ISSN:0003-2654    

DOI:10.1039/AN98712BP017

出版商:RSC    

年代:1987

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN98712FX021

出版商:RSC    

年代:1987

数据来源: RSC

摘要:

ANALAO 11 2(6) 717-932 (1987)The AnalystJune 198771 771972973975375776376777 177577978378779 179780380981 582 182583 1837The Analytical Journal of The Royal Society of ChemistryCONTENTS2ND INTERNATIONAL SYMPOSIUM ON KINETICS IN ANALYTICAL CHEMISTRY,PREVEZA, GREECE, 9-12 SEPTEMBER, 1986EDITORIALEnzymes as Analytical Reagents: Substrate Determinations with Soluble and with lmmobilised Enzyme Preparations.Selectivity and Kinetics in Analytical Chemistry. Plenary Lecture-Miguel ValcarcelCatalytic Titrations. Plenary Lecture-Ferenc F. GaalDetermination of Kinetic Parameters for 3a-Hydroxysteroid Dehydrogenase Using the Five Major Bile Acids and TheirConjugates as Substrates and Correlation with their Structure and Solubility-Evriklia S.Lianidou, Panayiotis A.SiskosKinetic - Potentiometric Determination of Amino Acids Based on Monitoring their Reaction with DinitrofluorobenzeneUsing a Fluoride-selective Electrode-Eleni At ha nasiou-M ala ki, Michael A. Ko u ppa ri sKinetic - Potentiometric Determination of Monosodium Glutamate in Soups and Soup Bases and of GlutamicDehydrogenase-Dimitrios P. NikolelisKinetic - Spectrophotometric Method for the Determination of Ascorbic Acid in Orange Juice, Parsley andPotatoes-M i I tiades I. Ka raya n nis, Despi na I. Fa rasog louStudy of the Mechanism of the Manganese-catalysed Oxidation of a Thiol-containing Organic Compound-JosePeinado, Fermin Toribio, Dolores Perez-BenditoKinetic - Fluorimetric Determination of Ascorbic Acid at the Nanomole Level-Jose Peinado, Fermin Tori bio, DoloresPerez-Bend itoEffect of Temperature on the Kinetics of Free Radical Formation During the Pyrolysis of Some Amino Acids-Boiidar Lj.MiliC, Sonja M.Djilas, Djordje S. VlaovicCP - MAS 13C NMR Spectral Study of the Kinetics of Melanoidin Formation-Boiidar Lj. MilicIndicator Reaction for the Kinetic - Spectrophotometric Determination of Nanogram Amounts of Iron-Anastasia Ch.Catalytic Determination of Nanogram Amounts of Iron(ll1) Using its Catalytic Effect on the Oxidation of ChromotropicKinetic Determination of Trace Amounts of Copper(l1) Using its Catalytic Effect on the Oxidation of Chromotropic AcidFlow Injection System for Kinetic Determinations Based on the Use of Two Serial Injection Valves-Alfonso Fernandez,Analytical Applications of Reversed-flow Gas Chromatography-N icholas A.Katsa nos, George Ka ra iska kisLeast-squares Fitting of Tabular Data t o Rational Functions in Bnslc-Emmanuel M. PapamichaelAutomation of a Stopped-flow Spectrophotometer for the Determination of Reaction-rate Parameters. Part 1 .-Norman C. Peterson, Panayotis A. Siskos, Miltiades I. KarayannisEffect of Hydroxylamine on Chemiluminescence Intensity Generated During the Oxidation of Pyrogallol withPeriodate-Nicholaos P. EvmiridisDetermination of Formaldehyde Using a Kinetic - Spectrophotometric Method. Part I. Oxidation of pphenylenedi-amine with Hydrogen Peroxide-Nicholaos P. Evmiridis, Miltiades I. KarayannisKinetic Determination of Trace Amounts of Nitrite Based on its Inhibitory Effect on the Photochemical ReactionBetween Iodine and Ethylenediaminetetraacetic Acid-Concepcion Sanchez-Pedrefio, M.Teresa Sierra, M. IsabelSierra, Antonio SanzPlenary Lecture-Horacio A. MottolaZotou, Constantine G. PapadopoulosAcid by Hydrogen PeroxideDemetrius G. Themelis, George S. Vasilikiotisby Hydrogen Peroxide-Demetrius G. Themelis, George S. VasilikiotisMaria Dolores Luque de Castro, Miguel Valcarcel841845849Monitoring of Microgram per Litre Concentrations of Trace Metals in Sea Water: the Choice of Methodology forSampling and Analysis-A. Ashton, R. ChanIon-selective Electrodes in Organic Analysis-Determination of Vanillin by a Vanillate-selective Electrodewing HongChan, Wai Ming Lee, Chuen Ley Foo, Wing Kai TangX-ray Fluorescence and Radiotracer Studies of Polyalkoxylate Interactions with Potentiometric Electrode PVC MatrixMembranes Containing Barium - Polyalkoxylate Complexes-P.H. V. Alexander, G. J. Moody, J. D. R . Thomas, 6. J.Birch855 Determination of Trace Amounts of Thallium in Commercial Radioactive 204TI Samples by the Redox Sub-superequivalence Method of Isotope Dilution Analysis-Hiroe Yoshioka, Kunihiko Hasegawa859 Determination of Acrolein and Crotonaldehyde in Automobile Exhaust Gas by Gas Chromatography withElectron-capture Detection-Harumitsu Nishikawa, Tomokuni Hayakawa, Tadao Sakaicontinued inside back coverTypeset and printed by Black Bear Press Limited, Cambridge, Englan86386787 187587988388789 189589990390991 391 7Application of Tryptamine as a Derivatising Agent for Airborne lsocyanates Determination.Part 1. Model ofDerivatisation of Methyl Isocyanate Characterised Fluorescence and Amperometric Detection in High-perfor-mance Liquid Chromatography-Weh S. Wu, Mark A. Nazar, Virindar S. Gaind, Louis CaloviniHigh-performance Liquid Chromatographic Determination of Mepyramine Maleate, Pheniramine Maleate andPhenylpropanolamine Hydrochloride in Tablets and Drops-Samia M. El-Gizawy, Abd-El-Hamed AhmedGas - Liquid Chromatographic and Ion-pair High-performance Liquid Chromatographic Determination of Pseudo-ephedrine Hydrochloride and Bromhexine Hydrochloride in Pharmaceuticals-E. Venkata Rao, G. Ramana Rao, S.Raghuveer, P.KhadgapathiRapid Method for t h e Direct Determination of Inorganic Iodine in Plasma Using Ion-exchange Chromatography and theSandell and Kolthoff Reaction-Gilles Aumont, Jean-Claude TressolUse of Post-column Ion-pair Extraction with Absorbance Detection for the Liquid Chromatographic Determination ofCyclamate and Other Artificial Sweeteners in Diet Beverages-James F. LawrenceFlow Injection Chemiluminescence Determination of Sulphite-S. A. At-Tamrah, Alan Townshend, Alan R. WheatleySpectrophotometric Determination of Trace Amounts of Germanium in Minerals and Ores with 9-(o-Chlorophenyl)-2,6,7-trihydroxyxanthen-3-one in the Presence of Cetyltrimethylammonium BromideHanxi Shen, ZhenqingWang, Guanghui XuReaction of Thorium( IV) with Tris[2,4,6-(2-hydroxy-4-sulpho-l -naphthylazo)]-s-triazine Trisodium Salt as a Spectro-photometric Method for the Determination of Phosphate and Thorium(1V)-lshwar Singh, Pratap Singh KadyanSpectrophotometric Determination of Chloramphenicol Using Orthogonal Polynomials-Mohamed E.Abdel-Hamid,Mustafa A. AbuirjeieDetermination of Sulphite and Hydrogen Peroxide in Pharmaceutical Matrices via Classical Spectrophotometry andFlow Injection-David S. Brown, Dennis R. JenkeInterference of Ascorbic: and lsoascorbic Acids in the Spectrophotometric Determination of Nitrite b y the Diazotisation - Coupling TechniqueGeorge Norwitz, Peter N. KeliherDetermination of Water-soluble Ammonium Ion in Soil by Spectrophotometry-Xing-Chu Qiu, Guo-Ping Liu,Ying-Quan ZhuSpectrofluorimetric Determination of Boron in Plants with Quinitarin-2-sulphonic Acid-Francisco Salinas, ArsenioMutioz de la Petia, Jose A. Murillo, Juan C.Jimenez SanchezEffect of TemDerature and Soil PhosDhorus Status on the Determination of Extractable Phosphorus by Olsen'sMethod-Development of a Correction Factor-Surinder Pal Singh Brar, Sukh Raj BishnoiJr., Marian TarverThompson.921 Fluorescence Inner Filtering in Double-pass Cell Configurations. Part 3. Secondary Inner Filtering-Kenneth W. Street,925 Determination of Octyl Nitrate in DERV Fuel by Gas Chromatography-Edward Searle, Michael W. Cass, Christopher M.927 BOOK REVIEWSIThe Chemical Analysis of Water:General Principles and Techniques2nd Editionby A. L. Wilson and D. T. E. Hunt, Water Research Centre, MedmenhamHardcover 704pp ISBN 0 85186 797 9Price f55.00 ($99.00) RSC Members f36.00This new edition covers the considerable developmentswhich have taken place in the eleven years since the firstedition was published, in the measurement of water qualitywith particular reference to methods for estimating andcontrolling possible errors in analytical results.Ordering:Non-RSC Members should send their orders to:The Royal Society of Chemistry, Distribution Centre,Blackhorse Road, Letchworth, Herts SG6 1 HN, UK.RSC Members should send their orders to:Brief Contents:Information Requirements of Measurement Programmes;Sampling; The Nature and Importance of Errors in AnalyticalResults; Estimation and Control of the Bias of AnalyticalResults; Estimation and Control of the Precision of AnalyticalResults; Achievement of Specific Accuracy by a Group ofLaboratories; Reporting Analytical Results; The Selection ofAnalytical Methods; General Precautions in Water-AnalysisLaboratories; Analytical Techniques; Automatic and On-LineAnalysis; Computers in Water Analysis.The Royal Society of Chemistry, Membership Manager,30 Russell Square, London WC1B 5DT, UK.ROYALSOCIETY OFCHEMISTRYlnformat ionService

