摘要:

THE ANALYSTTHE ANALYTICAL JOURNAL OF THE CHEMICAL SOCIETYEDITCIRIAL ADVISORY BOARD*Chairman: J. M. Ottaway (Glasgow, U.K.)R. Belcher (Birmingham, U.K.)L. J. Bellamy, C.B.E. (Waltham Abbey, U.K.)L. S. Birks (U.S.A.)E. Bishop (€xeter, U.K.)L. R. P. Butler (South Africa)'H. J. Cluley (Wembley, U.K.]E. A. M. F. Dahmen (The Netherlands)A. C. Docherty (Billingham, U.K.)D. Dyrssen (Sweden)'P. Gray (feeds, U.K.)J. Hoste (Belgium)H. M. N. H. Irving (South Alrica)M. T. Kelley (U.S.A.)W. Kemula (Poland)G. W. C. Milner (Harwell, U.K.)G. H. Morrison (U.S.A.)H. W. Nurnberg (West Germany)'G. E. Penketh (Wilton, U.K.)E. Pungor (Hungary)D. I. Rees (London, U.K.)'R. Sawyer (London, U.K.)P. H. Scholes (Middlesbrough, U.K.)"W. H. C. Shaw (Greenford, U.K.)S.Siggia (U.S.A.)*D. Simpson (Thorpe-le-Soken, U.K.)A. A. Smales, O.B.E. (Thornaby, U.K.)"A. Townshend (Birmingham, U.K.)A. Walsh (Australia)T. S. West (Aberdeen, U.K.)J . White head (Stockton - on - Tees,A. L. Wilson (Medmenham, U.K.)P. Zuman (U.S.A.)'J. H. Knox (Edinburgh, U.K.) U.K.)*Members of the Board serving on The Analyst Publications CommitteeREGIONAL ADVISORY EDITORSDr. J. Aggett, Department of Chemistry, University of Auckland, Private Bag, Auckland, NEW ZEALAND.Professor G. Ghersini, Laboratori CISE, Casella Postale 3986,201 00 Milano, ITALY.Professor L. Gierst, UniversitB Libre tie Bruxelles, Facult6 des Sciences, Avenue F.- D. Roosevelt 50,Professor R. Herrmann, Abteilung fur Med. Physik., 63 Giessen, Schlangenzahl 14, W.GERMANY.Professor W. A. E. McBryde, Faculty of Science, University of Waterloo, Waterloo, Ontario, CANADA.Dr. W. Wayne Meinke, KMS Fusion Inc., 3941 Research Park Drive, P.O. Box 1567, Ann Arbor,Dr. 1. Rubeika, Geological Survey of Czechoslovakia, Kostelni 26, Praha 7, CZECHOSLOVAKIA.Professor J. R&%ka, Chemistry Department A, Technical University of Denmark, 2800 Lyngby,Professor K. Saito, Department of Chemistry, Tohoku University, Sendai, JAPAN.Dr. A. Strasheim, National Physical Research Laboratory, P.O. Box 395, Pretoria, SOUTH AFRICA.Bruxelles, BELG I U M.Mich. 481 06, U.S.A.DENMARK,Published by The Chemical SocietyEditorial: The Director of Publications, The Chemical Society, Burlington House,London, W1 V OBN. Telephone 01 -734 9864. Telex No. 268001Advertisements: Advertisement Department, The Chemical Society, Burlington House, Piccadilly,London, W1 V OBN. Telephone 01 -734 9864Subscriptions (non-members): The Chemical Society, Distribution Centre, Blackhorse Road,Letchworth, Herts., SG6 1 HNVolume 104 No 1240 July 19790 The Chemical Society 197

ISSN:0003-2654    

DOI:10.1039/AN97904FX025

出版商:RSC    

年代:1979

数据来源: RSC

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ANALAO 104 (1 240) 593-704 (1 979)ISSN 0003-2654July 1979THE ANALYSTTHE ANALYTICAL JOURNAL OF THE CHEMICAL SOCIETYCONTENTS593601Simplification of the Mathematical Evaluation of Titration Results by RegardingComplexes of the Type A,B as n Complexes of the Type AB-Sten JohanssonAutomatic Titration by Stepwise Addition of Equal Volumes o f Titrant. Part IV.General-purpose Program for Evaluating Potentiometric Acid - BaseTitrations-Axel Johansson and Sten JohanssonPyridine-2-carbaldehyde 2-Hydroxybenzoylhydrazone as a Selective Reagent forthe Extraction and Spectrophotometric Determination of lron(l1)-M.Gallego, M. Garcia-Vargas and M. Valcarcel620 S pect r o p hotomet r i c Determination of Aceta m i no p hen, Sa I i cy I a m i d e andCodeine Phosphate in Tablets-M.Abdel-Hady Elsayed, Saied F. Belal, Abdel-Fattah M. Elwalily and Hassan Abdine626 Determination of Vitamin D2 in Multivitamin Tablets by High-performanceLiquid Chromatography-Christeen Mackay, J. Tillman and D. Thorburn Burns637 Modified Fluorimetric Procedure for the Simultaneous Determination o fThiamin and Riboflavin i n Cowpea-J. K. Edijala645 Determination of Noble Metals by Carbon Furnace Atomic-absorption Spectro-metry. Part I. Atom Formation Processes-W. B. Rowston and J. M.Ottaway660 Separation of Organotin Compounds by Using the Difference i n PartitionBehaviour Between Hexane and Methanolic Buffer Solution. Part 1.Determination of Butyltin Compounds i n Textiles by Graphite FurnaceAtomic-absorption Spectrophotometry-Shigeo KojimaMethod for the Rapid Detection o f Organic Pollutants i n Water by Vapour-phase Ultraviolet Absorption Spectrometry-K.C. Thompson and K. Wagstaff61 3668SHORT PAPERS680 Cathode-ray Polarographic Determination of Molybdenum in Serum, Plasmaand Urine-Gary D. Christian and Gaston J. Patriarche683 Improved Method for the Determination of Mercury in Fish Tissue Using 50%Hydrogen Peroxide and a Hot Block-J. W. Davidson687 Preparation and Operation of Selenium Electrodeless Discharge Lamps for Usei n Atomic-fluorescence Flame Spectrometry-R. G. Michel, J. M. Ottaway,J. Sneddon and G. S. Felllodimetric Determination of Milligram Amounts of Certain Aliphatic Acids-S. N. Nema and R. M. VermaA Variation of the Monier-Williams Distillation Technique for the Determinationof Sulphur Dioxide i n Ginger Ale-Bronislaw L. Wedzicha and Michael K.Johnson691694C 0 M M U N I CAT1 0 N S696 Sources of Error in the Flame Photometric Determination o f Sodium and698 Gas Analysis Using an Internal Standard in Adsorption Tubes-B. I. Brookes700 Book ReviewsPotassium in Portland Cements-P. G. Deane and T. P. LeesSummaries o f Papers in this Issue-Pages iv, v, vii, x, xii, x i i iPrinted by Heffers Printers Ltd Cambridge EnglandEntered as Second Class at New York, USA, Post Offic

ISSN:0003-2654    

DOI:10.1039/AN97904BX027

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

V SUMMARIES OF PAPERS I N THIS ISSUE July, 1979Summaries of Papers in this IssueSimplification of the Mathematical Evaluation of Titration Resultsby Regarding Complexes of the Type A,B as n Complexesof the Type ABWhen titration results are evaluated mathematically, the calculations can besimplified and generalised by regarding complexes of the type A,B as n complexesof the type AB. For example, a diprotic acid can be replaced by two mono-protic acids, each of the same molarity as thc diprotic acid but with otherequilibrium constants. The conditions to be fulfilled if such simplificationsare to give correct results are discussed in detail.Keywovds : Titration results ; mathematical evaluationSTEN JOHANSSONDepartment of Analytical Chemistry, The Royal Institute of Technology, S-10044 Stockholm 70, Sweden.Analyst, 1979, 104, 593-600.Automatic Titration by Stepwise Addition of Equal Volumesof TitrantPart IV.General-purpose Program for Evaluating PotentiometricAcid - Base TitrationsA computational procedure for evaluating titration results is described. Itutilises non-linear regression techniques to solve the equations for titrationgraphs from acid - base titrations. The procedure is based on the fact thatthe titration of an n-protic acid can usually be treated as a titration of nmonoprotic acids of the same molarity as the n-protic acid.In principle, the composition of an unknown mixture of acids can be deter-mined by non-linear regression if a sufficient number of measurements(millilitres of titrant uersus pH) are available.However, this assumes thatthe measured values are known with adequate accuracy, an assumption thatis not correct in practice. All measured values (especially the pH values)are imperfect, hence a conventional non-linear regression can give a largenumber of mathematically equivalent solutions. This approach may there-fore, by chance, produce a result that is mathematically correct but chemicallyimpossible. This problem is avoided by setting boundary conditions andcarrying out calculations in an appropriate sequence. Dominating termsare dealt with first, then the effects of the remaining terms are included. Acomputer program TITRA, capable of handling such a calculation scheme, ispresented. No preliminary estimates of the required concentrations orequivalence volumes are necessary in the program.The general equation for the titration graph derived is applicable to samplesthat may contain monoprotic acids (strong or weak), polyprotic acids,mixtures of acids, ampholytes, salts of weak acids and “abnormal” acids(acids with an abnormal sequence of stability constant, values).Byexchanging hydrogen-ion concentrations for hydroxide-iorr concentrationsand vice versa the equation is applicable to the titration of bases.Keywords : Acid - base titrimetry ; evaluation programAXEL JOHANSSON and STEN JOHANSSONDepartment of Analytical Chemistry, The Royal Institute of Technology, S-10044 Stockholm 70, Sweden.Analyst, 1979, 104, 601-612July, 1979 SUMML4RIBS OF PAPERS IX THIS ISSUEPyridine-2-carbaldehyde 2-I4ydroxybenzoylhydrazone as a SelectiveReagent for the Extraction and Spectrophotometric Determinationof Iron( 11)VPyridine-2-carbaldeliyde 2-h ydroxybenzoylhydrazone reacts with iron( 11) toproduce a green 2 : 1 complex ( A m a x .= 620 nm, E = 3.64 x lo3 1 mol-l cm-lin aqueous ethanolic solution, and A,,,. = 640 nm, E = 3.67 x 10”1 molk1 cn-l in chloroform). The grcen complex, extracted into chloro-form, has been used for tlie spectropliotometric determination of traceamounts of iron. The method has a high selectivity and has been appliedto the detcrniination of iron in different samples, such as industrial wastewatcr, non-ferrous materials and minerals.Keywovds I von detevnzination ; s~ectvo~JzotonzetvyM. GALLEGO and M.GARCIA-VARGASDcpartnient of Analytical Chemistry, I7aculty of Sciences, l’niversity of Seville,Scvillc, Spain.and M. VALCARCELDepartment of Analytical Chcmistry, 1;aculty o f Scicnccs, Iiniversity of Cdrdoba,C6rdoba, Spain.A n a l y s t , 1979, 104, 613-619.Spectrophotometric Determination of Acetaminophen, Salicylamideand Codeine Phosphate in Tablets,in accuratc and simple method is proposed for the analysis of a threc-coniponent mixture composed of acetaminophen, salicylamide and codeinephosphate, without the necessity for the previous separation of any com-poncnt. ‘L’he first two coniponcnts arc determined directly by independentspectropliotometric ~neasure~ncmts, based on the pH-induced spectral changes.Codcine phosphate is assayed by the formation of an ion pair with methylorange.The procedure has been applied successfully to the analysiso f known mixtures and commcrcial tablets.Keywovds 1 A cetaiizinoplien ; salicylamide ; codeine PJzospJiate ; spectvopJLoto-iizetvyM. ABDEL-HADY ELSAYED, SAIED F. BELAL, ABDEL-FATTAH M.ELWALILY and HASSAN ABDINEDcpnrtnicnt oi T’harniaceutical Analytical Chemistry, Faculty of I’harmacy, l - n i -a-crsity of Alexandria, Alexandria, Egypt.A n a l y s t , 1979, 104, 620-025J d y , 19 79 SUMMARIES OF PAPERS I N THIS I S S U EDetermination of Vitamin D, in Multivitamin Tablets byHigh- performance Liquid ChromatographyA procedure is described for the determination of vitamin D, in multi-vitamin tablets.The vitamin is released from the tablets by enzymicdigestion, which is followed by solvent extraction and chromatography on amicroparticulate silica column. Other fat-soluble vitamins do not interferewith the assay. The problem of defining and quantifying vitamin D, in orderto correlate results with biological potency is discussed. The procedure issuitable for stability studies.Keywords : Vitamin D, ; five-vitamin D, ; multivitamin tablets ; high-pevformance liquid chronzatogvaphyCHRISTEEN MACKAYDepartment of Chemistry, Loughborough Cnivcrsity of Technology, Loughborough,Leicestershire, LE11 3TU.J. TILLMANFisons Limited, Pharmaceutical Division, Research and Devclopment Laboratories,Bakewell Road, Loughborough, Leicestershire, LE 11 OQY.and D.THORBURN BURNSDepartment of Analytical Chemistry, The Qucen’s University of Belfast, Belfast,BT9 5AG, Northern Ireland.Analyst, 1979, 104, 626-636.Modified Fluorimetric Procedure for the SimultaneousDetermination of Thiamin and Riboflavin in CowpeaThe fluorimetric methods for the assay of thiamin and riboflavin have beenmodified so that the same sample extract may be used for the simultaneousanalysis of the two B vitamins. The procedure eliminates acid hydrolysisand specific pH adjustment for either vitamin; the sample extract is obtainedafter enzymic hydrolysis only with Clarase and has a pH of 4.5-4.8. Astandard additions method for thiamin determination is introduced. Acowpea sample analysed by this procedure gave mean recoveries andstandard errors of 103.9 -J= 1 and 100.5 & 2% for added thiamin and ribo-flavin, respectively; the detection limits for the aqueous solutions taken forfluorescence measurement according to the described procedures for these twovitamins are 0.002 5 and 0.0020 pg ml-l, respectively.Keywords : Thiamin detevmination ; Yibojlinvin determination ; Juovinzetvy ;Clavase ; cowpeaJ. K. EDIJALADepartment of Food Science and Nutrition, University of Strathclyde, 131 AlbionStreet, Glasgow, G1 ISD.Analyst, 1979, 104, 637-644.vi

ISSN:0003-2654    

DOI:10.1039/AN97904FP049

出版商:RSC    

年代:1979

数据来源: RSC

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x SUMMARIES OF PAPERS IN THIS ISSUEDetermination of Noble Metals by Carbon Furnace Atomic-absorption Spectrometry. Part 1. Atom Formation ProcessesEvidence is presented to support the theory that the formation of atoms ofnoble metals during carbon furnace atomisation proceeds via direct evapora-tion of the metal. The evidence includes (i) thermogravimetric investigationof noble metal salts and their aqueous solutions in an argon atmosphere, (iz)X-ray diffraction studies of the residues formed on heating aqueous solutionsof some noble metal salts in a carbon furnace atomiser and in the microfurnaceof a thermobalance, (iii) measurements of the appearance temperatures ofnoble metals in a carbon furnace atomiser using aqueous solutions andvacuum-deposited metal films and (iv) activation energies, Ea, and vapourpressure data relating to noble metals a t elevated temperatures.Experimental conditions giving the best sensitivity for the determinationof seven noble metals (osmium was not detected) in the Perkin-Elmer HGA-74carbon furnace atomiser are presented. On the basis of a 20-p1 samplevolume these gave sensitivities (1%) absorption) of 0.019, 0.005 8, 0.004 5,0.00021, 0.038, 0.023 and 0.00089 pg ml-l for ruthenium, rhodium,palladium, silver, iridium, platinum and gold, respectively.July, 1979Keywords Noble metal determination ; atomic-absorption spectvometvy ; carbonfurnace atomisationW.B. ROWSTON and J. M. OTTAWAYDepartment of Pure and Applied Chemistry, Cniversi ty of Strathclyde, CathedralStreet, Glasgow, G1 1XL.Analyst, 1979, 104, 645-659.Separation of Organotin Compounds by Using the Difference inPartition Behaviour Between Hexane and Methanolic BufferSolution.Part 1. Determination of Butyltin Compounds inTextiles by Graphite Furnace Atomic- absorptionSpectrophotometryA method for determining tributyltin conipounds (TBTs) and dibutyltincompounds (DBTs) in textiles has been studied by graphite furnace atomic-absorption spectrophotonietry. They are successfully extracted from textileswith methanol containing 0.050/, of hydrogen chloride. Their partitionbetween hexane and a methanolic buffer solution (pH 8.5) was studied byvarying the methanol concentration of the aqueous buffer phase. OnlyTBTs can be extracted with hexane from a methanolic buffer solution con-sisting nf 40 ml of methanol and 30 ml of buffer soliition.TBTs are adsorbedon an alumina column and eluted with a small volume of dichloromethane.DBTs remaining in the aqueous phase are extracted with dichloromethaneas a complex with 4- (2-pyridy1azo)resorcinol. After evaporation of thedichloromethane, t h e residue is heated with concentrated nitric acid and itstin concentration is determined by atomic-absorption spectrophotometry.Keywords : Organotin determination ; liquid - liquid partition ; butyltin com-pounds ; textiles ; graphite furnace atomic-absorption spectrophotometvySHIGEO KOJIMANational Institute of Hygienic Sciences, 1-18, Kamiyoga, Setagaya-ku, Tokyo,Japan.Analyst, 1979, 104, 660-667xii SUMMARIES OF PAPERS I N THIS ISSUEMethod for the Rapid Detection of Organic Pollutants in Water byVapour-phase Ultraviolet Absorption SpectrometryJ ~ l y , 1979A novel method for the rapid detection of organic pollutants in waterutilising vapour-phase ultraviolet absorption spectrometry is described.The water sample is extracted with hexane or chloroform and a small amount(0.1-10 pl) of the extract is placed in a small graphite tube, which is slowlyheated while the absorption of the sample is monitored.For optimumsensitivity for most substances a wavelength of 190 nm was used, and otherwavelengths can additionally be used t o characterise further and fingerprintthe sample. Each trace can be completed within 2 min and the techniqueresponds t o many substances that are difficult to characterise or detect bygas - liquid chromatography.Keywords : Organic pollutant detection ; uapouv-phase ultvaviolet absovptionspectvowaetry ; oil characterisationK.C. THOMPSON and K. WAGSTAFFMalvern Regional Laboratory, Severn-Trent Water Authority, 14 1 Church Street,Malvern, WR14 2AN.Analyst, 1979, 104, 668-679Cathode-ray Polarographic Determination of Molybdenum inSerum, Plasma and UrineShovt PaPevKeywovds : Molybdenum detevmintition ; blood ; atvine ; cathode-may polavo-SyaPlZYGARY D. CHRISTIAN and GASTON J. PATRIARCHEInstitut de Pharmacie, Universit6 Libre de Bruxelles, Campus Plaine %Is/ 1,Brussels, Belgium.A~zalyst, 1979, 104, 680-683.Improved Method for the Determination of Mercury in Fish TissueUsing 50% Hydrogen Peroxide and a Hot BlockShort PapevIleywovds : Mevcury detevvlzinntion ; fish ; 50% hydrogen pevoxide digestionJ.W. DAVIDSONDepartment of Fisheries ai;d Environmcnt, \Vest Vancouver, Uritish Columbia,Canada, V7V 1N8.Analyst, 1979, 104. 683-687.Preparation and Operation of Selenium Electrodeless DischargeLamps for Use in Atomic-fluorescence Flame SpectrometryShovt PapevKeywovds : Atomic flziovescence ; flame atomisation ; electsodeless disclzavgelamps ; seleniumR. G. MICHEL, J. M. OTTAWAY and J. SNEDDONDepartment of Pure and Applied Chemistry, University of Strathclycle, CathedralStreet, Glasgow, G1 1XL.and G. S. FELLDepartment of Biochemistry, Royal Infirmary, Glasgow, G4 USF.Analyst, 1979, 104, 687-691July, 1979 SUMMARIES OF PAPERS I N THIS ISSUEIodimetric Determination of Milligram Amounts ofCertain Aliphatic AcidsShort PaperKeywords : A liphatic acids deterwination ; solid iodate and iodide reagent ;iodimetric titrationS. N.NEMA and R. M. VERMADepartment of Post-Graduate Studies and Research in Chemistry, University ofJabalpur, Jabalpur, M.P., India.Analyst, 1979, 104, 691-693.A Variation of the Monier- Williams Distillation Technique for theDetermination of Sulphur Dioxide in Ginger AleShort PaperKeywords : Sulphur dioxide determination ; ginger ale analysisBRONISLAW L. WEDZICHA and MICHAEL K. JOHNSONProcter Department of Food Science, University of Leeds, Leeds, LS2 9 JT.Analyst, 1979, 104, 694-696.Sources of Error in the Flame Photometric Determination of Sodiumand Potassium in CementsCommunicationKeywords : Sources of erYor ; flame photometry ; sodium detzwnination ;potassium determination ; cement analysisP. G. DEANEBlue Circle Technical, Research Division, Greenhithe, Kent, DA9 9 JO.and T. P. LEESCement and Concrete Association, Research and Development Division, WexhamSprings, Slough, Buckinghamshire, SL3 6PL.Analyst, 1979, 104, 696-697.Gas Analysis using an Internal Standard in Adsorption TubesCommunicationKeywords ; Gas analysis ; adsorption tube ; internal standard; anisole ; gaschromatographyB. I. BROOKESDepartment of Regional Chemist, Strathclyde Regional Council, 8 Elliot Place,Clydeway, Glasgow, G3 8E J.Analyst, 1979, 104, 698-699.x ii

