 Level and a New Method of Refractometric Determinations D. Betteridge, E. L. Dagless," B. Fields and N. F. Graves* Department of Chemistry, University College of Swansea, Swansea, SA 2 8PP A simple photometric detector is described which, because of the high stability of the light source, permits determinations of metal ions at the parts per 1 O9 level with 4- (2-pyridy1azo)resorcinol as the spectrophotometric reagent. By virtue of the design of the transducer it also functions as a refractometer capable of determinations of solutions of organic and inorganic compounds down to a lower limit of approximately 0.01% nz/m.The theory of this function is discussed. is. gallium phosphide light-emitting diode and a silicon phototransistor act as light source and sensor, respectively. The output current from the photo- transistor is converted into voltage by the current t o voltage converter described. The transducer is designed as a flow-through cell which, when used in conjunction with standard unsegmented continuous-flow apparatus, is capable of sampling rates of up to 300 per hour with a relative standard deviation of the result of 1.5%. At slower flow-rates, with a sampling rate of 160 per hour, the relative standard deviation is less than 1 yo. Keywords : Flow injection analysis ; $ow cell ; photometric detector; refracto- metry ; light-emitting diode - photodiode Unsegmented continuous-flow analysis or flow injection analysis has been developed during the last 4 years as an automatic method of analysis that is simple, accurate and rapid, typical sampling rates being 120 per hour.In all such systems a stream of reagent or other carrier flows through a small-bore tube, the flow-rate being maintained by a constant-pressure or a constant-volume pump. At a point along the length of the tube an injection mechanism allows the sample to be injected into the stream and as the sample bolus passes down the tube it may react with reagent in the stream or undergo other reactions with the stream solution. Situated downstream from the injection point is a sensor, which measures the extent of these reactions.The detectors described in previous reports have been spectrophot~metric~-~ or potenti~metric.l-~~* If the unsegmented flow system is to be used for routine analyses on a specific chemical system in the way in which automatic methods are generally applied, then wavelength variability and many component parts of the spectrophotometer become superfluous. The apparatus in these circumstances is overdeveloped and the function of the machine no longer justifies its cost. The aim of this work was to provide a simplified photometric detector that can be connected into the system by push-fitting flow tubes and that increases signifi- cantly the over-all sensitivity of the method. In this paper such a detector is described. While it is simple, small and inexpensive, it allows sampling rates of up to 300 per hour and is capable of a lower detection limit of less than lOV9g of ions in solution.The light components are extremely cheap and robust and 897 * Department of Electrical Engineering.898 Analyst, VoE. 103 offer long life, typically 20 000-100 000 h. Interchangeable units permit most of the visible and near-infrared spectrum to be covered. With the associated electronics the total cost is about LlO. BETTERIDGE et al. : HIGHLY SENSITIVE FLOW-THROUGH Experiment a1 Reagents Reagent solutions for absorptiometry Prepared by dissolving the AnalaR salt in doubly distilled water. Trace impurities were extracted from 500ml of this solution with three successive 50-ml aliquots of 10-3 M 1-(2-pyridylazo)-2-naphthol (PAN) in chloroform followed by three 50-ml aliquots of chloroform to remove the slightly soluble PAN chelates.The solution was evaporated and the salt recrystallised from doubly distilled water and used to make up buffer solutions for the analysis of sample solutions having concentrations in the ranges given in Table I. Prepared by dissolving reagent-grade 4-(2-pyridylazo)resorcinol (PAR) disodium salt in doubly distilled water. The stock solution was used to prepare reagent solutions of the concentrations given in Table I. Disodium tetraborate solution, 0.1 M. 4-(2-Pyridylaxo)resorcinol disodium salt stock solution, 5 x loM3 M. TABLE I REAGENT AND BUFFER CONCENTRATIONS REQUIRED FOR GIVEN RANGES OF SAMPLE CONCENTRATION IN ABSORPTIOMETRY Sample Reagent Buffer concentration, p.p.b.concentration/M concentration/M 0-600 10-4 10-2 3-1 0 5 x lo-** 4.9 x 10-2* 0-100 4 x 10-5 10-3 * Concentration before addition to the sample. To each 20 ml of sample 0.5 ml was added. Sample solutions for absorptiometry AnalaR cobalt (11) nitrate was dissolved in doubly distilled water and the solution standardised by means of titration with EDTA. A stock solution containing 50 p.p.m. of cobalt was prepared. Sample solutions containing cobalt in the range 10-500 p.p.b. (parts per 109) were prepared from the stock solution. CoPpey(I1) sulphate solutions. AnalaR copper (11) sulphate was used to prepare sample solutions containing copper in the range 3-10 p.p.b. in the same way as for the solutions of cobalt nitrate. Cobalt(II) nitrate solutions. Sample solzttions for refractometry Standard solutions (35.0 g 1-1) of AnalaR sodium sulphate, potassium chloride and di- sodium tetraborate decahydrate were prepared and diluted to obtain solutions of concentra- tions 5, 10, 15 and 20 g 1-l.Sample solutions containing 1, 2, 3, 4 and 5 g 1-1 of sodium chloride and potassium chloride were prepared similarly. The refractive indices were measured by use of an Abb6 refractometer. Tabless of refractive index zlersm. concentration were used to deduce the concentrations of hydrochloric acid, lithium chloride, sodium chloride, potassium chloride and sodium sulphate that would give solutions of refractive index 1.3380 and these solutions were prepared accordingly. Apparatus Design and construction of transducer cell The mechanical construction of the transducer cell is shown in the sectional view in Fig.1. The cell was constructed by using Perspex. The light-emitting diode (LED) and photo- transistor are of a plastic encapsulated type and were glued in place by using an epoxy-resin adhesive. In this respect care was taken to form a watertight and rigid joint. Lead-in andSeptember, 1978 PHOTOTRANSDUCER FOR UNSEGMENTED CONTINUOUS-FLOW ANALYSIS 899 lead-out tubes were either glued in place or made to push fit. The whole transducer cell and 10 cm of the flow tubes on both sides of the cell were laquered black in order to exclude ambient light from the photodetector. Light cells of different dimensions have been constructed and tested. Fig. 1 shows the first construction: a cell with a 7-mm path length and a central bore of 1 mm i.d., which is slightly larger than the internal diameter of the tubing (0.86 mm).The transducer cell used to obtain the results published here was of path length 30 mm and internal bore diameter 2 mm. 4le 1 mm - + I + 1 mm t 7 mrn b Fig. 1. Sectional view of transducer cell. Cuwent to voltage converter The LED is driven from a constant 15-V source with current-limiting resistor R1. When biassed at a constant voltage a phototransistor appears as a current source whose output depends upon the incident illumination. This output current is converted into a voltage while maintaining a constant voltage bias across the phototransistor, using the current to voltage converter circuit shown in Fig. 2. The phototransistor is biassed at a constant 15 V as the inverting input to A1 (FET Operational amplifier, Radio Spares Components RS 305-456) is a virtual earth and the output voltage is given by v,, = R3 X IPh, where I p h is the phototransistor current.For R3 = loMQ, v o / I p h = 10 V pA-l. o v +15 V 560S2 k -15 V c1 22kS2 d?f - I1 1OMn Os1pF o v &b TIL78 phototransistor I -15v ov o'v 0- v Fig. 2. Transducer circuit.900 BETTERIDGE et aE. HIGHLY SENSITIVE FLOW-THROUGH Analyst, VOZ. 103 The band widths of both the LED and the phototransistor are of the order of hundreds of kilohertz. The band-width requirement of the flow injection system is dictated by the flow-rate of the carrier, sample volume and mixing-coil length, but it is generally less than 1 Hz. C1 in parallel with R3 limits the band width of the system, reducing noise (in particular 50-H~ mains interference).The potentiometer VR2 provides for gain adjustment while amplifier A2 (Operational amplifier, Radio Spares Components RS 741) is a fixed-gain stage, which serves to produce an output signal in the range -+lo V suitable for input to an analogue to digital converter and therefore a microprocessor. When correctly adjusted for use with a chart recorder the quiescent output when viewing the reagent stream is backed off, using potentiometer VR1. The output from amplifier A2 is then in the form of a positive going peak when the absorbance of the sample is greater than that of the carrier. Spectral considerations The spectral emission of a gallium phosphide LED centres on 565 nm with a band width of 30 nm.The spectral response of a silicon phototransistor has a maximum a t 800 nm. The gain at 565 nm is reduced to approximately 40% of the maximum but is sufficient for the device to be used as a detector of light at this frequency. A comparison of the range of frequencies emitted by the LED with the absorption spectrum of the cobalt - 2PAR complex is given in Fig. 3. The overlap of these curves is shown in practice to give reasonable results. Most metal - PAR complexes absorb in the region 550-580 nm, but their molar absorptivity at 565 nm varies and therefore the sensi- tivity of the transducer is not identical for every metal. 450 500 550 600 Wavelength/nrn Fig. 3. A, Absorption spectrum of the Co - SPAR complex. B, Emission spectrum of the gallium phosphide LED.Unsegnzeated continuous-flow apparatus The reagent stream was activated by a Schuco Pericyclic pump, the flow-rate being adjusted where necessary by braking coils connected to the end of the flow system in order to restrict the flow velocity. Pump. Mixing and braking coils. Sample injection. Sample solutions were injected from a 1-ml disposable plastic syringe without a hypodermic needle. Discharging the syringe depresses a rubber septum valve in an injection block,3 which allows passage of the sample into the stream. Back-flow of sample solution is prevented by maintaining pressure on the syringe until the injection is completed. All coils were of 0.86 mm i.d. polypropylene tubing. Refractometer refractive index relationship. A conventional Abb6 refractometer was used for the calibration of peak height versusSeptember, 1978 PHOTOTRANSDUCER FOR UNSEGMENTED CONTINUOUS-FLOW ANALYSIS 901 Procedures A bsorptiometry The flow system was purged of metal contaminants by pumping 1% m/V nitric acid through the system and leaving the acid in the system overnight.A diagram of the analytical system is presented in Fig. 4. ~ H ~ ~ / / ! ~ ~ / / / 100cm 215cm Phototransducer Waste * Chart recorder Fig. 4. Flow system for absorptiometric and refractometric determinations. FZow-rate. A flow-rate of 3.8 ml min-l was maintained for absorptiometric experiments in order to obtain calibration graphs. Flow-rates of up to 12 ml min-l were used to test the feasibility of high sampling rates. Sample volumes of 0.2 nil were injected at intervals such that an injection took place when the trace for the preceding sample had returned to less than 1% of its peak height.Three injections of each sample were performed and the mean value determined to give the result used in the calibration graphs. The concentration of reagent in the stream was chosen such that an excess of reagent ensured complete development of the colour for the most concentrated sample, while the colour of the reagent background was not so intense as to result in negative peaks for the most dilute samples. A suitable concentration of buffer must be chosen because the instrument is sensitive to differences in refractive index between the sample and the stream. In these experiments the concentration of buffer was kept as low as possible in order to keep the refractive index of stream and sample close to that of distilled water.Sample volume. Reagent concentration. B u f e r concentration. Procedure for sample solutions of concentration below 10 p.p.b. For very low concentrations of metal the sensitivity of the method is enhanced if the chelate is formed before injection into the system. For the determination of concentrations below 10 p.p.b. the following procedure was adopted. M in disodium tetraborate was prepared and a 0.5-ml aliquot was pipetted into each 20 ml of sample solution containing copper(I1) in the range 3-10 p.p.b. After 2min, allowed for colour formation, 0.4-ml aliquots of these solutions were injected into a stream of doubly distilled water buffered with the same concentration of buffer as in the final sample solutions, i.e., 1.22 x M disodium tetra- borate. The same procedure was used in the analysis of a solution containing 10 p.p.b.of cobalt(I1) for the assessment of precision. A reagent solution lo-* M in PAR and 5 x The mean of three results was recorded for each sample solution. Refractometry Flow-rates. A flow-rate of 2.9 rnl min-l was used in order to obtain the calibration graphs presented. Flow-rates of 2.9 and 2.2 ml min-l were used to demonstrate variations in the recorded response with flow-rate. Flow-rates of up to 12 ml min-l were used to assess the maximum sampling rate. Sample volume. Sample volumes of 0.2 ml were injected under the same conditions as for absorptiometry. The transducer response to refractive index (see under Results and Discussion) is to produce two consecutive peaks opposite in sign relative to the base line.The height from the base line of the first peak to appear after injection is the measured value and the mean peak height of two sample solutions is the value used in the calibrations.902 experiments. BETTERIDGE et al. : HIGHLY SENSITIVE FLOW-THROUGH Analyst, VoZ. 103 Carrier stream. A stream of distilled water was used as a carrier in the refractometry Results and Discussion Absorptiometry Figs. 5 and 6 show the transducer response for concentrations of cobalt(I1) injected in the ranges 0.1-0.5 p.p.m. and 10-100 p.p.b. The degree of linearity is good in view of the spectral range of the light source. As the spectrum of light emitted from the LED is constant and entirely reproducible it is valid to utilise a small range of frequencies, 550- 580 nm, provided the analyses are to be made through a calibration graph for each system of only one absorbing species.It is clear from these figures that the output from the phototransistor is in the range that is linearly related to incident light intensity and not in the range in which saturation of the phototran.sistor begins to occur. 90 80 5 5 70 60 - - - - 'z. 50- a 40- (D 30 - 20 10 u r V r cr, 1 Y Q .- - - 0 0.1 0.2 0.3 0.4 0.5 0 10 20 30 40 50 60 70 80 90100 Concentration of cobalt (I I), p.p.m. Concentration of cobalt (II), p.p.b. Fig. 5. Calibration graph for determinations of cobalt(I1) in the range 0-0.5 p.p.m. Fig. 6. Calibration graph for determinations of cobalt(I1) in the range 0-100 p.p.b.As the absorbance is measured over a range of frequencies, and a range over which the absorptivity of the chelate varies greatly, deviations from the Beer - Lambert - Bouguer law are to be expected. Nevertheless, these graphs are of equal quality and similar linearity to those obtained by using a flow cell in a spectrometer as detector and the sensitivity and resolution of intensity are greatly increased. This increase is due to the high stability of tamiccinn nf tho T F l 3 1ic~t-l ac 9 lirsht cniirrP Twhirh pn9hlpc D-rpatpr amnlifiratinn nf thp JfLL""cA""" V L CALL, b'"-'"' -lAL \ r l l l l . J d L V & A "I LLl" YYU UUYU wu u "a"' UVUSU", ..*LL"LL "*LWVI"U phototransistor signal to be used in order to resolve small differences in colour intensity between the dilute reagent and the chelate.which makes it possible to resolve parts per lo9 units at any absorbance level. For determinations at the parts per lo9 level (Fig. 7) the chelate is formed before injection. The flow injection system serves only to pass the pre-formed chelate through the photo- transducer. A large sample volume ensures minimum dilution of the chelate by the stream, which consists of buffer solution without reagent. Precision For samples containing more than 100 p.p.b. of cobalt(I1) the standard deviation in the result is about 1%. For samples containing 10 p.p.b. of cobalt(II), analysed by the pro- cedure involving pre-formation of the chelate, the relative standard deviation is 3%, this increase being due to noise on the base line.September, 1978 PHOTOTRANSDUCER FOR UNSEGMENTED CONTINUOUS-FLOW ANALYSIS 903 A theoretical detection limit can be deduced from the fact that a signal to noise ratio of 2 is given by 0.6 p.p.b.of cobalt(I1). This corresponds to an absorbance of 0.0006 if this were being measured in a conventional spectrophotometer in a l-cm cell. Refractometry By virtue of the cell design and its optical configuration the transducer is sensitive under dynamic conditions to change in the refractive index of colourless samples. The form of the curve is illustrated in Fig. 8 and the height from the base line of the first peak is a measure of refractive index under conditions of medium or high flow-rate. 1 2 3 4 5 6 7 8 9 1 0 Concentration of copper (I I), p.p.6.Fig. 7. Calibration graph for determinations of copper(I1) in c I D Start U E E E E the range 3-10 p.p.6.