ISSN:0003-2654    

DOI:10.1039/AN98712BX023

出版商:RSC    

年代:1987

数据来源: RSC

摘要:

ANALYST, JUNE 1987, VOL. 112 717 SECOND INTERNATIONAL SYMPOSIUM ON KINETICS IN ANALYTICAL CHEMISTRY Preveza Beach, Greece, 9-1 2 September, 1986 For three days at the Preveza Beach Hotel, facing the Ionian Sea, close to Preveza in picturesque northwest Greece, 77 scientists from 18 different nations actively participated in the Second International Symposium on Kinetics in Analytical Chemistry. Twenty-three lectures (four of them Plenary) and 64 poster presentations brought to the participants the present spectrum of active research and some general topics relevant to the impact that kinetics have on contempo- rary analytical chemistry. Some of these contributions are compiled in this issue of The Analyst and testify that the high standards set in Cordoba (Spain) in 1983, during the first international gathering of this nature, were maintained during this second symposium.The initiative for this publication resides in the late Professor John M. Ottaway , who diligently organised contacts between the Analytical Editorial Board of the Royal Society of Chemistry and the Scientific Committee for the Symposium. These efforts received endorsement and active participation from the Editor of The Analyst, Philip C. Weston; they are gratefully acknowledged by the Scientific Committee formed by Professors M. I. Karayannis, G. Werner, M. ValcArcel, N. Evmiridis and myself. As in Cordoba, local authorities provided generous support to the meeting, which opened the night of September 9,1986, with short welcoming speeches including one from the prefect of Preveza, Mr.Thomas Merentitis. Also, generous aid was received from the University of Ioannina, which provided the bulk of the Local Organising Committee (M. I. Karayannis, P. Isopoulos, A. Michaelidis, P. Veltsistas, Chr. Nanos and C. Konidari) , The Association of Greek Chemists (represented by N. Katsaros on the Local Organising Committee), the Laboratory of Analytical Chemistry of the University of Athens (represented on the Organising Committee by P. A. Siskos and D. P. Nikolelis), the municipality and Mayor of Preveza, Mr. Nikolaos Yannoulis, the Town Councillors and the Mayor of Ioannina, Mr. Charilaos Tolis, and the Ministry of Culture and Science of Greece. The efforts of the Local Committee provided a variety of social events, enhanced by the beauty of northwest Greece and the rich historical heritage of the area, which balanced and supplemented the scientific programme during the three days of the Symposium. The four Plenary Lectures focused on catalytic reactions and selectivity. It was emphasised that kinetics in analytical chemistry should not be restricted to reaction rate determina- tions and that both physical and chemical kinetics play important roles in the different phases that constitute every analytical process.Catalytic (non-enzymatic) methods were revisited with emphasis on the elucidation of the chemical role of the species involved in the determination and on the application of chemometrics for method optimisation. Analy- tical applications of soluble and insoluble enzyme prepara- tions and catalytic titrations were also covered in Plenary Lectures.The inclusion of enzymatic methods recognised the impetus given to kinetic-based determinations by the use of enzymes as analytical reagents; the level of maturity reached by catalytic end-point indication was recognised in the Fourth Plenary Lecture. Both chemical and instrumental aspects, in a balanced manner, were covered in the remainder of the oral and poster presentations. Following a trend already noticed during the First Symposium, instrumentation for fast kinetics and fast computer data acquisition retain their place in kinetic-based analytical practice, but the impact of continuous flow sample/ reagent processing (particularly flow injection) was again distinctly felt. Kinetic measurements have been made more reliable, relatively simple and instrumentally inexpensive by the implementation df unsegmented continuous flow sample/ reagent processing.The impact of flow injection was unveiled in about half a dozen oral presentations and a dozen posters and was also reflected, in an indirect manner, in some other presentations. A combination of physical and chemical dynamics makes continuous flow systems a fertile area for the exploitation of kinetic situations. The use of such systems for the study of the rate of dissolution of solid pharmaceutical preparations illustrated the application of this process approach to studies incidental to the analytical sub-discipline. Centrifugal sampleheagent processing was also illustrated in a poster presentation.The ability to record and process a large number of data points, if not the entire signal - time response, greatly mini- mises errors. Non-linear multi-point regression data handling is an unconventional approach to kinetic measurements for analytical purposes that exemplifies the advantages gained by use of digital computer data manipulation. The relative advantages of these methods was evident during the oral presentations. New approaches were not absent from the Symposium, demonstrating that creativity is not dormant in kinetic determinations. Examples of these contributions are a two- rate measurement approach to eliminate errors due to between-run fluctuations in rate coefficients, the application of factor analysis for multi-component rate evaluation, the potential usefulness of the Kalman filter in reaction-rate methods and differential approaches to catalytic determina- tions by continuous addition of catalyst to a reference solution.Many analytical techniques, if scrutinised closely, expose a kinetic nature or the presence of key kinetic components. Chromatography is a typical example of a totally dynamic analytical approach to separation and detection. This was emphasised during the Symposium as analytical applications of reversed-flow gas chromatography were considered. The details of many of these contributions made at the Second International Symposium on Kinetics in Analytical Chemistry can be found by the interested reader in this issue. The vitality of the area was evidenced in the urging by many of the participants that this series of symposia, initiated in 1983, be continued about every 3 years. As a result, in the general discussion session that closed the meeting, it was agreed that KAC’89 will have Yugoslavia as the host. This is a recognition of the rich tradition of Yugoslavia’s analytical chemists and their wide participation in the First and Second Symposia on Kinetics in Analytical Chemistry. Horacio A. Mottola Department of Chemistry, Oklahoma State University