ISSN:0003-2654    

DOI:10.1039/AN97904BP055

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

JULY 1979 The Analyst Vol. 104 No. 1239 Simplification of the Mathematical Evaluation of Titration Results by Regarding Complexes of the Type A,B as n Complexes of the Type AB Sten Johansson Department of Analytical Chemistry, The Royal Institute of Technology, S-100 44 Stochholnz 70, Sweden When titration results are evaluated mathematically, the calculations can be simplified and generalised by regarding complexes of the type A,B as n com- plexes of the type AB. For example, a diprotic acid can be replaced by two monoprotic acids, each of the same molarity as the diprotic acid but with other equilibrium constants. The conditions to be fulfilled if such simplifica- tions are to give correct results are discussed in detail. Keywords : Titration results ; inathematical evaluation Titrations may be carried out for different purposes.A physical chemist may perform a titration in order to determine equilibrium constants. He takes great pains to obtain the correct values for the constants, and titrations of this type may take a long time. An analytical chemist, on the other hand, performs a titration in order to determine the con- centration of one or several components in a sample. The equilibrium constants are often known, or they are determined by a separate titration. The exact value of the constant is of subordinate significance, the important thing being to obtain a correct value for the equivalence volume rapidly. The stepwise addition of equal volumes of titrant has proved to be a very useful method for obtaining quick and accurate results., A titration of this type cannot be terminated at the equivalence point, so the equivalence volume must be calculated from the results of the titration (e.g., millilitres of sodium hydroxide solution veysus pH values).In this department a computer program TITRA2s3 has long been in use for the evaluation of such acid - base titrations. In order to give the program a more general application, and to avoid the complications connected with chained equilibria, all acids are considered to be monoprotic. For example, a diprotic acid is replaced by two monoprotic acids, each of the same molarity as the diprotic acid but with different equilibrium constants. Certain conditions must be fulfilled if this simplified method of calculating equilibria is to be accurate, and these will be discussed in detail here.Simms’ Titration Constants His argument was more or less as follows. Suppose we have one solution containing a diprotic acid with the stability constants K, = [HA]/([H][A]) and K , = [H,A]/([H][HA]) and another solution containing the same concentrations of each of two monoprotic acids with the stability constants G, and G,. (The signs of charge are omitted in this paper except where necessary for clarity.) If we now titrate each solution with standard sodium hydroxide solution and after each addition measure the pH value, then we can draw up two series of paired values (millilitres of sodium hydroxide solution and pH). For these series to be identical at every point, the conditions are: Simms4 is apparently the first person to have dealt with this problem.K, = G, + G, .. . . . . * * (1) K, x K , = Gl x G, .. .. .. * (2) 1/K2 = l/Gl + 1/G2 . . * . . . - * (3) from which it follows that The above indicates that G, and G, are true constants. Simms calls the G constants 593594 JOHANSSON : SIMPLIFICATION OF THE MATHEMATICAL Analyst, VoZ. 104 titration constants. Although he refers to dissociation constants, the same term will be used in this paper for stability constants from now on. Simms continues his argument to prove that an n-protic acid with the constants K,, G,. K,, .... K , can be replaced by n monoprotic acids with the titration constants G,, G,, .... In this instance the following equations apply: n K, = 2 Gi i = l n-1 n K,K, = 2 2 GiGj n-2 n-11 n K1K2K3, ....K , = G,G,G,,. ... G, Kankare5 derived the same equations using a different approach. Klas6 used Simms’ formula in order to facilitate the calculation of the hydrogen-ion concentration of aqueous solutions of several polyprotic acids. Calculation of Titration Constants from the K values. calculate K , and K,. are known, is not easy to carry out. equations (1) and (3) G, m K, and G, m K2. constants are equal to the stability constants defined in the usual way. equations (1) and (2) yield G, = +Kl and G, == 2K, = +Kl. The more protons that are in the acid, the more difficult it is to calculate the G values If we take a diprotic acid, where G, and G, are known, it is easy to The converse calculation, i.e., to calculate G, and G, if K , and K , If G, is much greater than G, we obtain from the In this instance, therefore, the titration If G, = G, Thus, the titration constants will vary between - * (4) .... .. K, < G1, < K, . . * * (5) K, Q G , < 2K, .. .. .. .. If the program used for the evaluation of a. titration requires an approximate value for the titration constants, then it is usually sufficient to estimate this according to equations (4) and (5). It may be convenient to use some kind of standard procedure and the mid-point of the interval can then be taken as the initial value of the constant. The simple equations (4) and (5) are not applicable to acids with more than two protons. In those instances Table I can be used for the calculation of approximate log G values from known log K TABLE I VALUES OF ai FOR CONVERTING LOG Xi TO APPROXIMATE VALUES OF LOG Gi log Gi = log K, + a$.Number of protons in the acid a1 a2 a3 a 4 a5 a6 2 -0.12 +0.18 3 -0.18 +0.10 +0.30 4 -0.20 $0.07 t 0 . 2 4 +0.40 5 -0.22 +0.05 +0.22 +0.35 +0.48 6 -0.23 +0.04 +0.21 $0.34 +0.44 $0.54July, 1979 EVALUATION O F TITRATION RESULTS 596; values. Table I shows the values of ai to be added to the log K , values in order to obtain log G,. at the mid-point of the interval, according to the equation log Gi (mid-point) = log K, + a, If necessary, the values can be calculated more exactly by solving G1 and G, from equations (1) and (2) : G,,, = Kl/2 & $VK; - 4K1K2 The data on stability constants in the available literature, however, are often so unreliable that not much is gained from a calculation of this kind as a rule.The best results are obtained when the programs are so designed as to allow for a certain amount of error in the G values without causing an unacceptable degree of inaccuracy in the concentration deter- minations. It is also evident from equation (6) that G, and G, do not have red values if K , is less than 4K,. A titration of a diprotic acid with the quotient K,/K, < 4 or (log K, - log K,) < 0.60 cannot therefore be treated as a titration of two monoprotic acids. Significance of Titration Constants of a diprotic acid HB,-B2H can be illustrated as follows, mainly according to Adams7: Simms’ titration constants have a simple physical significance. The protolysis equilibrium .. .. * * (7) .. The constants k,, k2, k, and k, are stability constants defined by the following equations: k, [HI [-B,-B,] = [HB,-B2-] k, [HI [-B,-B,] = [-B,-B,H] k3 [HI [-B,-B,H] = [HBI-BZH] k, [HI [HB,-B2-] = [HB,-B,H] If K , and K , are the ordinarily defined stability constants then the following is valid: K , = k, + k, If the addition of a proton to the group B,- is independent of the presence or absence of a proton at the B2- group then k, = k3.In the same way k , = k , is obtained with the addition of a proton to the B,- group. In these circumstances we obtain Kl = k, + k , .. .. .. .. * - (8) Kl x K , = k, x k , . . .. .. .. .. (9) If one compares the equations (1) and (2) with (8) and (S), it can be seen that k, = G, and k, = G,. Simms’ titration constants can therefore be expressed as the intrinsic constants of the individual groups (also called microscopic constants) of the diprotic acid if the additions of hydrogen ions to the two groups are independent of each other.Similar reasoning can be applied to polyprotic acids. In his paper, Adams7 says that as a result of these statistical considerations KJK, > 4 is valid for the quotient between the two stability constants of a diprotic acid. The value 4596 JOHANSSON : SIMPLIFICATION OF THE MATHEMATICAL Analyst, VoZ. 104 is obtained if the acid is symmetrical and if the addition of hydrogen ions to both groups takes place independently of each other, i.e., k , = k , = k , = k,. For other polyprotic acids values for these statistical quotients are reported.* If Adams' argument was correct, it would mean that all n-protic acids could bt: replaced by n monoprotic acids with the same concentration.However, Bjerrum,S~lO Simms,4~11 Ricci,l2 Meites* and Kankare5 have pointed out that the real conditions cannot be expressed as simply as by these statistical quotients. To clarify this point, Adams' argument can be expanded and generalised. Development and Generalisation of Adarns' Ideas Consider the reaction A + IB = AB with the stability constant defined by the concentrations according to CAB]/( [A] [B]). In the solution there are ionic or molecular fornis of AB, A, B and other charged or uncharged molecules originating from reactions betwee:n these three and the solvent or other species present in the solvent. The probability of the reaction in question determines the extent to which the reaction takes place ; at equilibrium, certain definite proportions exist between the different species in the solution, and these proportions can be said to be controlled by the equilibrium constants.Each one of AB, A and B therefore occur in the solution in many different forms. When we determine a constant our methods of measurement are not precise enough to measure each separate constant, so that some forms are actually measured in the constant while other forms cannot be measured, for example, because they are affected by electrostatic forces in the solution. If the different measurable forms of AB are designated (AB),, (AB),, . . ., (AB), and the different measurable forms of A and B are designated (A)l, (A),, . . ., (A)m and (B)l, (B),, .. ., (B)n, then the stability constant has the following appearance : Reactions may occur between all of these molecules. 1 If we now especially consider the constants describing the reactions of (AB), to other measurable forms of AB etc., we obtain the following expressic n : where k , 1. In the same way we obtain, for A and BJuly, 1979 EVALUATION OF TITRATION RESULTS m 597 n u = l We then obtain the following expression for the constant K : If the reaction (A), + (B)l = W)l is the main reaction, then the constants kA, k, and k , should not be much greater than 1 in practice. These constants can be regarded as a sort of side-reaction coefficient (a-coefficient)13 that is used to adjust the main reaction constant in order to obtain a value for the constant being measured.It can be seen that if the transition (AB), to (AB),, etc., means that (AB), reacts with another ion, L, in the solution, then K,, has the same appearance as a normal a- coefficient : k,, = 1 + [L] X k:; f [LIZ x kzA + . . . + [LIZ-‘ x k S 1 ) The factor k , is still constant if the concentration of L is constant. This type of side reaction will probably have an effect at higher ionic strengths. The non-measurable forms of AB, A and B can be expressed, in the same way as the measurable forms, as (AB),, (A), and (B)l, respectively. The non-measurable forms that are thought to be measured in the constant are of special interest. They are designated by (AB),,, to (AB),, (A)m+l to (A), and (B),+l to (B)T, and can then be expressed as follows: w=Z+l The ratio between the measured species and the species that are thought to be measured is consequently k ~ ~ / ( 1 + g ’ A B ) .In the following, ( 1 + g’m) will be designated by gAB (gAB > 1 ) . This treatment gives rise to further correction factors for the main reaction constant. The concentrations (AB),, (A), and (B), must be divided by g-, gA and gB, respectively, so that the constant has the following appearance :598 JOHANSSON : SIMPLIFICATION OF THE MATHEMATICAL Analyst, Vol. 104 kaB . . g A gB The coefficients k=/gm, kA/gA and kB/gB should. be close to 1, but they can be both greater or less than 1. If the reaction (A), + (B), = (AB), is the main reaction for which the constant is to be measured, then they should be, in reality, a comprehensive expression for the activity coefficients and side-reaction coefficients for the side reactions that are not otherwise taken into account.They will be called $ from now on. These +-coefficients are constant if the conditions for the reaction are kept constant. The side reactions for which the constants are known, and for which it is also known that the reaction products are not calculated within the constant K, can be considered by ordinary a-coefficients. If, then, (AB), has a side reaction with L it is considered by CC(AB)~(L) so that instead of +D we obtain a- = $AB + ~ ( A B ) , ( L ) . - 1. The conditional constant now has the following appearance : The above suggests that all equilibrium constants can be regarded as concentration constants but as conditional13 concentration constants. As Simmsll and Ricci12 have shown, it still holds that polyprotic acids can be replaced by monoprotic acids.The condition for this is, however, that the +-coefficients and the or-coefficients are kept constant, and that the ratio between the constants is greater than the statistical quotient. If this reasoning is applied to Adams' diagratm [equation (7)], given the condition that the reaction via HB,-B,- is the main reaction (and that no reactions other than those in Adams' diagram occur) , the following expression is obtained for the constants (excluding the ionic charges) : where and [HBI-B;] = [HBI-BJ + [B,-B,H] If the reaction via B,-B,H occurs to an equally large extent as the reaction via HB,-B,, then k, = k, and $HBl-B2 = 2.If the reaction occurs to a lesser extent, &B1-B2 has a value between 1 and 2. The following is then valid for the quotient of the constants: where The size of K, and k, is of decisive importance for the size of the quotient K,/K2. then the maximum value of the quotient K1/K2 will be 4. 1 <+AB1--B2 <4 If k , = k ,July, 1979 EVALUATION OF TITRATION RESULTS 599 It is difficult to comment on the size of the quotient k,/k, other than to say that as a rule it should hold that k, > k,. However, Campbell and Meites,14 in a paper on titration graphs for acids with very small ratios of successive dissociation constants, have indicated a possible structure for such abnormal acids in which K , is greater than K,. An indicator that reacts like an abnormal acid has been discussed by Schwarzenbach.15 It is also conceivable that side reactions take place to such a large extent that the ratio between the conditional constants falls below the statistical quotients.This instance is dealt with more fully in the examples below. Examples of the Influence of Side Reactions Titration of adipic acid These problems occurred when we were titrating adipic acid. We often perform our acid - base titrations at an ionic strength of 0.5, using barium chloride as the ionic medium. The following constants are stated at p = 0 for adipic acid, H2A16J7: H + A =HA log Kl = 5.41 H + HA = H,A log K , = 4.42 Ba + A =BaA log KBaA = 1.85 Thus, when adipic acid is titrated in the barium chloride medium, a conditional constant Ki is valid for K, if the side reaction of A with Ba is taken into account.Taking these constants as a starting point, rough calculations indicate that Alog K = log Ki - log K , should be approximately 0.7 in the barium chloride medium when p = 0.5. Thus, we have come close to the critical value Alog K = 0.60 where, theoretically, the evaluation program TITRA no longer functions. These titrations were evaluated with regard to the constants in order to obtain the true value for Alog K. In this instance the computer program LETAGROP~* was used. The values obtained were log K; = 4.74 and log K , = 4.09, ie., Alog K = 0.65. Therefore, the evaluation of the concentrations by TITRA functioned normally and the relative error was less than 0.1 yo. Titrdion of the disodiwn salt of ethylenediaminetetraacetic acid (EDTA) In the previous example, the complexation with the ionic medium was assumed to be very weak.A stronger complexation results in more pronounced changes. This is true for, for example, EDTA. Titration of the disodium salt of EDTA with sodium hydroxide solution yields two steps on the titration graph, the second step being very small. At an ionic strength of 0.5 the following values for the stability constants can be assumed provided that no side reactions occur: H + Y = H Y H + HY = H,Y log K, = 10.2 log K , = 6.1 The two protons dissociate one after the other and as a result first HY3- and then Y4- are formed. If, however, 0.133 M barium chloride and 0.100 M sodium chloride solutions are used as the ionic medium the ionic strength will still be 0.5 but the conditions for the titration will be radically changed.The reasons for this are the reactions between barium chloride and EDTA according to the following formulae : Ba + Y = BaY log KBay = 7.4 Ba + HY = BaHY log KBaHP = 1.7 (The values of the constants given above are based upon those given by Schwarzenbach et al.19) The conditional acid constant values that can be calculated by using these values are log K; = 4.6 and log Ki = 5.2. It is evident that Ki has now become greater than K;, which means that both protons will react almost simultaneously during the titration.,O600 JOHANSSON Consequently, there will be only one step in the titration graph but this will not, however, be identical with the titration graph obtained for a monoprotic acid of twice the concentra- tion.14 (At the beginning of the titration giraph there is, of course, another difference, depending on the fact that Na,H,Y is an ampholyte.) In this instance the computer program TITRA~ will not work in its usual form.Non-acid Complexes The reasoning principally used for acids, i.e., proton complexes, can also be applied to other types of complexes. This has been partly carried out for metal complexes by Bjerrum,21 Fronaeus,, and K l a ~ . , ~ These workers have not, however, considered the applica- tion of their ideas within analytical chemistry. Even if the above principles can theoretically be applied to complexation titrations, in practice their application is limited. If in a titration of a metal ion M with a complexing agent L, [MI is measured by a metal ion sensitive indicator electrode, reasoning identical with the above can be used if only the complexes ML, M,L, etc., are formed.Usually, however, complexes of the type ML, ML,, etc., are formed. A solution con- taining only these complexes, by analogy with the above, can be replaced by another solution with complexes of the type ML. If these two solutions are titrated, the concentration of L must be the same in both solutions. If the titration is performed potentiometrically then the indicator electrode should respond to the L concentration. Such ligand-responsive electrodes are, however, rare. In these instances L must absorb light, or else an indicator must be used. Most of the indi- cators in use are metallochromic, and are therefore not suitable for studying variations in the ligand concentration.Similarly, it is rather unusual for such titrations to be performed photometrically. I am indebted to Professor Folke Ingman for valuable discussions and to Mrs. Alison Holmstrom for translating the manuscript. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. References Johansson, A., Analyst, 1970, 95, 535. Ingman, F., Johansson, A., Johansson, S., and Karlsson, R., Analytica Chim. Acta, 1973, 64, 113 Johansson, A., and Johansson, S., Analyst, 1979, 104, 601. Simms, H. S., J . A m . Chem. SOC., 1926, 48, 1239. Kankare, J . J., Talanta, 1975, 22, 1005. Klas, J., Analytica Chim. Acta, 1968, 41, 549. Adams, E. Q., J . A m . Chem. SOC., 1916, 38, 1503. Meites, L., J . Chem. Educ., 1972, 49, 682. Bjerrum, N., Z. Phys. Chem., 1923, 106, 219. Bjerrum, N., Ergedn. Exakt. Naturw., 1926, 48, 125. Simms, H. S., J . A m . Chem. SOC., 1926, 48, 12511. Ricci, J . E., “Hydrogen Ion Concentration,” Princeton University Press, Princeton, N. J., 1952. Ringbom, A., “Complexation in Analytical Chemistry,” Interscience, New York, 1963. Campbell, B. H., and Meites, L., Talanta, 1974, 21, 117. Schwarzenbach, G., Helv. Chim. Acta, 1943, 26, 418. Gana, R., and Ingold, C. K., J . Chem. SOC., 1931, 2153. Topp, N. E., and Davies, C. W., J . Chem. SOC., 1940, 87. Phyllis, B., SillCn, L. G., and Whiteker, R., Ark. Kemi, 1969, 31, 365. Schwarzenbach, G., and Ackermann, H., Helv. Chim. Acta, 1947, 30, 1798. Schwarzenbach, G., and Sulzberger, R., Helv. Chim. A d a , 1943, 26, 453. Bjerrum, J ., “Metal Ammine Formation in Aqueous Solution,” Haase, Copenhagen, 1941. Fronaeus, S., “Komplexsystem hos koppar,” Gleerupska Universitets, Bokhandeln, Lund, 1948. Klas, J., Analytica Chim. Acta, 1975, 74, 220. Received May 22nd, 1978 Amended September 1 lth, 1978 Accepted December 14th, 1978