- Copper Fig. 8. Typical peak profiles for refractive-index responses for is determined as the pre-formed sodium chloride solutions: A, 1; B, 2; C, 3; D, 4; and E, 6 g 1-l. chelate Cu - 2PAR. Carrier stream was distilled water. Principle of operation The principle of measurement of refractive index is based on the formation of a refractive- index gradient between the sample solution and a standard stream solution, the magnitude of which is a measure of the original refractive index of the sample. The high sensitivity of the system is due to the physical shape of this gradient. If a solution of high salt content and hence high refractive index is injected upstream from the transducer into a stream of distilled water, then when the sample passes through the transducer it will do so such that the lines of equal salt concentration (isohalines) are parabolic in shape owing to wall drag and according to the theory of flow in tubes under laminar conditions.Each isohaline will have a refractive index different to that of the adjacent one. The system can be regarded as a series of liquid lenses, which focus or diverge light in a direction and of a magnitude dependent on the dimensions of the parabola, the aspect of the parabola with respect to incident light, the direction of the refractive-index gradient and the magnitude of the refractive-index gradient. In practice the dimensions and aspect of the parabola are kept constant by maintaining a constant high flow-rate and current direction through the transducer.Also, the direction of the refractive-index gradient presents no problem if the direction of the peaks is taken into account: the system can be calibrated through zero, i.e., a sample of the same refractive index as the stream, to negative peaks, i.e., samples of refractive index less than the stream. The result, therefore, will depend only on the magnitude of the refractive-index gradient, which for a constant stream depends only on the original refractive index of the sample. The method is a relative one and calibration graphs need to be obtained. Consider the case of a sample of higher refractive index than the stream. When the leading interface passes through the transducer in the direction shown in Fig.9(a), light travels from isohalines of high refractive index to those of low refractive index. The light is therefore refracted away from the normal to the isohalines and construction of rays shows904 BETTERIDGE et al. : HIGHLY SENSITIVE FLOW-THROUGH Analyst, Vol. 103 the light to be focused toward the detector. Light leaving the LED is slightly divergent and hence the focusing effect leads to an increase in the amount of light reaching the photo- diode. A chart recorder records the peak in the direction of increased light (see Fig. s), the peak maximum being the point of maximum slope of the salt gradient. At the peak for the sample solution there is no change in salt concentration over a short distance such as that represented by the cell.The light is unrefracted and the trace passes through zero, the base line. As the trailing interface passes through the transducer, Fig. 9(b), light travels from a medium of low refractive index to one of high refractive index and is refracted towards the normal to the isohalines. It is therefore diverged from the path to the detector and a negative peak relative to that caused by the leading interface is observed. The over-all curve is therefore a differential curve of the concentration of the solute. Consider the case of a sample of lower refractive index than the stream. The leading and trailing interfaces are now similar to the trailing and leading interfaces, respectively, described in the first case.The trace obtained is similar to a mirror image of the former trace about ' Normal \ LED detector Light focused Direction of flow Light detector 77 77 > 71' Light diverged I Light detector 77 >v' Light focused Direction of flow- Fig. 9. Refraction by isohalines in trans- ducer cell: (a), leading interface for sample of higher refractive index than stream; (b) , trailing interface for sample of higher refractive index than stream; and ( G ) . leading interface for sample of higher refractive index than stream with direction of flow through the cell reversed; 7 = refractive index.September, 1978 PHOTOTRANSDUCER FOR UNSEGMENTED CONTINUOUS-FLOW ANALYSIS 905 the base line. Under conditions of high flow-rate the first peak height is greatest whatever the direction of the refractive index as under fast flow conditions the leading interface always has the steeper gradient.Consider the case of a sample of higher refractive index than that of the stream passing through the transducer in the opposite direction to that in the first instance. The gradient of the refractive index will be reversed but the aspect of the parabola is inverted such that light now refracted towards the normal to the isohalines is still focused on the detector for the leading interface, Fig. 9(c). Light now refracted away from the normal to the isohalines is still diverged from the path to the detectors for the trailing interface. The recorded peak, therefore, for a sample passed through the transducer is the same irrespective of direction of flow.An alternative theory is to consider that the sample is a cylindrical plug with isohalines normal to the direction of flow. If the rays leaving the LED were slightly convergent, refractive-index changes in the sample plug would give rise to peaks of the type observed. However, if the flow were reversed the peak direction would also be reversed, which is not the experimental result. Reflection from the cell walls and total internal reflection in the sample plug as well as refraction have to be considered. Refraction of light by the isohalines changes the angle of incidence of light on the cell walls and it is known that a greater angle of incidence leads to an increase in reflected light and hence in light reaching the photodiode. Construction of rays shows that reflections on the cell walls will always lead to an enhancement of the final signal whatever the flow direction and refractive-index gradient.If total internal reflection were a significant factor the signal for the mode shown in Fig. 9(a), in which total internal reflection occurs, would be significantly greater than for the mode in Fig. 9(c), Le., for the same sample with the flow direction reversed, as total internal reflection cannot occur in this mode. In fact the difference in peak heights is about 3%, suggesting that total internal reflection on the salt gradient either is small compared with the lens effect or does not occur. The theory was checked in a simple way by removing the LED and photodiode and viewing through the cell with the naked eye an object held at one end as the sample passed through. The object is magnified for one interface and diminished for the other if the difference in refractive index is not large.When it is large a shimmering effect is observed similar to that seen when brine is poured into distilled water. Fig. 10 shows the relationship between peak height and concentration expressed as molarity for solutions of potassium chloride, sodium sulphate and disodium tetraborate. A graph of peak height versus concentration in grams per litre similarly shows three separate curves. Fig. 11 shows the same peak heights, for the same samples as in Fig. 10, plotted against the refractive index of each of the samples measured on an Abbd refractometer. 0 0.1 0.2 0.3 ConcentratiodM 1.333 1.334 1.335 1.336 Refractive index Fie 10.Peak heights for samples of Fig. 11. Peak height Ztersus refractive disodium tetraborate decahydrate, A, sodium index for samples of disodium tetraborate sulphate, B, and potassium chloride, C, of decahydrate, A, sodium sulphate. 0, and concentration 5, 10, 15 and 20g 1-1 ex- potassium chloride, 0, of the same con- pressed as molarity. centration as in Fig. 10.906 BETTERIDGE ei! al. : HIGHLY SENSITIVE FLOW-THROUGH Analyst, VOE. 203 These figures demonstrate that refractive index is the cause of the phenomenon rather than a concentration effect. Fig. 12 shows the quality of such calibration graphs in concentration ranges which the Abbe refractometer cannot resolve. Clearly, the best precision can be obtained by calibrating the instrument for each solute to be determined rather than deducing the concentration of a solute from a general calibration graph of peak height zlersus refractive index.80 70 4 60 t 2 50 Y .- Q) 40 5 30 20 10 c1 z a r 2 The detection 0 1 2 3 4 5 Concentration/g I-' Fig. 12. Sample calibration graphs for potassium chloride, A, and sodium chloride, B. limit varies with the nature of the solute but is found t be of the order of 0.01%. The precision of the instrument is found to be dependent on flow-rate. At a flow- rate of 3 ml min-l (100 samples per hour) the relative standard deviation is 0.25% and at a flow-rate of 10.8 ml min-l (300 samples per hour) the relative standard deviation is 1.5%. The sensitivity of the apparatus is slightly poorer than that of a differential refractometer and at present requires samples of 50 pl or more compared with, typically, 5 pl.However, for samples in the range quoted, the method produces results in less than 10 s from injection and can be used for routine sampling at a rate of at least 320 samples per hour. Further, owing to the design of the transducer cell, air bubbles entering the system pass directly through the transducer, eliminating what can be a common and time-consuming problem with the conventional differential refractometer. Efect of pow-rate At low flow-rates different results are obtained for sample solutions of different solutes with the same refractive index, whereas at higher flow-rates the same results are obtained for these solutions (see Table 11). A partial explanation of this can be found by considering the different rates of diffusion of different solutes in solution.TABLE I1 EFFECT OF FLOW-RATE ON THE TRANSDUCER RESPONSE TO REFRACTIVE INDEX Peak height a t a flow-rate of- Concentration/ Refractive I A 'I Solute g 1-1 index 2.9 ml min-1 2.2 ml min-' HC1 21.8 1.338 0 59 55 LiCl 23.7 1.338 0 60 57 NaCl 28.0 1.338 0 61 60 KCl 37.5 1.338 0 62 49 Na,SO, 33.9 1.338 0 62 34 Diff usivit y (D) at 26 "C*/ cm2 s-1 3.05 1.27 1.48 1.84 1.12 * Figures for HCl, LiCl, NaCl and KCl represent the value for 0.1 M solutions. The figure for Na,SO, is for a 0.01 M solution.Septe?T%beY, 1978 PHOTOTRANSDUCER FOR UNSEGMENTED CONTINUOUS-FLOW ANALYSIS 907 For a component of high diffusivity net transport of solute occurs radially from the fast- moving parabolic head of the sample to the slower-moving regions at the walls, and from the tail of the sample, which recedes relative to the mean transport rate of the sample, to the more rapidly moving centre of the stream.This results in a movement of solute from the regions of high lateral spreading of the sample to those of low lateral spreading and the peak is sharpened. The effect is greater the greater the diffusivity. High diffusivity therefore results in a compacting of the isohalines and an increase in the principal focus of the isohaline parabola. The first factor increases the magnitude of the refraction and the second decreases it. If diffusion is allowed to occur, therefore, by using the system at low flow-rates, the result will be diffusivity dependent. In fact, some diffusion must occur a t all speeds to give the isohalines postulated in the theory.This is proved by the fact that colour development in absorptiometry occurs significantly even at very high flow-rates and with a short reaction time. The shape of the resulting peaks at high flow-rates proves to be the same, whereas at low flow-rates the peaks, which now tend to be Gaussian, have their profiles affected by diffusivity in the way spoken of in relation to colourless solutes. Hence the differential curves that are obtained by this method are the same for high flow-rates and different for low flow-rat es. Table I1 shows the transducer response to samples measured at high and low flow-rates. At the higher flow-rate, 2.9 ml min-1, the resulting peak heights for the different solutes vary only in accordance with the precision of the method. At the lower flow-rate, 2.2 ml min-l, the peak height for different solutes varies significantly and these variations are found to increase as the flow-rate is lowered.E f e c t of temperature As refractive index is temperature dependent the transducer is sensitive to temperature differences between the sample and the stream. Fig. 13 shows the transducer response for samples of distilled water at temperatures in the range 6 6 0 "C injected into a distilled- water stream a t 16 "C. The development of a high-precision instrument would necessarily include temperature control. Temperature/'C Fig. 13. Transducer response to temperature. Cell design It was anticipated that the sudden change in flow direction on leaving and entering the cell and the change in the cross-sectional area of flow of the stream on leaving and entering the larger type of cell might detract from the analytical precision of the instrument by disturbing the flow in a turbulent manner.Further, as the stream enters and leaves the cell normal to the light path, end errors seem inevitable. However, the precision attributed to flow injection analysis in recent publications7,* has been maintained. In fact, evidence is presented in the theory of refractometric determinations to suggest that the flow charac- teristics remain highly reproducible and laminar.908 BETTERIDGE, DAGLESS, FIELDS AND GRAVES Optimum Conditions for Absorptiometric and Refractometric Determinations Problems arise in absorptiometric determinations owing to the sensitivity of the trans- ducer to differences in refractive index between the sample and the stream.This presents no problem if the samples are all of similar refractive index as the stream can be adjusted by means of an inert solute to give no refractometric response. If, however, large variations in the sample refractive index do occur, there will be a detection limit set when the refracto- metric response becomes significant in the absorptiometric signal. By considering the theory of peak formation it should be realised that the maximum of the absorbance peak coincides with the point where the refractometric peak passes through zero, the base line. If this point on the over-all peak can be ascertained by accurate timing the result should be independent of refractometric considerations.Refractometry Similarly, problems arise in the refractometric method if the sample solutions are coloured. A suitable choice of the colour of the LED used from the wide range available will overcome this problem in most instances. Optimum conditions for high resolution in refractometric determinations occur when the refractive index of the carrier stream is chosen to be in the range of those of the samples. In the same way that differential spectrophotonietry is achieved for absorbance, so differential refractometry can be achieved for refraction. There is in this instance no need to back off the refractive index of the stream as the light reaching the photodetector when the cell contents are homogeneous is the same for all colourless solutes. Streams of all refractive indices are therefore all naturally at the same zero. Differential refractometry achieved in this way allows resolution of 0.1 g 1-1 in any concentration range. Maximum precision is obtained at a flow-rate of approximately 3mlmin-l when the larger type of cell is used. Higher sampling rates are provided a t the greater flow-rate, 320 per hour at 12 ml min-l, when the relative standard deviation is 2.1%. The conditions under which determinations are made should therefore depend on the requirement in terms of precision and speed. We are grateful to the Science Research Council for studentships for B.F. and N.F.G. and to Dr. D. R. Deans for helpful discussions. 1. 2. 3. 4. 5. 6. 7. 8. 9. References RbZiEka, J., and Hansen, E. H., Analytica Chim. Acta, 1976, 78, 145. RbiiEka, J., and Stewart, J. W. B., Analytica Chim. Acta, 1975, 79, 79. Stewart, J. W. B., RbiiCka, J., Bergamin Filho, H., and Zagatto, E. A., Analytica Chim. Acta RbiiEka, J., Stewart, J. W. B., and Zagatto, E. A., Analytica Chim. Ada, 1976, 81, 387. Stewart, J. W. B., and RbZiEka, J., Analytica Chim. Acta, 1976, 82, 137. Hansen, E. H., and RbZiEka, J., Analytica Chim. Acta, 1976, 87, 353. Hansen, E. H., RbiiEka, J., and Rietz, B., Analytica Chim. Acta, 1977, 89, 241. RbiiEka, J., Hansen, E. H., and Zagatto, E. A,, Analytica Chim. Acta, 1977, 88, 1. Weast, R. C., Editor, “Handbook of Chemistry and Physics,” 53rd edition, CRC Press, Cleveland, 1976, 81, 371. Ohio, 1972. Received February 13th, 1978 Accepted April 6th, 1978