ISSN:0003-2654    

DOI:10.1039/AN9871200717

出版商:RSC    

年代:1987

数据来源: RSC

摘要:

ANALYST, JUNE 1987, VOL. 112 719 Enzymes as Analytical Reagents: Substrate Determinations with Soluble and with lmmobilised Enzyme Preparations” Plenary Lecture Horacio A. Mottola Department of Chemistry, Oklahoma State University, Stillwater, OK 74078-0447, USA Two basic characteristics of enzymes as analytical reagents for the determination of substrates are the focal point of this Plenary Lecture, firstly the high degree of selectivity (or even specificity) offered by enzymes and secondly the regeneration afforded by enzymes as catalysts. Various approaches t o the monitoring of enzyme-catalysed reactions (e.g., direct monitoring, coupling t o indicator reactions and amplification by cycling) are considered in detail. Keywords: Enzymes; substrate determination; immobilised enzymes; enzyme regeneration; enzyme reactors The large number of enzymatic determinations performed daily in clinical laboratories around the world is responsible for a fact that comes as a surprise at first: the number of determinations carried out using kinetic-based methods exceeds those carried out by equilibrium-based methods or even direct instrumental measurement, Enzyme-catalysed reactions are used to determine both enzyme activities and substrate concentration, parameters of importance in clinical diagnoses.The use of enzymes in the diagnosis of disease is an important benefit derived from biochemical research that has intensified since the 1940s and enzymes have provided the avenue that introduced clinical analysis as a significant branch of analytical chemistry.Such an avenue began to be paved in the 1950s with contributions such as Stetter’s description of the application of impure enzyme preparations to a variety of analytical problems’ and by predictions such as that of Lee2 that the use of enzymatic methods would increase with advances in the study of enzymes and with the commercial availability of enzymes of known activity and purity. The real impact of enzymes in chemical analysis was not felt, however, until the mid-1960s and early 1970s.3 All known enzymes are protein materials of high relative molecular mass (104-20 x 105) made up primarily of chains of amino acids linked together by peptide bonds. Many enzymes need the presence of other species, known as cofactors, to act as catalysts. The enzyme - cofactor complex is called a holoenzyme; the protein portion of this is known as an apoenzyme.Apoenzymes may be associated with different types of cofactors such as: 1, a coenzyme (non-proteinic organic species loosely attached to the apoenzyme); 2, a prosthetic group (organic species firmly attached to the apoenzyme); or 3, a metallic ion. Of all enzyme properties, it is their high degree of selectivity or even specificity that singles them out as unique analytical reagents. Some enzymes are so specific that they catalyse only one given reaction. Other enzymes exhibit catalytic activity towards reactions involving only chemical species that have a given functional group (e.g., amino, phosphate or methyl groups) and have “group specificity. ” “Linkage specificity” is exhibited by enzymes that act only on a certain type of chemical bond and “stereochemical specificity” by enzymes that will exert catalytic action on reactions of particular steric * Presented at the 2nd International Symposium on Kinetics in Analytical Chemistry, Preveza, Greece, P-12 September, 1986.or chiral isomers. The high degree of selectivity of enzymes is not enjoyed by cofactors, which may interact with different apoenzymes. A classification of enzymes with respect to the type of chemical reaction that they catalyse is given in Table 1.4 Kinetics of Enzyme-catalysed Reactions The general (simplified) mechanism for a simple enzyme- catalysed reaction can be written as E+S,--[ES]sl’+E kl . . . . (1) k-1 k-2 with E = enzyme, S = substrate, [ES] = addition complex and P = products.Mechanistically, the scheme represented by equation (1) has been treated both as a pre-equilibriums and as a steady-state case.6 These kinetic treatments lead in essence to the following expression: where KM = Michaelis - Menten constant. The relationship given by equation (2) is fundamental in guiding determinations of enzyme concentration (activity) and substrate concentration. It indicates that, experimentally, the initial rate is directly proportional to the enzyme concentration when [S] >> K M , as in this instance the following applies for enzyme determination: (IR) = (1R)Inax. = k2[EIo * * . (3) where IR = initial rate. When [S] << KM, the initial rate is directly proportional to the substrate concentration and provides the basis for its determination: (IR) = - [sl0 = (a.[slo = constant. [sl0 (4) because substrate determinations are performed at constant enzyme concentration. Experimentally, at least in most instances, the initial rate, IR, of substrate transformation is found to be directly proportional to the enzyme concentra- tion; it follows “saturation kinetics” with respect to the substrate concentration (Fig. 1). Some considerations regarding the Michaelis - Menten con- stant are pertinent here. K M is equal to [S] when the initial rate is equal to half the maximum (saturation) rate; and since k2PIo KM K M K M = ( k - i + k2)/kl . . . . . . ( 5 ) under initial rate conditions, if k-,>> k2, KM = k-l/kl and the720 ANALYST, JUNE 1987, VOL. 112 Table 1.Classification of enzymes according to the type of chemical reaction that is catalysed Chemical change Enzymes Addition or removal of water . . (a) Hydrolases (e.g., esterases, carbohydrases, nucleases, deamineases, (b) Hydrases (e.g., fumarase, enolase, carbonic anhydrase) amidases, proteases) Electron exchange . . . . . . (a) Oxidases (e.g., glucose oxidase, galactose oxidase, uricase, amino acid oxidases) dehydrogenase, lactate dehydrogenase) Radical transfer . . . . . . . . (a) Transglycosidases (of monosaccharides) (b) Dehydrogenases (e.g., alcohol dehydrogenase, hydroxysteroid (b) Transphosphorylases and phosphomutases (of a phosphate group) (c) Transaminases (of amino groups) (d) Transmethylases (of a methyl group) (e) Transacetylases (of an acetyl group) Desmolases Breaking or forming of a C-C bond Concentration of substrate Fig.1. Reaction rate versus substrate concentration for an enzyme- catalysed reaction obeying saturation kinetics. The saturation point, characterised by (IR)max,, can be interpreted as all active sites occupied by the substrate at ever time t and substrate concentrations larger than the minimum needeifor realisation of (IR)max, Michaelis - Menten constant becomes the dissociation constant for the enzyme - substrate complex, ES. If, on the other hand, k2 >> k - l , KM = k2/kl and should be considered as a ratio of kinetic coefficients. A small value of KM (indicating that the enzyme requires a small amount of substrate to become saturated) results in a small linear concentration range in calibration graphs based on IR vs.substrate concentration graphs. Recently, however, Hamilton and Pardue7 described a data-processing method for the kinetic determination of substrates in enzyme-catalysed reactions that yields linear calibration graphs for up to 3.5 times the substrate concentration corresponding to the Michaelis - Menten constant value. In their method, data for absorbance and the rate of absorbance change with time are plotted vs. time from data collected up to about 70% of completion of the reaction and they are fitted to the rate expression [equation (2)] written as where AAf is the difference between absorbance at time t and time zero, AAm the absorbance change between t = 0 and t = 00, E the molar absorptivity of the monitored species and b the cell path length.The fitting parameters are (Rate),,,., the Michaelis - Menten constant and the total absorbance change, AA,. Data for absorbance vs. time collected over a portion of the course of the reaction are used to compute AAm. Enzyme Activity and Enzyme Concentration The catalytic activity of an enzyme is proportional to the number of operating active sites at the time of its measure- ment and is expressed in enzyme “activity units.” Although the catalytic activity is related to the analytical concentration of protein it may be different for different preparations of the same enzyme and consequently the analytical concentration is useless in describing the enzyme activity. Also, the activity of a given enzyme preparation may change at different rates with time as a result of processes such as denaturation (disruption of the tertiary structure of the protein).Several enzyme units have been used in the literature (e.g., Karmen, Buchner, Bodansky and Wroblewski units) as defined by various investigators. This has resulted in difficult- ies in the comparison of results and the Fifth International Congress on Biochemistry adopted the recommendations of the International Union of Biochemistry Commission on Enzymes.8 These recommendations include the definition of activity for international adoption: one unit (U) of an enzyme is that amount that will catalyse the transformation of one micromole of substrate per minute, or, where more than one bond