ISSN:0003-2654    

DOI:10.1039/AN9790400593

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, July, 1979, Vol. 104, pp. 601-612 601 Automatic Titration by Stepwise Addition of Equal Volumes of Titrant Part IV.” Acid - Base Titrations General-purpose Program for Evaluating Potentiometric Axel Johansson and Sten Johansson Department of Analytical Chemistry, The Royal Institute of Technology, S-100 44 Stockholm 70, Sweden A computational procedure for evaluating titration results is described. It utilises non-linear regression techniques to solve the equations for titration graphs from acid - base titrations. The procedure is based on the fact that the titration of an n-protic acid can usually be treated as a titration of n monoprotic acids of the same molarity as the n-protic acid. In principle, the composition of an unknown mixture of acids can be deter- mined by non-linear regression if a sufficient number of measurements (millilitres of titrant veYsz4.s pH) are available.However, this assumes that the measured values are known with adequate accuracy, an assumption that is not correct in practice. All measured values (especially the pH values) are imperfect, hence a conventional non-linear regression can give a large number of mathematically equivalent solutions. This approach may there- fore, by chance, produce a result that is mathematically correct but chemically impossible. This problem is avoided by setting boundary conditions and carrying out calculations in an appropriate sequence. Dominating terms are dealt with first, then the effects of the remaining terms are included. A computer program TITRA, capable of handling such a calculation scheme, is presented.No preliminary estimates of the required concentrations or equivalence volumes are necessary in the program. The general equation for the titration graph derived is applicable to samples that may contain monoprotic acids (strong or weak), polyprotic acids, mixtures of acids, ampholytes, salts of weak acids and “abnormal” acids (acids with an abnormal sequence of stability constant values). By exchanging hydrogen-ion concentrations for hydroxide-ion concentrations and vice versa the equation is applicable to the titration of bases. Keywords : Acid - base titrimetry ; evaluation program In Part IIIl of this series we described a method for calculating equivalence volumes in the titration of monoprotic acids or bases with almost any value of the stability constants.This procedure, which is based upon solving a set of linear equations, has the following advantages : it requires only a few pairs of measurements (millilitres of titrant and e.m.f. or millilitres of titrant and pH) ; no preliminary estimate of equivalence volume is necessary; the stability constants of the acid or the base need not be known; and exact pH values are not required. Its main disadvantage is the fact that it does not allow calculation of the individual com- ponents in a mixture of acids or bases. However, in some instances it allows calculation of the total concentration of components. In this paper we discuss a more general computer program, TITRA, which is capable of handling results from acid - base titrations of almost any kind; it is an extended version of a program that has been in use for several years at this Institute.It has not previously been published, although applications of this program have been previously reported.2 Programs based on similar principles have also been described by us in collaboration with Pehrsson and Ir~grnan.~,~ These papers describe in detail several special cases, such as the titration of diprotic acids and of a mixture of two monoprotic acids. They also include a literature review of earlier work in this field. Since then, other similar computational procedures have been described.5-’ * For Part I11 of this series, see reference list, p. 612.602 JOHANSSON AND JOHANSSON : AUTOMATIC TITRATION BY STEPWISE Analyst, VoZ.104 A set of m equations is solved for r unknowns (m > r ) . The equations are no't linear, so the equation set is solved by estimating approximate values of the unknowns and then using a least-squares procedure to obtain more accurate values. The general equation for the titration graph of a polyprotic acid is complex and thus difficult to handle. However, the titration of an n-protic acid can usually be treated as a titration of n monoprotic acids of the same molarity as the rc-protic acid. It is then necessary to use so-called titration constants rather than conventional stability constants.8 All components in a mixture of acids are treated as monoprotic acids, thus reducing the equations to a simpler and more easily handled form. Note that treating an wprotic acid as a mixture of monoprotic acids does not introduce approximations (except for so-called abnormal acids, which simultaneously release -two or more protonsQ; however, these can be handled by a modification of the program).TITRA is based on classical mathematical procedures. Equations of Titration Graphs Mixtures of Acids and Polyprotic Acids Consider Vo ml of a solution containing n acids, HA(,,, HA(,,, . . ., HA(,,, . . ., HA,,,, each at concentration Col, C,,,, . ., Coi, . . ., Con, and with stability constants K,, K,, . . ., K,, . . ., K,. This solution is titrated with a strong base of concentration C,. After each addition of an aliquot of base, the e.m.f. is measured and the hydrogen-ion concentration, [HI, is calculated. A total of m pairs of measurements are obtained, i.e., at least (m - 1) aliquots of base are added.The hydrogen-ion concentra.tions [H'] measured often include a systematic error, f, such that [HI = f[H'], where f is an unknown, but constant, factor. This error factor f arises due to the fact that the electrode response is changed by the transfer from one solution to an0ther.l~~ At each titration point, the following set of equations applies,2 assuming that V ml of base are added: n Kk = Kwlf where K , is the ionic product of water; K; = Kif where Ki is the stability constant of acid HA,,, ; and Ki = [HA(,,]/{ [HI [A(,,] ). is the titration constant of one of the monoprotic acids that replaces the polyprotic acid, all acids having the same concentration.) (If the acid is polyprotic, Ampholytes For example, consider a solution of potassium hydrogen phthalate.It. is necessary to assume that phthalic acid is equivalent to two monoprotic acids, HA,,, and HA,,,, each a t concentration C, (C,, = C,, = Co) and with stability constants K, and K,, respectively; the initial potassium-ion concentration is C,. The charge balance gives With ampholytes, equation (1) takes a slightly different form. The ampholyte is titrated with sodium hydroxide solution. or Thus, equation (2) includes the term -VoCo, which is not in equation (1). Samples Mixed with a Known Amount of Strong Acid chloric acid, to the sample solution before the titration is carried out. It may sometimes be advantageous to add a known amount of strong acid, e.g., hydro- An increase in theJuly, 1979 ADDITION OF EQUAL VOLUMES OF TITRANT. PART IV 603 hydrogen-ion concentration permits calculation of better estimates of f (see below).Addition of a known amount of strong acid may also be useful when a salt of a weak acid or an ampholyte is titrated. All samples can then be titrated with standard sodium hydroxide solution. It is not necessary to add an excess of the acid iff is not to be calcu- lated (see Table 11). With the addition of strong acid a term Vo[C1] must be added to the titration equation, where Vo[C1] is the number of milliequivalents of strong (hydro- chloric) acid added. Acids that Simultaneously Release Several Protons The program TITRA treats the titration of an n-protic acid as being equivalent to the titration of n monoprotic acids, each of the acids having the same total concentration as the n-protic acid.The stability constants of these component acids will not be the same as those of the n-protic acid (KgA, K;+, etc., where KgnA is a conventional stability constant). The stability constants (or titration constants) of these component acids are related to the intrinsic constants of the individual groups in the rc-protic acid.* Titration constants are usually designated as Gi, but in this paper we have used the symbol Ki for all monoprotic acids (including component acids). The following discussion is limited to diprotic acids (H,A), so as to avoid excessively lengthy equations. A diprotic acid can be treated as being two monoprotic acids if KgA > 4Kz2,. Thus, acid H2A must be stronger than acid HA-, and this is almost always true.When such an acid is titrated, protons are released sequentially with increasing pH, forming first HA- and then A,-. Each of these ions is the predominant species over a certain pH range. In the special case KgA = 4Kg2A, the titration constants are equal, i.e., I<, = K, and equation (1) becomes 2T;r,GCO/(l + [HI K,) - (Vo + V ) [HI + (Vo + V)KW/[Hl - vc, = 0 This is the same as for a monoprotic acid of twice the concentration. The condition KgA < 4Kg2A is almost never found with diprotic acids, but it can apply to conditional constants if complex formation occurs.8s10 In this last instance, the titration graph has only one step, indicating that the species H2A and A,- predominate, with very low HA- concentrations during the entire titration.Vo ml of a diprotic acid, H,A, of total initial concentration Co, is titrated with C, M sodium hydroxide solution. Assuming that [HA-] is negligible, and after considering charge balance, we obtain The titration graph equation for such an abnormal acid can be derived as follows. [H+] + [Naf] - 2[A2-] - [OH-] = 0 . . . . * * (3) Following the addition of V ml of sodium hydroxide solution, "a+] = VC,/(V, + V ) . total concentration of acid, CHZA, is given by The and by combining this equation with the stability constant we obtain where Co(ab) is total initial concentration of the abnormal acid. yields Substitution in equation (3) 2 v o c o ( a b ) / ( l + [HI2Kab) - (Vo + V ) [HI -k ( v o + V)Kw/[H] - ~ C B = 0 (4) Equation (4) differs from equation (1) only in the first term.604 JOHANSSON AND JOHANSSON : AUTOMATIC TITRATION BY STEPWISE Analyst, Val.104 General Titration Graph Equation (2) and (4), adding V,[Cl] and considering that [HI = f[H’]. The following general formula for a titration graph is obtained by combining equations (1) , n where a is the number of neutralised steps in an ampholyte of concentration Co(am) [k, at least one Coi value is equal to Co(am)], b is the number of protons that are simultaneously released during an abnormal protolysis step of an acid of concentration Co(ab), and K& = Kabfb where Kab is the product of the stability constants for the abnormal step. VoJ V , C,, a, b, [Cl] and [H’] in equation (5) are known, while &, K’ , K& andf are unknown (or only approximately known).In principle, the composition of an unknown mixture of acids can be determined if a sufficient number of measurements are available. However, this assumes that the measured values are known with adequate accuracy, an assumption that is not correct in practice. Further, the e.m.f. values measured correspond1 to a variation in the hydrogen-ion concentra- tion over many orders of magnitude. The equations can therefore be characterised as being ill-conditioned.11 From a computational viewpoint this ill-conditioning means that there are a large number of nearly correct solutions to the equation set (5). The computational procedure used to solve these equations is important. For example, consider the term (V, + V ) [H’lf. If (V, + V ) = 100, the teim has the value 1 x f when log [H’] = -2 and the value 10-6 x f when log [H’] = -8.Thus, f cannot be determined precisely from titration results obtained at high pH values. The computational procedure must therefore be designed first to utilise dominating terms arid then to include the effects of the remaining terms. Also, chemically impossible values of the parameters, e.g., negative stability constants and negative concentrations, must be rejected. Coi are the required unknown concentrations. Detailed Calculation Procedure 1. 2. Measured e.m.f. or pH values are converted into [H’] values, which are then used in equation (5). If the values of all constants were known, leaving Coi values as the only unknowns, this would result in a set of linear equations thai could be solved by a least-squares procedure.In practice, this is usually not the case and the equation set is not linear. Instead, the problem is solved by substituting estimated values of the parameters Coi, Ki, f, K& and, if necessary , K&, and then obtaining improved. values by successive approximation. After substituting these estimated values plus a data pair (V, [H’])j, the left-hand sides of these equations have values U j ( j = 1, 2, . . ., nz), where Uj = 0. The estimated values of parameters are then corrected so as to minimise the sum C Uj2. m j = 1 3, For this purpose, a further set of equations is developed, with the corrections as the unknowns. The coefficients in these equations are obtained by differentiating equation (5) , thus obtaining a set of linear equations.Operations 2 and 3 above are carried out using the subroutine DPV. 4. The number of measurement points (equal to the number of equations developed in paragraph 2 above) must be at least equal to the number of unknowns. If more results are available a least-squares procedure can be used to compensate for random errors in the measurements. This compensation is achieved by reducing the equations to the corre- sponding normal equations; they are then solved by the Gauss elimination procedure, using an IBM routine SIMQ. The roots are added to the estimates and then new Uj values are calculated by using DPV. This procedure is repeated until 2 Uj2 meets certain conditions m j = l (see flow diaaam. Fin. 1).July, 1979 ADDITION OF EQUAL VOLUMES OF TITRANT. PART IV 605 START 1-2 +- 1CYK:l HDT vector for storing the values of the parameters during the variation UMINSIZ em f.DATA ph‘ IJATA J 1 I number of protolysis steps, parameters and refinement - DT HDT=DT I REPET = TRUE 4 - b l ’ 1CYK:O I C Y K = ICYKi1 I DAMP.1 card OT vector for storing the best values of the parameters @ repetition begins @ next cycle begins A r ‘ the number of points considered after the last equation point is reduced to max. 3 (if not otherwise stated) U1 and U2 are error square sums final output obs. ph, calc. ph, Ve;, Coi, log Ki, log K, and f HROT vector for storing the calcu I ated corrections of the parameter @ continuation on right Fig. 1. Flow diagram of program TITRA. The constant data consist of NCYK = maximum number of calculation cycles, IE = 0 if ph’ data are used, otherwise IE = 1, Cg, VO’, pWw, (temp., E&ja) and pairs of V and e.m.f.or ph’. If ph’ values are used it is not necessary to give the constants in parentheses. If a base is titrated poh’ is calculated from poh’ = P K ’ ~ - ph’ (here poh’ = - log[OH’] and ph’ = - log [H’]) . 5 . The values of Coi obtained are used to calculate the equivalence volumes (VeJ of the acids and all measurement points after the last equivalence point, except the first three, are omitted. The reason for this omission is that the quality of the experimental values decreases when a glass electrode is used at a high pH value.606 JOHANSSON AND JOHANSSON : AUTOMATIC TITRATION BY STEPWISE ApzaZyst, VoZ. 104 If the error factor f is calculated the [H’] and K; values are divided by f and the K& value is multiplied by f.This operation gives the values of [HI, Ki and K,. These values are then used to repeat the calculations from para,graph 4. Finally, the obtained values of the parameters are substituted into equation (5) and -log[H] or -log[H’] are calculated for each measurement point and compared with observed values. The calculation can be carried out in many ways; the program TITRA performs it as follows. The value of -log[H’] is assumed to be within the interval 0-14. The hydrogen-ion concentrations corresponding to the interval boundaries are substituted in the left-hand side of equation (5), yielding expressions with negative and positive values of U,. As equation (5) is a single-valued function of [H’], the po1arit:y of the new value of U, can be used to select the half interval that includes the true value of [H’].The other boundary of this half interval will have opposite polarity. The procedure is iterated until -log[H’] is deter- mined with an error of less than 0.001. 6. The pH interval is then halved and a new value of Ui is calculated. Which estimated values aye required? KI, Kk, K&, f and COi are unknown, but estimated values are required for the calculation. It is inconvenient to have to choose estimates before each calculation, and standard numbers, say 1 or 0, would be preferable as initial approximations. It is often, but not always, possible to obtain standard values (see examples below). The need for accurate estimates increases with increasing sample complexity. The factor f has a value of about 1, which can always be used as an initial estimate.If the maximum error in EA (see Experimental below) is known, it is possible to set limits on possible f values. For example, experimental conditions can be chosen such that -log[H’] has a maximum error of rt0.02 unit, i.e., the error in EL is less than 51.2 mV; f can then vary between 0.95 and 1.05. If the computed value off lie; outside this range the experimental results have not been sufficiently accurate to allow e::act calculation of f. As was mentioned above, the term (V, + V ) [H’] f, from which f is calculated, may be small compared with other terms in the equation. The errors in these other terms may be greater than the value of (V, + V ) [H’] f.Hence, direct application of the equations may give very large or very small values of f, which is chemically impossible. If the error in f is great, the iteration procedures either do not converge or converge to a completely wrong result. The program TITRA is designed so that if calculated f values ever lie outside tlhe range 0.95-1.05 they are corrected to the nearest range boundary. It is also possible to repeat the titration after adding a known amount of a strong acid to the sample solution. This increases the value of the term (V, + V ) [H‘] f so that f can be calculated satisfactorily. Log K’ generally lies between 13.5 and 14.0,12 but a more accurate value can be obtained by calibrating the electrode pair. If the only measurements available are from acidic solutions, no attempt should be made to obtain a better estimate of K& because the K& term has little effect in these instances.It is more difficult to estimate K; values. In many instances a log K; value of -10 for strong acids and 0 for other acids can be used. In other instances estimates can be obtained from tables, but these will probably not be exact values. K; values must be corrected for the ionic strength of the solution. Also, sample components may form complexes with anions of the acid, with marked effect on the .K; values. The stability constants affect only the term VoCoi/(l + [H’IK;) in equation (5), and errors in [H’] can be partly compensated for by using appropriately corrected values of Ki.Thus, it is usually desirable to calculate stability constants rather than to use pre-selected values, except with complex samples. For polyprotic acids it may be difficult to convert literature values of stability constants into the corresponding titration constants.8 However, calculations with TITRA yield the appropriate values directly. In difficult instances, the constants should be determined by titrating a known amount of acid using the appropriate electrode pair. TITRA can be used to make the calculations. If the stability constants are nearly equal, the calculations only yield acceptable results if This value is then used in the next calculation cycle. An approximate K& value is chosen according to the ionic strength of the solution.July, 1979 ADDITION OF EQUAL VOLUMES OF TITRANT.PART IV 607 the stability constants are accurately known. Examples are given in Table I and in reference 2. The Coi values are unknown, but as a first approximation they can be set as where Vm is the total volume of titrant added and 9 is the total number of protolysis steps. All acids are thus assumed to have the same initial concentration. As the quantities V,, CB, Vo and p must always be entered into the program, this calculation mode does not entail additional work. Experimental Selecting an Electrode Pair Potentiometric acid - base titrations are usually carried out by using glass and reference electrodes, with the electrode pair responding to hydrogen-ion activity rather than hydrogen-ion concentration. With conventional sigmoidal titration graphs this is not a problem, as equivalence volumes are determined by detecting a point of inflection.Differ- ences between hydrogen-ion activity and hydrogen-ion concentration will shift the inflection point along the pH axis, but will not affect the equivalence volume. Also, the behaviour of the electrode pair at values remote from the equivalence volume is not critical. With numerical evaluation, titration results measured over almost the entire titration graph are used and if the sample contains a mixture of acids the titration results may cover a wide pH range. This necessitates control of the electrode pair performance over almost the entire pH scale. The computer procedure is designed so that all measured values of hydrogen-ion concentra- tion can include an error factor f.This error factor must be constant over the pH range used. If this is not the case, the calculated composition of the sample may be wrong. The hydrogen-ion concentration in a solution can be calculated from measured e.m.f. values (E in mV) using the equation E = Ed + Qlog[H] + E; where EA stands for the conditional standard potential and includes the glass electrode's normal potential, E&, the reference half-cell potential and that part of the activity co- efficients and the liquid-junction potential that is independent of acidity, [HI is the hydrogen- ion concentration (not activity) and Q = RT x lnlO/F = 59.158 mV at 25 "C. E; = j,[H] + joHIOH] accounts for the acidity-dependent part of the activity coefficients and the liquid junction potential. The factors j , and joH are constants at constant ionic strength.Also, -log [HI is designated ph in the following text. The value of jH can be determined by titrating a known amount of 0.02-0.05 M hydro- chloric acid with a 0 . 