ISSN:0003-2654    

DOI:10.1039/AN9780300897

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

Analjlst, September, 1978, VoL. 103, p@. 909-915 909 Determination of Lead in Irons and Steels by Atomic-a bsorption Spectrophotometry with the Introduction of Solid Samples into an Induction Furnace David Godfrey Andrews, Abdullah M. Aziz-Alrahman and James B. Headridge Department of Chemistry, The University, Shefield, S3 7HF Atomic-absorption spectrophototnetry with an induction furnace has been used for the determination of 0.1-100 p g g - l of lead in 1-12-mg samples of irons and steels dropped into the furnace. Calibration graphs of peak absorbance veYsus mass of lead have been constructed by using standard steels. Samples of alloys can be added to the furnace a t 4-5-min intervals. Information is presented on the calibration graphs and on the accuracy, precision and limits of detection of the method for 44 irons and steels.With steels containing more than 1 pg 8-l. of lead relative standard deviations are usually <lo%. The limit of detection for lead is ~ 0 . 0 5 pg g-l when using this method. Keywords : Lead determination ; iron analysis ; steel analysis ; atomic- absorption spectrophotometry ; induction furnace The presence of low concentrations of lead can cause problems with austenitic stainless steels. Edge cracking in ingots of 18Cr-8Ni steel results from the presence of 5Opgg-1 of leadl and to prevent hot cracking during forging and rolling of stainless steels a maximum lead content of 30 pg has been stipulated.2 Also, a reduction in notched tensile strength is produced by 5Opgg-1 of lead in maraging steel^.^ The presence of lead impairs the formation of spheroidal graphite in cast iron treated with magnesium and a maximum content of 20 pg g--l has been suggested.4 Therefore, there is a need for methods of deter- mination of lead in irons and steels at concentrations of less than 100pgg-l and many papers have been published on this subject.Information on methods for the determination of lead in steels is presented in Table I. TABLE I METHODS FOR THE DETERMINATION OF LEAD IN STEELS Method D.c. arc emission after pre-concentration by precipitation of PbS in CuS .. . . .. .. .. .. .. .. Absorption spectrophotometry of Pb - dithizone complex after solvent extraction . . .. .. .. .. .. .. Differential cathode-ray polarography after solvent extraction . . Anodic stripping voltammetry . .* . .. .. .. Flame atomic-absorption spectrophotometry of Pb(I1) in 4-methylpentan-2-one after solvent extraction . . .. * . Atomic-absorption spectrophotometry after hydride generation. . D.c. arc emission using Li,CO, flux . . .. .. .. .. Atomic-absorption spectrophotometry with carbon furnace atomisation after dissolution of the alloy . . .. .. Concentration range for lead/pg g-’ 0.5-50 0.4-30 6-500 -0.2-500 1-1 60 10-100 7-2 300 0.6-160 0.3-100 NO. 6-1 60 0.1-280 Reference 5 6 7 8 9 10 11 12 13 14 15910 ANDREWS et al. : DETERMINATION OF LEAD IN IRONS AND Analyst, VoE. 103 The methods involving a pre-concentration step are lengthy and, for very low levels of lead, the risk of contamination through lead pick-up increases as the method becomes more involved.Undoubtedly the most convenient method to date appears to be atomic- absorption spectrophotometry with carbon furnace atomisation after dissolution of the alloy in acidic solution. However, at very low trace levels problems can still arise from minute concentrations of lead in acid and in the plastic tips of rnicropipettes.l5 These problems are eliminated when a direct analysis is carried out on solid samples. Andrews and Head- ridge16 have determined bismuth in steels and cast irons by means of atomic-absorption spectrophotometry with the introduction of solid samples into an induction furnace. A similar procedure is now described for the determination of 0.1-100 pg gf of lead in 44 irons and steels. However, whereas standard alloys for bismuth were not available, many alloys standardised for lead content were used in this study and a proper assessment of the accuracy of the induction-furnace method was possible and is reported together with detailed information on precision.Experimental Reagents and Materials British Chemical Standards, alloys from National Bureau of Standards, USA, alloys from the Institutet for Metallforskning, Sweden, and cast irons from the British Cast Iron Research Association. These should preferably be millings or turnings of irons or steels so that no more than three pieces need to be added to the furnace core at the same time. Powdery millings are less suitable, for invariably such samples lead to greater scatter in the results. Lead nitrate. Analytical-reagent grade, dried overnight under vacuum at room tempera- ture.Nitric acid (sp. gr. 1.42). Analytical-reagent grade. Standard lead nitrate solution A (1 000 pg ml-1 of lead). Dissolve 1.599 g of lead nitrate Standard lead nitrate sohtion B (100 pg ml-l of lead). Dilute 10 ml of lead nitrate solution Standard irons and steels. Samples for analysis. in 100 ml of 1% V/V nitric acid and dilute to 1 1 with 1% V/V nitric acid. A to 100 ml with 1% VlV nitric acid. Apparatus and Method for Determining Absorbances for a Series of Solid Samples The apparatus and the method were identical with those previously describedls except that towards the end of the investigation the graphite core and side-arms were made from Ultra “F” Purity Graphite, type UF-BS (Ultra Carbon, USA). Graphite cores and side-arms were usually baked for 1 h under vacuum at about 1300 “C before use.The resonance lines at 261.4 and 283.3nm from a lead hollow-cathode lamp (Activion Glass Ltd.) were used with a slit width of 0.2 nm on a Perkin-Elmer 300s atomic-absorption spectrophoto- meter. The experimental conditions for the determination of lead are shown in Table 11. TABLE I1 EXPERIMENTAL CONDITIONS FOR THE DETERMINATION OF LEAD Concentration Mass range of Core temperature/ Wavelength/ Damping rangelpg g-l samplelmg O C nm position* 20-100 2-12 2 000-2 100 261.4 1 1-20 1-12 1 820-2 020 283.3 4 <1 4-12 1 950-2 050 283.3 1 * Damping positions 1 and 4 are for time constants of 0.2 and 10 s, respectively. Calibration Graphs For the determination of lead in steels containing 20-100pggl of lead, calibration graphs of peak absorbance versus amount of lead are obtained by dropping increasing amounts of mild steel BCS 327, which contains 105pgg-l of lead, into the graphite core under conditions capable of producing absorbances up to 1.0 (see Table 11).For irons and steelsSeptember, 1978 STEELS BY ATOMIC-ABSORPTION SPECTROPHOTOMETRE- 911 containing <20 pg g-l of lead, a calibration graph is prepared in a similar way using the standardised steels JK 2C (0.2% of carbon, 4.2 pg gl of lead), SRM 362 (low-alloy steel, 4.3 pg g--l of lead) or BCS 337 (austenitic stainless steel, 12 pg g-l of lead). BCS 337 is less suitable than JK 2C or SRM 362 for a calibration graph when the concentration of lead in the sample to be analysed is expected to be less than 1 pug g-1.Calibration graphs can also be obtained for the range 0-2 pg of lead using aqueous solu- tions, core temperatures of from 1750 to 2 150 "C and the lead line at 261.4 nm. Suitable volumes of standard lead nitrate solution B are dispensed from a 10-pl syringe on to carbon discs (3 mm thick x 6.5 mm diameter) cut from Specpure carbon rod, these discs having been previously baked under vacuum at 1400 "C. The discs are dried under an infrared lamp for 30 s and then dropped into the graphite core. Determination of Lead in Irons and Steels When a series of irons and steels is to be analysed, suitable masses are dropped into the graphite core over a period of 2-3 h and, during the same run, various masses of BCS 327, BCS 337, JK 2C or SRM 362 are also added, generally at the beginning of the run, for the purpose of constructing a calibration graph.During a run the temperature of the core should not alter by more than &lo "C. When the run is completed the calibration graph is drawn and the mass of lead in each sample is obtained from the graph. The concentrations of lead in the samples are then calculated. Results The method of direct analysis of solid samples was so sensitive that the normal resonance line at 283.3 nm could not be used in the construction of a calibration graph for the range 0-2 pg of lead for the determination of concentrations of lead in the range 20-100 pg g-l. The less sensitive line at 261.4nm was used instead. The resulting calibration graph passed through the origin and was slightly curved at the upper end.A typical calibration graph is shown in Fig. 1. The resonance line at 283.3 nm was employed with damping position 4 to construct a calibration graph for the range 0-100 ng of lead for the determination of concentrations of lead between 1 and 20 pg g-l. Because of the damping the calibration graph was a curve that passed through the origin. Calibration graphs constructed for the range 0-25 ng of lead, with no damping and using the 283.3-nm line, in Such a graph is shown in Fig. 2. /x L I 1 0.5 1 .o 1.5 2.0 Mass of lead/pg Fig. 1. Calibration graph for the range 20-100 pgg-1 of lead in steels prepared from BCS 327 using the 261.4-nm line and a core temperature of 2050 "C. 0.8 0.6 W S -F 2 0.4 2 0 20 40 60 80 Mass of leadhg Fig.2. Calibration graph for the range 1-20 pg g;1 of lead in steels prepared from J K 2C using the 283.3-nm line and a core temperature of 1970 "C.912 ANDREWS et aZ. : DETERMINATION OF LEAD IN IRONS AND Analyst, VoZ. 103 0.6 X X 8 i : 0.4 +? 2 a 52 0.2 - x/dx/ I I I 0.3 i-- $ 0.2 - 0.4 0 0.5 1 .o 1.5 2.0 Mass of lead/pg Fig. 5. Calibration graphs for A, 0.3-1.8 pg of lead as lead nitrate and B, 0.3-2.0 pg of lead from BCS 327 using the 261.4-nm line and a core temperature of 2 100 "C. Samples of several series of irons and steels were dropped into the furnace in order to determine their lead contents, using the conditions outlined in Table 11. Results are shown in Tables 111, IV and V. The limit of detection of the method was 0.05pgg1, calculated as twice the standard deviation obtained for the determination of 15 samples of SRM 365 using SRM 361 for the calibration graph and x 5 scale expansion (see Table V).Discussion The results in Table I11 for the determination of lead in mild steels are in good agreement with the certificate values. The same applies for results in Table IV when a comparisonSeptember, 1978 STEELS BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY TABLE 111 RESULTS FOR THE DETERMINATION OF LEAD IN MILD STEELS CONTAINING 20-100 pg 8-l OF LEAD Calibration graph prepared using BCS 327 (105 p g g-’ of lead). BCS alloy 271 272 273 274 275 276 277 330 456 457 459 460 Lead reported (certificate value) / 25 30* 30* 85 50 65* 65* 30 100 60 30 30 Pi3 g-’ Lead found/ 23 31 30 83 56 78 78 28 98 64 26 24 P8 g-’ Number of samples analysed 6 G 6 5 11 6 6 8 8 7 8 7 Relative standard deviation, % 3 5 10 5 5 7 4 6 6 0 11 13 *Not fully standardised.RESULTS FOR THE DETERMINATION OF LEAD IN IRONS AND STEELS CONTAINING 1-20 pg K1 OF LEAD Calibration graphs prepared using JK 2C (4.2 pg g-1) of lead except for results marked. Alloy BCS 320 BCS 321 BCS 322 ECS 323 BCS 324 BCS 325 BCS 331 BCS 332 BCS 333 BCS 334 BCS 335 BCS 336 BCS 338 BCS 339 BCS 340 BCS 341 BCS 342 BCS 451 BCS 452 BCS 453 BCS 454 BCS 455 SRM 362 SRRl 363 JK 2C D5 D8 Type Mild steel Mild steel Mild steel Mild steel Mild steel Mild steel Austenitic stainless Austenitic stainless Austenitic stainless Austenitic stainless Austenitic stainless Austenitic stainless Austenitic stainless Ferritic stainless Ferritic stainless Ferritic stainless Ferritic stainless Mild steel Mild steel Mild steel Mild steel Mild steel AISI 94B17 steel (modified) Cr-V steel (modified) 0.2% C steel Cast iron Cast iron 11 16 7 Lead reported (certificate value) / Lead found/ PLg i3-1 3.9 2.6 1.4 1.0 1.2 2.3 5.9 8.2 6.5 11* 13* 6.9 5.7 5.9 5.6 7.2 2.2 3.8 5.5 3.5 4.3t 3.8 4.2 11 13 22$ 19* 4 7 187 4.25 4.211 4.1* 19* 913 Number of samples analysed 6 G 6 5 6 6 5 5 6 6 6 6 6 6 6 6 5 6 6 6 6 6 G 10 5 10 G G Relative deviation, yo 14 5 6 5 15 6 6 5 2 5 8 13 3 4 4 2 3 9 5 5 8 15 10 6 4 9 4 9 * Calibration graph prepared using BCS 337 (12 pg g-l of lead).t In the form SKM 1262.’’ In the form SRM 1263.” 5 Not fully standardiscd. 7 Value from British Cast Iron Research Association. 11 Calibration graph prepared using SRM 362 (4.3 pg 8-l of lead).914 ANDREWS et at?.: DETERMINATION OF LEAD IN IRONS AND Analyst, Vol. 103 with certificate values is possible. There is every reason to believe that the other results for lead shown in Table IV are accurate. It will be seen from Tables I11 and IV that the relative standard deviation of the method at these levels is usually <loyo. The values of 105 pg c1 for lead in BCS 327 and of 4.2 pg g1 for lead in JK 2C are the averages of eleven and three determinations, respectively, reported on the certificates. TABLE V RESULTS FOR THE DETERMINATION OF LEAD IN IRONS AND A STEEL CONTAINING <1 pg g-l OF LEAD The numbers in parentheses are the numbers of samples analysed. Lead content Lead Relative Lead Relative Lead Relative (certificate found*/ standard found?/ standard found$/ standard Material Type value)/pg g-' pg g-l deviation, yo pg g-l deviation, "/o pg g-I deviation, % SRM 365 Electrolytic iron 0.1511 0.16 20 (8) 0.13, 0.15 12(6), 19(18) 0.14 JK 1C Iron 0.57 0.26 BCS 260/3 High-purity iron 0.38 19 (15) 0.22 15 (6) 0.24 IS (14) SRM 361 AISI 4340 steel 0.258 0.29 11 (10) 0.25 13 (6) ii &) 0.33 47 (6) BCS 14913 High-purity iron 1.0 15 (7) 0.78 18 (6) * Calibration graph prepared using SRM 362 (4.3 pg g-1 of lead).? Calibration graph prepared using JK 2C (4.2 pg g-l of lead). $ Calibration graph prepared using SRM 361 (0.25 pg g-l of lead). § In the form SRM 1261." 