of each substrate molecule is attacked, one micro- equivalent of the group concerned per minute.In reporting activity units, the experimental conditions for their measure- ment should be defined. Temperature should be stated and when possible it should be 30 “C. Other conditions (e.g., pH and substrate concentration) should be at an optimum. Initial rate measurements are recommended for the determi- nation of enzyme activity and substrate concentration should be sufficient for saturation. If concentrations below saturation must be used, the Michaelis-Menten constant should be known so that the observed rate can be converted to the value that would be observed under saturation conditions. Other recommended expressions are: 1, “specific activity,” expressed as units of enzyme per milligram of protein; 2, “molecular activity ,” defined as units per micromole of enzyme at the optimum substrate concentration (number of molecules of substrate transformed per minute per molecule of enzyme); 3, “catalytic centre activity,” defined as the number of molecules of substrate transformed per minute per catalytic centre (for enzymes that have a prosthetic group or catalytic centre whose concentration can be measured); and 4, “concentration,” expressed as units per millilitre.Determination of Substrates by Means of Enzyme-catalysed Reactions These determinations are of relevance not only to clinical chemistry but also, in many instances, to industrial (especially pharmaceutical) and environmental analytical chemistry.721 ANALYST, JUNE 1987, VOL. 112 Table 2. Typical determinations of substrates using enzyme-catalysed reactions Substrate Comments I.Clinical applications: Galactose . . Glucose . . . . Blood alcohol . . Enzyme, galactose oxidase (E.C. 1.1.3.9); reaction, D-galactose + O2 + D-galacto-hexodialdose + H202; use, diagnosis of galactosaemia Enzyme, glucose oxidase (E.C. 1.1.3.4); reaction, glucose + O2 + H20 + gluconic acid + H202; use, diagnosis of diabetes Enzyme, alcohol dehydrogenase (E.C. 1.1.1.1); reaction, ethanol + NAD+ acetaldehyde + NAD; uses, law enforcement laboratories, treatment of alcoholism allantoin + C02 + H,O,; uses, diagnosis of gout, chronic leukaemia, lymphoma, polycythaemia and myeloid metaplasia Uric acid . . . . Enzyme, uricase (E.C. 1.7.3.3); reaction, uric acid +02 + H20+ 11. Indwtrial applications: Penicillins Ethanol . .Maltose . . Sucrose . . Lactose . . Catechol . . . . Enzyme, penicillinase (E.C. 3.5.2.6); reaction, penicillins + H20 ---f penicilloic acid; uses, determination in pharmaceutical preparations and fermentation broths . . Enzyme, yeast alcohol dehydrogenase (E.C. 1.1.1.1); reaction, ethanol + NAD ---f acetaldehyde + NADH; use, determination in alcoholic beverages Enzyme, maltase [a-glucosidase (E.C. 3.2.1.20)]; reaction, maltose + H20 + 2~-glucose (glucose determined by use of glucose oxidase); use, determination in food products (e.g., corn and malt syrups) Enzyme, invertase (P-fructosidase (E.C. 3.2.1.26); reaction, sucrose + H20 + D-glucose + fructose (glucose determined by use of glucose oxidase); use, determination in food products (e.g., fruit juices, ice cream, condensed milk, vegetables and syrups) Enzyme, lactase [P-galactosidase (E.C. 3.2.1.23)]; reaction, lactose + H20 + D-glucose + galactose (glucose determined by use of glucose oxidase); use, determination in food products (e.g., milk, ice cream, yogurt and buttermilk) .. Enzyme, catechol1,Zoxygenase (E.C. 1.13.1.1); reaction, catechol + 02+ dicarboxylic acid resulting from ring cleavage between the two hydroxy groups monitoring O2 levels; uses, determination in dyes, drugs, rubbers, antioxidants for lubricating oils, chemicals used in photography and cigarette smoke . . . . . . 111. Environmental application: Phenol . . . . Enzyme, tyrosinase [polyphenoloxidase (E.C. 1.14.18.1)]; reaction, phenol + O2 + o-benzoquinone with monitoring of O2 levels; uses, determination in water and wastewater Max = 1.2287 A 0) C a -P 2 2 2 50 300 350 400 Wavelengthlnm Fig.2. Difference in absorbance at 340 nm between nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH). Concen- trations: NAD, 1.25 x 10-4 M; and NADH, 2.5 x M Table 2 provides some examples illustrating these types of applications. The EC number follows each enzyme according to the classification suggsted by the International Union of Biochemistry.9 A wealth of information pertinent to enzymes as analytical reagents can be found in the literature provided by commer- cial sources of enzymes. Reference 10, for instance, provides a comprehensive listing about the reaction being catalysed, characteristics of the enzyme ( e . g . , relative molecular mass, chemical composition, molar absorptivity, activators, inhibi- tors, selectivity, stability and optimum pH), practical direc- tions for the determination of the enzyme and pertinent references for slightly over 100 enzymes.Also included are properties of several related biochemicals of use in determina- tions involving enzymes. Approaches to the Monitoring of Enzyme-catalysed Reactions The approach to be used in monitoring the reaction rate is dictated primarily by the chemistry of the enzyme-catalysed reaction. There are, however, some common chemical charac- teristics in many enzyme-catalysed reactions and a few common ways of monitoring are worth mentioning. In spectrophotometric monitoring, one of the reaction products sometimes shows an absorption spectrum different from the substrate and other reactants or products involved in the reaction.This permits the monitoring of the course of the reaction in a simple manner. Nicotinamide dinucleotide (NAD) , a coenzyme for many dehydrogenase-catalysed reac- tions, does not absorb photons in the 340-nm region of the spectrum, whereas its reduced form does (Fig. 2). This difference is widely used to follow the progress of reactions involving dehydrogenases by direct monitoring at 340 nm. A typical example is the determination of lactic acid by means of the following reaction: CH~CHOHCOOH + NAD lactic acid CH3COCOOH + NADH pyruvic acid . . . . (7)722 ANALYST, JUNE 1987, VOL. 112 The structure of NAD is 0 \\ .CH2 ‘0’ 0 - / 0 p\ OH OH and it acts by accepting hydrogen (reversibly) at the 4-position of the nicotinamide ring: a-Ketoglutarate + NH: Glutamate R I R The coupling of the main enzyme-catalysed reaction with a second (indicator) reaction is a common practice in enzymatic methods.Perhaps the most popular of these couplings is the use of an organic compound that will react with a product (or the substrate); the appearance or disappearance (or other change) of colour is monitored spectrophotometrically . As reactions involving oxidases yield as one of the products H202, the oxidising power of this species is exploited in the coupled scheme. This is aided by the action of a second enzyme (peroxidase) on the indicator reaction; a typical example is Glucose + 0 2 + H20 glucose oxidase + gluconic acid peroxidase H20 + reduced form of a dye ----+ H 2 0 + oxidised form of the dye A typical example of a chemical species used in the coupled reaction is o-dianisidine (3,3’-dimethoxybenzidine) , which is colourless in solution, whereas its oxidised form absorbs photons at 460 nm.Other species used are o-tolidine and leuco bases of triphenylmethane dyes (e.g., leuco crystal violet). The oxygen consumption and H202 formation in oxidase- catalysed reactions make them amenable to the amperometric detection of either oxygen consumption or H202 formation. The use of amperometric detection of oxidase-mediated reactions, with emphasis on applications using continuous- flow sample processing, has been reviewed by Gulberg et al. l1 and Mottola et ~ 1 . 1 2 Species such as glucose, D- and L-amino acids, uric acid, amylase, maltose, sucrose, lactose, NADH and serum lactate dehydrogenase have been determined by the amperometric monitoring of changes in dissolved oxygen levels due to oxidase-catalysed reactions. Several commonly used enzyme-catalysed reactions involve proton exchange with the background electrolyte and pH sensing is the simplest monitoring approach as it does not require additional coupling to an indicator reaction. Hydrogen ion sensing does, however, entail some incon- veniences as the pH change, if large enough, may inhibit the enzyme activity.Buffering species present in the medium may also affect the measurement. Keeping the pH change small, however, allows the reliable monitoring of the reaction rate. Alternatively, a pH-stat approach may be adopted to circum- vent the problem.An example of its use is in the hydrolysis of acetylcholine in presence of cholinesterase, which results in NADPH NADP+ 4 1 GGPDH x 6-P-Gluconate Glucose-6-phosphate Fig. 3. Amplification in NADP determination by means of enzy- matic cycling (for explanation see text) the formation of acetic acid. Acetylcholinesterase activity is very sensitive to pH and the medium must be held very close to pH 7.4 for the reaction to proceed conveniently.13 A careful design of buffer system and buffer capacity allows, in certain instances, a direct monitoring of ~H.14~15 Enzyme-catalysed reactions leading to the release of ammonia, which in aqueous solution generates ammonium ions, are amenable to