1 - 0 . 4 ~ solution of sodium hydroxide. The base is added stepwise and E is measured after each addition of titrant. Plotting a graph of E - Q x ph versus [HI yields jH as the slope and EA as the intercept with the y-axis.13 The term j,, has such a small value that it is usually unnecessary to consider it if ph < 12. The value of jH for a given electrode pair is stable, but it should be checked if the electrode is used under extreme conditions. Eh values vary from day to day. A constant error AE& introduces an error, Aph, in all ph values, corresponding to the error factor f in all hydrogen-ion concentrations.However, a change in EA may take place in some glass electrodes going from the acid to the alkaline ph range. The value of EL in acidic solution, Eda, may differ from the value in alkaline solution, Edb, by several millivolts. According to Ciavatta14 the difference is to be ascribed to the change of the E,$ occurring in the ph range 5-8.5. He explains the change by assuming that the protective layer of hydrated silica formed in acidic solutions on the surface of the glass electrode will begin to dissolve as ph becomes greater than 7. Thereby, the surface structure of the glass membrane, which is in contact with the test solution, will be modified.In order to avoid any influence of the difference between the Ed values that are valid in acidic and alkaline solutions on the results of the titrations the results for e.m.f. obtained at ph less than 5 and ph greater than 8.5 are usually treated608 JOHANSSON AND JOHANSSON : AUTOMATIC TITRATION BY STEPWISE Analyst, VoZ. 104 separately.3J4 This treatment is not practical in routine analysis and therefore we have studied the influence of a change in Eh on the results. The effect was investigated by use of synthetic titrations in which 100.0 ml of a 0.01 M solution of an acid were titrated with 1.00-ml aliquots of 0.1 M strong base, Log K values of the acid varied between -10 and +lo. ph values were calculated and then altered SO as to simulate an electrode with Eda differing from E&.In one series of titrations ph values between 6.5 and 7.5 were increased by 0.01 per 0.1 unit while ph values above 7.5 were increased by 0.1. This is equivalent to a change in E, of 6 mV and of the hydrogen-ion concentration by a maximum of 21%. The equivalence volumes were calculated by different methods. When correct stability constants were used the greatest error was 5%. An error in the equivalence volume caused by an error introduced in changing [H'] can, as mentioned above, be reduced by using appropriate corrected values of Ki. Similarly, it is preferable to calculate K& for use in the term (V, + V ) K ' / [ H ] . At high ph values an error in [H'] can be compensated for by an equivalent change in K;. These procedures result in a maximum relative error of 1.1% for acids with 6.3 < log K' < 7.6.For other acids the relative errors were <0.1%. If the change in Ed is 3 mV the error never exceeds o.3y0, and if the change is 1.2 mV the maximum error is 0.2%. If a titration is performed rapidly it can be assumed that the change of E;, being rather slow, will occur over a wider pH range than if the electrode pair is allowed to attain equi- librium after each addition of titrant. If the change in EL appears, for example, in the pH range 6.5-9.5, the error will not exceed 0.1 yo. The optimum titration speed for a given glass electrode is the fastest speed that allows the concentration term Q x log[H] to reach equilibrium. From these synthetic titrations it can be concluded that the change in the conditional standard potential of a glass electrode pair must never be greater than 1.2 mV, corresponding to 0.02 ph unit if the greatest tolerable relative error in the equivalence volume is 0.2%.The difference Ed, - E,6 can be determined by comparing the glass electrode with a hydrogen electrode but this method of calibration is time consuming. It is sufficient to titrate a known solution consisting of a mixture of acids with log K evenly distributed over the ph range. An example of such a solution is hydrochloric acid (log K = -lo), phthalic acid (log K = 2.59 and 4.37), p-nitrophenol (log K = 6.88) and barbital (log K = 7.61). If the difference between calculated and experimentally determined ph values never exceeds 0.01 ph unit the electrode pair is acceptable.For each new electrode pair it is necessary first to determine jH by titration of a known 0.02-0.05 M solution of hydrochloric acid.l3 Then, an approximate value of EA should be obtained by measuring the e.m.f. of a 0.01 M hydrochloric acid solution at the appropriate ionic strength (2% = E + 2Q - O.OljH). It is not necessary to determine an exact value for J% as this value can change by about 0.1 mV when the electrode is transferred from one solution to another. Temperature measurements are required for determining the constant Q. The value of Q can be assumed to be sufficiently precise not to contribute significantly to the over-all error. Apparatus E.m.f. measurements were carried out with a micro-sized combination glass and silver - silver chloride electrode pair (AH-401 M5, Ingold, Zurich, Switzerland) and a digital volt- meter (Model S1016H, AB Systemteknik, Lidirtgo, Sweden).Titrant aliquots were added with a pneumatically driven pipette (AutoChem Instrument AB, Bromma, Sweden). The delivery volume of usually 1.001 1 & 0.0005 ml (mean & standard deviation) was determined by weighing ten consecutive aliquots of water. The titration vessel was a Metrohm glass vessel, E;A 875-200, equipped with an EA 880 cover and immersed in a thermostatically controlled bath maintained at 25.0 & 0.1 "C. A stream of nitrogen (purified by passing it through 2 M potassium hydroxide solution and pre-saturated by passing it through distilled water and the ionic medium) was passed over the titration solution in order to prevent absorption of atmospheric carbon dioxide.The titrations of monoprotic acids were carried out by using the simpler procedure described in Part 111.1July, 1979 ADDITION OF EQUAL VOLUMES OF TITRANT. PART IV 609 Hydrochloric acid was used as a primary standard and was prepared from Titrisol ampoules (Merck) and checked by gravimetric determination as silver chloride. The sodium hydroxide solution that was used as titrant was prepared by diluting a 50% solution and adding barium chloride. After separation of the barium carbonate formed from carbonate present as an impurity in the sodium hydroxide, the solution was diluted and the barium chloride content adjusted so that the ionic strength was 0.5. This solution was then standardised by titration against hydrochloric acid.There is no interference from carbonate formation with this titrant but many acids form complexes with barium ions. Thus, both titrant and titrand should have the same barium-ion concentration. The ionic strength of the titrand solution was adjusted to 0.5 by addition of sodium chloride. (In principle, higher ionic strengths are desirable but this requires the addition of large amounts of very pure neutral salts. When the use of barium chloride would result in precipitate formation with the sample, sodium chloride only was used as the ionic medium. Merck pro analysi grade barium chloride and sodium chloride were used in order to adjust ionic strengths. This procedure is impractical for routine analysis.) TABLE I TITRATION OF VARIOUS ACIDS, AMPHOLYTES AND BASES Figures in parentheses show the order in which estimates of parameters were adjusted: (0) no change in parameter value; (1) adjusted from first computation cycle; and (2) adjusted from second computation cycle.Sample 1. Hydrochloric acid 2. Picric acid . . 3. Sulphanilic acid 4. Acetic acid . . 5. Nitrophenol . . 6. Barbital Diprotic mi&- Monoprotic acids- 7. Adipic acid . . 8. Succinic acid . . 9. Phthalic acid . . 10. Malonic acid . . 11. Tartaric acid . . Triprotic acids- 12. Phosphoric acid 13. Boric acid . . Tetraprotic acids- 14. Diphosphoric acid 15. EDTA .. .. Pentaproiic acid- 16. Triphosphoric acid Mixtures of acids- 17. Hydrochloric acid + Phthalic acid +Barbital . . 18a. Phthalic acid . . I Nitrophenol +Barbital .. 1Sb. Phthalic acid + Nitrophenoi . +Barbital 19. Hydrochloric acid . . +Excess of acetic acid 20. Hydrochloric acid . . +Excess of acetic acid A mpholytes- 21. 22. Potassium hydrogen phthalate 24. Alanine 25. Disodiurn'EDTA' :: 1: Bases- 26. Sodium acetate . . . . . . 27. Sodiumhydroxide . _ .. +Sodium acetate . . .. Potassium hydrogen tartrate . . 23. Glycine .. .. .. Initial value of log K 3.1 (2), 7.2 (2), 12.7 (2) 9 (a), 12.5 (0), 14.0 (0) 1.0 (2), 2.5 (2), 6.1 (2), 8.5 (2) 2.3 (2), 2.3 (2), 0 (1) 0 (2), 2.0 (2), 2.2 (2), 5.0 (2), 7.0 (2) -10 (0) 2.5 ( l ) , 4.5 (1) 2.5 (2), 4.5 (2) 7.0 (2) 7.5 (1) 7.5 (2j 2.5 (2), 4.5 (2) 6.88 (0) 7.61 (0) -10 (0) 4.4 (0) -10 (0) 0 (1) f 1.0 (1) 1.0 (2) 1.0 (2) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (2) 1.0 (2) 1.0 (2) 1.0 (2) 1.0 (2) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (2) 1.0 (2) 1.0 (2) 1.0 (0) 1.0 (2) 1.0 (2) 1.0 (0) 1.0 (1) 1.0 (0) 1.0 (0) 1.0 (1) 1.0 (2) 1.0 (2) Amount added/ mmol 1.0000 0.5260 0.3950 0.685 3 1.0823 0.5046 0.9120 0.7706 0.9370 1.0289 0.483 0 1.0894 0.778 4 0.9155 0.9376 0.436 9 1.000 1.0025 1.001 1 1.023 4 0.4858 0.4934 1.0234 0.485 8 0.493 4 0.096 0.2400 1.815 7 0.7074 0.5167 1.2302 0.6074 0.9310 1.000 0.600 0.600 Amount found/ mmol 1.0004 0.5280 0.3955 0.6852 1.0778 0.5044 0.9130 0.7702 0.9374 1.026 4 0.4849 1.0877 0.7826 0.9124 0.9386 0.4349 1 .ooo 0.9992 1.0055 1.024 9 0.5295 0.4547 1.0249 0.4829 0.496 1 0.095 0.2381 1.5132 0.7076 0.5189 1.233 7 0.606 7 0.9308 1.001 0.598 0.602 Recovery, Yo 100.0 100.4 100.1 100.0 99.6 100.0 100.1 99.9 100.0 99.8 100.4 99.8 100.5 99.7 100.1 99.5 100.0 99.7 100.4 100.1 109.0 92.2 100.1 99.4 100.5 09.0 99.2 99.8 100.0 100.4 100.3 99.9 100.0 100.1 99.7 100.610 JOHANSSON AND JOHANSSON : AUTOMATIC TITRATION BY STEPWISE Analyst, VoZ.104 Evaluation of Program TITRA The program was tested by using results obtained from titrations of a considerable number of acids and mixtures of acids. A few titrations of bases and mixtures of bases were also used. Column 2 of Table I lists values of the stability constants that were used as initial approximate values, with the number in parentheses after each parameter referring to the order in which the parameters were adjusted. Adjustment from the first calculation cycle is designated by (1) and from the second cycle by (2), with no adjustment shown as (0).Approximate values of concentrations were never used and the program calculated all initial values by use of equation (6). An initial value of log K& = -13.61 for the parameter K' was used, and it was only adjusted when measurements at high pH were $available. The error factor f in hydrogen-ion concentration was assumed to be 1.00 and was adjusted only if two of the initial pH values were below 4. Also, f was not adjusted when Ei was determined in direct connection with the sample titration. The results are summarised in Tables I and 11. Comments on Table I The evaluation of titrations of single monoprotic acids is straightforward, particularly because knowledge of the initial values of stability constants and concentrations is unnecessary. (a) For strong acids, a low K value, typically 10-lo, should be selected and then not adjusted.All measurements after the equivalence volume has been reached should be rejected. If values beyond the equivalence volume are included, the sample should be treated as a mixture of a strong and a weak acid. Sample or titrant may contain small amounts of weak protolytes, e.g., carbonate. The contaminating acid can be assumed to have a log K value of 6-8. It is not necessary to correct this value during the calculations. (b) For weak acids, the error factor f should not be adjusted. ( c ) For acids of intermediate strength, Ir: should only be adjusted from the second The program EKVOL~ performs these operations automatically and is For diprotic acids, it is not necessary to provide initial values of stability constants unless For titrations of polyprotic acids, approximate values of the constants are required.Diphosphoric acid and triphosphoric acid were prepared by passing weighed amounts of the sodium salts through an ion-exchange coluinn (Dowex 50LvX8 [H+]) . The titration (No. 15) of EDTA (ethylenediaminetetraacetic acid) should be discussed in more detail. It was carried out in a barium chloride (0.133 M) and sodium chloride (0.100 M) solution, with the titrant having the same barium-ion concentration. Because of complex formation between the acid and the barium-ion, the conditional protonation constants for EDTA change, giving values of log K i y = 4 . 5 , log K&Y = 5.2 log KdSy = 2.4 and log KkdY = 1.9. The two equally strong carboxylic acid groups are titrated in the first step, followed by the remaining acid groups.(As the value of log KLy is less than log KL3. the two last groups are titrated simultaneously.) The titration is also affected by the fact that EDTA is so insoluble that it does not completely dissolve until about half of the total titrant has been added. In sample No. 25 the disodium salt of EDTA is titrated in the same ionic medium. In this instance the calculations will be further complicated by the salt being an ampholyte. Table I also summarises two different calculations (18a and 18b) of a titration of a mixture of phthalic acid, p-nitrophenol and barbital. The two last acids are of about equal strengths, with stability constants differing by only 0.8 logarithmic unit. A conventional titration graph of a similar sample (Fig.2) does not show a step between the two acids (point 3, Fig. 2), therefore graphical evaluation has little value. However, numerical evaluation can give good results if accurate values of the stability constants are available. Hence, the stability constants of the two acids, p-nitrophenol anid barbital, were determined by titration of known amounts of the separate acids. Correct concentrations were entered into the program TITRA, which was only used to calculate the constants. Use of the values obtained (log K = However, the following points should be noted. calculation cycle. preferred for these titrations. they are approximately equal (e.g., adipic acid). Under these conditions the titration graph shows only two steps.July, 1979 ADDITION OF EQUAL VOLUMES OF TITRANT.PART IV 61 1 9 - a - 7 - 0 2 4 6 8 10 12 14 16 18 20 2 2 k k k k Volume of titrant/ml ! Fig. 2. Titration of a mixture of phthalic acid, nitrophenol and barbital with sodium hydroxide solution. The graph was recorded with a Metrohm Potentiograph E336. The equivalence points are indicated by arrows. Numbers 1 and 2 refer to phthalic acid, number 3 to nitrophenol and number 4 to barbital. 6.88 for P-nitrophenol and 7.61 for barbital) gave a titration error of less than 0.5% for the mixture. An error of 0.1 logarithmic unit in the constants resulted in a titration error of about 10%. With both calculation procedures, there is excellent agreement between observed and calculated hydrogen-ion concentrations, showing that in this instance equation (5) has several mathematically equivalent solutions.Murtlow and Meites' reported on the potentiometric titration of solutions containing small amounts of hydrochloric acid together with much larger amounts of acetic acid. They titrated with a strong base, and used a non-linear regression technique in order to evaluate the titration graph equations. Titration of a solution containing 4.7 x lo-* M hydrochloric acid and 3 x 10-3 M acetic acid yielded errors of +3.3 and -1.3%, respectively. We have repeated Murtlow and Meites' titration using our experimental and evaluation techniques. Table I shows two sets of titration results. In the first set (No. 19), five 0.25-ml aliquots of 0.1 M sodium hydroxide solution were used for each 100-ml sample, so that only hydro- chloric acid was titrated.Duplicate analyses yielded hydrochloric acid recoveries of 99.0 and 101.5~0, respectively. In the second set of results (No. 20), a 500-ml sample was titrated with 40 0.5-ml aliquots of base. This titration yielded recoveries of 99.2 and 99.9% for hydrochloric and acetic acid , respectively. TABLE I1 TITRATIONS CARRIED OUT WITH ADDITION OF STRONG ACID Initial value of Amount Amount Recovery, Sample ph added/mmol found/mmol % Acetic acid .. .. .. 0.780 0 0.7775 99.7 0.7800 0.7787 99.8 0.780 0 0.780 7 100.1 Sodium acetate . . .. . . 2.32 0.551 7 0.550 2 99.7 2.40 0.625 1 0.621 2 99.4 3.14 0.993 7 0.992 6 99.9 3.27 1.0292 1.0307 100.1 4.13 1.5693 1.5720 99.8 Potassium hydrogen phthalate .. 2.15 0.437 1 0.435 7 99.7 2.16 0.435 4 0.4353 100.0 Log K 4.415 4.414 4.410 4.420 4.417 4.406 4.441 4.340 2.598, 4.371 2.593, 4.375612 JOHANSSON AND JOHANSSON Comments on Table 11 The titrations listed in Table I1 illustrate the importance of adding a known amount of strong acid to the sample solution. In all examples the volume V , was 100.0ml and the concentration of added hydrochloric acid was 0..01 M. The amounts (in mmol) of the samples are listed in column 3. These solutions were titrated with l-ml aliquots of 0.1 M sodium hydroxide solution until the last equivalence point was passed. In instances where the initial hydrogen-ion concentration was high f could be accurately calculated, which made it possible to calculate the stability constants. From the first five titrations in Table I1 the log K value of acetic acid was calculated as 4.415 & 0.003 (valid in 0.133 M barium chloride and 0.1 M sodium chloride solution).The last three titrations of sodium acetate give varying values of log K ; the initial ph values are too high to allow a correct evaluation off. Conclusions The results given in Tables I and I1 show t:hat the experimental and computational pro- cedures described in this paper can yield very accurate analytical results. A wide variety of analyses have been carried out: monoprotic to pentaprotic acids; mixtures of acids; ampholytes; acids with an abnormal sequence of stability constant values; and bases. The precision was determined by running duplicates (5-10 analyses) of hydrochloric acid, acetic acid and phthalic acid. The relative standard deviation was found to be less than 0.2%. The method of evaluation described in the present study is characterised as follows. (a) All normal acids are treated as if they were a mixture of monoprotic acids. This necessitates use of titration constants rather than conventional stability constants (or dissociation constants). (b) The procedure utilises non-linear regression techniques. (c) The parameter calculations are performed in an appropriate sequence. (d) Preliminary estimates of the required concentrations or equivalence volumes are not (e) Results from practically all types of acid - base titrations can be handled. necessary. The authors are indebted to Professor Folke Ingman for stimulating discussions and to Dr. Roland Ekelund for valuable assistance in preparing the computer program. They also thank Dr. Douglas Mitchell for translating the manuscript and Mrs. Karin Lindgren for skilful experiment a1 assistance. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. References Johansson, A., and Johansson, S., Analyst, 19713, 103, 305. Ingman, F., Johansson, A., Johansson, S., and Karlsson, R., Analytica Chim. Acta, 1973, 64, 113. Pehrsson, L., Ingman, F., and Johansson, A., Talanta, 1976, 23, 769. Pehrsson, L., Ingman, F., and Johansson, S., Talanta, 1976, 23, 781. Nowogrocki, G., Cannone, J., and Wozniak, M. Bull. SOC. Chim. Fr., 1976, 5. Bos, M., Analytica Chim. Acta, 1977, 90, 61. Murtlow, D., and Meites, L., Analytica Chim. Acta, 1977, 92, 286. Johansson, S., Analyst, 1979, 104, 593. Campbell, B. H., and Meites, L., Talanta, 1974, 21, 117. Schwarzenbach, G., and Ackermann, H., Helv. (Shim. Acta, 1947, 30, 1798. Sullivan, J . C., Kydberg, J., and Miller, W. F., Acta Chem. Scand., 1959, 13, 2023. Ilarned, H. S., and Gary, C. G., J . Am. Chem. SOC., 1937, 59, 2032. Biedermann, G., and SillCn, L. G., Ark. Kemi, 11963, 5, 425. Ciavatta, L., Ark. Kemi, 1963, 20, 417. 1L'oTE-Reference 1 is to Part 111 of this series. Received May 22nd, 1978 hmended December Sth, 1975 Accepted December 14tla, 1978