11 In the form SRM 1265." fi Single determination. The results in Table V for the determination of lead in SRM 361 and SRM 365, using both SRM 362 and JK 2C for the calibration graphs, are in good agreement with thecertifi- cate values.The values reported in the table for the determination of lead in JK lC, using the proposed method, are in satisfactory agreement with a value of 0.3 pgg-l recently reported by Frech.13 The precision of the method is not as good for levels of lead below 1 pg g-l as it is for higher levels of lead, but it is still acceptable except for BCS 260/3. The relative standard deviations of 46 and 47% for this iron suggest that the lead is inhomo- geneously distributed in the material. The limit of detection of 0.05 pg g1 reported above is almost certainly too conservative. Absorbances in the region of 0.04 were obtained for the 15 samples of SRM 365 that were analysed in order to calculate the limit of detection.How- ever, no sample of iron could be found that contained less than 0.13 pg g-l of lead. If a sample had been available to produce absorbances in the region of 0.01, it seems likely that the limit of detection would have been found to be 0.02 pg g-l of lead or even lower. The amount of lead producing 1% absorption of the 283.3-nm fine was approximately 0.17 ng at 2000 "C. The method is straightforward. Weighed turnings or millings of an iron or steel are added to the furnace at 4-5 min intervals. Most of the work was done with cores and side- arms machined from the same batch of AGW-grade graphite (British Acheson Electrodes), which, after heat treatment in a vacuum, showed no lead background. However, the next batch of AGW-grade graphite produced a high lead background, which could not be removed even after extensive baking under vacuum at 2 200°C.Therefore it was necessary to finish the work with a core and side-arms made from Ultra "F" Purity Graphite, type UF-4S (Ultra Carbon). All the results reported in Tables 111, IV and V were obtained from calibration graphs prepared by using standardised irons or steels. However, in the absence of a suitable iron or steel standard, it should be possible to construct a calibration graph from standard solutions of lead nitrate at 2 120-2 150 "C. We are indebted to the BSClBISPA Chemical Analysis Committee and the British Steel Corporation for a grant to buy the Perkin-Elmer 300s atomic-absorption spectrophoto- meter.September, 1978 STEELS BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY References 915 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Bergh, S., Iron Age, 1949, 164, 96. Pruger, T. A., Blake, F., and Valley, J. A., Spec. Tech. Publs Am. SOC. Test. Mater., 1967, 418, 24. Mayer, G., and Clark, C. A., Metall. Mater. Technol., 1974, 491. Donoho, C. K., Mod. Cast. (New Technol. Sect.), 1964, 608. Balfour, B. E., Jukes, D., and Thornton, K., AppZ. Spectrosc., 1966, 20, 168. Atwell, M. G., and Golden, G. S., AppZ. Spectrosc., 1973, 27, 464. Postlethwaite, R. T., Kidman, L., Bagshawe, B., Bills, K. M., Harrison, T. S., and Watt Smith, hlaienthal, E. J., Am. Lab., 1973, 5, 25. Metters, B., and Cooksey, B. G., Analyst, 1974, 99, 457. Hofton, M. E., and Hubbard, D. P., Analytica Chim. Acta, 1970, 52, 425. Fleming, H. D., and Ide, R. G., Analytica Chim. Acta, 1976, 83, 67. Shaw, F., and Ottaway, J. M., Analyst, 1974, 99, 184. Frech, W., Analytica Chim. Acta, 1975, 77, 43. Barnett, W. B., and McLaughlin, E. A., Analytica Chim. Ada, 1975, 80, 285. Dulski, T. R., and Bixler, R. R., Analytica Chim. Acta, 1977, 91, 199. Andrews, D. G., and Headridge, J. B., Analyst, 1977, 102, 436. “Catalog of NBS Standard Reference Materials,” 1975-76 Edition, NBS Special Publication 260, Received February 23rd. 1978 Accepted March 30th, 1978 J. A., J . Iron Steel Inst., 1970, 208, 500. National Bureau of Standards, Washington, D.C.

ISSN:0003-2654    

DOI:10.1039/AN9780300909

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

916 Analyst, September, 1978, Vol. 103, pp. 916-920 Measurement of Silver in Blood by Atomic-absorption Spectrophotometry Using the Micro-cup Technique Catherine Howlett and Andrew Taylor" South West Thames Regional Heavy Metals Reference Laboratory, Department of Biochemistvy, Univevsity of Surrey, Guildford, Surrey, GU2 5XH A rapid and precise method for the measurement of silver in blood by atoniic- absorption spectrophotometry using the micro-cup (Delves) technique is described. The limit of detection is 2.7 ng ml-l and the calibration graph is linear up to a concentration of 400 ng ml-l. In 30 subjects industrially exposed to fine silver dust the silver concentration in their blood was less than 2.7 ng ml-l, so that the principal use of the method will be in detecting cases of excessive exposure to this metal.P' Keywords : Blood aizalysis ; silver determination ; micro-cup atomic-absorption spectrophotornetry ; battery plate manufacture There is little information about the effects of silver in man apart from the production of a characteristic blue - grey skin coloration in argyria, which is the condition produced by acute or chronic exposure to gram amounts of silver. Experimental studies with animals have indicated that silver has varied toxic effects, causing muscular dystrophy, microcytic anaemia and heart enlargement in the turkey.l It is deposited in the kidney of the rat.2 With the increasing awareness of its possible toxicity and the use of silver in industry, of silver amalgams in dentistry3 and of silver-containing ointments (such as Viacutan and silver - zinc allantoin powder) in the topical treatment of burns,4 it may become important to monitor human blood levels.Exposure to silver is uncommon but in the manufacture of silver plates, required for certain types of specialised batteries, the surfaces of work benches and tables become covered with a fine silver dust. Persons working with this manufacturing process are liable to contaminate their fingers and clothes with silver dust and run a considerable risk of ingestion. We were requested, therefore, to examine blood or plasma for its silver content in order to assess the extent of exposure. Published methods for the determination of silver in biological materials involve the use of equipment that is not generally available in clinical laboratories, although atomic- absorption spectrophotometry is frequently used for the measurement of silver in metals and in chemicals.General methods for the determination of trace metals in biological materials by atomic-absorption spectrophotometry involve time-consuming sample-prepara- tion procedure~.~$6 As a large number of samples was expected, a rapid method with minimum sample preparation was desirable and, as silver has a relatively high vapour pressure, the micro-cup technique described by Delves' was investigated. Experimental Apparatus An Instrumentation Laboratory 353 atomic-absorption spectrophotometer fitted with the micro-cup assembly was used without background correction. Nickel cups were obtained from Electronic Development Co., Compton, Surrey, and an alumina absorption tube with a square hole from Thermal Syndicate, Wallsend, Tyne and Wear.The instrument conditions were as follows: wavelength, 328.1 nm; lamp current, 6 mA; slit width, 320 pm; photomultiplier voltage, 530 V; scale expansion, x a; and recorder voltage, 20 mV. Reagents Standard silver solutions. * To whom correspondence should be addressed. Prepare from silver nitrate a stock solution containing 500HOWLETT AND TAYLOR 91 7 pg ml-l of silver in 1% V/V nitric acid. Dilute the stock solution with distilled water to give an intermediate standard containing 5 pg ml-l of silver and, by further dilution, working standards containing 10-100 ng ml-l. Control blood samples. To well mixed normal human blood add silver nitrate solution to give silver concentrations of 10 and 50 ng ml-l.Procedure Preparation of calibration solutions a hot-plate at 150 "C. cup and again dry the cups at 150 "C. Pipette 50 pi of well mixed normal human blood into each nickel cup and dry the cups on Add 50 p1 of a working standard silver solution to the blood in each Preparation of samples and controls Pipette 50 p1 of well mixed blood into a nickel cup and dry the cup at 150 "C. Preliminary studies indicated an equal distribution of silver between blood cells and plasma. Either material could, therefore, be used for the measurement of silver but with a blood matrix the signal returns to the base line more quickly and smoothly than with a plasma matrix. Whole blood was, therefore, used for all measurements of silver.Analysis Place the cups in the holder and introduce it into the stoicheiometric air - acetylene flame, recording the signal on a chart recorder with a chart speed of 20 mm min-l. Assay all standards, controls and samples in triplicate. Animal Experiments Six male Wistar albino rats weighing about 200 g were given 1, 4 or 8 mg of silver (in a volume of 50 p1) on each of two days by subcutaneous injection of silver nitrate solution. Blood was collected by cardiac puncture on the third day and the silver concentration assayed by the micro-cup technique. Fig. 1. Absorption signals showing the identical atomisation of silver from prepared standards (peaks A and B; silver concentration 100 and 60 ng nil-l, respectively) and from silver-treated rats (peaks C and D; silver concentration 124 and 110 ng ml-l, respectively).Non-atomic absorption due to combustion products is indicated by the arrows.918 HOWLETT AND TAYLOR: MEASUREMENT OF SILVER IN Results Analyst, VoZ. 103 Recordings of the absorption signals for calibration standards and blood from silver- treated rats are shown in Fig. 1. There was a clear separation of the absorption due to silver atoms from the non-atomic absorption due to combustion products of the sample. Background correction was, therefore, unnecessary. Endogenous silver atomised in a manner identical with that in which the silver used to prepare standards atomised. Recordings of the absorption signals obtained for a calibration standard, untreated blood and an empty nickel cup are shown in Fig.2. The raised base line following atomisation of silver as seen in A is also seen in B and C, indicating that the “tail” following the peak is due to the presence of the nickel cup and not to simultaneous volatilisation of matrix material and silver. 0.5 0.4 8 0.3 S m -P a n 0.2 0.1 0 Measurements of peak height were therefore made from this base line. Fig. 2. Absorption signals obtained by heating a nickel cup containing prepared standard (A; silver concentration 100 ng ml-l), untreated blood (B; no detectable silver) and an empty nickel cup (C). (The expected position of a silver peak is marked with an arrow.) Recovery was between 98 and 110% (Table I). The recovery of silver added to whole blood to give concentrations of 10,20 and 50 ng ml-1 TABLE I RECOVERY OF SILVER FROM BLOOD Silver added/ Silver measured/ Recovery, ng ml-1 ng ml-l % 10 11 110.0 30 31 103.3 50 49 98.0 Precision samples of blood with added silver are given in Table 11.The results of 15 replicate determinations, within batch and between batch, for two Linearity of Calibration Graph The calibration graph was linear up to a concentration of 400 ng ml-l (Fig. 3).September, 1978 BLOOD BY ATOMIC-ABSORPTION SPECTROPHOTOMETRY TABLE I1 919 PREC: ;ION OF REPLICATE ANALYSES OF BLOOD WITH ADDED SILVER Amount of silver added: sample 1, 10 ng ml-l; sample 2, 60 ng ml-1. Recovery of silver Within batch (n = 16) Between batch (n = 16) ' A h r \ I Standard Coefficient of Standard Coefficient of Sample Mean/ deviation/ variation, Mean/ deviation/ variation, 1 9.8 0.68 6.9 10.6 0.93 9.0 2 50.9 2.96 5.8 48.3 2.85 6.0 No.ngml-1 ngml-l % ng ml-l ng ml-1 % Limit of Detection Using a sample with a silver concentration of 10 ng ml-l, the limit of detection, defined as twice the standard deviation of replicate determinations of a sample with a low concentration of silver, was 2.7 ng ml-l or 1.35 x g of silver. Age of Cups The signals obtained by repeated measurement of silver in blood using a single nickel cup showed a gradual decrease in sensitivity. The decrease in peak height was not sufficient to render the cup useless but it did mean that it was essential to match cups in order to achieve an acceptable precision. Discussion Methods that have been used to measure silver in organic material include atomic- absorption spectrophotometry, plasma emission spectrophotometry,* neutron-activation analysis0 and spectrophotometry.1° Other methods used for the determination of trace metals in biological fluids could be adapted for the measurement of silver in blood, but all require some form of sample preparation prior to presentation to the instrument.This 150 E E -. + L m r Y (IJ a 'a, 100 50 0 200 400 600 8C Concentration of silver/ng rnI--' Fig. 3. Calibration graph showing linearity of response to concentration of silver.920 HOWLETT AKD TAYLOR preparation varies from lengthy and intricate procedures for X-ray fluorescence analysis to rapid and simple extractions for anodic-stripping voltammetry, but with all of these pro- cedures there are risks of sample contamination. The method described here is both rapid and simple, requiring no preparation except for pipetting and drying the sample.Further, it requires neither highly specialised nor very expensive equipment and can be performed on any atomic-absorption spectrophotometer with a Delves micro-cup attachment. The published limits of detection for measurements of silver in biological fluids vary considerably, the lowest being 2.5 ng ml-l using plasma emission spectroscopy.8 The limit of detection of the method described here is 2.7 ng ml-l, which compares favourably with the above. The few results available for silver concentrations in the blood of unexposed persons show a wide variation. Using plasma emission spectroscopy, Nakashima et aZ.8 reported 10-20 ng ml-1, whereas Addink,ll using a spectrophotometric technique, suggested 190 ng ml-1.We were unable to confirm these results; our series of 30 subjects who had been industrially exposed to fine silver dust had silver levels in their blood below the limit of detection of the method. Conclusion The micro-cup technique affords a rapid and precise method for the measurement of silver in blood. Although it is not sufficiently sensitive to determine the concentration of silver in normal blood, the low limit of detection indicates that when the exposure has been significant this method is satisfactory for the measurement of silver in blood and its main use will therefore be in detecting cases of excessive exposure. 1. 3. 4. 5. 6. 7. 8. 9. 10. 11. r) Y. References Jensen, L. S., Peterson, R. P., and Falen, L., Poult. Sci., 1974, 53, 57. Creasey, M., and Moffat, D. B., Experientia, 1973, 29, 326. Dubrow, H., J . Am. Dent. Ass., 1976, 93, 976. Klippel, A. P., Margraf, H. W., and Covey, T. H., J . Am. Coll. Emergency Physicians, 1977, 6, 184. Gorsuch, T. T., Analyst, 1959, 84, 135. Rooney, R. C., Analyst, 1975, 100, 471. Delves, H. T., Analyst, 1970, 95, 431. Nakashima, R., Sasaki, S., and Shibata, S., Analytica Chim. Acta, 1975, 77, 65. Kawabuchi, K., and Riley, J. P,, Analytica Chim. Acta, 1973, 65, 271. Lai, M. G., and Weiss, H. V., Analyt. Chem., 1962, 34, 1012. Addink, N. W. H., Red Trav. Chim. Pays-Bas Belg., 1951, 70, 168. Received February lStA, 1978 Accepted April 24th, 1978