direct monitoring by ammonium ion-selective electrodes.Guilbault et aZ.,16 for instance, have described the determination of a series of substrates (urea, glutamine, asparagine, amino acids and glutamic acid) by monitoring the reaction with a cationic electrode responding to the ammonium ion in enzyme-cataly- sed reactions. Amplification by Cycling of Enzyme-catalysed Reactions An interesting exploitation of enzyme catalysis is an amplifica- tion approach centred on the cycling of two reversibly interrelated chemical species (e.g., the oxidised and reduced form of a given chemical structure) sequentially acting on two enzyme-catalysed reactions. The cycling results in an accumu- lation of product produced and, if side reactions do not interfere with the cycling process, the resulting amplification is large.Cofactors such as NAD - NADH or NADP - NADPH (NADP = nicotinamide adenine dinucleotide phosphate) are ideally suited to this approach. An example, illustrated in Fig. 3, is the cycling of NADP - NADPH between two enzyme- catalysed reactions resulting in the accumulation of 6-phosp- hogluconate, which is determined by a third enzyme-catalysed reaction.17 The sample containing NADP is added to a mixture containing three chemical species, ketoglutarate, ammonium ion and glucose-6-phosphate, in non-limiting concentrations, and controlled amounts of the enzymes glutamate dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G6PDH). Both 6-phosphogluconate and glutamate build up at a rate related to the original concentra- tion of NADP in the sample.Once cycling has resulted in a measurable concentration of 6-phosphogluconate, the reac- tions are all stopped by destroying the enzymes by heatingANALYST, JUNE 1987, VOL. 112 723 (denaturing). The accumulated 6-phosphogluconate is then determined by the addition of a known amount of NADP and 6-phosphogluconate dehydrogenase: 6PGDH 6-P-gluconate + NADP - NADPH + ribulose-5-P + C02 + H+ (9) If the concentrations of the enzymes are high enough, cycling rates of 20 X l o 3 per hour can be accomplished. If the NADPH formed in a first step is entered in a second cycling stage, another 20 x l o 3 cycles per hour can be implemented; this would result in an amplification factor of 108, allowing the detection of as little as 10-19 mol of NADP. A short but thought-provoking account of the potential of amplification by cycling enzyme-catalysed reactions has been given by Lowry.18 Catalysis in general is an amplification approach as sub-stoicheiometric amounts of catalysts yield a product accumulation by the action of the catalytic cycle; this and other aspects of chemical amplification have been discussed by Blaedel and Bogulaski.19 Enzyme Regeneration in Homogeneous Systems When enzymes are used as analytical reagents for the determination of substrates, their catalytic nature suggests that the repeated use of the same enzyme preparation should be possible.This results in a better utilisation of relatively expensive reagents. Reagent recirculation in closed flow- through systems20 permits the recycling of soluble enzyme preparations.Moreover, and in principle, the use of enzymes introduces sufficient selectivity (specificity) that inherently non-specific methods of detection (e.g., glass pH-sensing electrodes, conductimetric cells and thermistor-based detec- tors) can be used. Several substrate determinations, for example, are based on the general scheme Substrate + 02% Product(s) + H202 . . (10) in which E is an appropriate enzyme. The determination of substrates such as glucose, uric acid, galactose and D- and L-amino acids are among those based on the chemistry of equation (10). An example of recycling of the enzyme solution, sample injection into a closed flow-through system and amperometric monitoring of the change in dissolved oxygen levels as a result of the over-all reaction of equation (10) has been offered for glucose determination.20 As already indicated, the advantage of enzyme recirculation is obvious; sample injection with flow-through systems allows the pro- cessing of a large number of samples per unit time, a feature of interest in clinical laboratories with heavy sample-handling demands.The over-all approach is an example of the analytical use of transient signal measurement under dynamic conditions in a system approaching equilibrium; therefore it qualifies as a kinetic method of determination. The hydrogen peroxide released in the reaction of equation (10) would interfere with the electrochemical determination of dissolved oxygen. This problem can be circumvented by using catalase- impurified glucose oxidase, which is inexpensive and ensures the instantaneous destruction (for all practical purposes) of the hydrogen peroxide: catalase 2H20 + 0 2 A diagram of the closed flow-through system is shown in Fig. 4.Because the amperometric response of the platinum electrode to oxygen is very sensitive to changes in flow-rate,z1 Air ample i l l el Potentiostat Fig. 4. Schematic diagram of closed flow-throu h system for the determination of glucose. WE, Working electrofe (Pt wire); CE, counter electrode [Pt/NaCl, 7.0 g 1-11; RE, reference electrode SCE). Arrows indicate the direction of flow. For details see ref. 20. Copyright American Chemical Society, 1978; reproduced with permission) Fig. 5. Oscilloscopic traces of ty ical transient signals obtained by repetitive injection of three lO-pf aliquots of a D-glucose solution containing 10 g 1-1 of sugar.Y axis: current, 1 division = 2 pA. Xaxis: time, 1 division = 2 s. From ref. 20. (Copyright American Chemical Society, 1978; reproduced with permission) the flow system is divided into two hydrodynamically indepen- dent sections. As a result of difference in elevation between points A and B, the solution flows by gravity (at a constant rate) from A to B. A peristaltic pump takes the solution back from B to the reservoir. The flow between B and C (0.5-0.6 ml s-1) is slightly over twice the flow-rate between A and B (0.20-0.25 ml s-1) so as to aspirate the air and bubble it through the reservoir solution to keep a constant oxygen level responsible for the base-line signal. The reservoir solution, located between branches AB and BC, acts as a pool, the large volume of which dissipates electrostatic charges generated by the friction of the pump rollers on the plastic tubing transporting the solution. Fig.5 shows typical traces obtained with the set-up of Fig. 4. The base line corresponds to oxygen saturation; after injection, the glucose sample travels through the coil and reacts with the dissolved oxygen in the circulating reagent solution, producing a segment or “plug” in which the oxygen level is lower than outside the plug. Because of the predominant laminar nature of the flow, this plug retains its boundaries (little dispersion) and reaches the working elec- trode that senses the oxygen level in the flowing solution. The restoration to the base line is the result of the imposed flow and the bubbling of air into the reservoir vessel.The small positive signal preceding the actual peak profile (Fig. 5 ) is the result of an artifact due to the sudden change of the flow produced by the injection. The height of the negative peak724 provides the analytical information for substrate determina- tion. As many as 700 measurements h-1 (with some precau- tions even about 1700 measurements h-1) can be performed and more than l o 4 serum samples (10 pl each) can be processed with the same reservoir solution (200 ml initial volume). The recycling of both enzyme and coenzyme was demon- strated in the determination of glucose using glucose dehy- drogenase.22 Dehydrogenases need nicotinamide adenine dinucleotide (NAD) or its reduced form (NADH) as a coenzyme.The recirculation of glucose dehydrogenase, NAD and glutamate dehydrogenase (for the slow oxidation of NADH back to NAD) and photometric monitoring of NADH (340 nm) permitted the selective determination of glucose in human blood serum at a rate of 120 samples per hour. As many as 300 determinations are possible with the same 40-ml (initial volume) circulating solution and injected sample volumes of 23 p1. Enzyme Regeneration in Heterogeneous Systems: Use of Immobilised Enzyme Preparations Glucose oxidase is a relatively inexpensive enzyme that also offers relatively reasonable retention of activity with time in solution. It takes about a month at room temperature for the average activity of glucose oxidase solution to decrease to 20% of its original value.Storage at +4 “C reduces the same value to about 30%. Some important clinical determinations make use of enzymes that are not sufficiently stable and/or inexpensive to be used in solution for relatively long periods of time and at high activity levels. For such enzymes immobilisa- tion offers a competitive alternative way to use these enzymes as analytical reagents in batch and continuous-flow systems. Immobilisation refers to the localisation or confinement of an enzyme molecule in such a manner that it remains physically separated from the substrate and the products of the enzyme- catalysed reaction. An ever-increasing trend in studies using immobilised enzyme preparations, particularly in analytical chemistry, has been