ISSN:0003-2654    

DOI:10.1039/AN9790400601

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, July, 1979, Vol. 104, pp. 613-619 613 Pyrid i ne-2-ca r ba Ide h yde 2-Hyd roxy benzoy I hyd razone as a Selective Reagent for the Extraction and Spectrophotometric Determination of Iron( I I) M. Gallego and M. Garcia-Vargas Department of Analytical Chemistry, Faculty of Sciences, University of Seville, Seville, Spain and M. Valcarcel Depadmen.t of Analytical Chemistry, Faculty of Sciences, University of Cdvdoba, Cdvdoba, Spain Pyridine-2-carbaldehyde 2-hydroxybenzoylhydrazone reacts with iron (11) to produce a green 2: 1 complex (Amax. = 620 nm, 4 = 3.64 x lo3 1 mol-l cm-1 in aqueous ethanolic solution, and A,,,. = 640nm, E = 3.67 x lo3 1 mol-l cm-l in chloroform). The green complex, extracted into chloro- form, has been used for the spectrophotometric determination of trace amounts of iron.The method has a high selectivity and has been applied to the determination of iron in different samples, such as industrial waste water, non-ferrous materials and minerals. Keywords Iron determination ; spectroplzotometry Hydrazones have been used widely for the spectrophotometric determination of metal ions and several papers have dealt with the use of aroylhydrazones as analytical reagents. The most studied examples have been derived from 2-hydroxybenzaldehyde,lp2 4-dimethyl- aminobenzaldehyde, 2-hydroxynapht halene-1 -carbaldehyde*s5 and pyridine-2-carbaldehyde.' Aroylhydrazones behave as bidentate,798 tridentate,gs10 or tetradentatell ligands, forming coloured complexes with transition metal ions. In moderately acidic media or alkaline solution the hydrogen atom of the -CONH- group can split off and neutral metal complexes are formed.gJ0 Pyridine-2-carbaldehyde 2-hydroxybenzoylhydrazone (PAHB) has been used in spectro- photometric determinations of nickel and zinc.10 In this paper, the characteristics and properties of the iron(I1) complex with PAHB are described and a sensitive and very selective photometric method for determining trace amounts of iron is proposed.The determination of small amounts of iron in different .materials is described. Experimental Apparatus A Pye Unicam SP800 spectrophotometer was used for recording spectra in the ultraviolet and visible regions of the spectrum and a Coleman 55 (digital) instrument was used for measurements at fixed wavelengths, equipped with 1-cm glass or silica cells.Pye Unicam SPlOOO infrared and Perkin-Elmer 460 atomic-absorption spectrophotometers were also used. A Philips PW 9408 pH meter, with glass - calomel electrodes, was used for pH measure- ments. Reagents All solutions were prepared with analytical-reagent grade chemicals using distilled water. Pyridine-2-carbaldehyde 2-hydroxybenzoylhyd~azone reagent solution. A 0.075% m/V solu- tion was prepared by dissolving 0.075 g of recrystallised reagent in 3 ml of NN-dimethyl- formamide and diluting to 100ml with chloroform. This solution was stable for at least 1 week. The reagent was prepared in the pure form from 2-hydroxybenzoylhydrazide and pyridine-2-carbaldehyde as detailed in reference 10. This solution was prepared by dissolving ammonium iron( 11) sulphate hexahydrate in dilute sulphuric acid and standardising it gravimetrically. This solution was prepared fresh daily.Iro.n(I1) standard solution, 2.0436 g 1-1 of iron(1I). Ascorbic acid, 5% m/V.614 GALLEGO et al. : PYRIDINE-2-CARBALDEI-IYDE 2-HYDROXYBENZOYL Analyst, VOJ. 104 Bufer solution, pH 4.35. This solution was prepared by dissolving 105 g of sodium acetate trihydrate in distilled water, adding 100 ml of glacial acetic acid and diluting the mixture to 1 1. Procedure for the Determination of Iron( 11:) To 10-250 ml of sample solution in a separating funnel containing up to 120 pug of iron(II), add 1 ml of 5% m/V ascorbic acid solution and 5 ml of buffer solution and extract the mixture with one 10-ml volume of PAHB reagent solution.Shake the funnel vigorously for 2 min, allow the phases to separate and transfer the llower (organic) layer into a 10-ml flask con- taining anhydrous sodium sulphate. Measure the absorbance of the green chloroform extracts against water at 640 nm. The calibration graph is prepared by using standard solutions of iron(I1) treated in the same way. Results and Discussion Study of Iron - PAHB System Formation of iron complex in aqueous ethanolic solution green complex is formed immediately. Fig. 1. iron(I1) .12J3 When dilute iron(I1) solutions and a 0.1% m/V solution of PAHB in ethanol are mixed, a Absorption spectra of the complex are shown in PAHB differs from other related ligands in that a complex is formed only with 1.0 - 400 500 600 700 Wavelength/nm Fig. 1.Absorption spectra of green iron complex. Concentration of iron(I1) 10 pg ml-1. A, Green com- plex extracted into chloroform a t pH 4.35; B, green complex in aqueous ethanolic medium a t pH 7 ; and C, reagent blank. I 0.4 -0 -=f 'i 0.2 0 2 A 4 6 8 10 PH Fig. 2. Influence of pH on formation of iron complex. A, In aqueous ethanolic solution at 620 nm; and B, extracted into chloroform, at 640 nm.JuJy, 1979 AS A SELECTIVE SPECTROPHOTOMETRIC REAGENT FOR IRON(II) 61 5 Volumes (10ml) of a 0.1% m/V solution of PAHB in ethanol were allowed to react with 2.5ml of a 100.0pgml-l solution of iron(I1) at different pH values in a series of 25-ml calibrated flasks. The absorbances were measured at 620 nm after a 15-min reaction and are shown in Fig. 2. The green complex is formed immediately in aqueous media and is stable for 2 h in the optimum pH range, but high pH causes numerous interferences by metal ions that precipi- tate as hydroxides or basic salts.It is concluded that in aqueous media the complex of iron(I1) with PAHB is not of great analytical interest. The optimum pH range is 6.0-8.0. Stoicheiometry of the complex Job’s curves were plotted for the complex. The absorbance of the green complex obtained at pH 7.0 was measured at 620 nm immediately after preparation. Results showed [Fig. 3 (A)] a stoicheiometric ratio of metal to ligand of 1:2. The samples were then extracted with PAHB in chloroform at pH 4.35 and the absorbance was measured a t 640 nm; the same molar ratio (1 : 2) was found [Fig. 3 (B)]. 0 0.2 0.4 0.6 0.8 I F e ( l l ) l IFe(ll)l + IPAHBI Fig.3. Composition of iron(I1) - PAHB complex by the continuous variation method. A, In aqueous ethanolic solution (pH = 7, h = 620 nm, 2 x 10-3 M) ; and B, extracted into chloroform (pH = 4.35, h = 640 nm, 3 x 10-3 M). Oxidation state of iron and structure From experimental evidence it was concluded that the reagent reacts with iron in the bivalent state to give the green complex. In order to ensure that iron is present as iron(II), ascorbic acid was selected for use as a reducing agent. The green complex was not retained on either a cationic (Dowex 50-X8, sodium form) or an anionic (Dowex 1-X8, chloride form) ion-exchange resin, indicating that it was uncharged. In order to check the co-ordination in the complex, other related ligands have been tested.Benzaldehyde 2-hydroxybenzoylhydrazone does not form a green complex with iron( 11) , as the ligand does not contain the -N =C-C=N- chromophore group. Pyridine-2-carbaldehyde benzoylhydrazone forms a green 3 : l complex with iron(I1). The molecule contains the ferroin group, but not the hydroxide group. It is apparent that this reagent acts as a bidentate ligand. On the other hand, infrared spectra of the iron(I1) - PAHB complex in the solid state (using potassium bromide discs) show that the shift of the carbonyl stretching mode to a lower frequency is in accord with the co-ordination through oxygen; moreover, no imine stretching mode appears in the 3000 cm-1 region, owing to the de-protonation of61 6 GALLEGO et d. : PYRIDINE-2-CARBALDEHYDE 2-HYDROXYBENZOYL Analyst, VOl.104 the -CONH- group. The exact configuration of a complex of this type, bis(N-pyrid-2- ylidene-N‘-salicyloylhydrazinato)nickel( 11) , has been establi~hed.~ It is evident that PAHB acts as a tridentate ligand that forms an octahedral complex and four five-membered rings are produced. 1:2 iron- aroylhydrazone green complex Choice of extracting solvent, and absorption spectra When a solution of PAHB reagent in an organic solvent is shaken with a weakly acidic aqueous solution of iron(II), the green complex is formed immediately, in the organic phase. Solutions of the complex in chloroform are very stable, whereas those in aqueous ethanol are less stable. When benzene is used as the organic solvent the resulting complex is stable; however, the interferences of foreign ions are high.In oxygenated solvents, such as higher alcohols and 4-methylpentan-2-one, the colour system is less sensitive and less stable than in chloroform. The absorption spectrum of the iron(I1) - PAHB complex in chloroform is shown in Fig. 1 (A). It presentis two bands in the visible region, at 380 and 640nm. The latter wavelength was used in all subsequent measurements of absorbance because the reagent itself does not absorb at this wavelength. It is stable for at least 7 h. In$uence of p H The maximum constant absorbances were obtained in the pH range 4.04.7 [Fig. 2 (B)]. A decrease in absorbance below pH 4.0 can be attributed to incomplete formation of the iron - PAHB complex, owing to protonation of the pyridine nitrogen atom.1° A decrease in absorbance above pH 4.7 is probably caused by the precipitation of iron(I1).Influence of reagent concentration and shaking time An aliquot of 10-250ml of solution (acetate buffer) containing 40pg of iron(I1) was extracted with a solution of O.Ol-O.lyo m/V PAHB in chloroform (10 ml). The extraction was quantitative from 0.05% m/V of the reagent solution and remained constant with increasing concentration. Therefore, 10 ml of (3.075y0 m/V reagent solution was adopted as the concentration of solution containing the coinplexing ligand to be used. The shaking time was varied from 0.5 to 5 min, while the other variables were kept constant. These variations in shaking time did not produce any change in absorbance if the volume ratio Vors. : Vaq.was between 1 : 1 and 1 : 5. On the other hand, shaking for 2 min was neces- sary for the complete extraction of iron(I1) if tlhe volume ratio Vors. : Vaq. was 1 : 25. Spectrophotometric Determination of Iron( 11) with PAHB Based on the experimental work, a method. is proposed for the determination of trace amounts of iron involving the formation of the green complex with PAHB and its extraction into chloroform. Beer’s law is obeyed betweein 2 and 12 pg ml-l of iron(I1) in the organic phase at 640 nm. The optimum concentration range, evaluated by Ringborn’s method, is 3-10 pg ml-l of iron. The green complex gave a molar absorptivity of E = 3.67 x lo3 1 mol-l cm-l a t 640 nm [in the chloroform phase (10 ml)]. The sensitivity of the method, according to Sandell, is 0.015pgcm-2 of iron.The precision was estimated for 10-250-ml aliquots of 4Opg of iron(I1) solution, and the relative error of the method is 0.26%. For the determination of 40 pg of iron by this method, the foreign ions can be tolerated at the levels given in Tables I and 11. Alkali and alkaline earth metals, chloride, nitrate, sulphate, carbonate, perchlorate, thiocyanate , hydroxylamine and ascorbic acid can be tolerated at levels of 0.4 g. The Thioglycollic acid i(TGA) can be tolerated a t the l-g level.July, 19 79 AS A SELECTIVE SPECTROPHOTOMETRIC REAGENT FOR IRON(II) TABLE I TOLERANCE LIMITS OF EXTRACTIVE DETERMINATION OF IRON (11) 61 7 Results obtained using a 40-pg sample of iron(I1). Amount tolerated/ Ion added pg ml-l La(III), Mn(II), Cr(III), Hg(I), Hg(II), Se(IV), Ce(IV), Tl(I), Al(III), As(III), As(V), ammonium, alkali and alkaline earth metals, S20s2-, I-, Br-, C1-, PO,3-, NO,-, SO,%-, SOSB-, CO,a-, ClO,-, ClO,-, SCN-, Sa-, tartrate, dimethylglyoxime (DMG), thioglycollic acid (TGA), hydroxylamine and ascorbic acid .. .. .. 10 000 In(III),t 10,- . . .. .. .. .. .. .. .. .. .. .. 1000 Th(IV), Te(IV), Pt(IV), Bi(III),* citrate . . .. .. .. .. .. .. 5 000 F-, borate . . .. .. .. .. .. . . . . .. .. .. 2 500 * Centrifuged. t 0.1% m/V PAHB solution in chloroform. limiting value of the concentration of foreign ions was taken as that value which caused an error of not more than 2.5% in the absorbance. The method is remarkably free from interferences because most of the metallic chelates of PAHB are not extracted into chloroform and present their absorption peaks in the 350- 400-nm region.The good results obtained by masking were due to the fact that the precipitates remained in the aqueous phase. Further, the method has been compared, advantageously, with the other method previously reported from this laboratory with the use of related reagents (Table 111). Applications materials. The method has been applied satisfactorily to the determination of iron in different Determination of iron in minerals and non-ferro~s alloys Results of the analysis of iron in mineral and non-ferrous alloy samples from the Bureau TABLE I1 ELIMINATION OF INTERFERENCES BY ADDITION OF MASKING AGENTS Amount tolerated/pg ml-l Without With masking r L 3 Foreign ion masking agent Pd(I1) .. .. 1000 .. 5 000 V(V) . . .. 750 Bi(II1). . .. 2 500 In(II1). . .. 750 Cu(I1) . . .. 50 Cu(I1) . . .. 50 Sn(I1) . . .. 50 Mo(V1) .. 2 500 Ti(1V) . . .. 15 U(V1) . . .. 250 W(V1) .. .. 250 2[?ni * . . 1000 Zr (IV) .. 75 Co(I1) . . .. 20 Sb (111) .. 75 Zn(I1) . . .. 100 Ni(I1) . . .. 100 Cd(I1) . . .. 2 500 Pb(I1) . . .. 7 500 CN- .. .. 12 * Shaking for 5 min. t NH,OH.HCl; 0.3 g. Heating gently before extracting. agent 10 000 10 000 5 ooo* 6 000 10000 10000 2 500 10 000 5 OOOt 6 ooo* 5 000 10000 10000 2 500 5 000 2 500: 10000 10000 10000 10000: 10 ooot Masking agent NH,OH.HCl; 1 ml of 10% m/V solution NaCl; 0.2 g NH,OH.HCl; 1 ml of 10% m/V solution NH,OH.HCl; 1 ml of 10% m/V solution TGA; 1 ml of solution TGA; 1 ml of solution KSCN; 0.3 g TGA; 1 ml of solution TGA; 1 ml of solution NH,OH.HCl; 1 ml of 10% m/V solution F-; 2500 pg ml-l F-; 2500 pg ml-l F-; 2600 p g ml-l PO,s- + Ca2+; 10000 pg ml-' TGA; 1 ml of solution S,OS2-; 10000 pg ml-l TGA; 1 ml of solution DMG; 0.05 g TGA; 1 ml of solution TGA; 1 ml of solution Hg2+; 10000 pg ml-l61 8 GALLEGO et al.PYRIDINE-Z-CARBALDEHYDE 2-HYDROXYBENZOYL Analyst, VOE. 104 TABLE I11 COMPARISON WITH EXISTING METHODS Compound Di-2-pyridyl ketone azine . . Pyridine-2-carbaldehyde azine .. .. .. Di-2-pyridyl dihydrazone . . 2-Benzoylpyridine hydrazone Pyridine-2-carbaldehyde Z-hydroxybenzoyl- Diacetyldihydrazone .. hydrazone . . .. .. Optimum PH 4.2-5.2 4-5 4.7-7.8 4-7 4-7 4.0-4.7 Molar absorptivity/ X,,,./nm 1 mol-1 cm-1 Interferences* Reference 750 6.367 Cu(I1) 12 660 2.900 Au(III), Pd(II), Se(1V) 13 488 8.240 Mn(II), Co(II), Cu(I1) 14 490 11.500 Co(II), Cu(II), Cr(V1) 15 480 9.550 Co(II), CU(II), V(V) 16 640 3.670 - - * Cations that interfere a t an identical concentration. to iron.of Analysed Samples Ltd. and British Chemical Standards (Table IV) support the precision and reliability of this method. In order to prevent interferences due to foreign ions, 0.3 g of potassium thiocyanate was added to solutions of H.T. brass and 0.3g of potassium thiocyanate and 0.05 g of dimethylglyoxime (D:MG) were added to solutions of cupro-nickel. Each cupro-nickel solution was then heated gently. TABLE IV ANALYSIS OF MINERALS AND NON-FERROUS ALLOYS Sample Dolomite (BAS, No. 9h) . . . . Brass (BAS, No. 5g) .. .. .. H.T. brass (BAS, No. 10e) .. .. .. Cupro-nickel (BAS, No. 19e) . . .. Aluminium alloy (BAS, No. 20b) . . .. Portland cement (BCS, No. '372) .. Zinc concentrates (BAS, No. 4ldGj * . . * Average of five separate determinations. t Percentage as Fe,O,. Iron content, yo f A \ Reported value Found* 0.21t 0.207 f 0.002 2.491. 2.489 f 0.002 0.32 0.320 f 0.001 1.38 1.376 f 0.003 0.83 0.85 f 0.02 0.43 0.431 f 0.001 10.0 10.04 & 0.04 Determination of iron in milk, blood, tobacco, garkc and water Milk and blood were treated with equal voluines of 20% mlV trichloroacetic acid solution for 30 min at 30 "C in a water-bath and then the solid residues were removed. Fermented tobacco, in powder form, and garlic were ashed and then treated with a mixture of con- centrated perchloric, nitric and sulphuric acids (1 + 10 + 1) and boiled to ensure complete dissolution. The water from a sulphuric acid plant (pyrites process) was filtered through an TABLE V COMPARISON OF RESULTS FOR THE ANALYSIS OF DIFFERENT SAMPLES BY 1 ,lo-PHENANTHROLINE AND PAHB METHODS Iron content* r A 'L 11,lO-Phenanthroline Sample method PAHB method Milk ... . . . . . .. .. 1.38 p.p.m. 1.42 & 0.03 p.p.m. Bloodserum . . . . .. .. .. 0.93 p.p.m. 0.95 f 0.01 p.p.m. Tobacco . . . . . . . . . . .. 0.062% 0.061 f 0.001% Garlic . . . . . . .. .. .. 0.01 4 yo 0.014 f 0.001% Waste water (from sulphuric acid plant) . . 60.30 p.p.m. 60.25 f 0.02 p.p.m. * Average of six determinations.July, 1979 AS A SELECTIVE SPECTROPHOTOMETRIC REAGENT FOR IRON(II) 619 asbestos mat contained in a Gooch crucible.The results are compared with those obtained by the spectrophotometric method using 1 ,lo-phenanthroline in Table V. Determination of iron by the standard additions method in waters and analytical reagents The PAHB method can also be applied to the determination of iron a t parts per billion (loQ) levels, which decreases the lower limit of the iron determination by taking a larger volume of the aqueous phase in relation to the chloroform volume and applying the standard additions method. The method consists in adding several increasing known amounts of iron(I1) (0, 5 , 10, 15 and 20 pg) to several aliquots of sample solution: river water (200 ml of sample solution, 25 ml of acetate buffer) ; sea water (200 ml of sample solution, 25 ml of acetate buffer) ; water from a fertiliser plant (100 ml of sample solution, 15 ml of acetate buffer) ; concentrated nitric acid (5 ml of sample solution, 50 ml of acetate buffer) ; and sodium hydroxide solution (100 ml of 0.1545 N sample solution, 50 ml of acetate buffer).Then, all prepared solutions, after the extraction procedure described above, are measured at 640 nm. The procedure is repeated for blank solutions (distilled water) treated in the same way. The absorbances are plotted against the concentrations of the four iron-containing solutions of each sample. The straight lines (one for the sample solutions and the other for blank solutions) are extrapolated until they intersect the abscissa. The segment between the points of intersection give the concentration of the sample solution. The results are compared in some instances with those obtained by atomic-absorption spectroph~tometryl~ in Table VI.TABLE VI DETERMINATION OF IRON IN SAMPLES BY STANDARD ADDITIONS METHOD Iron content I A 1 Sample Reported value Found Concentrated nitric acid (Panreac, PRS) . . o . o o o l ~ o 0.000 15% Sodium hydroxide pellets (Merck, GR) . . 0.0005% 0.000 64% River water . . .. . . .. . . - 29.3 p.p.b. Sea water . . .. .. .. .. . . 15.7 p.p.b.* 13.5 p.p.b. Waste water (from fertiliser plant) . . . . 71.3 p.p.b.* 72.5 p.p.b. * Results from atomic-absorption spectrophotometry. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. References Katiyar, S. S., and Tandon, S. N., Talanta, 1964, 11, 892. Vasilikiotis, G. S., and Tossidis, J. A., Microchem. J., 1969, 14, 380. Uno, T., and Taniguchi, H., Bunseki Kagaku, 1971, 20, 997. Odashima, T., and Ishii, H., Nippon Kagaku Kaishi, 1973, 729. Vasilikiotis, G. S., Microchem. J., 1968, 13, 526. Capitan, F., Salinas, F., and Gimenez Plaza, J., A r s Pharm., 1975, 16, 293. Aggarwal, R. C., and Rao, T. R., Transition Met. Chem., 1977, 2, 21. Aggarwal, R. C., and Rao, T. R., Transition Met. Chem., 1977, 2, 59. Domiano, P., Musatti, A., Nardelli, M., and Pelizzi, C., J . Chem. SOC., Dalton Trans., 1975, 295. Gallego, M., Garcia-Vargas, M., Pino, F., and Valcarcel, M., Microchem. J., 1978, 23, 353. Rastogi, D. K., Sahni, S. K., Rana, V. B., and Dua, S. K., Transition Met. Chem., 1978, 3, 56. Valcarcel, M., Martinez, M. P., and Pino, F., Analyst, 1975, 100, 33. Luque de Castro, M. D., and Valcarcel, M., Analyt. Lett., 1978, 11, 1. Graciani Constante, E., An. Quim., 1971, 67, 607. Graciani Constante, E., and Olias Jimenez, J. M., An. Quim., 1971, 67, 615. Graciani Constante, E., An. Quim., 1974, 70, 695. Nix, J., and Goodwin, T., Atom. Absorption Newsl., 1970, 9, 119. Received December 29th, 1978 Accepted January 15th, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790400613