ISSN:0003-2654    

DOI:10.1039/AN9780300916

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

Analyst, September, 1978, Vol. 103, pp. 921-927 921 Determination of Copper in Plasma Ultrafiltrate by Atomic-a bsorption Spectrometry Using Carbon Furnace Atomisation H. Kamel,* J. Teape, D. H. Brown, J. M. Ottawaytand W. E. Smith Department of Pure and Applied Chemistry, University of Strathclyde, Cathedral Street, Glasgow, GI 1XL A method for the direct determination of the level of copper in plasma ultra- filtrate using atomic-absorption spectrometry with carbon furnace atomisation is reported. The relative standard deviation and the detection limit expressed as 26 were 3.6% (50 pl of 0.05 p g ml-l copper solution) and 0.0036 p g ml-l, respectively. Interferences from the salt matrix, the use of background correction and the problems of contamination caused by the low level of copper are discussed.The method has been applied to the determination of the level of copper in the plasma ultrafiltrate of patients with rheumatoid arthritis. Keywords : Copper determination ; plasma ultrafiltrate ; atomic-absorption spectrometry ; carbon furnace atomisation Plasma copper levels have been measured in many studies concerned with metabolism and disease.l Although it is more difficult to determine the copper distribution in various plasma fractions, enough results have been reported to enable a general picture of the distribution to be obtained.lS2 The major copper-containing fraction in plasma is the a2 globulin, ceruloplasmin, which contains about 90% of the copper. The remainder is present mainly in ultrafiltrate and on albumin, although in some animal studies copper bound to other plasma fractions has been rep~rted.~ Changes in the plasma copper level are dominated by changes in ceruloplasmin copper and the effect of disease on ultrafilterable copper has to be studied by analysis of the ultrafilterable fraction itself.The low level of copper makes such analysis difficult and many previous studies have relied on a subtraction of the plasma and ceruloplasmin levels* or a measurement of the total copper that will react with thio- carbamates5 in order to obtain a level that may approximate to that in the ultrafiltrate. The indications from these studies are that such levels do change appreciably in disease processes. This paper reports a relatively simple, sensitive and reliable method for the determination of copper in plasma ultrafiltrate by using atomic-absorption spectrometry with carbon furnace atomisation.Atomic-absorption spectrometry using flame atomisation has been used for the deter- mination of copper in urine samples.6 The method requires pre-concentration of the copper in the samples and extraction into organic solvents prior to aspiration into the flame. Another flame atomic-absorption spectrometric method for the direct determination of copper in urine has been reported' that requires 15 x scale expansion to achieve an appreciable signal. Special procedures and rigid control of the instrument are required in order to suppress the noise signal during the measurement. A related measurement, that of direct- reacting copper, taken to be all copper in plasma except that in ceruloplasmin, can be obtained by chelation of the copper with ammonium tetramethylenedithiocarbamate, extrac- tion of the chelate into n-butyl acetate and aspiration of the organic extract into the The use of acid is avoided in this technique in order to prevent splitting of tightly bound copper from copper proteins.In spite of their simplicity and sensitivity, the use of electrothermal atomisers in atomic- absorption spectrometry has not so far been reported for the direct determination of copper in plasma ultrafiltrate, possibly because of interference problems caused by the highly concentrated salt matrix. Separation of the analyte from the salt matrix prior to analysis is possible either by electrochemical deposition of the required element on either graphite8 * Present address : Water Resources Development Centre, P.O.Box 12020, Kuwait. To whom all correspondence should be addressed.922 KAMEL et al. : DETERMINATION OF COPPER IN PLASMA Analyst, VoZ. 103 or platinum electrodes: followed by direct insertion of the electrodes into the furnace, or by the use of a higher ashing temperature so that much of the matrix is volatilised out of the furnace prior to atomisation of the analyte.10 The former method is complex and the latter results in a marked decrease in sensitivity, probably owing to co-volatilisation of the trace elements. Determination of copper at trace concentrations in biological fluids is difficult, particularly because of problems due to contamination. Consequently, pre-concentration steps implicit in most of the above methods of analysis are undesirable and a direct analysis technique using carbon furnace atomisation would have a distinct advantage, provided that the problems of matrix interference can be overcome.Recently, a study of the interferences from a salt matrix in the determination of copper and manganese in biological fluids by using carbon furnace atomic-absorption spectrometryll suggested that such a method might be possible, provided that a controlled heating stage was included in the procedure prior to atomisation in order to remove the non-atomic absorption signal due to the biological matrix. However, in the determination of copper in biological calcification and tissue samples11 a chemical extraction of the elements or a standard additions technique was required and sensitivity was not as important as in the determina- tion of copper in plasma ultrafiltrate.Experimental Reagents Aqueous standard solutions were prepared with de-ionised water. Copper contamination is always a possibility in the determination of trace amounts and therefore care should be taken in handling the reagents and regular checks of the level of copper in the reagents and in the de-ionised water should be carried out. Prepared from AnalaR copper nitrate diluted with de-ionised water. All the materials used were of the highest purity available. Copper stock solution, 1000 pg ml-l. Medical saline solution (BDH) containing 0.9% m/V of sodium chloride. Nitric acid, 0.2 M . Prepared from Aristar nitric acid and de-ionised water.Preparation of Aqueous Standard Solutions of Copper A 0.5-ml volume of the 1000 pg ml-1 stock solution of copper was transferred into a 100-ml calibrated flask and made up to the mark with Of this solution 0, 0.05, 0.1, 0.15 and 0.2-ml volumes were transferred into 10-ml calibrated flasks and made up to the mark with This procedure gave aqueous standard solutions containing 0, 0.025, 0.05, 0.075 and 0.1 pg ml-l of copper, respectively. A set of standard solutions containing 0, 0.05, 0.1, 0.15, 0.2 and 0.25pgml-1 of copper in 1 0 - 2 ~ nitric acid was prepared in a similar manner. M nitric acid. M nitric acid. Preparation of Saline Standard Solutions of Copper A 0.5-ml volume of the 1000 pg ml-1 stock solution of copper was transferred into a 100-ml calibrated flask and made up to the mark with de-ionised water to give a working standard solution of copper with a concentration of 5 pg ml-l.Of this solution 0, 0.05, 0.1, 0.15 and 0.2-ml volumes were transferred into 10-ml calibrated flasks and made up to the mark with medical saline solution (0.9% m/V sodium chloride solution), giving standard solutions with concentrations of 0, 0.025, 0.05, 0.075 and 0.1 pg ml-l of copper, respectively. A similar procedure was adopted to prepare a second set of standard solutions of concentrations 0, 0.05, 0.1, 0.15, 0.2 and 0.25 pgml-l of copper. Plasma Ultrafiltrate Solutions for Standard Additions Technique Volumes of 0, 0.025, 0.05 and 0.075 ml of the 5 pg ml-l working standard solution of copper were transferred into 5-ml calibrated flasks and made up to the mark with ultra- filtrate from the same plasma sample.September, 1.978 ULTRAFILTRATE BY ATOMIC-ABSORPTION SPECTROMETRY 923 Ultrafiltration A standard Millipore ultrafiltration cell, which utilises a disc-shaped membrane (Millipore PSAC type) 25 mm in diameter, of nominal molecular mass cut-off 1000, was used.All parts of the apparatus, including the gaskets, ultrafiltration cell and screen, were soaked in 1 M nitric acid for 24 h and then washed thoroughly with de-ionised water. The filter- membranes and apparatus were soaked in de-ionised water overnight and the solution was measured for copper by injection into the carbon furnace. Negligible amounts of copper were found ((0.005 pg ml-l). Apparatus The instrument used was a Perkin-Elmer 306 atomic-absorption spectrometer equipped with an HGA-72 heated graphite tube atomiser and a deuterium-arc background corrector.Atomisation signals were measured on a Servoscribe RE 511 strip-chart recorder. A Perkin- Elmer manganese hollow-cathode lamp was used as the source. Samples were transferred into the centre of the furnace by using 2,10,20 or 50 -p1 Oxford micropipettes. The gas-stop facility at the atomisation stage and the background corrector were used throughout. The operating conditions were as follows: wavelength, 324.7 nm; lamp current, 8 mA; spectral band width, 0.7 nm; drying temperature, 120 "C; drying time, 30 s ; ashing tempera- ture, 800 "C; ashing time, 30 s; atomisation temperature, 2 550 "C; atomisation time, 10 s; argon flow-rate, 1.5 1 min-l at 40 p.s.i.g.Sample concentrations were read from a calibration graph constructed from the results obtained when 20 or 50p1 of the calibration solutions were sequentially injected into the carbon furnace. The standard additions technique was used to investigate the precision of the method and to study interference effects. Analysis of Plasma metry with carbon furnace atomisation. for copper12 and, using the equipment described here, for gold? fold with lov2 M nitric acid. sequentially into the carbon furnace. filtrate. Analyses of plasma samples were also carried out by means of atomic-absorption spectro- Similar methods have been described previously Samples were diluted ten- Five-microlitre aliquots of the diluted samples were injected Instrument parameters were the same as for ultra- Results and Discussion Recent studiesll of interferences in the determination of gold and copper plasma levels by means of atomic-absorption spectrometry using carbon furnace atomisation demonstrated that these interferences were very much reduced by a ten-fold or greater dilution of the sample and that the sensitivity of the method was not seriously affected.However, the lower concentration of copper in plasma ultrafiltrate makes a dilution step less acceptable, both because the level of copper is closer to the detection limit of the method and because the problem of contamination from a dilution procedure is greater at the lower concentration. Blood plasma contains high concentrations of salts, present mainly as sodium chloride.Some cations (Ca2+ and Mg2+) are bound to plasma proteins of relative molecular mass greater than 1000 (the relative molecular mass cut-off of the membrane) and therefore they are separated with the protein during the ultrafiltration process. It seems that the largest components of the salt matrix in ultrafiltrate are sodium chloride and, to a lesser extent, potassium chloride and magnesium chloride.14 The depression of the copper signal caused by chlorides was studied using the atomisation conditions given under Apparatus by injecting 20-p1 aliquots of 0.05 pg ml-1 copper solutions containing various amounts of sodium chloride, potassium chloride and magnesium chloride (Fig. 1 ) . The results of a previous study at lower concentrations of chloridell agree well with those of the present study but in this instance the range is extended to cover the concentrations expected in ultrafiltrate. The depression of the copper signal caused by a mixture of sodium chloride, potassium chloride and magnesium chloride in solution a t concentrations expected in ultrafiltrate (25 pg ml-l of magnesium, 170 pg ml-l of potassium and 3500 pg ml-1 of sodium as chlorides) was found to be between 25 and 30% of the signal924 KAMEL et al.: DETERMINATION OF COPPER IN PLASMA Analyst, VoZ. 103 and a similar figure was obtained for sodium chloride at concentrations of about 9000 pg ml-1 (medical saline solution concentration). Therefore, standard solutions prepared in medical saline solution are preferred to plasma ultrafiltrate standard solutions, for which there is a storage problem, or to aqueous standard solutions.The standard additions method was used to confirm that the decreased copper signal caused by the ultrafiltrate matrix is similar to that with medical saline solutions. u- 30 o 50 250 1001 - 2000 6000 10000 Concentration of saIt/pg rnl-' Fig. 1. Depression of the copper atomic-absorption signal as a function of the concentration of interfering salts: 0, NaC1, A, KC1; and 0, MgCl,. A 2 0 4 aliquot of saline solution containing 0.15 pg ml-l of copper was injected into the carbon furnace and atomised under the conditions described under Apparatus. Without background correction the signal as a function of time consisted of a broad band which, as well as the copper atomic-absorption signal, appeared to include a non-specific absorption signal.When 20 pl of copper-free saline solution was injected into the carbon furnace and atomised under the same conditions, a similar signal was observed but with much smaller peak height. This background absorption is probably due to the overlap in time of the sodium chloride molecular-absorption band with the copper atomic-absorption signal and/or scattering of the radiation from the hollow-cathode lamp by smoke formed by condensation of vaporised salt particles at the colder ends of the carbon tube. These effects were minimised by using background correction and charring of the sample prior to atomisation. Curve A shows the signal obtained during the atomisation of 50 p1 of saline solution without background correction.Curve B shows the result obtained when using the same solution under the same conditions The effect of both is shown in Fig 2. Time/s Fig. 2. Interference effects from 60-pl saline solutions at the copper atomic-absorption line (324.7 nm) : A, without background correction; B, with background correction; C, as B but with charring for 30 s a t 600 "C prior to atomisation; and D, ashing temperature increased to 800 "C for 30 s. Chart speed, 2 cm s-l. Atomisation starts at time zero.September, 1978 ULTRAFILTRATE BY ATOMIC-ABSORPTION SPECTROMETRY 925 as for A but with background correction. Curve C shows the result obtained under the same conditions as for B but including a charring step of 30 s at 600 "C prior to atomisation.The small residual background signal was further reduced by increasing the ashing temperature to 800 "C for 30 s (curve D). A previous study using aqueous standards for plasma analysis indicated that after ashing at 800 "C some loss of signal would be expected and therefore a comparative study of the effect of charring temperatures on the copper atomic-absorption signal was carried out in order to ascertain the temperature at which copper in the salt matrix starts to volatilise. Aliquots of 20 p1 of saline and aqueous solutions containing 0.025 pg ml-l of copper were injected into the carbon furnace and atomised as above using background correction and ashing at different temperatures for 30 s prior to atomisation. The non-specific absorption signal obtained during atomisation of the saline standard solutions of copper after ashing at lower temperatures was measured by setting the monochromator on the copper non-absorbing line at 322.5nm.This signal was subtracted from the signal obtained at the copper absorbing line in each instance. Fig. 3 shows the effect of ashing in aqueous and saline solutions. Comparatively little change occurs in the copper atomic-absorption signal in saline solution between 600 and 800 "C, indicating that 800 "C is a safe, if maximum, tempera- ture for ashing. As the salt matrix is either completely removed by this procedure or reduced to a level at which it is accurately compensated for by the background corrector, direct analysis for copper in this matrix is straightforward.A calibration graph for copper standard solutions added to saline solution was linear over the range used of 0.02-0.1 pg ml-l of copper. A reproducible depression of about 25% compared with aqueous copper standard solutions was obtained at all levels. 6 1 1 1 0 ~~ 400 800 1200 1600 Ash ing tern perat u rePC Fig. 3. Peak height of the copper atomic-absorption signal in aqueous solution, A, and in 0.9% m/V saline solution, B, as a function of ashing temperature. The ultrafiltrate from plasma samples obtained from five different patients with rheumatoid The results, given in Table I, A standard additions check (Table I, Fig. 4) on arthritis was analysed by the procedures described above. are an average of three measurements. TABLE I COMPARISON OF RESULTS OBTAINED BY USING SALINE STANDARD SOLUTIONS AND STANDARD ADDITIONS FOR THE DETERMINATION OF COPPER I N PLASMA ULTRAFILTRATE FROM RHEUMATOID ARTHRITIS PATIENTS Concentration of copper in plasma ultrafiltrate/pg ml-I Sample Using saline standard (patient) solutions Using standard additions 1 0.080 0.078 2 0.110 0.118 3 0.076 0.079 4 0.091 0.092 5 0.124 0.128926 KAMEL et &?. DETERMINATION OF COPPER I N PLASMA Analyst, VOZ.103 81 I 1 0.100 0.0750.050 0.025 0 0.025 0.050 0.075 0.100 Concentration of copper/pg mI-' Fig. 4. Example of the standard additions method for the determination of copper in plasma ultra- filtrate: A, sample 2, 0.12 pg ml-' of copper; B, sample 4, 0.091 pg ml-l of copper; and C, saline copper standards. each sample gave results that were in good agreement.Consequently, the use of O.9y0 m/V saline standard solutions appears to be reasonable as a compensation for the matrix depression, and the procedures described here give a simple and accurate method for the determination of copper in ultrafiltrate. Corroborative analysis carried out by means of standard additions is not a satisfactory check for residual background signal interference, as this would appear as an addition to both sets of results. Therefore, the procedure described here was compared with a procedure using flame atomisation. The ultrafiltrate level of copper in eight separate samples and in eight separate aliquots from a pooled sample was determined by means of both techniques. With flame atomisation the signal size was small even with scale expansion, and a large sample volume (about 1 ml), which was not always available from individual samples of ultrafiltrate, was required.The results were not of sufficient accuracy to make a detailed comparison with those obtained by the carbon furnace technique worthwhile but they were of the same order of magnitude as the latter results. As a further check on the possibility of residual background signal interference, measurements were made at the non- absorbing copper line. No signal was detected for any sample, indicating that the procedure adopted satisfactorily avoids this interference. The procedure adopted appears to be advantageous in view of the major problem presented by contamination in the measurement of low levels of copper. No dilution, pre-concentration or extraction steps are required and the equipment used to separate the ultrafiltrate contains a minimum of biochemical support media from which it is difficult to remove copper contami- nation.The reproducibility obtainable with the instrument for the determination of copper in plasma ultrafiltrate was tested by repeated single analysis for one sample, injecting 20 p1 of the solution into the carbon furnace ten times. The relative standard deviation and the detection limit expressed as 28 were 3.6% (50 ,ul of 0.05 pgml-l copper solution) and 0.003 6 pg ml-l, respectively. Six samples were from normal subjects and 12 were from rheumatoid arthritics undergoing treat- ment at the Centre for Rheumatic Diseases, Baird Street, Glasgow. With one exception, the level of ultrafilterable copper was between 1 and 9% of the plasma value and the average value was 5.3%.Therefore, the level of ultrafilterable copper is a significant component of the non-ceruloplasmin blood copper. The difference between normal subjects (range 0.092- 0.010, mean value 0.041 pg ml-l of copper) and rheumatoid arthritis patients (range 0.124- 0.003, mean value 0.062 pg ml-l of copper) was smaller than that obtained in previous determinations1 and is not significant. However, all the rheumatoid arthritis patients were undergoing drug therapies and much more must be done before the role of small copper molecules in inflammatory processes can be determined. This paper reports a comparatively simple and reliable technique for use in such studies.Delves15 has recently described an electrothermal atomic-absorption procedure for the determination of copper in plasma protein fractions after separation by electrophoresis. Combination of a procedure such as Using the carbon furnace technique, 18 analyses of ultrafiltrate were performed.September, 1978 ULTRAFILTRATE BY ATOMIC-ABSORPTION SPECTROMETRY 927 that developed by Delves and the method described in this paper will allow the concentra- tion of copper in all the major copper-containing fractions of serum to be measured by means of atomic-absorption spectrometry. We thank Professor W. W. Buchanan and Dr. El Ghobarey of the Centre for Rheumatic We thank the Royal Society for a grant Diseases for the samples used and for discussion. in aid to one of us (J.M.O.) for the purchase of the HGA-72. 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. References Sorenson, J . R. J .. 1 l t h Annual Conference on Trace Elements in Environmental Health, Columbia, Henkin, R. I., in Mertz, W., and Cornatzer, W. E., Editors, “Newer Aspects of Trace Elements in Bremner, I., Proc. Nutr. Soc., 1976, 35, 21A. Sorensen, J. R. J., and DiTommaso, D., Ann. Rheum. Dis., 1976, 35, 186. Blomfield, J,, and Macmahon, R. A., J . Clin. Path., 1969, 22, 136. Bayer, W., Analytica Chim. Acta, 1972, 38, 119. Dawson, J. B., Ellis, D. J., and Newton-John, H., Clinica Chim. Acta, 1968, 21, 33. Lund, W., and Larsen, B. V., Analytica Chim. Acta, 1974, 70, 299. Thomassen, Y., Larsen, B. V., Lang, F. J., and Lund, W., Analytica Chim. Acta, 1976, 83, 103. Segar, D. A., and Gonzalez, J . G., Analytica Chim. Acta, 1972, 58, 7. Verbeke, J. S., Michotte, Y., Winkel, P. V., and Massart, D. L., Analyt. Chew., 1976, 48, 125. Muzzarelli, R. A. A., and Rochetti, R., Talanta, 1975, 22, 683. Kamel, H., Brown, D. H., Ottaway, J. M., and Smith, W. E., Analyst, 1976, 101, 790. Diem, K., and Lentner, C., “Documentia Geigy Scientific Tables,” Geigy I.R., Basle, 1974. Delves, H. T., Clinica Chim. Acta, 1976, 71, 495. Missouri, June 1977, to be published. Nutrition,” Marcel Dekker, New York, 1971, p. 255. Received January 24th, 1978 Accepted March 16th, 1978