documented in the past two decades.This surge in interest results from the advantages that immobilised enzyme preparations offer: 1, easy separation and recovery from reactants or products of the enzyme-catalysed reaction (which obviously leads to the possibility of enzyme re-use); and 2, easy adaptation of immobilised enzyme reactors for the continuous monitoring of substrate levels by continuous-flow analysis. Enzyme immobilisation can generally be achieved by chemically or physically attaching the enzyme to a support or by confining the enzyme to a restricted volume by means of a semipermeable membrane. Containment by a membrane or entrapment in a polymeric gel are applied in the construction of so-called “enzyme electrodes”; physical adsorption of enzymes has found little use in chemical analysis, but covalent binding is extensively used in the preparation of enzyme rectors for use in continuous-flow.systems.23 ANALYST, JUNE 1987, VOL.112 It appears that the first report of enzyme immobilisation was by Nelson and Griffi11.2~ They observed that invertase (extracted from yeast) was adsorbed on charcoal and that the adsorbed enzyme preparation showed catalytic action similar to that of the native enzyme. The first successful covalent binding of a variety of proteins (including enzymes) was carried out after World War 11. Michael and Ever95 described such linkage of proteins to carboxymethylated oxidised cellulose in 1949. Enzyme immobilisation did not become popular, however, until the 1950s when Grubenhofer and Schleith26J7 reported coupling carboxypeptidase and amylase to diazotised polyb-aminostyrene).The theoretical and practical aspects of immobilised enzymes have been covered in several reviews such as Konecny28 and monographs centred on analytical applications are also available . 2 9 3 In chemical immobilisation, covalent bond formation by the reaction of some functional groups of the support material and the enzyme (or two or more enzymes) is involved. The covalent attachment must be, of course, via amino acid residues not essential to the catalytic function of the enzyme and usually depends on the amino rather than the carboxyl groups of such residues. A usual feature of chemical immobil- isation is the irreversibility of the reaction. The original enzyme molecules cannot be separated from the matrix and used as free enzymes again.There are, however, some instances in which the enzyme can be liberated from the immobilising support. The physical immobilisation of enzymes depends on their entrapment within microcompartments, containment within special membranes or physical interactions such as those due to ionic forces (electrostatic immobilisation), hydrogen bonds or enzyme - enzyme interactions. In theory, physical immobi- lisation should be completely reversible, but there are exceptions. As heterogeneous catalysts, immobilised enzymes differ in many respects (e.g., optimum pH, Michaelis - Menten con- stant and stability) from their soluble counterparts. An exception to this is, however, immobilisation by the contain- ment of the enzyme solutions by membranes. This mild and widely applicable method of immobilisation has received little analytical attention, probably because no enhancement of stability can be achieved by this method, a drawback for the analytical utilisation of some expensive enzymes.Depending on the enzyme, immobilisation at surfaces may result in a remarkable increase of activity retention with time. The two main areas of analytical application of immobilised enzyme preparations are “enzyme electrodes” and “enzyme reactors” for use in continuous-flow systems. Most applications of so-called “enzymic electrodes” involve the physical entrapment of the enzyme and measurements at steady-state conditions by the use of an electrochemical sensor (amperometric or potentiometric) in contact with a layer of entrapped enzyme preparation.As they do not monitor ~~ ~ Table 3. Comparative data of analytical interest extracted from calibration graphs of the determination of urea with nylon-immobilised urease14 Equilibrium determination* Kinetic determination? Linear concentration rangeh . . . . . . . . 1 .O x 10-6-5.0 x 1.0 x 10-6-1.0 x lo-’ Correlation coefficient of calibration graph . . 0.999 0.998 Slope of calibration graph (method sensitivity) . . 0.433 pH unit decade-’ of urea concentration$ concentration min-1$ 0.375 pH unit decade of area concentration§ 0.395 pH unit decade-1 of urea Absolute standard deviation (at least three * Measurement of equilibrium pH after completion of reaction. t Initial rate in pH min-l extracted from the first 10% of reaction.$ Standards in Tris buffer. 0 Standards in physiological buffer. Smaller slope may be due to a higher ionic strength. replicates per concentration value) . . . . 0.010-0.050 pH 0.010-0.060 pH min-lANALYST, JUNE 1987, VOL. 112 725 enzyme activity (concentration) and the electrode per se is not made of enzyme, the name “enzyme electrode” for substrate determination is open to criticism. The author prefers the name of “enzyme reactor,” although he recognises the popular use of the “electrode” denomination. In reporting the performance of an enzyme reactor constructed around a flat-surface pH electrode and a chamber filled with urease covalently immobilised on nylon shavings, the analytical performance by initial rate measurements was seen to be competitive with equilibrium (steady state) measurements.14 The comparison is shown in Table 3.Covalent bonding on the surface of an inert matrix (e.g., glass or nylon) offers the best immobilisation approach for preparations to be used in continuous flow? This type of immobilisation takes advantage of the exposed groups on the enzyme surface. The most commonly used reactions are those involving primary amino groups or the phenolic hydroxyl of tyrosine. The choice of method depends on the stability of the enzyme at the pH at which coupling is performed, the stability of the linkage at the pH at which the immobilised enzyme preparation will be used and also on the carrier. Background information on the reactions involved can be found in monographs by Means and Feeney31 and by Zab0rsky.~2 For analytical applications, one of the most widely used methods involves alkylamino glass and glutaraldehyde. Although the detailed chemistry of this reaction is not well understood, the individual steps include the reaction of the silica framework with an aminosilane, modification of the product of this reaction with glutaraldehyde and finally immobilisation of the enzyme.These steps can be symbolised as follows: I I I - 0- Si - OH + (C2H50)3Si(CH2)3NH2 0 -0-S- OH + ( C ~ H S O ) ~ S ~ ( C H ~ ) ~ N H ~ NH2 + OHC - (CH213-CHO column enzyme reactors and amperometric detection of the hydrogen peroxide produced in the following reactions: invertase Sucrose + H20 ---+ D-glucose + fructose glucose oxidase D-Glucose + O2 + H 2 0 + D-gluconic acid + H202 The use of open tubes with the enzyme immobilised at the inner wall seems to have originated in the work of Hornby and co-worker~35~36 with polystyrene and nylon tubes.These tubes contained a monomolecular enzyme layer covalently bound to the etched inner wall, with rather low local activity. Horvath and co-workers37J8 developed an alternative providing a thick porous enzymatic annulus in a tubular envelope. The enzyme was bound to a polycarboxylic gel layer attached to the inner wall of narrow-bore nylon tubing. Horvath and Peder~en3~ have reviewed the fundamental aspects of this type of reactor and presented a mathematical model describing coil perfor- mance in air-segmented continuous-flow systems. By combining enzyme immobilisation with the technology developed in capillary gas chromatography, Iob and Mottolam and Kojima et aZ.,41 independently, introduced glass open tubular reactors with the enzyme chemically bonded to the inner wall of narrow-bore glass tubing.In order to increase the surface area available for immobilisation, silica “whiskers” (filaments) were grown by the adaptation of an ammonium hydrogen fluoride treatment. Typical applications of this type of reactor are the determination of uric acid by means of immobilised uricase and amperometric monitoring of dis- solved oxygen levels“ and L-lactate with immobilised lactate I I I I I I - 0 -Si - 0 - Si(CH2I3NH2 - 0 0 - 0 -Si- 0- Si(CH2I3NH2 ---+ t- N = CH - (CH,), - CHO F C H O + H2N-E - - - + k C H = N - E Immobilisation on glass via glutaraldehyde attachment is attractive owing to 1, its simplicity and mild operating conditions, 2, its success with a variety of enzymes and 3, the good mechanical and chemical stability of glass.Several types of reactor configurations can be adapted to continuous-flow operations; the most common are packed columns, open-tube wall reactors and single-bead-string reactors. Packed-column reactors are the most commonly reported in the literature, with enzyme preparations made using the glutaraldehyde bridging linkage and controlled-pore glass as the inert matrix. Different analytical aspects of packed- column immobilised-enzyme reactors with the emphasis on thermochemical detection have been discussed by Schifreen et al. 33 Their observations indicate that the rate of