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

620 Analyst, July, 1979, VoL 104, $9. 620-625 Spectrophotometric Determination of Acetaminophen, Salicylamide and Codeine Phosphate in Tablets M. Abdel-Hady Elsayed," Saied F. Belal, Abdel-Fattah M. Elwalily and Hassan Abdine Defiartment of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt An accurate and simple method is proposed for the analysis of a three- component mixture composed of acetaminophen, salicylamide and codeine phosphate, without the necessity for the previous separation of any com- ponent. The first two components are determined directly by independent spectrophotometric measurements, based on the pH -induced spectral changes. Codeine phosphate is assayed by the formation of: an ion pair with methyl orange.The procedure has been applied successfully to the analysis of known mixtures and commercial tablets. Keywords Acetaminophen ; salicylamide ; codeine phosphate ; spectrophoto- metry Acetaminophen (paracetamol; N-acetyl-p-amjnophenol) and salicylamide (o-hydroxy- benzamide) occupy prominent positions among the extensively employed antipyretic - analgesics. They are frequently prescribed in admixture with each other or with other related drugs. Prior to its spectrophotometric determination, acetaminophen was separated from combination with other antipyretic - analgesics by column chromatography using purified siliceous earth,1-3 a cation-exchange resin4-6 or an anion-exchange resin.' Recently, high- performance liquid chromatographp was utilised in the determination of an acetaminophen - methaqualone (2-methyl-3-o-tolylquinazolin-4-one) mixture. Spectrophotometric methods9P for the determination of acetaminophen in Combination with acetylsalicylic acid (aspirin) have been proposed.In biological fluids, acetaminophen was extracted and determined by spectrophotometry,ll fluorimetry,12 differential spectrophotometry13J4 and gas - liquid chromatography.16Js Spectrophotometric determinations have been described for salicylamide in combination with caffeine and phenacetin,l' papaverine, phenobarbitone and guaiphenesine carbamate (methocarbamol) ,l* acetaminophen and caffeine,19 chloroquine phosphate,20 and phenacetin, caffeine and chlorpheniramine maleate.21 Salicylamide and acetaminophen, in combina- tion, could be determined by the use of an ion-exchange thin-layer chromato- graphy23 or differentiating non-aqueous titration.24-26 Hence the independent spectrophotometric *analysis of a ternary mixture containing the strongly absorbing compounds acetaminophen and salicylamide and the weakly absorbing compound codeine phosphate is challenging. The problem is made more difficult when the dose ratio is not in favour of the codeine phosphate.In this instance the ratio of acetaminophen to salicylamide to codeine phosphate is 25 : 30 : 1. In this investigation the orthogonal function method27,2* and the absorbance-diff erence meth0d~~~~0 are used for the determination of strongly absorbing components without prior separation. Codeine phosphate is assayed by the ion-pair method.31 In the application of the orthogonal function method to spectrophotometric analysis, the absorbance A is replaced by the coefficient $j, which is proportional to concentration.In order to extract the coefficient of a given pcllynomial from an absorption graph, it is necessary to obtain absorbances at a number of equally spaced wavelengths. Thus, to extract the coefficient of a quadratic polynomial $,, we need, for example, eight absorbances at eight equally spaced wavelengths [equation (3)]. By plotting the quadratic coefficient * Present address : Department of Pharmacy, University of Nigeria, Nsukka, Nigeria.ELSAYED, BELAL, ELWALILY AND ABDINE 62 1 p2, or its substitute he2, at different intervals versus [whereA, = ()(initial + hrina1)/2] a convoluted graph is obtained (Fig.2). The absorbance-diff erence method is based on the absorbance measurement of the ionic form (in alkaline medium) against the molecular form (in neutral medium) of the drug. This permits the determination of the active components without any interference from other co-existing components, not sensitive to pH change. Experimental Materials and Reagents All reagents were of analytical-reagent grade and the solvents were of spectroscopic grade. Acetaminophen standard solution, 1 mg ml-1. Obtained from the Alexandria Company, Salzcylamide standard solution, 1 mg ml-1. Codeine phosphate standard solution, 1 mg ml-1. Obtained from E. Merck, Darmstadt, Codacetine tablets. Obtained from Elkahira Co., Egypt. Each tablet contained 0.25 g Methyl orange solution.B u f e r solz&ion. Egypt. Obtained from the CID Company, Egypt. Germany. of acetaminophen, 0.3 g of salicylamide and 0.01 g of codeine phosphate. hydrochloric acid (36.3 ml) and diluting to 100 ml with water. solution is adjusted potentiometrically (using a Pye pH meter). A 0.1% m/V solution in 20% V/V aqueous ethanol. Prepared by mixing 1 M sodium acetate solution (63.7 ml) with 1 M The pH 3.5 of the prepared Sodium hydroxide solution, 0.1 M. Apparatus A Prolabo photoelectric spectrophotometer with 1-cm silica cells was used. Analysis of Codacetine Tablets Twenty tablets were powdered and mixed. one tablet was extracted with three 60-ml portions of hot water. and diluted to 200 ml with water (solution X). An accurately weighed mass equivalent to The extracts were collected Determination of acetaminophen component Two equal volumes of solution X were accurately measured and identical dilutions were made.One volume was diluted with water and the other with 0.01 M sodium hydroxide solution, so as to obtain a concentration of approximately 1.0% m/m. The absorbance of the alkaline solution was measured at 262.5nm using the aqueous solution as a blank (AA method). The absorbance of the alkaline solution was measured in the range 256-270 nm, at 2-nm intervals using 0.01 M sodium hydroxide solution as a blank. The absorbance of the aqueous solution was measured in the range 256-270 nm at 2-nm intervals using water as a blank. The coefficients p , for the alkaline and the aqueous solution were calculated and A$, (p2alk - Pzaq) was then computed (Ap2 method).Determination of salicylamide component Two aliquots of solution X were accurately measured and identical dilutions were made with water and 0.01 M sodium hydroxide solution to give concentrations of approximately 1.8% m/m. The absorbance of the alkaline solution was measured at 329nm using the aqueous solution as blank (AA method). Both solutions (alkaline and aqueous) were measured in the wavelength range 324-338nm at 2-nm intervals using 0.01 M sodium hydroxide solution or water, respectively, as the blank. The coefficient 9, for each solution was calculated and Ap2 (p2alk - Pz,,) was then computed. Determination of codeine Phosphate component An aliquot (5.0 ml) of solution X was accurately measured and transferred into a 100-ml separating funnel, containing 3 ml of the 0.1% m/V methyl orange solution and 3 ml of pH 3.5 acetate buffer solution. The mixture was extracted three times with chloroform (10, 10 and then 5 ml). The extracts were pooled in a 25-ml calibrated flask and diluted to622 ELSAYED et al.: SPECTROPHOTOMETRIC DETERMINATION OF Analyst, VOl. 104 volume with chloroform. The absorbance of the combined extracts was measured at 418 nm against a blank, prepared in a similar manner to that described above but omitting the addition of drug solution. Results and Discussion The assay of acetaminophen and salicylamide: in combination with codeine phosphate in the ratio 25: 30: 1 is based on the use of their pH-induced spectral changes.19 Codeine phosphate shows negligible interference, as it exists as a minor component and its differential curve exhibits negligible absorbance (Fig.1). I 230 250 270 290 310 330 Wavelength/nm Fig. 1. A A curves of 1% xn/m acetaminophen (solid line), 1% m/m salicylamide (broken line) and 10% m/m codeine phosphate (dotted line). From the differential curve (Fig. 1) acetaminophen and salicylamide can be determined at 262.5 and 329 nm, respectively. Salicylamide shows an isosbestic point of zero absorbance at 262.5 nm, acetaminophen (in a concentration equal to salicylamide) exhibits no absorbance at 329 nm. The calibration graphs for acetaminophen and salicylamide were prepared by the application of the procedure mentioned above, using a concentration range of 0.2-2.0% m/m of the former and 0.3-2.4y0 m/m of the latter.Using the method of least squares,32 the data from the absorbance measurements give the following regression equations : .. (1) AA262.5 = 0.0147 + 0.3863C . . .. . . * (2) AA,,, = 0.0043 + 0.3856C . . .. for which the percentage fiW3 are 99.87% and 99.94y0, respectively. and salicylamide. (0.01 M sodium hydroxide solution and water11 were measured at 2-nm intervals. coefficient pz for each solution was calculated using the following equation: Alternatively, the A$2 method can be applied to the determination of acetaminophen The absorbances A,, A,, A,, . . ., A8 of the compound in different solvents TheJuly, 1979 ACETAMINOPHEN, SALICYLAMIDE AND CODEINE PHOSPHATE 623 The subscripts 1-8 refer to A&,, respectively. The figures in parentheses are given in standard books3* and 168 is the normalisation factor.Apz ($2,1k - p2,,) was computed. A graph of Afi, versZts Am [where h, = (A1 + &)/2] was a convoluted curve35 (Fig. 2). From the curve, a of 263nm (i.e., the absorbance measurement from the wavelength range 256-270 nm a t 2-nm intervals) was chosen for the determination of acetaminophen without the interference from salicylamide. The latter can be determined, with no interference from the co-existing components, by absorbance measurement in the wavelength range 324-338 nm at 2-nm intervals (A, = 331 nm). Using the procedure mentioned above, a linear relationship between AP2 and C is obtained in a concentration range 0.2-1 .6y0 m/m of acetaminophen and 0.3-2.4% m/m of salicylamide.The corresponding curves can be described by the following regression equations: Ap, x lo3 = -0.1713 - 4.8881C .. .. * - (4) A$2 x lo3 = 0.0643 - 3.2253C . . .. .. * . (5) for which the percentage fits are 99.56% and 99.Slyo, respectively. 6.0 3.0 0.0 E) z &d X 4 -3.0 -6.0 -9.0 I I I I I I I \ I \ I \ I \ 1 \ I \ I \ I \ I \ ' '.=< 270 290 310 330 350 Wavelength/nm Fig. 2. Apz curves of acetaminophen (solid line) and salicylamide (broken line), derived from their absorption spectra, and 10% m/m codeine phosphate (dotted line). The ion-pair method was utilised in the determination of codeine phosphate without any interference from acetaminophen or salicylamide, as they have no basic centres. Codeine with methyl orange, in a pH 3.5 acetate buffered medium, gave an ion pair, easily extracted in chloroform and the absorbance was measured at 418 nm.The absorbance of the chloro-624 ELSAYED et al. : SPECTROPHOTOMETRIC DETERMINATION OF Analyst, VoZ. 104 form extract is linear over a concentration range of 0.4-3.2% m/m of codeine phosphate, calculated in the final solution. The regression equation of the calibration graph is .. ' * (6) A,,, = 0.0133 + 0.3815C . . .. for which the percentage fit is 99.82%. Equation (1) or (4) was used for the determination of acetaminophen. salicylamide was calculated using equation (2) or ( 5 ) . employing equation (6). The validity of equations (l), (2), (4) , (5) and (6) was checked by assaying a known mixture. The concentration of Codeine phosphate was determined The results obtained (Table I) are accurate and reproducible.TABLE I[ DETERMINATION OF ACETAMINOPHEN, SALICYLAMIDE AND CODEINE PHOSPHATE I N A KNOWN MIXTURE The composition of the mixture is similar to commercial tablets; 0.25 g of acetaminophen, 0.3 g of salicylamide and 0.01 g of codeine phosphate to which a tablet base consisting of lactose, starch, talc and magnesium stea.rate was added. Codeine - 7 f 7 ion-pair AA method Ap2 methold AA method Ap2 method method Acetaminophen Salic ylamide phosphate : t A A No. of experiments . . .. 12 9 12 12 10 Mean recovery, yo . . . . 100.03 99.91 100.00 100.02 100.44 Coefficient of variation, % . . 0.50 1.77 0.62 1.26 0.32 Calculated t . . * . .. 0.22 0.05 Theoretical to.,, . . .. 2.09 2.07 Subjecting the results of the AA and AP2 methods to statistical analysis, they are of equal accuracy, because the calculated t does not exceed the theoretical t32 (Tables I and 11).Consequently, both methods proved to be equally effective in discounting the absorbance from the co-existing components as well as tablet excipients. The use of this procedure in the assay of commercial tablets (Table 11) gave results comparable to those obtained in analysing a known mixture. TABLE I1 DETERMINATION OF ACETAMINOPHEN, SALICYLAMIDE AND CODEINE PHOSPHATE I N COMMERCIAL TABLETS Each tablet contains 0.25 g of acetaminophen, 0.3 g of salicylamide and 0.01 g of codeine phosphate. Codeine Acetaminophen Salicy lamide phosphate : A ion-pair -7 I AA method Ap2 method AA method Ap2 method method No. of experiments . . .. 12 12 12 12 10 Mean of results as a Coefficient of variation, % .. 0.46 1.30 0.93 1.54 0.45 Calculated t . . .. .. 0.38 1.25 Theoretical to.ss . . .. 2.07 2.07 percentage of label claim 99.49 99.34 99.04 98.40 99.73 Conclusion Use of the AA method or the A@2 method in the determination of acetaminophen (25 parts) and salicylamide (30 parts), and the ion-pair method in the assay of codeine phosphate (1 part) illustrates the suitability of the proposeld procedure for the analysis of a three- component mixture without the necessity for the previous isolation of any componentl-8,22@ or complex mathematical treatment.36 Utilisatian of the AA method for routine qualityJuly, 1979 ACETAMINOPHEN, SALICYLAMIDE AND CODEINE PHOSPHATE 625 control has two advantages over the Ap2 method: (a) low coefficient of variation (Tables I and 11) with a consequent increase in the reproducibility of the results; this is due to the absorbance measurement at only one wavelength in the AA method and 16 wavelengths in the Ap2 method, giving rise to an increase in imprecision in the latter method owing to the increased wavelength setting error entailed; and (b) calculation in the AA method is direct and simple. 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. 35. 36. References Koshy, I<. T., J . Pharm. Sci., 1964, 53, 1280. Levine, J., and Hohmann, J. R., J . Ass. Off. Analyt. Chem., 1966, 49, 533. Hohmann, J. R., J . Ass. 08. Analyt. Chem., 1970, 53, 591. DeFabrizio, F., J .Pharm. Sci., 1968, 57, 644. Koshy, K. T., and Lach, J . L., Drug Stand., 1960, 28, 53. Koshy, K. T., and Lach, J. L., Drug Stand., 1960, 28, 86. Dibbern, H. W., and Scholz, G., Arch. Pharm., Berl., 1965, 298, 175. Caude, M., and Le Xuan, P., Chromatographia, 1976, 9, 20. Mouton, M., and Masson, M., Annls Pharm. Fr., 1960, 18, 759. Deodhar, R. D., Shastri, M. R., and Mehta, R. C., Indian J . Pharm., 1976, 38, 18. Gurtoc, H. L., and Phillips, B. M., J . Pharm. Sci., 1973, 62, 383. Dolegeal-Vendrely, M., and Guernet, M., Analusis, 1976, 4, 223. Routh, J. I., Shane, N. A., Arrendondo, E. G., and Paul, W. D., Clin. Chem., 1968, 14, 882. Scemama, M., Annls Pharm. Fr., 1972, 30, 861. Windorfer, A., Jun., and Roettger, H. J., Arzneimittel-Forsch.. 1974, 24, 893.Prescott, L. E., J . Pharm. Pharmac., 1971, 23, 111. Machek, G., Scientia Pharm., 1961, 29, 73. Kluczykowska, B., and Krowczynski, L., Dissnes Pharm. Pharmac., 1972, 24, 507. Shane, N., and Kowblansky, M., J . Pharm. Sci., 1968, 57, 1218. Wahbi, A. M., and Ebel, S., J , Pharm. Pharmac., 1974, 26, 317. Szekeres, L., Harmon, R. E., and Gupta, S. K., Microchem. J . , 1973, 18, 101. Haberli, E., and Beguin, E., Pharm. Acla Helv., 1959, 34, 65. Pfandl, A., Dt. ApothZtg, 1974, 114, 325. Agarwal, S. P., and Walsh, M. T., Indian J . Pharm., 1974, 36, 47. Blake, M. I., and Shumaker, L. B., J . Ass. Off. Analyt. Chem., 1973, 56, 653. Rhodes, H. J., Denardo, J. J., Bode, D. W., and Blake, M. L., J . Pharm. Sci., 1975, 64, 1386. Glenn, A. L., J . Pharm. Pharmac., 1963, 15, 123T. Abdine, H., Wahbi, A. M., and Korany, M. A., J . Pharm. Pharmac., 1972, 24, 518. Aulin Erdtman, G., Chemy Ind., 1955, 74, 581. Juenjo, G. M., and Glenn, A. L., Chemy Ind., 1956, 75, 813. Xbdine, H., Elsayed, M. A., and Ibrahim, S. A,, Pharmazie, 1974, 29, 614. Spiegel, M. R., “Theory and Problems of Probability and Statistics,” McGraw-Hill, New York, Davies, 0. L., and Goldsmith, P., “Statistical Methods in Research and Production,” Oliver and Fisher, R.A., and Yates, F., “Statistical Tables for Biological Agriculture and Medical Research,” Hackman, R. B., and Tukey, J . W., “The Measurement of Power Spectra,” Dover, New York, Clayton, A. W., and Theirs, R. E., J . Pharm. Sci., 1966, 55, 404. 1975, pp. 229 and 270. Boyd, Edinburgh, Fourth Edition, 1972, p. 178. Fourth Edition, Oliver and Boyd, Edinburgh, 1953, pp. 80 et sep. 1958, p. 72. Received May 16th, 1978 Accented Taizuarv 1st. 1979