ISSN:0003-2654    

DOI:10.1039/AN9780300921

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

928 Analyst, September, 1978, Vol. 103, $9. 928-936 Technique for the Determination of Fluorescence Quantum Efficiencies: a Method Avoiding Direct Measurement of Absorbance Adam Britten John Archer-Hall Department of Pharmacy, University of Aston in Birmingham, Gosta Green, Birmingham, B 4 7ET Department of Physics, University of Aston in Birmingham, Gosta Green, Birmingham, 1124 7ET and Geoffrey Lockwood Department of Chemistry, University of Shefield, Shefield, S10 2TN A method is described for the determination of the absorbance of a fluorescent solution by recording the fluorescence intensities at two points along the excitation path. An expression is developed that relates the ratio of these two fluorescence intensities to the absorbance of the solution. Thereby both the fluorescence and the absorbance of a solution can be obtained by recourse only to a spectrophotofluorimeter.This approach is extended to the deter- mination of fluorescence quantum efficiences and an expression is presented that relates the fluorescence intensities at two points along the excitation path to the quantum efficiency of the fluorescent material. Quantum efficiences can therefore be determined without the need for an accurate separate measurenient of the absorbances of solutions of standard and sample materials. A cuvette with a nitrogen deoxygenation facility is described for use in these and other related determinations. Keywords : Fluorescence eficiency ; absorbance ; nitrogen deoxygenation The method most commonly used and accepted for the determination of fluorescence quantum efficiencies, an important photophysical property of materials, is that described by Parker and Rees.l This is a comparative method that relies on the previous knowledge of the quantum efficiency, C$s, of some standard material.The fluorescence intensities, F , and FU, and the absorbances, escsl and cucul for the standard and the unknown, respectively, where E is absorptivity, c concentration and 1 path length, are determined and used in equation (1) to evaluate the fluorescence efficiency, C$u, for the unknown. Fu/Fs = IeuCu~+u/IgEsCsJ+s . . ,. .. * . (1) The limitations associated with the use of this expression have been fully re~iewed.~J For a number of reasons, such as absorption - emission overlap of spectra, for example, it has been recommended, and found to be true in practice,2 that the most precise fluorescence efficiencies are obtained if optically dilute solutions are used in spectrophotofluorimeters with right-angle optical configurations.The use of optically dilute solutions, whose absorbances are of the order of 0.01-0.05, presents the problem of the accurate and precise measurement of such low absorbances. Even when this is achieved with an acceptable degree of error the effect of the difference in optical performance of an absorption spectro- photometer and the fluorimeter on the absorbances of the solutions of standard and unknown materials may be such that the actual absorbance differs by lOOyo from the determined “true” value. This effect can be brought about by the different wavelength band pass of the two instruments and by the profile and width of the absorption bands of the spectra of the standard and unknown material.It is a frequently encountered situation because it is rarely possible to find a standard substance that has an absorption spectrum identical with that of the unknown. A correction for this type of error can be made, but it requires the determination of the slit function of the excitation monochromator of the fluorimeter and the convolution of this function with the absorption spectrum of the substance being examined. Such an approach requires extensive digitisation and computation and is itselfBRITTEN, ARCHER-HALL AND LOCKWOOD 929 sensitive to errors arising from differences in the wavelength calibration of the excitation monochromators of the absorption spectrophotometer and the fluorimeter.It appeared that the accurate and precise measurement of the absorbances of optically dilute solutions could be made routinelymore reliable by the use of an absorbance - fluores- cence cuvette with a long path length. Thus, if the transmittance path of the cell was 4.0 cm a solution with an absorbance of, say, 0.05 cm-l would give a reading of 0.2 absorbance unit. This value having been determined in an absorption spectrophotometer, the cuvette could then be used in the fluorimeter by utilising its 1.0-cm path length for a right-angle optical configuration. Such a cuvette was obtained and further modified so as to allow nitrogen deoxygenation to be used. The cuvette is shown in Fig.2. A t this stage it was realised that if fluorescence intensities were measured at different points along the excitation path, then these intensities at the different points would be related to the absorbance of the solution. In this event, the absorbance could be determined from two such fluorescence intensities obtained from the spectrophotofluorimeter. Such an approach would eliminate the problems referred to in the paragraph on measurement of the absorbance of optically dilute solutions. Clearly, this would be a desirable development. Accordingly, equation (8) * was derived to relate absorbance and fluorescence intensities F, and F , at two points along the excitation path separated from the front surface of the cuvette by L, and L, cm, respectively: A natural progression from this method of absorbance determination was to apply this approach to the measurement of fluorescence efficiencies.Using equation (8) and the known dependence of fluorescence intensity on excitation intensity, absorbance and quantum efficiency, +, equation (17)" was derived, which relates the fluorescence intensities F , and F , at L , and L,, as defined above, with the fluorescence and instrumental constants K and S. Fl(l + L1Y)/F2L1Y = K410g(F,/F2) .. . . .. (17) A full derivation of these expressions and their application to quantum yield determinations are described in subsequent sections of this paper. Experimental Apparatus Two spectrophotofluorimeters of different design but both with right-angle optical configurations were used.One was the American Instrument Company (Aminco-Bowman) instrument, which was fitted with a 250-W xenon-arc excitation source and an EM1 9871B, modified S5 response, photomultiplier tube. The standard cuvette holder was replaced with a square plate made of Perspex and having a centrally milled-out channel in which a rectangular cuvette was located. The cuvette holder fitted the base of the sample compart- ment. The cuvette, in this instance with a 4.0 x 1.0cm internal path structure, was placed with its long path length along the direction of excitation and was used in two extreme locations. In one position its front face came in contact with the exit slit of the excitation monochromator and in the other position its rear face came in contact with the side of the sample compartment opposite the exit slit from the excitation monochromator.This gave the distances of observation of emission from the front surface of the solution in the cuvette exactly at 3.50 and 0.50 cm. Fig. 1 illustrates the holder and the movement of the cuvette corresponding to the two distances, L, and L,. The cuvette was adapted to take a hollow-key stopcock with inlet and outlet vents for nitrogen deoxygenation. The design of the cuvette and stopcock system is shown in Fig. 2. In this instance two separate 1.0-cm square cuvettes were used to provide the two measurements of fluorescence intensities at two points along the excitation path. * These equations appear in the correct sequence in the Discussion. The stopcock is rotated to close the vents.The second instrument was purpose-built and has been described previo~sly.~930 BRITTEN et a,!. : DETERMINATION OF FLUORESCENCE QUANTUM Analyst, VOI?. 103 n I I I 1 Fig. 1. Diagram showing the two positions of the cuvette in the cuvette holder. Reagents The compounds used were purified, commercially available products and consisted of indole, 3-indolylacetic acid, yohimbinic acid, 9-terphenyl and 9-methylanthracene. Methanol was used as the solvent and was purified so that it was spectroscopically pure in the wavelength region of interest (250450 nm). Procedure (a) Validation of equations (8) and (17). The cuvette was filled with pure methanol at room temperature and its absorbance at 285 nm was determined on a Beckmann Acta V spectro- photometer using air as a reference.The cuvette was then filled with a solution of a fluorescent material and its absorbance again determined on the spectrophotometer. The concentration of the solution was adjusted by dilution or otherwise until its absorbance Using a 4.0 x 1.0 cm cuvette in the Aminco-Bowman spectrophotojuorimeter Excitation Fig. 2. Diagram of the cuvette showing the stopcock system.September, 1978 EFFICIENCIES AVOIDING DIRECT MEASUREMENT OF ABSORBANCE 931 was of the order of 0.1-0.15 cm-l. The cuvette was then fitted with the hollow-key inlet - outlet stopcock and deoxygenated by bubbling nitrogen through it for 10 min. The stop- cock was closed and the cuvette placed in the fluorimeter at position L,. The monochromator slit widths were 1.Omm and the photomultiplier entrance slit width was 2.0mm.The excitation and emission wavelengths in all instances were 285 and 340 nm, respectively. The fluorescence intensity, F,, was recorded. The cuvette was then moved into position L, and the new fluorescence intensity, F,, recorded. This procedure was repeated three times for each solution used in order to determine any error that might arise from inexact positioning. It was found that errors were either absent or less than 1% due to some small imprecision in cell location. The solution in the cuvette was serially diluted to give a range of absorbances down to 0.02 cm-1 and the fluorescence intensities, F, and F,, in each instance were recorded. The procedure was carried out for $-terphenyl, indole, 3-indolylacetic acid and yohimbinic acid.As pointed out earlier, the two fluorescence intensities F, and F , correspond to distances from the front surface of the solution of exactly 0.50 and 3.50 cm, respectively. This was verified by vernier caliper measurement of the outer dimensions of the cuvette, which were 4.230 cm, and of the interior separation between the walls of the sample compartment, which was 7.230 cm. As the emission is viewed at the centre of the compartment, which corre- sponds to 3.615 cm from the inside of the compartment walls, and the cuvette wall thickness is 0,115 cm, then the two configurations correspond exactly to 0.50 and 3.50 cm for L, and L,, respectively. (b) Validation of equations (8) and (17). Stock solutions of each compound were pre- pared in air-saturated methanol at room temperature, with absorbances of about 0.5 cm-l at the maximum of the absorbance band used.For each compound F , and F , were measured on the stock solution and on solutions by serial dilution of the stock solution to two or three other arbitrary absorbances down to a final value of about 0.05cm-l. For each dilution an aliquot of the solution was placed in a 1.0-cm “fluorescence” cuvette and a similar amount of the identical solution was placed in a 1.0-cm “absorbance” cuvette. A matched “absorbance” cuvette was filled with solvent alone. The intensity F , was measured with the fluorescence cuvette in the sample position and in contact with the solvent-containing cuvette which was juxtaposed in the excitation path ; replacing the solvent-containing cuvette with the solution-containing “absorbance” cuvette gave F,.Several changes of cuvettes were made for each solution so as to eliminate any random error due to inconsistent positioning of the cuvette. The distances L, and L, in this instance were 0.50 and 1.50 cm, respectively. Emissions were measured at the maximum wavelength or, for anthracene, at the first maximum emission wavelength removed from the excitation wavelength. The compounds examined were P-terphenyl, indole, 3-indolylacetic acid and 9-methylanthracene (seven anthracene derivatives containing the pyridinium or imidazolium residue were also examined and are the subject of another publication*). (ii) Validation of equation (8). A solution of 9-methylanthracene in methanol was prepared with an absorbance of 0.275 absorbance unit a t the 366-nm peak.The absorbance was determined on a Perkin-Elmer 137 UV spectrophotometer using a band width low enough to cause no distortion to the absorption. F , and F , values for this solution were obtained as described under procedure (b)(i). The excitation was at 366 nm a t different excitation slit widths. The results obtained were compared with calculated absorbances that were evaluated by convoluting the absorption spectrum with the slit function by using a published pr~cedure.~ The slit function was found by scattering light with a Ludox suspension from the excitation monochromator at various slit widths into the emission monochromator. Using two 1.0 x 1.0 cm cuvettes in a custom-built spectrophotoJEuorimeter (i) The latter scanned the band at high-resolution slits of 0.25 mm.Results When using procedure (a) to verify equation (8) by confirming the correctness of equation (17) [which is based on equation (S)] with the distances L, and L, equal to 0.50 and 3.50 cm, respectively, a plot of the ratio of (F,7/F2)& against log(F,/F,) was obtained. The results are presented in Fig. 3.932 BRITTEN et al. : DETERMINATION OF FLUORESCENCE QUANTUM Analyst, VoZ. 103 /n I: 0 0.1 0.2 0.3 0.4 x % Ic 20 N w- - 0 0.1 0.2 0.3 0.4 0.5 LogFl IF2 LogFl IF2 Fig. 3. Graph of (F,7/F,)i versus log Fig. 4. Graph of (FIa/F,)3 vevsus Fl/Fa for: A, 9-terphenyl; B, yohimbinic logF,/F, for: A, p-terphenyl; B, acid; and C, indole. 3-indolylacetic acid ; and C, indole. When using procedure ( b ) ( i ) the choice of L, and L, was 0.50 and 1.50 cm, respectively, The results from this pro- The verification of the validity of equation (8) and its application to the measurement of The results are presented in which led to a plot of the ratio of (FI3/F,)* against log(F,/F,).cedure are presented in Fig. 4. absorbances was also carried out by using procedure (b)(ii). Table I. TABLE I RESULTS OF MEASUREMENT OF ABSORBANCES USING PROCEDURE (b) (ii) Fl* F 2 l Excitation slit width/mm arbitrary arbitrary Convoluted (entrance = exit) units units Log(F,/F,) absorbance 0.5 41 22 0.270 0.270 1 .o 67.7 37 0.262 0.261 2.0 68.5 39.5 0.239 0.240 3.0 57 34 0.224 0.220 Discussion The determination of the absorbances of solutions in the course of fluorescence efficiency measurements on an instrument other than a spectrophotofluorimeter can result in large errors.The complete avoidance of this determination or its performance on the same instrument used for fluorescence intensity measurements is therefore highly desirable. The fluorescence intensity of a solution at any point along the excitation path is directly proportional to the intensity of the radiation incident upon the fluorescent species at that point. The incident intensity of the exciting radiation at any point is dependent on the intensity of the source, the distance of travel along the excitation path and the absorbance of the solution. It follows that fluorescence intensities F, and F , at two points L, and L,, respectively, along the excitation path are related to the absorbance of the solution and the separation between L, and L,.An expression relating these parameters can be derived as follows. The exciting radiation has a constant incident intensity, lo, at the front surface of the solution. The fluorescence intensities Fl and F , are observed at right-angles to the direction of excitation radiation over a constant viewing path A at two points L, and L, cm, respec- tively, from the front surface of the solution. The fluorescence intensity, F,, at a point L cm along the excitation path and viewed over a field A at right-angles to it can be expressed by the integral equation Fig. 5 illustrates the situation.September, 1978 EFFICIENCIES AVOIDING DIRECT MEASUREMENT OF ABSORBANCE 933 Fig. 5. Observation of fluorescence intensity at points along the excitation path.where /I is an instrumental constant accounting for the geometry of the sample position and the excitation and viewing angles and E is the molar absorptivity per centimetre. The distance traversed by the radiation of incident intensity I . is Zcm and the fluorescence efficiency is 4. Integration of equation (2) between the limits L and L + A gives .. - * (4) = I , e-C1 (1 - e-eA) . . .. Therefore, the ratio FJF2 of two fluorescence intensities F, and F, at L, and L,, respectively, is given by But . . - * (7) doge = A . . . . .. ( A = absorbance per centimetre) and therefore If L, - L, is defined as . . - * (9) L2 -L1 = 11s . . .. . . . . then A = Slog(F,/F,) . . .. .. . . . . (10) Equation (8), expressing the ratio of two fluorescence intensities at two points along the excitation path and the absorbance, is therefore fundamental and should hold true for all instruments at all absorbances.This will be true provided that there is no significant over- lap between the emission and absorption bands. Should such an overlap be present, then the basic equation (2) will not apply for all absorbances. The degree of departure from equation (2) will be a function of the spectral overlap and the viewing width, A. The latter is generally kept small in all instruments so that errors due to spectral overlap are kept to a minimum. In practice it was found that for two instruments of different design, solutions of several934 BRITTEN ei! d. : DETERMINATION OF FLUORESCENCE QUANTUM Analyst, vd.103 compounds with absorbances up to 0.5 absorbance unit could be used without appreciable departure from linearity of equation (17) (see below), which is based on equation (8). In the first method, described under procedure (b) (ii) , a solution of 9-methylanthracene of a known accurately measured absorbance was used as a test material. The fluorescence intensities F , and F2 were measured for this solution by the given procedure a t four different excitation mono- chromator slit widths. This had the effect of changing the absorbance of the solution owing to an increased band width of the exciting radiation in the spectrophotofluorimeter. The apparent absorbances operative in the spectrophotofluorimeter were then determined from equation (8) using the F , and F , values obtained at L, and L, of 0.5 and 1.0 cm, respectively.The absorbances were also calculated by a known procedure5 from the convolution of the absorption spectrum of 9-rnethylanthracene and the spectrophotofluorimeter slit function. The results are compared in Table I and show that the absorbances as measured by the fluorescence method employing equation (8) are in agreement with the "true" absorbances operative in the spectrophotofluorimeter. The second method, which validates equation (8), is based on the application of equation (8) to derive an expression relating the fluorescence quantum efficiency of a solution to two fluorescence intensities F , and F , measured at two points along the excitation path at L, and L,cm, respectively, from the front surface of the solution.The validation of the resultant expression validates equation (8). The validity of equation (8) was verified by two approaches. Determination of Fluorescence Efficiencies without Measurement of Absorbance From equations (5) and (9) we can obtain E = Sln(F,/F,) .. . . . . .. . . (11) Substituting equation (11) for E in equation (4) and letting F , = F , at L , gives F , = I , p+e-LlflncF,/P,) [1 - e-A3"'qF2)] . . .. .. . . (12) This simplifies to Expanding e-As*n(Fllp2) gives Neglecting all terms raised to powers greater than 1 gives Substituting equation (15) into equation (13) we obtain F , = Io~~(F1/F2)-L~8ASln(Fl/F2) .. . . . . (16) Replacing the product 10/34S/2.303 by a constant K and simplifying gives F1'1+L1S'/F2L1S = l<C$log(F,/F,) .. .. .. . . (17) Therefore, a plot of F11+L19/F2L2S against log(F,/F,) for solutions of different absorbances should yield a straight line with a slope KC$. The results from two separate determinations on two different instruments are shown in Figs. 3 and 4 for several fluorescent substances. The plots are linear and in good agreement with equation (17). The plot in Fig. 3 was obtained using distances L, and L, of 0.50 and 3.50cm, respectively, which give a plot of (F,7/F2)+ against log(F,/F,). The graph in Fig. 4 was obtained using 0.50 and 1.50 cm for L, and L,, respectively. The latter conditions give a plot of (Fl3/F,)+ against log(F,/F,).SC’tembC7,1978 EFFICIENCIES AVOIDING DIRECT MEASUREMENT OF ABSORBANCE 935 It can be seen that this approach can be used for the determination of quantum efficiencies.Using some standaxd substance of known #, the instrumental constant K can be evaluated and so other quantum efficiencies can be measured. Alternatively, the simple ratio of the plots of F11+L1s/F2L1s of the unknown fluorescence efficiency material to that of the standard substance will give equation (18), from which the unknown #u values can be calculated: Slope of plot of F11+QS/F2L1S of unknown - $u Slope of plot of F11+L1B/F2L1S of standard & - . . (18) It must be pointed out that this method only removes errors due to absorbance determina- tions, and does not account for errors that arise from the overlap of absorption and emission spectra or those which arise from a difference in the wavelength range of the emission spectra of the standard and unknown materials.These remain to be corrected by known procedures. Some quantum yields determined by this method are presented in Table 11. TABLE I1 QUANTUM YIELDS Quantum yield A f \ Compound This work Reported Anthracene . . .. .. . . 0.24 0.27,2 0.226 9-Methylanthracene . . .. . . 0.25 0.29,6 0.336 9,lO-Dimethylanthracene . . . . 0.69 0.63,6 0.82’j 3-Indolylacetic acid . . .. . . 0.23 0.196 The quantum yields of seven other anthracene compounds were determined by the method described in this paper and have been published el~ewhere.~ The practical application of the proposed method rests on the choice of three parameters, viz., the distances L, and L, and the absorbance of the solutions.The choice of L, and L, determines the exponents m and n in the ratio F,”/F,“. The exact values of these exponents cause no mathematical problem if a scientific calculator is available. However, simple exponents can be obtained by a judicious choice of L, and L,, provided that the internal layout of the instrument sample compartment allows it. In general, relatively large values of L, and L, are to be preferred in order to reduce errors that might arise from imprecise location of the cuvette a t the two positions and also to ensure that FJF, is considerably greater than unity. The choice of absorbance is also important. At low absorbances, of the order of 0.01-0.05 absorbance unit, and a t a separation of 2.0cm between L, and L,, FJF, will be between 1.047 and 1.259.Hence F , and F , are close in value and errors in their determination will produce a significant error in the calculation of the absorbance and in log(F,/F,). It was found that absorbances of the order of 0.1-0.2 absorbance unit can be used and give results in complete agreement with the results expected from equations (8) and (17). At these absorbances and a separation of 2.0 cm between L, and L,, F,/F, is between 1.58 and 2.51. At the same time, the absolute values of F , and F , are such that little amplification of the corresponding signals is required. This results in errors of 1.0% or less in the recording of F , and F,, which can result in a maximum error of about 2.0% in F,/F,. The choice of the value of the absorbance has no bearing on the validity of equation (8), as this is derived without any approximations.However, equation (17) is based on the approximation expressed in equation (15). This approximation is valid for small values of A, S and ln(F,/F,). The last parameter is directly proportional to the absorbance used and inversely proportional to the separation between L, and L,. It can be calculated that for a solution with an absorbance of 0.2 absorbance unit, a separation between L, and L, of 2.0 cm and a viewing path, A, of 0.2 cm, this approximation would introduce an error of about 5.0% in the use of equation (17). If the absorbance is reduced to 0.1 absorbance unit and the separation between L, and L, is increased to 4.0 cm, then this error is reduced to less than 3% while F J F , is still maintained at the same value as in the previous example.936 BRITTEN, ARCHER-HALL AND LOCKWOOD It is apparent, therefore, that minimum errors in the use of equations (8) and (17) arise if absorbances of the order of 0.05-0.10 are used in conjunction with a separation of the order of 2.04.0 cm between the points used for determining the fluorescence intensities F , and F , corresponding to L, and L,, respectively.In conclusion, it can be said that the proposed technique for the determination of the absorbance of a fluorescent solution by measuring its fluorescent intensity at two points along the excitation path has a sound theoretical basis, the result of which is expressed in equation (8). This relationship leads to equation (17), which can be used to determine fluorescence quantum efficiencies, avoiding the measurement of the absorbances of the fluorescent solutions. Therefore, only a spectrophotofluorimeter is required and errors arising from the use of an absorption spectrophotometer are absent. The technique is simple and is applicable to all spectrophotofluorimeters that have a right-angle optical configuration. It requires a simple modification in the sample compartment to allow move- ment of a long-path fluorescence cuvette to permit the observation of fluorescence emission intensity by the detector at two points along the excitation path. A rectangular fluorescence cuvette having one path of the order of 2.04.0 cm and fitted with an inlet - outlet hollow- key stopcock is an ideal cuvette for fluorescence solutions that require nitrogen deoxygenation prior to quantum efficiency determinations. It can be used in the standard procedure for this determination with an increase in the accuracy of the required absorbance measure- ment, or it can be used in the new procedure described in this paper, which avoids the absorbance measurement. The assistance of Mr. C. Linskill in modifying the sample compartment of the Aminco- Bowman spectrophotofluorimeter is gratefully acknowledged. References 1. 2. 3. 4. 5. 6. Parker, C. A., and Rees, W. T., Analyst, 1960, 85, 587. Demas, J. N., and Crosby. G. A., J . Phys. Chem., 1971, 75, 991. Parker, C. A., “Photoluminescence of Solutions,” Elsevier, Amsterdam, 1968. Blackburn, G. M., Lockwood, G., and Solan, V., J . Clzem. Soc., Perkin Trans. 11, 1976, 1452. Lockwood, G., Plz.D. Thesis, University of Aston in Birmingham, 1974. Birks, J. B., “Photophysics of Aromatic Molecules,” Wiley, Chichester, 1970. Received December 14tlz, 1977 Accepted February 27th, 1978