product formation is controlled by the mass transfer of the substrate to the surface of the carrier or to diffusion within the porous glass rather than by the kinetics of the enzyme-catalysed reaction.They also indicate that to a first approximation a packed-bed reactor can be modeled in terms of “reaction plates” instead of “separation plates” as with chromatographic columns. The same mathematics that apply to chromatographic columns can be used for packed-column enzyme reactors. A typical example of packed-column enzyme reactors in continuous-flow systems is shown in Fig. 6 adapted from Masoom and Townshend.34 It illustrates manifold configura- tions for the simultaneous and sequential determination of two species (sucrose and glucose) by the use of two packed- dehydrogenase and photometric monitoring of NADH.42 Although whisker growth increases the surface area by about three orders of magnitude, the process is elaborate, involves the use of corrosive chemicals and frequently results in non-uniform surface coverage because of a strict requirement of temperature control. In a quest for a simpler mode of increasing the surface area of open tubular reactors that would also result in more uniform surface coverage, Gosnell et aZ.43 prepared open tubular reactors by thermally embedding controlled-pore glass chips on the walls of plastic tubing.The higher local activity of these reactors when compared with “whisker”-modified reactors is of analytical interest; their simplicity and fast preparation is of additional appeal.Fig. 7 illustrates the topography of this type of reactor. Penicillinase [E.C. 3.5.2.61-coated glass beads formed by glutaraldehyde attachment after ammonium hydrogen flu- oride etching were made part of a single-bead-string reactor used for the determination of penicillins in pharmaceutical tablets, injectable solutions and fermentation broths.15 A coupling of a controlled-pore glass embedded on plastic tubing-etched glass beads single-bead-string reactors may well be the optimum configuration for immobilised-enzyme reac- tors to use in continuous-flow sample processing. Batch determinations using immobilised enzymes do not seem to have been reported outside of the use of “enzyme electrodes.” Closer to this type of application seems to be the innovative use of a tubular reactor coupled to the observation726 Phosphate buffer -- pH 6.8 Waste - buffer r Fig.6. Flow systems arrangements for the (a) simultaneous and ( b ) sequential determination of sucrose and glucose. Column A contained invertase and mutarotase immobilised on controlled-pore glass and column B glucose oxidase immobilised on the same support. Column dimensions: A, 25 x 25 mm, B, 50 x 2.5 mm (adapted from ref. 34 with permission) Waste Fig. 7. Typical scanning electron micrograph of the interior wall of an open tubular Tygon reactor containing embedded controlled-pore glass. 35 x magnification cell in a stopped-flow system.# This configuration, making use of enzymes immobilised on the walls of nylon tubing, has been used to determine glucose and lactate.45 In this type of reactor arrangement, the enzyme-catalysed reaction occurs under static conditions and the kinetics are controlled by diffusion and by the reaction rate of the reaction in question.Amplification has also been carried out with enzyme- immobilised reactors. An enzyme electrode with a chemically amplified response for L-lactate has been used by Mizutani et ~ 1 . ~ The electrode system consisted of an “oxygen electrode” (Pt cathode and a gas permeable membrane) and an immobi- lised enzyme layer in contact with the test solution and the gas permeable membrane. The immobilised enzyme layer con- tained lactate oxidase (to oxidise L-lactate) and lactate dehydrogenase (to regenerate the L-lactate); the regeneration process permits oxygen consumption beyond the stoicheio- metric yield and results in an electrode response amplified 2-250 times, depending on the characteristics of the immobi- lised enzyme layer (e.g., layer thickness, value of the apparent KM, etc.) A detection limit of 5 x 10-9 M is reported for lactate by this system.As a result of time limitations, this paper is focused on substrate determinations. Analytical applications of enzymes extend, however, to the determination of enzyme activators and enzyme inhibitors although these applications are con- siderably more limited and considerably less routinely used than those of substrates. Research support from the National Science Foundation has directed the author’s attention to the use of enzymes as analytical reagents and such support is gratefully acknow- ledged.The University of Ioannina (Ioannina, Greece), the Greek Chemist Association and the Ministry of Culture and Science of Greece are also acknowledged for making possible the Second International Symposium on Kinetics in Analytical Chemistry and this Plenary Lecture. References 1. Stetter, H., “Enzymatische Analyse,” Verlag Chemie, Wein- heim, 1951. 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. 37. 38. 39. ANALYST, JUNE 1987, VOL. 112 Lee, T. S., in Mitchell, J., Jr., Kolthoff, I. M., Proskauer, E. S . , and Weisseberg, A., Editors, “Organic Analysis,” Volume 2, Interscience, New York, 1954, p. 242. Guilbault, G. G., “Enzymatic Methods of Analysis,” Per- gamon Press, Oxford, 1970.Harrow, B., and Mazur, A., “Textbook of Biochemistry,” Saunders, Philadelphia, PA, 1958, p. 109. Michaelis, L., and Menten, M. L., Biochem. Z . , 1913,49,333. Briggs, G. E., and Haldane, J. B. S., Biochem. J., 1925, 19, 338. Hamilton, S. D., and Pardue, H. L., Clin. Chem. (Winston- Salem, N. C . ) , 1982, 28,2359. International Union of Biochemistry, “Report of the Com- mission on Enzymes,” International Union of Biochemistry Symposium Series, Pergamon Press, Oxford, 1961, Volume 20, Chapter 9, p. 45. IUPAC and the International Union of Biochemistry, “Enzyme Nomenclature, Recommendations (1972),” Elsevier, Amsterdam, 1973, Chapter 4. Decker, L. A., Editor, “Worthington Enzyme Manual,’’ Worthington Biochemical Corporation, Freehold, NJ, 1977.Gulberg, E. L., Attiyat, A. S., and Christian, G. D., J. Autom. Chem., 1980, 2, 189. Mottola, H. A., Wollf, Ch.-M., Iob, A., and Gnanasekaran, R., in Pungor, E. and Buzds, I., Editors, “Modern Trends in Analytical Chemistry,” Analytical Chemistry Symposia Series, Volume 18, Elsevier, Amsterdam, 1984, p. 49. Jensen-Holm, J., Lausen, H. H., Milthers, K., and Moller, K. O., Acta Pharmacol. Toxicol., 1959, 15,384. Begum, K. D., and Mottola, H. A. ,Anal. Biochem., 1984,142, 1. Gnanasekaran, R., and Mottola, H. A., Anal. Chem., 1985, 57, 1005. Guilbault, G. G., Smith, R., and Montalvo, J., Anal. Chem., 1969, 41, 600. Lowry, 0. H., Passonneau, J . V., Schulz, D. W., and Rock, M. K., J. Biol. Chem., 1961, 236, 2746 and 2756. Lowry, 0. H., Acc. Chem. Res., 1973,6,289. Blaedel, W. J., and Bogulaski, R. C., Anal. Chem., 1978, 50, 1026. Wolff, Ch.-M., and Mottola, H. A,, Anal. Chem., 1978,50,94. Wolff, Ch.-M., and Mottola, H. A., Anal. Chem., 1977, 49, 2118. Roehrig, P., Wolff, Ch.-M., and Schwing, J. P., Anal. Chim. Acta, 1983, 153, 181. Mottola, H. A,, Anal. Chim. Acta, 1983, 145, 27. Nelson, J. M., and Griffin, E. G., J . Am. Chem. SOC., 1916,38, 1109. Michael, F., and Evers, J., Makromol. Chem., 1949,3, 200. Grubhofer, N., and Schleith, L., Naturwissenschaften, 1954, 40, 508. Grubhofer, N., and Schleith, L., 2. Physiol. Chem., 1954,297, 108. Konecny, J., in “Survey of Progress in Chemistry,” Academic Press, New York, 1977, Volume 8, pp. 195-251. Carr, P. W., and Bowers, L. D., “Immobilized Enzymes in Analytical and Clinical Chemistry,” Wiley, New York, 1980. Guilbault, G. G., Editor, “Analytical Uses of Immobilized Enzymes,” Modern Monographs in Analytical Chemistry, Volume 2, Marcel Dekker, New York, 1984. Means, G. E., and Feeney, R. E., “Chemical Modification of Proteins,” Holden Day, San Francisco, CA, 1971. Zaborsky, 0. R., “Immobilized Enzymes,” CRC Press, Cleveland, OH, 1973. Schifreen, R. S . , Hanna, D. A., Bowers, L. D., and Carr, P. W., Anal. Chem., 1977,49, 1929. Masoom, M., and Townshend, A., Anal. Chim. Acta, 1985, 171, 185. Hornby, W. E., Inman, D. J., and Mcdonald, A., FEBS Lett., 1970, 9, 8. Inman, D. J., and Hornby, W. E., Biochem. J . . 1972,129,225. Horvath, C., and Solomon, B., Biotechnol. Bioeng., 1972, 14, 885. Horvath, C., Sardi, A., and Woods, J. S., J . Appl. Physiol., 1973, 34, 181. Horvath, C., and Pedersen, H., in “Advances in Automated Analysis, Proceedings of the 7th Technicon International Congress, December 1976, New York City,” Technicon Instrument Corporation, Tarrytown, NY, 1977.ANALYST, JUNE 1987, VOL. 112 40. Iob, A., and Mottola, H. A., Clin. Chem. (Winston-Salem, N.C.), 1981,27, 195. 41. Kojima, T., Hara, Y., and Morishita, F., Bunseki Kagaku, 1983,32, E101. 42. Morishita, F., Hara, Y., and Kojima, T., Bunseki Kagaku, 1984,33,642. 43. Gosnell, M. C., Snelling, R. E., and Mottola, H. A., Anal. Chem., 1986,544 1585. 727 44. Thomson, R. Q., and Crouch, S. R., Anal. Chim. Acta, 1982, 144, 155. 45. Thompson, R. Q., and Crouch, S. R., Anal. Chim. Acta, 1984, 159,337. 46. Mizutani, F., Yamanaka, T., Tanabe, Y., and Tsuda, K., Anal. Chim. Acta, 1985, 177,153. Paper A61262 Received August 8th, 1986