ISSN:0003-2654    

DOI:10.1039/AN9790400620

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

626 Analyst, July, 1979, Vol. 104, pp. 626-636 Determination of Vitamin D2 in Multivitamin Tablets by High-performance Liquid Chromatography* C hristeen M ackay Department of Chemistry, Loughborough University of Tec.hnology, Loughborough, Leicestershire, L E l l 3 T U J. Tillman7 Fisons Limited, Pharmaceutical Division, Research and .Development Laboratories, Bakewell Road, Lough- borough, Leicestershire, L E l l OQY and D. Thorburn Burns Department of Analytical Chemistry, The Queen’s Universi:ty of Belfast, Belfast, BT9 5AG, Northern Ireland A procedure is described for the determination of vitamin D, in multi- vitamin tablets. The vitamin is released from the tablets by enzymic digestion, which is followed by solvent extraction and chromatography on a microparticulate silica column.Other fat-soluble vitamins do not interfere with the assay. The problem of defining and quanti:fying vitamin D, in order to correlate results with biological potency is discussed. The procedure is suitable for stability studies. Keywords : Vitamin D, ; pre-vitamin D, ; multivitamin tablets ; high- performame liquid chromatography Vitamins D, (ergocalciferol) and D, are the most active of the antirachitic vitamins and are the forms most commonly used in pharmaceutical preparations. They are usually incorpora- ted in tablets in the form of starch-coated, gelatin-protected beadlets. The assay of these vitamins in multivitamin preparations is difficult for three main reasons: firstly, vitamin D is a heat-labile molecule, which, in solution, readily isomerises to its precursor, pre-vitamin D ; secondly, vitamin D is usually present in micro-amounts relative to other fat-soluble vitamins, such as vitamin A and vitamin E, which affects the assay; and thirdly, complex matrices contain various excijpients and antioxidants, which may also have an effect.Laborious clean-up procedures prior to determination are usually required, as many of the chemical methods of assay are non-specific.lS2 This difficulty of accurate measurement of vitamins D, and D, is illustrated by the variety of techniques examined and by the increasing number of papers being published on the subject. The original method for the determination of vitamin D is a biological assay, which evaluates the curative response to vitamin D when administered to rachitic rats., Physico- chemical methods have also been reported.Direct ultraviolet spectrophotometry is only applicable to relatively pure, concentrated preparations. The most commonly used chemical procedure involves reaction with antimony( 111) chloride, commonly known as Nield’s reagent.4 This method is based on the ability of antimony(II1) chloride to form a transitory complex with the vitamin D C,-C,, seco-sterol dieine system. However, any other seco-sterol or unsaturated system will also react (e.g., vitamin A). Hence, extensive purification stages, such as saponification, solvent extraction and clerm-up on several columns, may be required prior to the colorimetric determination of vitarnin D.596 Thin-layer chromatography has also been employed, usually as a clean-up procedure prior to colorimetric Sheppard et aZ., have reviewed the application of gas - liquid chromatography to the deter- mination of fat-soluble vitamins, including vitamin D.Cyclisation of vitamin D and pre- vitamin D was a problem at the high column temperatures necessary for their elution, and this problem is now usually overcome by isomerisation and derivatisation after separation from other fat-soluble vitamins.1°-12 Determinations can take 2 d to complete ; however, the method does distinguish between vitamins D,; and D,. High-performance liquid chromatography (HPLC) provides an alternative to derivatisation gas - liquid chromatography for the separation of labile, heat-sensitive compounds, such as * Presented at the Fourth SAC Conference, Birmingham, July 17th to 22nd, 1977.t To whom correspondence should be addressed.MACKAY, TILLMAN AND BURNS 627 vitamins D, and D,. Reversed-phase chromatography has been used in order toseparate the D vitamins from other fat-soluble vitamins.l3-15 The separation of vitamin D, from D, is possible, especially in the presence of silver nitrate in the mobile phase.16 In general, adsorption chromatography will not separate vitamin D, from vitamin D,, but it has been used for the separation of the D vitamins from other fat-soluble vitamins.17-19 Adsorption chromatography has the advantage of providing a better separation of pre-vitamin D, from vitamin D3,l*-l9 compared with reversed-phase systems.15 Independent collaborative studies comparing the AOAC chemical method with HPLC and rat bioassay20,21 and also the AOAC method with HPLC and gas - liquid chromatography22 have recently been reported.It was recommended that HPLC should be adopted as the first official action for D, in resins and oils,21 with the reservation that thermal isomerisation to the pre-vitamin could occur in sample pre-treatment. This aspect of vitamin D chemistry is important and it implies that any quantitative analytical procedure for the determination of vitamin D must be performed under conditions where the equilibrium ratio is not altered. This paper describes a reliable, quantitative chromatographic method for the determination of vitamin D and pre-vitamin D, and a sample pre-treatment procedure that takes cognisance of, and resolves, this problem of the thermal isomerisation of vitamin D,.Experimental Reagents and Stock Solutions All reagents are of analytical-reagent grade unless otherwise stated. Cyclohexane (laboratory grade). Isopro$yl alcohol. n-Hexane (spectroscopic grade). Trypsin with 85% lactose as diluent. 4-Hydroxybiphenyl. Vitamin D,, pure, crystalline. Dilute sodium hydroxide solution (BP). Phosphate bufler (pH 7). Obtained from BDH Chemicals Ltd. Obtained from BDH Chemicals Ltd. Obtained from Koch-Light Laboratories. A 5% m/V solution in water. Dissolve 8.28 g of sodium dihydrogen phosphate (NaH,PO,.H,O) and 19.88 g of disodium hydrogen phosphate (Na,HPO,) (anhydrous) in distilled water and dilute to 11. Dissolve 20 mg of 4-hydroxybiphenyl in 5 ml of ethyl alcohol, then dilute to 100 ml with cyclohexane.Dilute 10 ml of stock solution to 100 ml with cyclo- hexane. Dissolve 50 mg of vitamin D, in 5 ml of ethyl alcohol and dilute the solution to 100 ml with cyclohexane. Dilute 2-, 4-, 6-, 8- and 10-ml aliquots of the stock vitamin D, standard solution, together with 5 ml of stock internal standard solution, to 50 ml with cyclohexane. These solutions cover the range 20-100 p g ml-l of vitamin D,. Internal standard stock solution. Dilute internal standard solution. Vitamin D, standard stock solution. Working standard solutions. Apparatus The apparatus used consists of a liquid chromatograph equipped with a constant-flow pump (Waters Associates, Model M 6000), an untraviolet absorbance detector (Waters Associates, Model 440) and a syringe - loop injection system (Waters Associates, Model U6K). Chromatographic Conditions Column.Stationary phase. Mobile phase. Flow-rate. 0.8 ml min-l. Detector. Chart speed. 0.2 cm min-l. Stainless-steel, 250 x 4.6 mm i.d. Microparticulate silica, 10 pm (Partisil 10). Cyclohexane containing 1.25% V/V isopropyl alcohol. An ultraviolet detector operated at 254 nm, with sensitivity of 0.02 a.u.f.s. Procedure Calibration graph Allow sufficient time for the column to equilibrate with the solvent in order to obtain a628 MACKAY et d.: DETERMINATION OF VITAMIN D, IN MULTIVITAMIN Analyst, VOl. 104 steady base line (15-30min). Then inject 5-pl aliquots of the dilute working standard solutions. Calculate the peak-height ratio for th.e vitamin D, peak (retention time approxi- mately 20 min) relative to the internal standard peak (retention time approximately 24 min) and construct a calibration graph of peak height ratio against vitamin D, concen- tration (in pg ml-1).Procedure for tablets Sample preparation. Weigh 20 tablets and grind them to a fine powder in a pestle and mortar. Accurately weigh duplicate portions, each equivalent to 50 pg of vitamin D,, into 100-ml stoppered conical flasks. Digestion process. To the sample add 500 mg of trypsin and 50 ml of phosphate buffer and digest in a water-bath at 37 "C for 1 h, swirling occasionally to release the vitamins from the tablet matrix. Transfer the digestion mixture into a 250-ml separating funnel, washing it in with two 10-ml portions of water, then two 5-nd portions of ethyl alcohol.Finally, rinse with two 40-ml portions of hexane, collecting a'U of the extracts in the separating funnel. Shake the funnel vigorously for 1 min, allow the two phases to separate and run off the lower, aqueous phase into a second separating funnel. Extract the aqueous phase with a further three 40-ml portions of hexane, combining each hexane extract with the extract in the first separating funnel. Next wash the bulked hexane extracts with 20ml of distilled water containing 2 ml of dilute sodium hydroxide solution. A white gelatinous precipitate (from stearate excipients) might be fonned; this should be run off and rejected with this alkaline solution. Wash the hexane extract with two 20-ml volumes of distilled water (or until it is free from alkali), then dry it by shaking with about 5 g of anhydrous sodium sulphate.Filter the extract into a 250-ml round-bottomed flask and evaporate it under vacuum to a low volume in a rotary evaporator a t 37 "C. Transfer the residual liquid quantitatively into a 25-ml pear-shaped flask and continue the evaporation to dryness. Dissolve the residue in 2 ml of dilute internal standard solution. Use 5 pl of this extract for chromatography under the same conditions as in the calibration procedure and calculate the peak-height ratio of vitamin D, to internal standard. From the calibration graph, determine the concentration of vitamin D, in the sample solution. Calculation Extraction. M Micrograms of vitamin D, per tablet = X x V x - m where X is the concentration of vitamin D, in the extract (pg ml-l), V the volume of extract (normally 2 ml), M the mean mass of the tablet (g) and m the amount taken (g).Investigation of Experimental Variables Mobile phase Laboratory-grade cyclohexane was chosen as the solvent as it is inexpensive and readily available and was shown to have negligible absorlbance at 254 nm. Cyclohexane itself offers no de-activation of the silica and hence the vitamin D is strongly absorbed on to the column, leading to long retention times and broad peaks. The addition of small volumes of iso- propyl alcohol to the mobile phase produces dramatic de-activation of the silica, and by increasing the concentration of isopropyl alcohol the retention of vitamin D is reduced (Fig. 1) and sharp peaks are obtained. However, if excessive amounts of isopropyl alcohol are added then column efficiency is reduced and separation of vitamin D from vitamins A and E is not achieved.It is recommended that the concentration of isopropyl alcohol necessary to produce sharp peaks and adequate separation from interfering vitamins be determined for each column. With the column used in this work 1.25% V/V was the optimum con- centration of isopropyl alcohol in the cyclohexane that was necessary to give a reasonably sharp vitamin D, peak and adequate separation from pre-vitamin D, and from vitamins A and E. Vitamins A acetate and E acetate were eluted at about 8min, pre-vitamin D, at about 15 min and vitamin D, at about 20 min. A further advantage of this system is that vitamin A alcohol (a possible degradation product of vitamin A acetate) is eluted after vitamin D,.Jab, 1979 TABLETS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 60 50 C .- 5 4 0 - - 30- .- E 0 .- + : 20- c.' U 10 , - - 0 629 0 0.5 1 .o 1.5 2.0 Concentration of isopropyl alcohol in cyclohexane, % V/V Fig.1. Effect of isopropyl alcohol concentrations on retention time of vitamin D,. Choice of internal standard For quantitative work, using syringe injection, an internal standard is desirable. A suitable compound was found to be 4-hydroxybiphenyl, which has a retention time of about 25 min. A base-line separation from vitamin D, was obtained and there was also no inter- ference from the pre-vitamin or vitamin A alcohol. A typical chromatogram is shown in Fig. 2. 4 5 1 L - Time/min Fig. 2. Chromatogram of vitamin mixture in tablet pro- portions.1, Vitamin A acetate (17.2pg) andvitamin E (250pg); 2, unknown; 3, pre-vitamin D,; 4, vitamin D, (0.125 pg); 5, 4- hydroxybiphenyl; and 6, vit- amin A alcohol. Conditions as in Procedure.630 Linearity of response working range 0-0.5 pg of vitamin D,. Reproducibility The two main factors that affect precision in quantitative liquid chromatography are reproducibility of the peak height and reproducibility of the retention time. A series of eight replicate 5-p1 injections of a standard solution containing 40 pg ml-l of vitamin D, gave a mean peak-height ratio of 0.525 with a coefficient of variation of 0.93%. A greater problem with de-activated silica columns is the: reproducibility of retention time from day to day.The absolute retention times of the pre-vitamin D,, vitamin D, and the internal standard varied, but the relative retention times remained constant. MACKAY et ai. : DETERMINATION OF VITAMIN D, I N MULTIVITAMIN Analyst, voi. 104 The measurement of peak-height ratios gave (consistent linear calibration graphs over the Digestion and extraction conditions Many procedures involve digestion of the vitamin from the beadlets with water or dimethyl sulphoxide17 at 60-70 "C or saponification with alcoholic potassium hydroxide solution20 at a similar temperature. It was considered essential to avoid the risk of formation of pre- vitamin D, at these elevated temperatures and an alternative procedure was sought. Digestion with trypsin in a phosphate buffer at 37 "C for 1 h was shown to be a most effective procedure for breaking up the gelatin beadlets.This procedure also avoided the formation of vitamin A alcohol and hence reduced the analysis time. Hexane was chosen as the solvent with which to extract the fat-soluble vitamins from the aqueous digest as it gave minimum problems with emulsion formation. However, four extractions were required in order to obtain a quantitative recovery from aqueous so'lutions, as shown. Volume of hexane Recovery of vitamin D,, yo 1 x 100ml + 1 x 50ml 82 1 x 100ml + 2 x 50ml 92 1 x 8 0 m l + 3 x 40ml 96 Efect of heating vitamin D, solutions at 37 "C In solution, vitamin D, forms an equilibrium mixture with the pre-vitamin and the ratio, at equilibrium, is solely temperature dependent. The proportion of pre-vitamin increases with increasing temperat~re,,~ hence the need to :maintain low-temperature reaction conditions in order to minimise the formation of pre-vitamin during the analytical procedure. The total time at 37 "C for the analytical procedure described is of the order of 1+2 h.A standard solution of vitamin D,, with internal standard, was therefore maintained at 37 "C and 5-pl injections made after various periods of time. There was no appreciable change in the peak-height ratio over a period of at least 4 h and no increase in the small (not greater than 5%) pre-vitamin peak in the initial sample. After 3 d at 37 "C the pre-vitamin peak had increased and the change in peak-height ratio of vitamin D, indicated a loss of 10%. These results gave confidence that no additional pre-vitamin would be formed during the analytical procedure.Recovery from gelatin beadlets Recovery experiments, using the recommended procedure, were then performed on the vitamin D, beadlet concentrate and in the presence of tablet excipients, including the theoretical proportion of other vitamins. Vitamin D, was first assayed in the beadlets by use of ultraviolet absorption (due allowance being made for the presence of the antioxidants butylated hydroxyanisole and butylated hydroxytoluene) . The sample size taken for the ultraviolet assay was 100 mg, in an attempt to obtain as precise a result as possible, against which to compare the HPLC results. Ten 2-mg samples were taken for the HPLC assay, which corresponded to the assay requirement of about 50pg of vitamin D,.The results are shown in Table I. The slightly low recovery in the presence of tablet components was believed to be due to the slight emulsion problems that were encountered because of the bulk of the insoluble matter present ; however, it was considered to be acceptable for a micro-assay of this nature.J d y , 1979 TABLETS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY TABLE I 63 Z RECOVERY OF VITAMIN D, FROM BEADLET CONCENTRATE HPLC assay r A \ Ultraviolet Beadlets + tablet assay Beadlets components Mean vitamin D,/mg g-l . . .. .. . . 26.5 26.7 25.2 Recovery with respect to ultraviolet assay, % . . 100.5 95.1 Relative standard deviation, % . . .. . . 0.8 2.4 3.5 A fialysis of multivitamirt tablets A sample of 20 tablets containing theoretically 500 I.U.of vitamin D, per tablet was ground up and four replicate analyses were performed. A mean result of 513 I.U. per tablet was obtained, with a range of 478-532 I.U. per tablet. A typical chromatogram is illustrated in Fig. 3. It will be observed that there is some interference with the pre-vitamin D, peak from degradation products of vitamin A or vitamin E. In this instance it would be necessary to determine the pre-vitamin content by reference to the vitamin D beadlet concentrate as described later under Discussion of Potency Measurement. 1 3 - Tirne/min Fig. 3. Chromatogram of multivitamin tablet. 1, Vitamins A acetate and E; 2, vitamin D,; 3, internal standard; and X, retention of pre-vitamin D,. Conditions as in Pro- cedure. Relative detector Yesfionse for vitamin D, and $re-vitamin D, In order to quantify any pre-vitamin that may be present in beadlets or concentrate, the detector response to the pre-vitamin needs to be determined, as this has a much lower absorbance at 254 nm than vitamin D, itself.In the absence of a pure sample of pre-vitamin the detector response relative to vitamin D, was determined in the following manner. A 100-ml volume of a cyclohexane solution containing 10 mg of pure vitamin D, and 2.5 mg632 MACKAY et al. DETERMINATION OF VITAMIN D, I N MULTIVITAMIN Analyst, VOl. 104 of internal standard was isomerised by refluxing at 80 "C for 2 h. factor (R,) for the pre-vitamin is given by The relative response RP (isom.) R - - RD (init.) - RD (isom.) where RP (isom.) = peak-height ratio of pre-vitamin to internal standard in isomerised solution; RD (init.) = peak-height ratio of vitamin D, to internal standard in initial solution; and RD (isom.) = peak-height ratio of vitamin D, to internal standard in isomerised solution.Reproducible, but different, values were obtained for two different detectors: 0.797 for detector A (Varian) and 0.65 for detector B (Waters Associates, Model 440). This variation in response between detectors is contrary to the findings of Hofsass et aL21 and emphasises the need to calibrate each detector. Stability Aspects In order to determine whether or not the procedure would be suitable for stability studies of formulations, various samples of pure crystalline vitamin D, were subjected to exaggerated conditions designed to cause degradation.Efect of ultraviolet light A solution of vitamin D, (0.1 mg ml-1) in cyclohexane and pure solid vitamin D, were sealed in glass ampoules and placed in the sample compartment of a xenon-arc fade tester (Xenotest). The solution and solid had discoloured after 5 d. The solution showed a loss of 86% of the vitamin while the solid assayed at 53%. Efect of heat (on solid material) Assay after 1 week gave the following results: Pure crystalline vitamin D, was heated at 60 and 100 "C in sealed and unsealed ampoules. Vitamin remaining, yo Temperature/"C Sealed ampoule Unsealed ampoule 60 41 24 100 5 4 For samples stored at both temperatures there was considerable darkening and the solid had partly melted to form a resin-like mass.A11 samples showed the presence of pre-vitamin as well as other more polar degradation peaks. Typical chromatograms are shown in Fig. 4. Saponijcation A sample of vitamin D, concentrate in beadlet form was saponified by refluxing on a water-bath, using 5 ml of 50% m/V aqueous potassium hydroxide solution in 70 ml of 70% V/V aqueous ethanol in the presence of sodium ascorbate as antioxidant. A considerable amount of pre-vitamin (about 15%) is formed during this procedure (Fig. 5). Stability of vitamin D, in solution at elevated tem$eratuures Solutions of pure vitamin D, and internal standard were retained at various temperatures for different periods of time in order to determine the rate of formation of pre-vitamin: solutions in cyclohexane were stored at 20 and 4.0 "C; a solution in cyclohexane was refluxed (at 80 "C); and a solution in toluene was refluxed (at 102 "C).In all instances the only degradation peak to be produced was that due to the pre-vitamin (see Fig. 6 for an example) and an equilibrium was attained. It was therefore possible to determine the equilibrium ratio of vitamin to pre-vitamin and the time taken to reach equilibrium. These results are summarised in Table I1 and compared with the figures calculated by Keverling Buisman et aL2, for vitamin D,. Previous workers' have indicated that equilibrium and isomerisation rates of vitamin D, and D, are similar, as is confirmed by the above results.July, 1979 TABLETS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 2 I - Time/min - Time/min Fig. 4. Chromatograms showing degradation of vitamin D,: (a) 60 OC and (b) 100 "C, after 1 week in unsealed ampoules.1, Pre-vitamin D,; 2, vitamin D,; and 3, internal standard. Other peaks unidentified. Discussion of Potency Measurement - Time/min Fig. 5. Chromatogram of vitamin D, concentrate in beadlet form after saponifica- tion. 1, Pre-vitamin D,; and 2, vitamin D,. 633 Attempts to correlate bioassay results with physico-chemical assay results have led to confusion in defining the vitamin D content of a product. The biological assay measures I Time/min - Fig. 6. Chromatograms showing the rate of formation of pre-vitamin D, from solutions in cyclohexane of pure vitamin D,, refluxed at 80 "C for different periods of time: (a), initial; (b) after 15 min; and (c) after 2 h. 1, Pre-vitamin D,; 2, vitamin D,; and 3, internal standard.634 MACKAY et al.: DETERMINATION OF VITAMIN D, I N MULTIVITAMIN Analyst, VOl.104 TABLE I1 RATE OF FORMATION OF PRE-VITAMIN D, AT DIFFERENT TEMPERATURES Temperature/"C A r \ 20 410 80 ---+-- +- - Theoreti- Theoreti- Theoreti- cal* HPLC cal HPLC cal HPLC pre-vitamin D, . . 93:7 95:5 89: 11 92:s 78:22 78:22 Ratio vitamin D, : Time to reach equilibrium . . .. 30d 14d 3.5d 2 d 2.4 h 2 h * According to Keverling Buisman et aLe3 102 - Theoreti- cal HPLC 7 2 ~ 2 8 74:26 30min 10min the vitamin D in terms of units of potency, the International Unit (I.U.), which was defined by the World Health Organization as being equivalent to 0.025 pg of pure, crystalline vitamin D, or D,; this is the amount of material required to produce a minimum prophylactic response in rachitic rats or chicks.The major problem in all assay procedures arises from the fact that vitamin D readily isomerises in solution to form the pre-vitamin. The rate of isomerisation and the equi- librium ratio appear to be solely temperature dependent and unaffected by solvent, light or catalysts. 24 Vitamin D2 Pre-vitamin D2 Pre-vitamin D has about 3540% of the biological activity of vitamin D itself. This corresponds to the percentage formation of vitamin D from the pre-vitamin at 40 "C in 9 h, as deduced from kinetic studies on the pre-vitamin - vitamin isomerisation. It can therefore be concluded that the apparent biological activity of the pre-vitamin is a function of its in vivo conversion to vitamin D at body temper(ature.25 Thus, any assay method that measures actual vitamin D only, and that causes conversion to the pre-vitamin during the procedure, will yield falsely low results.In order to over- come this problem, Keverling Buisman et aL2, and Mulder et aLZ4 have defined two terms: Actual vitamin D Potential vitamin D = vitamin D + pre-vitamin D = vitanlin D only It was shown that in many analytical procedures the actual vitamin D content could vary owing to formation of the pre-vitamin, but that the potential vitamin D content remained unaltered. It has therefore been suggested that the potential vitamin D content is the only usable measure of vitamin D activity. Under specified conditions colorimetric methods, thin-layer chromatography, gas - liquid Chromatography and HPLC procedures are all capable of yielding the potential vitamin D content of a preparation.Three approaches have been suggested.27July, 1979 TABLETS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 635 Purification without separating vitamin and pre-vitamin and determination by a reaction that gives the same response to both compounds (e.g., Nield's reagent). Purification and separation of the vitamin and pre-vitamin followed by (a), analysing the fractions separately and adding the results, or ( b ) , re-uniting the fractions and analysing them together (e.g., thin-layer chromatography followed by extraction and Nield reaction on the separate or combined fractions). 3. Comparison of a measurable property of a purified sample solution and a standard solution after equilibration of both under identical conditions. These approaches would give no indication of the pre-vitamin content of the original sample because the vitamin and pre-vitamin are either measured in total or the ratio is altered during the analytical procedure.As the pre-vitamin is less active than the actual vitamin a false indication of the potency of a preparation could be obtained. These approaches would only be valid for preparations containing no pre-vitamin initially. The only universally valid approach to obtaining good correlation with biological potency is to quantify the vitamin and pre-vitamin separately and not to alter the proportion of each during the process. This implies that saponification or digestion at elevated temperatures prior to measurement would render a method invalid for preparations that contain an appreciable amount of pre-vitamin.The concept of potency measurement has been extended by Tartivita et aZ.18 and Vanhaelen- Fastre and Vanhaelen19 to make allowance for the lower biological activity of the pre- vitamin by inclusion of a bioactivity factor. Vitamin D potency = actual vitamin D content + BF x pre-vitamin content where BF is a factor to allow for the bioactivity of pre-vitamin D relative to actual vitamin D. Based on the work of Hanewald et ~ 1 . ~ ~ this can be taken to be 0.35, but it is obviously empirical and difficult to measure precisely and there may be a species variation. In the work reported in this paper, sample pre-treatment is carried out at less than 40 "C and it has been shown that no isomerisation of vitamin D occurs during the analysis time.Isomerisation of vitamin D in the solid state does not occur and is unlikely in solid multi- vitamin formulations. Thus, if the proportion of pre-vitamin present in the beadlets formed as a result of the manufacturing process is measured, then by the determination of the actual vitamin content of the tablet, allowance can be made for the pre-vitamin content and a valid determination of the vitamin D potency of the formulation obtained. With solution formulations of vitamin D, where isomerisation is a possibility, it would be necessary to quantify both the vitamin D and the pre-vitamin peaks separately. Conclusions In order to obtain good correlation between chemical assays and biological potency it is essential that actual vitamin D and pre-vitamin D should be measured separately and that the ratio should not be altered during the analytical procedure; allowance can then be made for the lower activity of the pre-vitamin.The proposed procedure for the determination of vitamin D in multivitamin tablets has several advantages and has proved to be satisfactory in routine use. Saponification is avoided by means of an enzymic digestion, and solvent extraction is carried out at low temperatures. This procedure eliminates the risk of pre-vitamin formation during the assay, as would ultrasonic vibration.19 The formation of the pre-vitamin is unlikely in tablet formulations at ambient temperature, so that the actual vitamin D content is also equal to the vitamin D potency (provided that pre-vitamin is excluded or limited in the beadlet concentrate incorporated in the tablet).For liquid formulations, where isomerisation is a possibility, it is also necessary to quantify the pre-vitamin content. The chromatographic conditions are such that other fat-soluble vitamins do not interfere ; this avoids laborious column clean-up procedures and allows at least four assays per day to be completed. The procedure was developed using vitamin D,, but should be equally applicable to preparations containing vitamin D, as their chromatographic and thermal properties are almost identical. The method is also suitable for stability studies providing there is no interference from degradation products of other fat-soluble vitamins.The authors thank Dr. A. G. Fogg, Loughborough University, for kind liaison and helpful discussions during the course of this work. 1. 2. Thus, the following term can be defined636 MACKAY, TILLMAN AND BURNS References Freed, M., “Methods of Vitamin Analysis,’’ Interscience, New York, 1966. Gyorgy, P., and Pearson, W. N., “The Vitamins,” Academic Press, New York, 1967. “Official Methods of Analysis of the Association of Official Analytical Chemists,” Twelfth Nield, C. H., Russell, W. C., and Zimmerli, A., J . BioZ. Chem., 1940, 136, 73. “United States Pharmacopeia XIX,” USP Convention Inc., Rockville, Md., 1975, p. 633. Mulder, F. J., and de Vries, E., J . Ass. Ofi. Analyt. Chem.. 1974, 57, 1349. Hanewald, K. H., Mulder, F. J., and Keuning, K. J., J . Pharm. Sci., 1968, 57, 1308. Johnson, G. W., and Vickers, C., Analyst, 1973, 98, 257. Sheppard, A. J., Prosser, A. R., and Hubbard, W. D., J . Am. Oil. Chem. Soc., 1972, 49, 619. Murray, T. K., Erdody, P., and Panalaks, T., J . Ass. 08. Analyt. Chem., 1968, 51, 839. Bell, J. G., and Christie, A. A., Analyst, 1973, 98, 268. Bell, J. G., and Christie, A. A., Analyst, 1974, 9’9, 385. Williams, R. C., Schmit, J. A., and Henry, R. A., J . Chromat. Sci., 1972, 10, 494. Osadca, M., and Araujo, M., J . Ass. OH. Analyt. Chem., 1977, 60, 993. Wiggins, R. A., Chemy Ind., 1977, 841. Tscherne, R. J., and Capitano, G., J . Chromat., 1977, 136, 337. Tompkins, D. F., and Tscherne, R. J., Analyt. (?hem., 1974, 46, 1602. Tartivita, K. A., Sciarello, J. P., and Rudy, B. C., J . Pharm. Sci., 1976, 65, 1024. Vanhaelen-Fastre, R., and Vanhaelen, M., J . Chromat., 1978, 153, 219. Hofsass, H., Grant, A., Alicino, N. J., and Greenbaum, S. B., J . Ass. 08. Analyt. Chem., 1976, 59, Hofsass, H., Alicino, N. J., Hirsch, A. L., Ameilta, L., and Smith, L. D., J . Ass. OH. Analyt. Chem. Borsje, B., Craenen, H. A. H., Esser, R. J. E., Milder, F. J., and de Vries, E. J., J . Ass. Ofl. Analyt. Keverling Buisman, J. A., Hanewald, K. H., Mulder, F. J., Roborgh, J. R., and Keuning, K. J., Mulder, F. J., de Vries, E. J., and Borsje, B., J . Ass. Off. Analyt. Chem., 1971, 54, 1168. Hanewald, K. H., Rappoldt, M. P., and Roborgh, J. R., Recl Trav. Chim. Pays-Bas Belg., 1961, Edition, Association of Official Analytical Chemists, Washington, D.C. , 1975, Section 43.166. 251. 1978, 61, 735. Chem., 1978, 61, 122. J . Pharm. Sci., 1968, 57, 1326. 80, 1003. 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. Received December 13th, 1978 Accepted February 7th, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790400626