ISSN:0003-2654    

DOI:10.1039/AN9780300928

出版商:RSC    

年代:1978

数据来源: RSC

摘要:

AIzalyst, September, 1978, Vol. 103, $9. 937-949 937 Some Fluorescent Derivatives of the Drug Phenelzine 8 . Caddy and A. H. Stead Forensic Science Unit, Department of Pharmaceutical Chemistry, University of Strathclyde, Glasgow, G1 1XW Pyridazine, phthalazinone and some substituted phthalazinone derivatives have been prepared from the drug phenelzine. The application of these fluorescent compounds to the analysis of the drug has been explored. A sensitive and specific test for phenelzine in urine has been developed. Keywords : Phenelzine determination ; fluorescence ; spot test Analysis to determine the substituted hydrazine drug phenelzine has been problematical because of the drug’s instability in basic solutions. Gas-chromatographic methods have been de~elopedl-~ for the analysis of the drug, which depend either on its conversion in s i h into a stable acetonide derivative1 or on its controlled breakdown by ~ x i d a t i o n .~ , ~ While these procedures are precise and their detection limits adequate for the determination of phenelzine in the urine of patient^,^ it was felt that, as phenelzine does not possess a suffici- ently conjugated system to produce an analytically useful fluorescence, the use of some fluorescent derivative might lead to an improvement of the limit of detection, allowing blood as well as urine samples to be analysed and also enabling more samples to be analysed per unit time. Further, by careful choice of the derivatising agent it was hoped that greater specificity would be conferred on any such assay procedure. Derivatives that were thought to be appropriate for this investigation were the pyridazines and phthalazines and reagents investigated for their ability to form fluorescent products with phenelzine were p-dimethyl- arninobenzaldehyde and Fluram.Experimental Reagents Eastleigh, H ant s. analytical-reagent grade unless otherwise stated. Phenelzine sulphate. Drug standards were as supplied by the manufacturer. This material was supplied by W. R. Warner and Company Limited, All other reagents were of Apparatus Fluorescence measurements were recorded uncorrected on a Perkin-Elmer - Hitachi, Model MPF 3A, recording spectrophotofluorimeter, using a quartz cell of 1-cm path length. Examination of the Fluorescent Properties of l-Phenethylpyridazine-3,6-dione Prepare a solution (1 pg ml-l) of the pure pyridazinedione in chloroform by suitable dilution of a stock solution (100 pg ml-l) and determine its wavelengths of maximum excita- tion and emission.Record the intensity of fluorescence of the solution a t these wavelengths and compare it with a standard quinine sulphate solution (1 pg ml-l) prepared in 0.1 N sulphuric acid. Quantification of Dilute Solutions of Phenelzine by Reaction with Maleic Anhydride Prepare standard aqueous solutions of phenelzine sulphate in the concentration range 0.2-1.0 mg ml-l by suitable dilution of a 10-mg ml-1 stock solution. Take 1 ml of each of these standards containing 0.2, 0.4, 0.6, 0.8 and 1.0 mg, respectively, and a blank sample consisting of 1 ml of water, and reflux them for 90 min in acetic acid (9 ml) in which an approximately ten-fold excess (5 mg) of maleic anhydride has been dissolved.After cooling, dilute 1 ml of the mixture to 8 ml with water and extract this solution with chloroform (5 ml). Next separate the organic layer and wash it, in turn, with distilled water (2 ml), sodium hydrogen carbonate solution (10% m/V, 2ml) and water (2ml). Examine the intensity of fluorescence of 4 ml of this chloroform solution at 393 nm (excitation 330 nm).938 CADDY AND STEAD: SOME FLUORESCENT Artajyst, Vol. 103 From this set of standards construct a calibration graph of concentration plotted against the uncorrected fluorescence intensity. Repeat this procedure three times for the purposes of statistical evaluation. Thin-layer Chromatographic Examination of the Products of the Reaction Between Phenelzine Sulphate and Maleic Anhydride in Dilute Solutions Evaporate a sample (2 ml) of a 1.0 mg ml-l chloroform extract of the reaction mixture almost to dryness under a stream of nitrogen while heating gently at 40 "C on a water-bath.Spot the residue on to a thin-layer chromatographic plate coated with silica gel, together with standards of maleic anhydride, maleic acid, phenelzine and pyridazinedione, and a concentrated blank extract. Develop the plate with a solution of methanol - acetone (9 + 1 V / V ) until the solvent has travelled a t least 15 cm. Remove the plate from the tank, dry and examine it under long-wavelength (365 nm) ultraviolet light. Improved observation of the spots is effected by the use of bromocresol green reagent spray.Examination of the Fluorescent Properties of 2-Phenethyl-(3W)-phthalazine- 1,4-dione Determine the wavelengths of maximum excitation and emission of a 1 pgml-1 solution of the pure phthalazinedione in chloroform that has been prepared by dilution of a stock solution (100 pg ml-l), and calculate the relative intensity of fluorescence from the recorded fluorescence spectrum a t these wavelengths by comparison of the intensity obtained with those of standards of quinine sulphate prepared in the range 0.2-1 pg ml-1. Effect of pH on the Intensity of Fluorescence of 2-Phenethyl-(3H)-phthalazine- l,$-dione Dissolve the 2-phenethyl-(3H)-phthalazine-l,4-dione (50 mg) in the minimum volume of ethanol and dilute to 100 ml with water to give a stock solution (500 pg ml-l). Just prior to use, dilute a 2-ml aliquot of this solution to 100 ml with water to give a working solution containing 10 pgml-l.Examine the intensity of fluorescence of this dilute solution a t 468 nm (excitation 354nm). Return the examined sample to the working solution and, after acidification with dilute (0.05 M) sulphuric acid (2 drops), record the intensity of fluorescence of a second aliquot. Repeat this procedure at increasing pH values, altering the pH by the dropwise addition of dilute alkali solution (either 0.1 or 1 M) to the working solution. The measured intensities of fluorescence are not corrected for any change in volume (approximately 5%) of the working solution caused by the addition of either acid or alkali.Quantification of Dilute Solutions of Phenelzine by Reaction with Phthalic Anhydride Prepare standard aqueous solutions of phenelzine sulphate in the range 2-10 mg ml-l by suitable dilution, where necessary, of a 10 mg ml-l stock solution. Take 1 ml of each of the standard phenelzine sulphate solutions containing 2, 4, 6, 8 and 10 mg of drug, respec- tively, and reflux them for 2 h with acetic acid (9 ml) in which an approximately ten-fold (60 mg) excess of phthalic anhydride has been dissolved. After cooling, dilute 1 ml of the mixture to 8 ml with water and extract with chloroform (5 ml). Remove the organic layer, wash it with water (2 ml) and dilute sodium hydrogen carbonate solution (10% m/V, 2 ml), then remove the organic layer (4.5 ml) and dry it over anhydrous sodium sulphate.Treat 1 ml of water in the same way in order to obtain an appropriate blank. Determine the intensity of fluorescence of 4ml of the dried chloroform solutions a t 397nm (excitation 318nm) and plot a calibration graph of concentration against the measured fluorescence intensity corrected for reagent blanks. Repeat the procedure three times for the purposes of statistical evaluation. Thin-layer Chromatographic Examination of Products of the Reaction Between Phenelzine Sulphate and Phthalic Anhydride in Dilute Solutions By means of a water-bath maintained at 40 "C and a stream of nitrogen, concentrate 2 ml of the 10 mg ml-1 chloroform extract obtained in the above experiment. Spot this con- centrated residue on to a silica gel plate, together with standards of pure phthalic anhydride,September, I978 DERIVATIVES OF THE DRUG PHENELZINE 939 phthalic acid, phenelzine and 2-phenethyl-(3H)-phthalazine-l,4-dione.Develop the plate for a distance of 15 cm in a solvent mixture composed of acetic acid - benzene - diethyl ether - methanol (18 + 120 + 60 + 1 V/Y). After drying, examine the plate under long- wavelength (365 nm) ultraviolet light for the presence of fluorescent or absorbing spots. Examination of the Fluorescent Properties of 5,8-Dihydroxy-2-phenethyl-(3W) - phthalazine- 1,4-dione Solutions with Changes in pH This was carried out as described for 2-phenethyl-(3H)-phthalazine-l,4-dione. Variations of the Intensity of Fluorescence of 5,8-Dihydroxy-2-phenethyl-(3W)- phthalazine- 1,4-dione Solutions with Changes in pH Dissolve 5,8-dihydroxy-2-phenethyl- (3H)-phthalazine-1 ,4-dione (50 mg) in a minimum amount of ethanol (less than 1 ml) and dilute the solution to 100 ml with water.Further dilute 2ml of this solution to 100ml with water to give a working solution containing 10pgml-l. Prior to preparing these solutions, all liquids should be deoxygenated by flushing with nitrogen gas for a period of 10 min (see below). Adjust the pH of this solution to values greater than 7 by the dropwise addition of deoxy- genated 0.1 or 1 0 ~ sodium hydroxide solution. Samples are withdrawn from the stock solution a t regular pH intervals, flushed with nitrogen for 3 or 4 min and returned to the stock solution after reading their fluorescence intensity. Prepare samples for examination at pH 13 and 14 in deoxygenated 0.1 and 1 M sodium hydroxide solution, respectively.Below pH 7, adjust the pH similarly by adding 0.05 M sulphuric acid solution. The total volume change during these procedures is less than 5% and no correction is made for this. Stabilisation of the Intensities of Fluorescence of Solutions of 5,8-Dihydroxy-2- phenethyl-(3H)-phthalazine-1,4-dione by Removal of Oxygen Prepare solutions of 5,8-dihydroxy-2-phenethyl-(3H)-phthalazine- 1,4-dione (10 pg ml-l) in 0.1 and 1 M sodium hydroxide solutions (pH 13 and 14, respectively) that have been deoxy- genated by bubbling oxygen-free nitrogen through each for 10 min prior to the addition of pure solid phthalazinedione. The addition of the phthalazinedione has no observable effect on the pH of these solutions.Prepare non-deoxygenated solutions of 5,8-dihydroxy-2-phenethyl-( 3H)-phthalazine- 1,4-dione (10 pg ml-l) in 0.1 and 1 M sodium hydroxide solutions. Measure the intensities of fluorescence of different aliquots of these solutions at 475 nm (excitation 396 nm) after various times (0, 2, 5, 10 and 30min) of exposure to bubbling with oxygen-free nitrogen, and compare these intensities of fluorescence with those obtained from the first two solutions, in which completely deoxygenated sodium hydroxide solutions are used. Re-measure the intensity of fluorescence of each sample 30 min after the original measurement. Preparation of Calibration Graphs for 5,8- Di h ydroxy -2-p henethyl - (3H) -p hthalazine - 1,4-dione at pH 13 Make suitable dilutions of a stock solution containing 100 pg ml-l of 5,8-dihydroxy-2- phenethyl-(3H)-phthalazine-l,4-dione in 0.1 M sodium hydroxide solution just prior to examination, to give working solutions containing 2, 4, 6, 8 and 10 pg ml-1. Measure the intensities of fluorescence of these previously deoxygenated solutions at 475 nm (excitation 396 nm) after a further flushing with nitrogen for 3 or 4 min.Construct a calibration graph by plotting intensity of fluorescence against concentration (in pg ml-l) of phthalazinedione. Repeat this procedure five times for statistical evaluation. Quantification of Dilute Solutions of Phenelzine Sulphate by Measurement of the Intensity of Fluorescence of the 3,6-Dihydroxyphthalic Anhydride Reaction Product Prepare a stock solution of phenelzine sulphate in water at a concentration of 100 pg ml-l and from this prepare working solutions containing 20, 40, 60, 80 and 100 pg ml-l by making suitable dilutions.Prepare a stock solution containing 3,6-dihydroxyphthalic anhydride (250 mg) in glacial acetic acid at a concentration of 10 mg ml-l. Dilute this solution with glacial acetic acid to give a working solution containing 1 mg ml-l.940 CADDY AND STEAD: SOME FLUORESCENT Analyst, VoE. 103 Add 2 ml of each of the standard phenelzine sulphate solutions containing 40, 80, 120, 160 and 200 pg of the drug to 1 ml of the anhydride solution (1 mg ml-l) in acetic acid. Dilute each solution to 20 ml with acetic acid and reflux it under nitrogen for 2 h. Then cool it, add deoxygenated water (20 ml) and continue to reflux under nitrogen for a further 30 min.Again cool, add deoxygenated water (50ml) and extract a 20-ml aliquot of the reaction mixture with 10ml of chloroform. Next wash the chloroform layer with hydro- chloric acid (0.1 M, 2 x 10 ml) and dilute sodium hydrogen carbonate solution (5% m/V, 2 x 10 ml) and dry it over anhydrous sodium sulphate. Examine the intensity of fluores- cence of the chloroform solution at 415 nm (excitation 365 nm) and plot a calibration graph of the phenelzine concentration against intensity of fluorescence after correcting for reagent blanks . Repeat the procedure three times for the purposes of statistical evaluation. All working solutions should be prepared immediately before use and deoxygenated by flushing with nitrogen for 5min.The stock solution of the anhydride, stored at room temperature in the dark, was observed to lose 5% of its fluorescence over a period of 1 week. Thin-layer Chromatogra2;lhic Examination of the Products Obtained from the Reaction Between Phenelzine Swlphate and 3,6-Dihydroxyphthalic Anhydride in Dilute Solution A 2-ml aliquot of a chloroform extract from a reaction mixture containing 200 pg ml-l of phenelzine sulphate is evaporated to a small volume on a water-bath at 40 "C in a stream of nitrogen. The residue is spotted on to a silica gel thin-layer plate, together with standards of authentic phthalazinedione, the anhydride, the acid, a reagent blank and a 2-ml sample of pure chloroform evaporated to a small volume. Develop the plate with a solvent com- posed of acetic acid- benzene-diethyl ether-methanol (18 + 120 + 60 + 1 V / V ) for a distance of 15 cm in an atmosphere of nitrogen and examine it under long-wavelength ultra- violet light (365 nm) for the presence of fluorescing and absorbing spots. Use of p-Dimethylaminobenzaldehyde for the Fluorimetric Analysis of Phenelzine Sulphate Thoroughly mix 1 ml of a dilute solution of phenelzine sulphate (1 pg ml-1) with 3 ml of an aqueous solution of trichloroacetic acid (10% V / V ; reagent grade) and centrifuge the mixture at 1500 rev min-l for 15 min.Tilt-shake 3 ml of this mixture with 3 ml of chloro- form for 15min, remove the aqueous layer and mix thoroughly with 2ml of an ethanolic solution of 9-dimethylaminobenzaldehyde (0.4% m/ V ; reagent grade).Heat this mixture in a stoppered tube at 70 "C for 45 min, then cool, tilt-shake it with 3 ml of chloroform for a further 10 min, remove the chloroform layer and dry it over anhydrous magnesium sulphate (10 mg): Centrifuge and measure the intensity of fluorescence of the extract at 546 nm (excitation 466 nm). Stabilisation of the p-Dimethylaminobenzaldehyde Derivative of Phenelzine Carefully prepare a solution containing 2.5 g of p-dimethylaminobenzaldehyde and 2.5 g of trichloroacetic acid in 10ml of water. Apply this reagent to a Whatman No. 1 filter- paper and allow it to dry. Prepare a standard solution of phenelzine sulphate in water (100 pg ml-l) and dilute suitable aliquots in order to obtain working solutions in the concentration range 2-10 pg ml-l.Prepare similar working solutions in urine and also standard solutions of the appropriate salts of hydrazine, methylhydrazine, isoniazid, nialamide, isocarboxazid, nicotine and caffeine in water a t a concentration of 10 pg ml-1. With thinly drawn out Pasteur pipettes apply 2 drops of each of the standard solutions in the range 2-10 pg ml-l (equivalent to approximately 0.1 ml of each test solution) to the filter-paper at separate points, such that fluorescent spots equivalent to each of the 2, 4, 6, 8 and 10pgml-l solutions are formed. Also apply aqueous and urine reagent blanks, a positive hydrazine control solution (2 pg ml-l) and solutions of methylhydrazine, isoniazid, nialamide, isocarboxazid, nicotine and caffeine. Leave the paper at room temperature in the air for approximately 5 min, until all of the reagents have reacted and dried.Moisten the filter-paper with water and examine it forSeptember, 1978 DERIVATIVES OF THE DRUG PHENELZINE 941 pink fluorescent spots under ultraviolet light (365nm). Allow the filter-paper to stand at room temperature for 18 h and then re-examine it. Use of Fluram (Fluorescarnine) for the Fluorimetric Analysis of Phenelzine Sulphate To a suitable aliquot of this solution add, dropwise, sufficient 0.5 M sulphuric acid to bring the pH to 3.0. Similarly prepare a solution of pH 10 by the dropwise addition of 1 M sodium hydroxide solution. Add 0.5 ml of Fluram reagent (280 pg ml-1 in acetone) to each of four test-tubes containing water (aml), an acidic solution of phenelzine sulphate (pH 3.0, 4ml), a neutral solution of phenelzine sulphate (4ml) and a basic solution of phenelzine sulphate (pH 10, 4 ml).Immediately mix the contents thoroughly, wait for 1 min for the excess of Fluram reagent to hydrolyse and record the intensity of fluorescence of these four solutions at 480 nm (excitation 390 nm). As a spot test apply, in turn, to the same area of a buffered Whatman No. 1 filter-paper from a Pasteur pipette, 1 drop of a phenelzine sulphate solution (10 pg ml-l) and 1 drop of Fluram reagent, allowing each application to dry before the second application. View the paper under long-wavelength ultraviolet light after 5 min. It may be necessary to repeat the application of the phenelzine sulphate solution and reagent in order to obtain the best results.The Whatman No. 1 filter-papers are buffered by applying, dropwise, the appropriate buffer solutions (citrate - phosphate for pH 3-7, sodium hydrogen carbonate for pH 8.5, trisodium orthophosphate for pH 11 and potassium chloride - sodium hydroxide for The lower limit of detection is determined by the application of 1 drop of a series of phenelzine sulphate solutions prepared in the range 0.2-1.0 pg ml-l to separate areas of a Whatman No. 1 filter-paper buffered to pH 6. After drying, the Fluram reagent (1 drop) is applied and the intensities of fluorescence of any spots are observed after 5 min. Prepare an aqueous solution of phenelzine sulphate (50 pg ml-1). pH 12-13). Results and Discussion Analytical Application of 1 -Phenethyl-3,6-pyridazinedione Prepared from Phenelzine Sulphate and Maleic Anhydride A common and versatile method used for the preparation of pyridazine and the related pyridazones and pyridazinediones consists in the cyclisation between hydrazines and deriva- tives of maleic acid.It is usually the anhydrides that are used, although esters and acid halides, etc., have also been employed. All of these and many other methods used for synthesis of pyridazines have been thoroughly reviewed by Mason and A l d o ~ s . ~ Mason and Aldous5 reported that the choice of reaction conditions (solvent, temperature, rate of addition of reactants, etc.) is critical if a maximum yield of the desired product is to be obtained, as many side reactions have been reported. The reaction media that have been described are caustic alkali,6 mineral acid,' alcohol* and glacial acetic acid.9 Because of the instability of phenelzine in sodium hydroxide, the method of Amatsu and Karasawa6 cannot be used.No reaction product was isolated on using the method of Harris and Schoene,' while relatively low yields (40%) were obtained when using ethanol as solvent. Reaction in glacial acetic acid gave a 72% yield of the hydrazide, which was reduced to 65% and 67% when carried out in 80% and 62% aqueous acetic acid, respectively. However, this procedure was adopted as the standard method for the reaction of phenelzine with maleic, phthalic and substituted phthalic anhydrides. The relative intensity of fluorescence of (I) in chloroform (1 pg ml-1) at its maximum excitation and emission wavelengths of 330 and 393 nm, respectively, was found to be equivalent to that of 519 ng ml-1 of quinine sulphate.While a rectilinear relationship could be established for the concentration of pure (I) plotted against measured fluorescence intensity over the concentration range 2-10 pg ml-1, the reaction of phenelzine sulphate with maleic anhydride to produce (I) as the sole product showed linearity only over the range 0.2- 1.0 mg ml-l (Table I) with a theoretical limit of detectionlo of 91 pg ml-l. Because the lower limit of detection is insufficiently sensitive to use in analytical procedures for the determination of phenelzine in body fluids,4 the use of maleic anhydride was abandoned in favour of a derivative with a more extensive conjugated system.942 CADDY AND STEAD: SOME FLUORESCEMT TABLE I Analyst, VoL.103 RELATIONSHIP BETWEEN PHENELZINE SULPHATE CONCENTRATION AND THE FLUORESCENCE INTENSITY OF ITS REACTION PRODUCT WITH MALEIC ANHYDRIDE AND THE ASSOCIATED REGRESSION LINE Phenelzine sulphate Fluorescence intensity concentration/mg ml-l corrected for reagent blank Standard deviation 0.2 17 7 0.4 35 8 0.6 46 4 0.8 73 5 1.0 93 5 All data: y = 91.8% - 2.02, where y = measured fluorescence intensity and x = concentration (mg ml-l). Correlation coefficient = 0.986. Analytical Application of the Reaction Between Phenelzine Sulphate and Phthalic and Substituted Phthalic Anhydrides Many phthalazinones have been shown to possess luminscent propertiesl1*l2 but luminol (11), which exhibits chemiluminescence when oxidised in alkaline solution, is the most well known.13 It gives a blue fluorescence in neutral or acidic, but not alkaline, solution.A number of other dihydroxyphthalazinones have been synthesised14 and their fluores- cence characteristics reported. For example, (111) exhibits a very intense green fluore~cence~~ while N-substituted derivatives of some 4-hydroxy-l-(2H)-phthalazinones, such as (IV), have also been shownls to give rise to strong fluorescence at selected pH values. The fluorescence intensities of solutions of both phthalic hydrazide (V) and its N-methyl deriva- tive (VI) have been shown to be proportional to their concentrations.17 Their ease of preparation, coupled with their potential as fluorophores, makes the phthalazinones pre- pared from phenelzine well qualified for study.I II I l l Fhorescence analysis of 2-phenethyl-( 3H)-p hthalaxine- 1,4-dione The wavelengths of maximum excitation and emission of the phthalazinone (VII) in chloroform are 318 and 397 nm, respectively, and the relative fluorescence intensity of a 1 pg ml-l solution in this solvent was calculated to be equivalent to 169 ng ml-1 of quinine sulphate at these wavelengths. As evidence is available indicating the pH dependence of the fluorescence intensities of some of the phthalazinones,14 the effect of different pHs on the fluorescence intensity of (VII) was measured at a concentration of lopgml-l, using an excitation wavelength ofSeptember, 1978 DERIVATIVES OF THE DRUG PHENELZINE 943 354 nm and recording the emission at 468 nm within 5 min of the preparation of the solutions. These wavelengths were chosen because they were found to be the optimum wavelengths for aqueous solutions (cf., chloroform solutions).The results from three replicate analyses are given in Table 11. These results show that, although the intensity of fluorescence is greatly TABLE II EFFECT OF pH ON THE INTENSITIES OF FLUORESCENCE OF SOLUTIONS OF 2-PHENETHYL- (3H) -PHTHALAZINE-1,4-DIONE PH 3 7 10 11 12 13 14 Fluorescence intensity corrected for reagent blank Standard deviation None - 3 1 77 2 82 2 85 2 86 3 92 3 reduced in acidic or neutral solution, changes at pH values greater than 10 make little or no difference to the measured intensity. White, Roswall and Zafirioul' examined the fluores- cence characteristics of phthalic hydrazide in aqueous solutions at optimum wavelengths for excitation (360 nm) and emission (467 nm).They found that the N-methyl derivative exhibited the same excitation and emission wavelengths. The present work shows that the phenelzine derivative of phthalic anhydride follows the same pattern (excitation 354 nm and emission 468 nm). The fact that little or no fluorescence is observed under neutral or acidic conditions can be explained by a low fluorescence yield at these pHs, or by the insolubility of the phthalazinedione in aqueous solutions. The latter explanation is probably true because the phthalazinone derivative arising from phenelzine and phthalic anhydride was shown to be not significantly soluble in dilute (10% m/V) sodium hydrogen carbonate solution.In basic solution the phthalazinone might be expected to exist in the tautomerk form (VIII). The intensity of fluorescence measured at the optimum wavelengths for the isolated phthalazinone (in solution in chloroform) was plotted, after correction for reagent blanks, against the concentration of dilute solutions of phenelzine (2-10 mg ml-l) added to a ten- fold excess of phthalic anhydride in acetic acid. Statistical evaluation of three sets of results showed that a linear relationship exists between the two parameters (Table 111). TABLE I11 RELATIONSHIP BETWEEN PHENELZINE SULPHATE CONCENTRATION AND FLUORESCENCE INTENSITY O F ITS REACTION PRODUCT WITH PHTHALIC ANHYDRIDE Phenelzine sulphate Fluorescence intensity Standard deviation concentration/mg ml-l corrected for reagent blank (analyses in triplicate) 2 17 8 4 34 7 6 59 2 8 75 8 10 92 5 All data: y = 9.55% - 1.967, where y = measured fluorescence intensity and x = concentration (mg ml-1) .Correlation coefficient = 0.980. Examination by thin-layer chromatography on .silica gel of the extract of a 10 mg ml-l reaction mixture gave two spots, one fluorescing (R, 0.63) and one absorbing (& 0.19). The previously characterised 2-phenethyl-(3H)-phthalazine-l,4-dione gave a spot identical with the former, while the latter could not be identified with standards of phenelzine, phthalic anhydride, phthalic acid or pure phthalazinone. It is suggested that the non-fluorescing reaction product could be the imide (IX) as it is known that this type of compound is formed944 CADDY AND STEAD: SOME FLUORESCENT Analyst, VoZ.103 if the reaction conditions and stoicheiometry are not optimised, and especially if the anhydride is in greater concentration than the hydrazine.l8*l9 Although methods are available for converting such imides into phthalazines, these would make any analytical procedure tedious and unwieldy for routine work. The absorbing spot was not characterised. The reduced relative intensity of fluorescence for the phthalazinone (169 ng ml-l) compared with that of phenelzine with maleic anhydride (569 ng ml-1) is reflected in the limit of detection of the drug with phthalic anhydride. The detection limit for phenelzine,1° assum- ing all of the reaction product is extracted as its phthalazinone derivative, was found to be 40 pg ml-l compared with approximately 10 pg ml-1 for the derivative with maleic anhydride.Variations in the reaction times of up to 16 h, and in the extraction procedures, failed to give any lowering of this level of detection. Increasing the ratio of anhydride to phenelzine sulphate to 100: 1 (mlm! resulted in a decrease in the measured intensity of fluorescence. Fl,uorescence analysis of 5,8-dihydroxy-2-phenethyl- (3H) -j5lztlaaZazine- 1,4-dione As an alternative to extending the conjugation as a means of increasing the fluorescence of a compound, the introduction of electron-donating groups, such as hydroxyl and amino, can prove helpful. For this reason, the use of dihydroxyphthalic anhydride for the production of hydroxylated phthalazinone derivatives of phenelzine was investigated.Although 3,4- 33- 3,6- and 4,5-dihydroxyphthalic anhydrides have been ~repared,~O-~* a symmetrically substituted molecule is desirable if the formation of isomeric phthalazinone mixtures is to be prevented. This restriction limits the useable anhydrides to the 3,6- and 4,5-dihydroxy compounds. However, the latter anhydride is much more difficult to prepare than the former and consequently the present work has been confined to the reaction between phenelzine sulphate and 3,6-dihydroxyphthalic anhydride. The strong fluorescent character of 3,6-dihydroxyphthalic anhydride has already been reported,22 but this is perhaps not unexpected in view of its structural similarity to salicylaldehyde, a well known fluorophore. The relative intensity of fluorescence of the phthalazinone (X) in chloroform (1 pg ml-l) at its maximum wavelengths of excitation (368 nm) and emission (415 nm) was shown to be equivalent to 921 ng ml-l of quinine sulphate.IX X The fluorescence of compounds with ionisable groups attached to the n-electron system can be greatly influenced by pH. Preliminary work, particularly with solutions at the higher pH values, suggested that the hydroxyphthalazinedione had only limited stability. As well as the fluorescence intensity falling drastically with time, the solutions were observed to change from colourless to yellow and then to brown over a period of about 1 h when stored in dilute alkali. Thiele and GiinterZ2 noted similar colour changes with alkaline anhydride solutions. Observations, obtained in triplicate, showing the fall in fluorescence intensity with time for a 10 pgml-l solution of the pure hydroxyphthalazinone, are given in Table IV.The fall in fluorescence intensity may be due to photolytic decomposition, oxidative breakdown or alkaline hydrolysis or any combination of these. Because similar falls in fluorescence intensity were observed when dilute solutions were stored at a high pH in the dark, it was felt that oxidation and alkaline hydrolysis were the more likely causes. However, all subsequent fluorescence analyses were carried out using a minimum exposure to the high- intensity xenon source. The effects of the exclusion of oxygen from samples were examined in triplicate for a 10 pg ml-l solution of the dihydroxyphthalazinone in sodium hydroxide (0.1 M, pH 13.0).Because, within the limits of experimental error, no fall in the intensity of fluorescence was observed over a 30-min period, it can be inferred that the presence of oxygen in a solutionSeptmber, 1978 DERIVATIVES OF THE DRUG PHENELZINE TABLE IV FALL IN INTENSITY OF FLUORESCENCE OF DILUTE ALKALINE SOLUTIONS OF 5,8-DIHYDROXY-2-PHENETHYL-(3H)-PHTHALAZINE-1,4-DIONE WITH TIME 945 Values are percentage intensity of fluorescence F,*/Fo. f p,H 7 Tinie/min 8 12 13 14 0 100 100 100 100 5 - - 42.1 19.5 10 92.7 80.2 20.1 7.1 30 88.4 65.6 15.4 3.5 40 79.3 25.0 9.6 2.3 180 75.7 8.8 2.8 1.8 * F J F , = Ratio of fluorescence intensity a t time t and time 0 of a 10 p g ml-l solution of 5,8-dihydroxy-2-phenethy1-(3H)-phthalazine- l,4-dione, expressed as a percentage. of the phenelzine derivative at pH 13 has a marked effect on the intensity of fluorescence after a period of time.In particular, after 5 min under normal conditions (Table IV) the intensity of fluorescence has fallen to 42% of its original value, while after deoxygenation over a 5-min period there is still lOOyo of the original fluorescence present. Table IV would also seem to support the view that a pH of 14 is also responsible for the fall in fluorescence intensity while at a pH of 13 (X) is more stable. All of the following experiments were, therefore, carried out using solutions that were deoxygenated for 2-5 min by flushing with nitrogen. The effect of pH on the intensity of fluorescence was further investigated by using deoxygenated solutions of pure (X) (10 pg ml-1) at various pH values.The results, which are given in Table V, clearly show that the optimum fluorescence conditions exist at a pH of 13 in deoxygenated solutions. Under these conditions, a calibration graph was constructed by plotting the intensity of fluorescence of solutions of pure (X) against con- centration in the range 2-10 pg ml-l (Table VI). Experience with phthalic anhydride shows that it is convenient to extract the product of the reaction between the anhydride and dilute solutions of phenelzine sulphate into chloro- form. With the dihydroxyphthalic anhydride, it would also appear to be advantageous to adopt the same procedure because the intensity of fluorescence of the dihydroxyphthalazinone formed is greater in chloroform than in aqueous solution at pH 13.However, initial experi- TABLE V EFFECT OF pH ON THE INTENSITIES OF FLUORESCENCE OF DEOXYGENATED SOLUTIONS OF 5,8-DIHYDROXY-%PHENETHYL-(3H)-PHTHALAZINE-1,4-DIONE (10 pg m1-l) PH 2.0 4.0 6.0 7.0 9.0 10.2 11.0 11.8 12.0 12.2 12.4 12.6 12.8 13.0 13.2 13.5 14.0 Fluorescence intensity* 4 4 4 11 27 47 155 350 376 452 626 836 1041 1411 849 779 759 Standard deviation from five sets of results 1 1 1 3 3 16 27 52 36 29 47 51 49 60 46 45 67 * Expressed as ng ml-l of quinine sulphate.946 CADDY AND STEAD: SOME FLUORESCENT Analyst, Vol. 103 TABLE VI RELATIONSHIP BETWEEN THE CONCENTRATION OF 5,8-DIHYDROXY-%PHENETHYL- (3H)-PHTHALAZINE-1,4-DIONE AND THE INTENSITY OF FLUORESCENCE OF ITS SOLUTIONS, TOGETHER WITH THE ASSOCIATED REGRESSION LINE Concentration of dihydroxy- Fluorescence Standard deviation phthalazinedione/pg ml-l intensity* of five determinations 2 286 17 4 538 26 6 867 42 8 1090 3 1 10 1373 39 * Expressed as ng ml-l of quinine sulphate.All data: y = 1 3 6 . 1 6 ~ + 13.36, where y = measured fluorescence intensity and x = concentration ( p g ml-l). Correlation coefficient = 0.996. ments showed that the reagent blanks were relatively high, this being attributable to the presence of residual anhydride or acid. The method used successfully with maleic and phthalic anhydrides was modified to incorporate an additional 30-min reflux with water in an atmosphere of nitrogen. This step ensured that any excess of anhydride was converted to the acid and this could then be removed from the final chloroform extract with a sodium hydrogen carbonate wash, thus reducing the background fluorescence.Dilute solutions of phenelzine in the range 40-200pgml-l were made to react with dihydroxyphthalic anhydride in an atmosphere of nitrogen. The dihydroxyphthalazine- dione product was extracted into chloroform and its fluorescence intensity measured (excitation 368 nm, emission 415 nm). The results of three sets of determinations at different concentrations are given in Table VII with the corresponding regression line. Experiments in which the reaction apparatus was not first flushed with nitrogen gave irreproducible measurements for the fluorescence intensity. Although phenelzine is known to undergo serious auto-oxidation in solution,25 this factor is unlikely to be the cause of the present problem, as the same effect would have been expected from the reaction with phthalic anhydride. TABLE VII RELATIONSHIP BETWEEN PHENELZINE SULPHATE CONCENTRATION I N DILUTE SOLUTION AND THE INTENSITY OF FLUORESCENCE OF ITS REACTION PRODUCT WITH 3,6-DIHYDROXY- PHTHALIC ANHYDRIDE (6,8-DIHYDROXY-%PHENETHYL-(3H)-PHTHALAZINE- 1 ,4-DIONE), AND THE ASSOCIATED REGRESSION LINE Phenelzine sulphate Fluorescence intensity Standard deviation concentrationlpg ml-l corrected for reagent blank of three determinations 40 17 7 80 32 7 120 52 0 160 60 4 200 74 5 All values: y = 0 .3 5 1 7 ~ + 4.667, where y = fluorescence intensity and x = concentra- Thin-layer chromatographic analysis on silica gel of the chloroform extract of the 200 pgml-1 reaction mixture was carried out in an atmosphere of nitrogen by using standards of authentic (X), 3,6-dihydroxyphthalic acid and anhydride, a reagent blank and a sample of chloroform evaporated to a small volume.The fluorescent spot corresponding in RF (0.58) to the value given by authentic (X) was extracted into chloroform and its fluorescent spectrum shown to be identical with that of the authentic compound. A fluorescent spot (R, 0.99) and an unidentified absorbing spot (RF 0.75) were also observed. The same pattern was observed when a 10 pgml-l reaction mixture was examined, but not when a similar 5 pg ml-l mixture was run, there being no fluorescent spot which corresponded to that of authentic (X). tion of phenelzine sulphate. Correlation coefficient = 0.958 3.September, 1978 DERIVATIVES OF THE DRUG PHENELZINE 947 As the relative intensity of fluorescence of a solution in chloroform of a dihydroxyphthalic anhydride derivative is five times greater than that of the equivalent phthalic anhydride derivative, the limit of detection was found to be lower. The limit of detectionlo when all of the reaction mixture was extracted was found to be 4.6 pg ml-l, which is nine times the corresponding limit for the phthalic anhydride derivative.However, the thin-layer chroma- tographic evidence would indicate that the level is more likely to be of the order of 10 pg ml-l. Variations in reaction times and relative concentrations of reactants failed to improve on this detection limit. Use of p-Dimethylaminobenzaldehyde for the Analysis of Phenelzine @-Dimethylaminobenzaldehyde has been widely used as an analytical reagent for primary alkylamines, arylamines, amino acids and indole derivativesz6 It has also been used for the colorimetric determination of hydrazineZ7 and monosubstituted hydrazinesBSm by measurement of the orange coloured aldazine produced under acidic conditions.The coloured product arising from the reaction of hydrazine and 9-dimethylaminobenzaldehyde gives an intense orange coloured fluorescence when stabilised by adsorption on to filter- paper. This fluorescent spot, while not producing a fluorescent solution when extracted into common solvents, can be stabilised by extraction into chloroform saturated with trichloroacetic acid. In this way, Vickers and Stuart30 were able to determine hydrazine in plasma at the 10 ng ml-l level.Applying the procedure of Vickers and Stuart30 to phenelzine in aqueous solution did not produce any fluorescent species, even at the 10 pg ml-l level, while under aqueous conditions liydrazine (1 pg ml-l) gave an intense fluorescence at the expected wavelength maxima (excitation 466 nm, emission 546 nm). However, a bright pink fluorescent spot developed when solutions of the phenelzine derivative were applied to filter-paper and allowed to dry. This procedure enabled 10 pg ml-l of phenelzine to be detected with ease. The sensitivity of the test could be further increased, down to levels of 1 pg ml-l (100 ng applied to the paper), by impregnating a filter-paper with a concentrated solution of p-dimethylamino- benzaldehyde in trichloroacetic acid and applying droplets of the test solution containing phenelzine, drying the paper between each application.The optimum reaction time was shown to be 5min. Repeating the analyses with urine containing phenelzine sulphate in the concentration range 2-10 pg rnl-l and with blank samples of urine gave a similar detection limit of 1 pg ml-l (100 ng applied to the filter-paper). With respect to the specificity of the reaction at the 10 pg ml-l level, only isoniazid, of those compounds examined, gave a coloured reaction product which exhibited fluorescence of the same colour. Both hydrazine and methylhydrazine gave orange fluorescent products while nialamide and isocarboxazid only gave a yellow complex and nicotine and caffeine or blank samples of urine gave neither a coloured nor a fluorescent product.Reaction of Fluram with Phenelzine Fluram (XI) has become widely used as a fluorescence label,31 particularly for amines and amino acids,32 because of its ease of reaction at room temperature in aqueous solution and because the reagent and its hydrolysis product are non-, or only weakly, fluorescent. XI XI I Following the method of de Bernado et aqueous solutions of phenelzine were examined at various pH values and concentrations up to 50pgml-l, but no fluorescing products could be obtained. A similar observation has been made on some unnamed monosubstituted948 CADDY AND STEAD: SOME FLUORESCENT Analyst, Vol. 103 hydrazines by de Silva and Strojny.33 The reason for this may lie in the fact that, whereas primary amines react with Fluram to form a stable, rigid, five-membered ring compound, hydrazines, as a result of possessing adjacent active nitrogens, can more readily form the more flexible six-membered substituted pyrazine (XII) .The formation of this compound can be rationalised by the mechanism shown below. If this is correct, it might be expected that the physical imposition of ridigity to this structure may produce a fluorescent species. By dropping a solution of Fluram in acetone on to an air-dried sample of phenelzine solution on a filter-paper, a fluorescent product can be observed when the paper is examined under long-wavelength ultraviolet light. Because pH is known to be important in the reaction of Fluram with a m i n e ~ , ~ ~ , ~ ~ a series of buffered filter-papers (pH 3-12) were prepared and phenelzine (10 pg ml-l) and Fluram (3 mg ml-l) solutions added in turn to produce the fluorescent species.Visual examination of the intensity of fluorescence showed that a pH of 5-6 was the optimum. Under these conditions, the limit of detection was shown to be 0.2 pg ml-l using 50 p1 of solution (equivalent to 10 ng). By use of a similar procedure, Weeks et aL3* have reported a detection limit of 5 ng for hydrazine. H R I NH 0 8 0 l/,H I & COOH While it is recognised that the reaction of phenelzine with Fluram to give a fluorescent compound is not specific, some selectivity can be imposed on an assay for phenelzine, which assay makes use of this fluorescent product in thin-layer chromatography.However, as the compound only fluoresces on a thin-layer plate and not in solution, the assay requires the use of a photodensitometer with the appropriate facilities for measuring fluorescence. Conclusions The potentials of these fluorescent compounds have been explored as a means of in situ derivatisation in the assay of the drug. While linear relationships between the intensity of fluorescence and concentration for the pyridazine, the phthalazinedione and the dihydroxyphthalazinedione derivatives have been established, the limits of detection preclude the use of these com- pounds for the assay of the drug in urine4 and were not therefore investigated as procedures that might be used for the much lower levels of phenelzine expected to be present in blood.The methods described may have some application for solid dosage forms. A fairly specific and sensitive test, using @-dimethylaminobenzaldehyde, has been developed for the identification of phenelzine in urine. A similar test based on the development of a fluorescent product with Fluram is also sensitive but lacks the specificity of the former test. A series of chemical derivatives of the drug phenelzine have been prepared. One of the authors (A.H.S.) acknowledges support from the Medical Research Council during the tenure of which this work was carried out.Sebtember, 1978 DERIVATIVES OF THE DRUG PHENELZINE 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 949 References Caddy, B., Tilstone, W.J., and Johnstone, E. C., Br. J . Clin. Pharmac., 1976, 3, 633. Caddy, B., and Stead, A. H., Analyst, 1977, 102, 42. Gelbicova-Ruzickova, J., Novak, J., and Chundela, B., Biochem. Med., 1971, 5, 537. Caddy, B., Stead, A. H., and Johnstone, E. C., in the press. Mason, J. W., and Aldous, D. L., in Castle, R. N., Editor, “The Pyridazinones, Alkoxy- and Aryloxy-pyridazines, and Related Compounds,” in “The Chemistry of Heterocyclic Compounds , Volume 28, ‘Pyridazines’,” John Wiley, New York, London, Sydney and Toronto, 1973, pp. 23- 218. Amatsu, H., and Karasawa, T., Jap. Pat. 7208, 1955; Chem. Abstr., 1957, 51, 18014~. Harris, W. D., and Schoene, D. L., US Pat. 2575954, 1951; Chem. Abstr., 1952, 46, 6161g. Arndt, F., Loewe, L., and Ergener, L., Rev.Fac. Sci. Univ. Istanbul, 1948, 13A, 103; Chem. Abstr., Fever, H., White, E. H., and Wyman, J. E., J . Amer. Chem. Soc., 1958, 80, 3790. Wilson, A. L., Analyst, 1961, 86, 72. Simpson, J . C. E., “Phthalazines,” in “The Chemistry of Heterocyclic Compounds,” Volume 5 , Patel, N. R., in Castle, R. N., Editor, “Phthalazines,” in “The Chemistry of Heterocylic Compounds,” Simpson, J. C. E,, “Phthalazines,” in “The Chemistry of Heterocyclic Compounds,” Volume 5 , Simpson, J. C. E., “Phthalazines,” in “The Chemistry of Heterocyclic Compounds,” Volume 5 , Drew, H. D. K., and Pearman, E. H., J . Chem. SOL, 1937, 586. Gleu, K., and Pfannsteil, K., J . Prakt. Chem., 1936, 146, 137; Chem. Abstr., 1936, 30, 82OEi5. White, E. H., Roswell, D. F., and Zafiriou, 0. C., J . Org. Ckem., 1969, 34, 2462. Drain, D. J., and Seymour, D. E., J . Chem. Soc., 1955, 852. Chattaway, F. D., and Tesh, W., J . Chem. Soc., 1920, 711. Freund, M., and Horst, F., Ber. Dt. Chem. Ges., 1894, 27, 355. Birkinshaw, J. H., and Bracken, A., J . Chem. Soc., 1942, 368. Thiele, J., and Giinther, F., Justus Liebigs Annln Chem., 1906, 349, 59. Helferich, B., B e y . Dt. Chem. Ges., 1921, 54, 157. Bruck, O., Ber. Dt. Chem. Ges., 1901, 34, 2743. Daly, R. E., Analyt. Profiles Drug Subst., 1973, 2, 383. Pesez, M., and Bartos, J., “Colorimetric and Fluorimetric Analysis of Organic Compounds and Drugs,” Marcel Dekker, New York, 1974, p. 199. Pesez, M., and Petit, A., Bull. Soc. Chim. Fr., 1947, 122; Chem. Abstr., 1947, 41, 5820i. Feigl, F., “Spot Tests in Organic Analysis,” Seventh Edition, Elsevier, Amsterdam, London and New York, 1966, pp. 227-278. Satoh, T., and Moroi, K., Analytica Chim. Acta, 1970, 51, 449. Vickers, S., and Stuart, E. K., Analyt. Chem., 1974, 46, 138. Bridges, J. W., in Reid, E., Editor, “The Luminescence Assay of Drugs,” in “Assays of Drugs and Other Trace Compounds in Biological Fluids,” North-Holland, Amsterdam and New York, 1976, pp. 25-38. De Bernado, S., Weigele, M., Toome, V., Manhart, K., Leimgruber, W., Bohlen, P., Stein, S., and Udenfriend, S., Archs Biochem. Biophys., 1974, 163, 390. de Silva, J. A. F., and Strojny, N., Aaalyt. Chern., 1975, 47, 714. Weeks, R. W., Yasuda, S. K., and Dean, B. J., Analyt. Chem., 1976, 48, 159. 1949, 43, 579a. Interscience, New York and London, 1953, pp. 69-200. Volume 27, John Wiley, New York, London, Sydney and Toronto, 1973, pp. 323-760. Interscience, New York and London, 1953, pp. 153-155. Interscience, New York and London, 1953, pp. 147-150. Received February 2nd, 1973 Accepted April 14t?z, 1978

ISSN:0003-2654    

DOI:10.1039/AN9780300937

出版商:RSC    

年代:1978

数据来源: RSC