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Analyst, July, 1979, Vol. 104, pp. 637-644 637 Modified Fluorimetric Procedure for the Simultaneous Determination of Thiamin and Riboflavin in Cowpea J. K. Edijala" Department of Food Science and Nutrition, University of Strathclyde, 131 Albion Street, Glasgow, G1 1SD The fluorimetric methods for the assay of thiamin and riboflavin have been modified so that the same sample extract may be used for the simultaneous analysis of the two B vitamins. The procedure eliminates acid hydrolysis and specific pH adjustment for either vitamin; the sample extract is obtained after enzymic hydrolysis only with Clarase and has a pH of 4.5-4.8. A standard additions method for thiamin determination is introduced. A cowpea sample analysed by this procedure gave mean recoveries and standard errors of 103.9 & 1 and 100.5 f 2% for added thiamin and ribo- flavin, respectively; the detection limits for the aqueous solutions taken for fluorescence measurement according to the described procedures for these two vitamins are 0.002 5 and 0.002 0 pg ml-l, respectively.Keywords : Thiamin determination ; riboflavin determination ; fluorimetry ; Clarase ; cowpea The fluorimetric methods for thiamin and riboflavin determination described by the AOACl are time consuming, tedious and costly in chemicals. Basically, the extraction procedure for the test sample involves acid hydrolysis. In the case of thiamin, extraction is followed by enzymic hydrolysis and a chromatographic purification using Decalso resin. The thiamin content of the extract is calculated by comparison with a thiamin standard solution analysed concurrently with the extract, but the use of a standard additions method is advocated for riboflavin.Two groups of workers have previously recommended modifications to improve the above procedure. Pelletier and Madere2 suggested: (i) omitting acid hydrolysis for both plant and animal products; (ii) incubating the sample - enzyme mixture for 20 h (overnight) instead of the recommended 3 h used for the sake of convenience; and (iii) the possibility of using the same enzymic (Clarase) extract for the automated analysis of thiamin and ribo- flavin. Edwin et aZ.3 recommended modifying the column-chromatographic stage in the thiamin analysis by adding the Decalso resin directly to the reaction tube. The feasibility of the suggested modifications was investigated.The studies that were undertaken included: (i) preparation of thiamin calibration graphs (with 0.02-0.10 pg ml-l thiamin standard solutions) during which the extractability of thiochrome using anhydrous and water-saturated isobutyl alcohol was determined; (ii) extraction of the two vitamins under different conditions; (iii) determination of the vitamins present in the dephosphoro- lytic enzymes Clarase and Taka-Diastase; (iv) evaluation of the methods of determination and calculation for thiamin; and (v) recovery of the added vitamins. From analysis of the findings, a satisfactory, straightforward and reproducible procedure was obtained. Experimental Reagents Cowj5ea JEouur. The cowpeas (sample ACC 73001, a brown variety obtained from the National Cereals Research Institute, P.M.B.5042, Ibadan) were milled to produce a fine unfractionated powder. Taka-Diastase. Obtained from Sigma Chemicals. Clarase. Obtained from MKC Enzymes, Miles Laboratories, Slough. Prepare a 2% m/V aqueous solution fresh for each assay. Resent address : National Cereals Research Institute, P.M.B. 6042, Ibadan, Nigeria.638 EDIJALA: MODIFIED FLUORIMETRIC PROCEDURE FOR THE SIMULTANEOUS Analyst, VoZ. 104 Adjust the sodium acetate solution to pH 4.5 with 0.1 N hydrochloric Dissolve 10 mg in 1 1 of 0.1 N sulphuric Activate the cation-exchange resin of 60-80 mesh The aqueous solution is stable for Sodium acetate solution, 2 N. B u f e r solution. acid. Quinine sulphate stock solution, 10 pg ml-l.acid and store in a light-resistant bottle. Decalso F (sodium aluminosilicate). (BDH) as described in the AOAC meth0d.l Potassium hexacyanoferrate(III), 1 yo m/V solution. 6 months if kept in a light-resistant bottle.* Sodium hydroxide solution, 15% m/V. Oxidising reagent. with 15% sodium hydroxide solution. Prepare immediately before use. Neutral potassium chloride solution, 25% mlV. Acidic potassium chloride solution. Add 8.5 nil of concentrated hydrochloric acid to 1 1 of neutral potassium chloride solution. Water-saturated isobutyl alcohol. Shake anhydrous isobutyl alcohol with de-ionised water (5 + 1) and allow it to equilibrate for at least 48 h before use. The used solvent can be redistilled for repeated use. Prepare a 0.25 pg ml-l dilution weekly. Store at 10 "C.Dilute 1 ml of 1% potassium hexacyanoferrate(II1) solution to 25 ml Glacial acetic acid. Potassium permanganate solution, 4% m/V. Hydrogen peroxide solution, 3%. Sodium dithionite (sodium hydrosulphite) . Sodium Jhorescein stock solution, 50 pg ml-l. Dilute 10 ml of 30% m/V hydrogen peroxide solution to 100ml. Dissolve 50 mg in 1 1 of de-ionised water and store in a light-resistant bottle. Prepare a 01.05 pg ml-l dilution weekly. Preparation of standards Thiamin hydrochloride stock solution, 100 pg ml-l. Dry the thiamin crystals for 5 h at 100 "C, dissolve 50 mg in 500 ml of 20% ethyl alcohol acidified with hydrochloric acid (pH 3.54.3) and store at 10 "C in a light-resistant bottle. Dilute 1.0 ml of the stock solution to 1OOml with acidified 20% V/V ethyl alcohol solution.Store at 10 "C and prepare weekly. Thiamin standard solution, 0.1 pg ml-l. Dilute 5.0 ml of the intermediate standard solution to 50 ml. RiboJlavin stock solution, 100 pg ml-l. Dry the riboflavin crystals for 5 h at 100 "C and dissolve 50 mg in warm 0.02 N acetic acid. Mak:e up to 500 ml on cooling and store under toluene in a light-resistant bottle at 10 "C. RibofEavin intermediate standard solution, 1.0 pi; ml-l. Dilute 1.0 ml of stock solution t o 100 ml. Store at 10 "C and prepare weekly. RiboJlavin standard solution, 0.2 pg ml-l. Dilute 10.0 ml of the intermediate standard solution to 50 ml with 0.02 N acetic acid. Prepare daily. Thiamin intermediate standard solution, 1.0 pg ml-l. Prepare daily. Apparatus Chromatographic columns. Glass tubes (9 x 260 mm) with a capillary tip.Automatic pipette. Alternatively use a graduated 5-ml pipette with a broken tip to deliver the oxidising reagent in 1 s. Glass-stop$ered test-tubes. Approximately 17-nil capacity. Mechanical shaker. pH meter. Model PW 9410 digital (Pye Unicam). Fluorimeter. Locarte, single side, Model MK4:, using cylindrical cuvettes of about 2-ml capacity. Fluorimeter Jilters. (a) Primary: LF2 to select an excitation wavelength of 365 nm for thiochrome and LF3 to select an excitation wavelength of 440nm for riboflavin. (b) Secondary: LF3 plus LF14 (gelatin) to select a transmission wavelength of 436 nm for thiochrome and LF8 plus LF14 to select a transmission wavelength of 565 nm for riboflavin.July, 1979 DETERMINATION OF THIAMIN AND RIBOFLAVIN I N COWPEA General Procedure 639 Extraction and dephosphorylation Mechanically shake 1.0-1.5 g of cowpea flour with 10-15 ml of 0.1 N hydrochloric acid in a 100-ml glass-stoppered conical flask for 10min.Rinse the sides of the flask with 10- 20 ml of buffer solution and adjust the pH of the mixture to 4.5 with 2 N sodium acetate solution (0.7-1.4 ml). Add 5 m l of the 2% enzyme solution and rinse the sides of the flask with 5-10ml of buffer solution (total volume being 45-55 ml). Incubate the flour - enzyme mixture at 45 "C for 20 h. Allow the flask to cool and carry out preliminary filtration with the water washings through glass-wool to yield a cloudy filtrate free of coarse particles. Re-filter through fluted filter-paper (Whatman No. 41) to give a clear filtrate and make up to 100 ml with de-ionised water (Vl), The pH of the filtrate will vary between 4.5 and 4.8.Use this filtrate for subsequent thiamin and riboflavin assays. Thiamin assay PuriJication. Insert a plug of glass-wool at the base of the chromatographic column and pack the column with 2 g of activated Decalso suspended in de-ionised water. Allow the water to drain to 5 mm above the resin level and pass 10 ml (V,) of the original extract (VJ through the column (rate of flow = 2 ml min-l); wash three times with 5 ml of hot de- ionised water. Elute the thiamine from the column with 4.04.5 ml of hot acidic potassium chloride solution (five times) and make the eluate up to 25 ml on cooling (V3). The alternative method3 of direct addition of Decalso to the reaction tube was beset with operational problems during the purification and subsequent thiochrome formation and was abandoned.Thiochrome formation. Transfer 2.5 ml of the eluate (V,) into six separate glass-stoppered test-tubes each containing 0.75 g of potassium chloride. To two tubes (A) add 1 ml of 0.1 pg ml-1 thiamin standard solution and to the remaining four tubes (B and C ) add 1 ml of de-ionised water. Shake all six tubes gently to mix the contents. To each of the two tubes (A) containing the standard add, with gentle shaking, 1.5 ml of the oxidising reagent. Immediately add 6.5 ml of water-saturated isobutyl alcohol, shake vigorously for 15 s and place in the dark. Treat two of the remaining tubes (B) the same way. The last two tubes (€) serve as blanks and are similarly treated except that 1.5 ml of 15% sodium hydroxide solution replaces the oxidising reagent.Shake all six tubes vigorously again for about 2 min and centrifuge at low speed for 5-10 min to produce a clear upper isobutyl alcohol extract. Operate the fluorimeter at half range with the diaphragm open to 80% of the maximum aperture. Set the galvanometer to maximum deflection with 0.25 pg ml-1 quinine sulphate solution and to zero with water-saturated isobutyl alcohol before reading the fluorescence of the three sets of tubes. Fluorescence measurement. Calculation. Fluorescence of extract + 1 ml of standard Fluorescence of extract + 1 ml of de-ionised water = A = B Fluorescence of sample blank = c B - c 0.1 v V1 100 x - x -3 x - x - 2.5 V , m 1000 Thiamin, mg per 100 g of sample = where m = grams of sample taken for analysis.NOTE- The problem of crystallisation of the potassium chloride in the column during the elution of thiamin3 is overcome by intermittently bringing the acidic solution to the boil just before the application of the next eluting portion ; evaporation of the solution and potential crystallisation on cooling are thereby prevented.640 EDI JALA : MODIFIED FLUORIMETRIC PROCEDU:RE FOR THE SIMULTANEOUS Analyst, VoZ. 104 Ribojavin assay Acidi$cation. Transfer 10 ml of the original extract (V,) into four separate glass-stoppered test-tubes. (The recommended adjustment1 of this extract to pH 6.5 and back to pH 4.5 has been omitted.) To each of two tubes (A) add 1 ml of 0.2 pg ml-1 riboflavin standard solution and to each of the remaining tubes (B) add 1 ml of de-ionised water.Acidify with 1 ml of glacial acetic acid, stopper the tubes andl shake. Oxidation of interfering substances. Add 0.5 ml of 4% potassium permanganate solution to each tube, mix and allow to stand for 2min. Add 0.5ml of 3% hydrogen peroxide solution (the extract should decolorise in less than 10 s). Stopper the tubes and shake vigorously for 15 s, removing the stopper intermittently to de-gas the test solutions. Using the appropriate filters, operate the fluorimeter as described for thiamine. Set the galvanometer to maximum deflection with 0.05 pg ml-l sodium fluorescein solution and to zero with de-ionised water. Transfer some of the assay solutions into the cuvettes and measure the fluorescence.To the remaining solution in tube B, add 20-30 mg of sodium dithionite, sha.ke vigorously and measure the fluorescence within 10-20 s; this reading serves as the blank (C). Fhorescence measurement. Calculation. Fluorescence of extract + 1 ml of standard = A Fluorescence of extract + 1 ml of de-ionised water = B Fluorescence of sample blank = c 1 100 - x - 1 m 1000 x - x (V,-Y) 19 - c 0.2 A - B lo Riboflavin, mg per 100 g of sample = where Y = micrograms of riboflavin contributed. by Clarase and m = grams of sample taken for analysis. Results and Discussion Thiamin Calibration Graphs During the preparation of calibration graphs, the extractability of thiochrome by anhydrous and water-saturated isobutyl alcohol and the optimum delivery time of the oxidising reagent were determined.The regression line parameters (Table I) indicated that isobutyl alcohol equilibrated with water for at least 48 h gave the most satisfactory results and it was used thereafter. A delivery time of 1 s, in preference to 5 s, for the addition of the oxidising reagent gave better results (Table I). To achieve this time of 1 s, an automatic pipetting device, or a 5-ml graduated pipette with a broken tip and standardised for this purpose, should be used. TABLE I REGRESSION LINE DATA FOR THIOCHROME EXTRACTION WITH ISOBUTYL ALCOHOL SUBJECTED TO VARIOUS AQUEOUS TREATMENTS Period of water Correlation saturation/h coefficient Intercept Slope 0 (anhydrous) 0.990 7 - 0.362 92.1 2 0.990 7 -0.021 93.9 24 0.995 0 0.252 102.3 48 0.999 7 0.088 100.6 48* 0.999 7 0.024 134.9 * Used as the reference calibration graph in later work and obtained when the delivery time of the oxidising reagent was 1 s compared with 5 s for the preceding calibration graphs.Thiamin and Riboflavin Content of Cowpea Flour The two B vitamins were determined after varying the extraction conditions (Table 11). Thiamin values for the cowpea flour were calculated by the AOAC comparison method1 and from the previously established regression equation for the calibration graph.July, 1979 DETERMINATION OF THIAMIN AND RIBOFLAVIN I N COWPEA TABLE I1 THIAMIN AND RIBOFLAVIN CONTENTS OF COWPEA FLOUR DETERMINED UNDER DIFFERENT EXTRACTION CONDITIONS 641 Milligrams of vitamin per 100 g dry mass* A fl \ Treatment Acid hydrolysis ( 2 ) 100°C .... .. .. (ii) 121 "C (autoclaving) . . .. Acid hydrolysis + enzymic hydrolysis (10% Taka-Diastase solution), 3 h incubation a t 45 "C . . .. .. Acid hydrolysis + enzymic hydrolysis (2% Taka-Diastase solution), 3 h incubation a t 45 "C . . .. .. Enzymic hydrolysis (2% Taka-Diastase solution), 3 h incubation a t 45 "C . . Enzymic hydrolysis (2% Taka-Diastase solution), 20 h incubation a t 45 "C . . Enzymic hydrolysis (2% Taka-Diastase solution), 20 h incubation a t 45 "C * Means of a t least 4 redicates. . . pH of extract 3.5 4.5 3.5 4.5 3.5 4.5 3.5 4.5 3.5 4.5 3.5 4.5 4.5-4.83 Thiamin Comparison Calibration - method graph 0.23 0.21 0.221 0.22: 0.27 0.26 0.28: 0.27: 0.92 0.93 0.90: 0.871 0.83 0.88 0.89: 0.86: 0.85 0.89 0.89: 0.912 0.84 0.86 0.90: 0.91: 0.89 0.85 Riboflavin : standard additions method 0.21t 0.22 0.20t 0.22 0.261.0.27 0.251. 0.24 0.26t 0.25 0.28t 0.31 0.32 t Riboflavin determined in thiamin extract. $ Thiamin determined in riboflavin extract. 5 No specific pH adjustment of the enzymic digest for either thiamin or riboflavin. Acid hydrolysis at 100 and 121 "C yielded low values for thiamin but the riboflavin values were similar to published data for cowpeas.5s6 Subjecting the sample to both acid and enzymic hydrolyses using Taka-Diastase (10 and 2% enzyme solutions and 3 h incubation at 45 "C) significantly increased yields of both vitamins but the higher level of Taka-Diastase conferred no extra advantage. These results indicated the necessity to include enzymic hydrolysis rather than the previously recommended1 acid hydrolysis for efficient conversion of the complex forms of riboflavin to the free state.However, enzymic hydrolysis alone (2y0 enzyme solution) converted the complex forms of both vitamins into their respective free states as efficiently as acid - enzymic hydrolysis and was more efficient than acid hydrolysis alone, as was also found by Pelletier and Madere.2 In the current investigation, the pH of the extracts did not affect the vitamin determinations as similar values were obtained for thiamin and riboflavin in the extracts a t both pH 3.5 and pH 4.5 as recom- mended in the AOAC methods1; both vitamins may therefore be determined at either pH value. Extending the hydrolysis time to 20 h for the 2% enzyme solution did not increase the yield of thiamin over the pH range 3.54.8, but the yield of riboflavin was consistently higher than the yield after only 3-h incubation (Table 111); thus, the riboflavin value obtained for this sample of cowpea was higher than the published data.5~6 The final adjustment of the enzymic extract to pH 6.5 and then back to pH 4.5, previously recommended for riboflavin assays1 for precipitation of interfering substances, appears to be unnecessary for cowpea flour.The unadjusted enzymic extract at pH 4.54.8 is therefore recommended for the assay of the two vitamins.642 EDIJALA MODIFIED FLUORIMETRIC PROCEDUIRE FOR THE SIMULTANEOUS Analyst, V d . 104 Taka-Diastase, which was used initially, and Clarase, which was introduced in later investigations, were analysed as potential contributors of thiamin and riboflavin to the over-all sample; the enzymes were found to contain 2.94 and 1.41 pg of riboflavin per 0.1 g of enzyme, respectively, but no thiamin.Using these values, appropriate corrections have been applied to the riboflavin values obtained for the cowpea flour. TABLE I11 EVALUATION OF THE EFFICIENCY OF TAKA-DIASTASE AND CLARASE AND THE METHODS OF CALCULATING THE THIAMIN COXTENT WHEN ANALYSING COWPEA FLOUR Milligrams of vitamins per 100 g dry mass Thiamin A 7 'r Riboflavin : Standard standard pH of Comparison Enzyme extract method Taka-Diastase . . . . 4.5-4.8 0.85 0.98 0.75 0.96 0.90 - - - - Mean f SE* .. .. 0.89 f 0.041 c v , t % * - .. .. 10.4 Clarase . . .. . . 4.5-4.8 0.69 0.85 0.98 0.94 - - - - - - - Calibration graph method 0.88 0.82 0.80 0.86 0.89 - - - - 0.85 f 0.017 4.6 0.73 0.77 0.82 0.93 - - - - - - - Mean & SE* .... c v , t % * * .. .. * Standard error. t Coefficient of variation. 0.87 f 0.064 0.81 f 0.043 additions method - 0.96 0.97 0.92 0.94 0.96 0.95 f 0.009 2.1 - 0.97 0.93 0.96 0.91 0.98 1.01 0.98 0.96 additions method 0.28 0.35 0.35 0.36 0.28 0.36 0.32 0.28 0.28 0.32 f 0.013 0.26 0.28 0.27 0.28 0.28 0.25 0.25 0.26 0.27 0.26 0.26 11.8 0.96 & 0.011 0.27 + 0.003 ~ - 1 4:s 1 0.7 3.2 4.2 Takadiastase and Clarase as Dephosphoroljrtic Enzymes Analysis of cowpea flour with 2% solutions of both enzymes, and incubated for 20 h at 45 "C, to give final extracts of pH 4.54.8, yielded similar thiamin values (Table 111). The two enzymes, therefore, proved to be equally suitable for thiamin determination.How- ever, the riboflavin values (Table 111) obtained from the Taka-Diastase extracts were beset with a high degree of variation (coefficient of variation 11.8%) compared with Clarase- treated extracts (coefficient of variation 4.2%) ; further evidence of the variability is shown by variance ratio analysis, which provided a significant F value of 10.8 (Table IV). The high riboflavin content of Taka-Diastase and the variable blanks of these extracts were probably responsible for the poor reproducibility of the results. Consequently, Clarase, which gave reproducible results for riboflavin and thiamin determinations, was preferred. Methods of Thiochrome (Thiamin) Determination and Calculation The results obtained with the AOACl comparison and calibration graph methods of calculation (Table 111) were not satisfactorily reproducible, with coefficients of variation of 10.4 and 4.6y0, and 14.8 and 10.7%, respectively, when using Taka-Diastase and Clarase.The standard additions method that was introduced gave higher and more reproducible results than the two former methods (Table 111). Variance ratio analysis ( F ) of theJuly, 1979 DETERMINATION O F THIAMIN AND RIBOFLAVIN I N COWPEA 643 TABLE IV VARIANCE RATIO ANALYSIS FOR THIAMIN AND RIBOFLAVIN VALUES OBTAINED WITH TAKA-DIASTASE AND CLARASE Enzyme Vitamin determination freedom (f) ( V ) * ( F ) Method of Degrees of Variance Variance ratio Taka-Diastase . . Thiamin Comparison 4 0.0069 (V,) V,/V, = 21.67 Calibration graph 4 0.0012 (V,) V 2 / V 3 = 3.8 Standard additions 4 0.0003 (V,) Clarase ., . . Thiamin Comparison 3 0.0124 (V,) VJV, = 15.57 Calibration graph 3 0.0056 (V,) V5/v(3 = 7.0 Standard additions 7 0.0008 (V,) Taka-Diastase . . Riboflavin Standard additions 8 0.0013 (V7) V7/v8 = 10.8: Standard additions 10 0.0001 (V8) * Variance V = SD2 (SD = standard deviation). T Significant a t 5% level. 1 Significant a t 1% level. comparison and standard additions methods with Taka-Diastase (F = 21.6) and with Clarase (F = 15.5) were significant at the 5% level (Table IV); the F values of the calibration graph and the standard additions methods with both enzymes ( F = 3.8 and F = 7.0) were not significant but the results for the vitamin analyses of cowpea flour obtained with the calibration graph method were the lowest (Table 111).The standard additions method was therefore adopted for thiamin assays. Recovery of Added Thiamin and Riboflavin Having eliminated Taka-Diastase, owing to poor reproducibility, in riboflavin analyses Clarase was used for recovery determinations of the added vitamins. The average recovery values of 103.9 & 1.0% and 100.5 & 2.0% for thiamin and ribo- flavin, respectively, were very satisfactory (Table V). For thiamin, the result was better than the 90.7 & 8.97% obtained with the AOACl comparison method using Taka-Diastase.' TABLE V RECOVERY OF THIAMIN AND RIBOFLAVIN ADDED TO COWPEA FLOUR Vitamin Thiamin . . Riboflavin . . .. Vitamin in cowpea*/pg 17.17 18.15 17.32 17.32 4.74 4.83 4.60 4.60 Vitamin added/pg 15 15 15 15 5 5 5 5 Vitamin in fortified sample of cowpea*/pg Recovery, % 33.04 105.8 33.04 99.3 33.04 104.8 33.04 104.8 Mean f SEt = 103.9 f 1.0 9.83 101.0 10.03 104.0 9.68 101.6 9.12 95.4 Mean f SET = 100.5 f 2.0 * Fresh mass of cowpea flour varied between 2.05 and 2.07 g.Standard error. Conclusion Although cowpea flour was selected as the test material, the proposed procedure should be applicable to all plant products. The modification has eliminated acid hydrolysis of the test sample and introduced a single hydrolytic treatment with Clarase at a lower concentra- tion than in previously reported enzymic procedures, not only for thiamin but also for riboflavin assays. The incubation period of 20 h (Pelletier and Madere2) proved to be more convenient for the organisation of bench work and also ensured the full release of free ribo-644 ED1 JALA flavin. The use of water-saturated isobutyl al.coho1 for thiochrome extraction, and the standard additions method for thiamin detennination, yielded higher and more repro- ducible results than the conventional AOAC meth0d.l Another major advantage of this modified procedure is that the same extract of the test sample at pH 4.54.8, obtained after enzymic hydrolysis only, permitted the simultaneous analysis of the two B vitamins. Thus, the use of the same enzymic extract for the determination of the two vitamins, as suggested by Pelletier and Madere2 for a possible automated procedure, has been applied successfully to manual techniques with a reduction of labour, time and cost of chemical reagents. 1. 2. 3. 4. 5. 6. 7. References Horwitz, W., Editor, “Official Methods of Anatlysis of The Association of Official Analytical Chemists,]’ Twelfth Edition, Association of Official Analytical Chemists, Washington, D.C., 1975, pp. 823 and 826. Pelletier, O., and Madere, R., J . Fd Sci., 1975, 40, 374. Edwin, E. E., Jackman, R., and Hebert, N., Analyst, 1975, 100, 689. Pearson, W. N., in Gyorgy, P., and Pearson, W. N., Editors, “The Vitamins: Chemistry, Physiology, Ogunmodede, B. K., and Oyenuga, V. A., J . Sci. Fd Agric., 1969, 20, 101. Watt, B. K., and Merril, A. L., “Handbook of Nutritional Contents of Foods,” Dover Publications, Defibaugh, P. W., Smith, J. S., and Weeks, C. E., J . Ass. Off. Analyt. Chem., 1977, 60, 522. Pathology, Methods,” Volume VII, Academic Press, New York, 1968, p. 53. New York, 1975, p. 28. Received October 9th, 1978 Accepted January 24th, 1979

ISSN:0003-2654    

DOI:10.1039/AN9790400637

出版商:RSC    

年代:1979

数据来源: RSC