ISSN:0003-2654    

DOI:10.1039/AN9871200719

出版商:RSC    

年代:1987

数据来源: RSC

摘要:

ANALYST, JUNE 1987, VOL. 112 729 Selectivity and Kinetics in Analytical Chemistry* Plenary Lecture Miguel Valcarcel Department of Analytical Chemistry, University of Cordoba, Cordoba, Spain The positive influence on selectivity of physical, chemical and physico-chemical kinetics in the different steps of the analytical process is presented and discussed through representative examples. The selectivity levels achieved in situations of increasing dynamism are critically compared. Keywords : Kinetics; selectivity The role of kinetics in analytical chemistry teaching and research is frequently misunderstood. To many analytical chemists, kinetic aspects are secondary and are relevant only to the development of reaction rate methods. Kinetics has scarcely been used systematically to develop new methods or to improve existing analytical techniques, despite the fact that a large number of determinations are currently based on measurements made on transient signals.1 Moreover, only chemical kinetics has received due attention, to the detriment of physical and physico-chemical kinetics, which have been the subject of significantly less discussion.As pointed out earlier by Mottola2 and Pardue and Fields3 and also in the course of the First International Symposium on Kinetics in Analytical Chemistry held in Cdrdoba (Spain) in 1983,475 kinetics can and do play a major role in the over-all context of analytical chemistry. In fact, the significance of kinetics in some analytical methods and techniques was not acknowl- edged until later than they were developed-such is the case with flow injection analysis (FIA).Kinetics is relevant to analytical chemistry in at least four respects: (a) it allows the elucidation of the physical, chemical and physico-chemical mechanisms on which analytical processes are based and hence their rational optimisation; (b) it facilitates the development of new analytical methods and techniques that are otherwise unattainable if the dynamic aspects are not dealt with; (c) it is the foundation of reaction rate methods (kinetic determinations); and (d) it contributes to the improvement of significant analytical parameters such as sensitivity, selectivity and precision. The purpose of this paper is to demonstrate the influence of kinetics on selectivity. Chemical, physical and ph ysico-chemical kinetics can decisively influence one or more stages of the analytical process (sample handling and treatment, analytical reaction, signal measurement and data collection processing).Any of these stages can endow the analytical determination with a kinetic character. As a rule, kinetics is considered to affect only the development of the analytical reaction, although as will be shown here, it can also have a determining influence on the other stages. Selectivity is one of the cornerstones on which the analytical process relies, together with sensitivity and precision. Whenever the development of new methods or the improve- ment of existing techniques is considered as a response to particular problems, selectivity can be dealt with in two manners: by avoiding or lessening the influence of other species in the determination of the analyte, or by developing a simultaneous or sequential determination for several analytes in the same sample.* Presented at the 2nd International Symposium on Kinetics in Analytical Chemistry, Preveza, Greece, 9-12 September, 1986. Unlike sensitivity and precision, it is difficult to determine completely and accurately the selectivity of a given analytical process as it requires a knowledge of the influence of a large number of species and the determination of the maximum tolerated level, which usually involves arduous experimental work to obtain data usually unavailable in the literature. It is more common to study a small number of species and to use standard samples or recovery procedures to determine the matrix effect.Selectivity can be expressed according to various criteria,6?7 which have been systematically compiled by IUPAC.8 As far as possible, only two such ways of describing selectivity will be used in this paper, namely (a) the tolerance ratio, i. e . , the maximum concentration ratio of foreign species to analyte resulting in no interference, the chief use of which is to establish the selectivity of a given analytical method, and (b) the selectivity factor, which is defined as the relationship between the tolerance ratios for a given foreign species in the determination of the same analyte by two different methods and is commonly used to compare the selectivity of two analytical procedures. The selectivity of an analytical procedure can be achieved or improved in several ways: (a) through the chemical or physico-chemical reactivity; in this instance, the reagent used is active only against a limited number of species and the degree of selectivity can be improved by using further chemical processes (pH changes, masking reaction, etc.); reaction rate differences also allow one to distinguish between the different analytical behaviours of various species in the same sample; (b) by material isolation, i.e., through the use of both chromatographic and non-chromatographic separation techniques; this is by far the commonest way of increasing selectivity; and (c) by use of differential signals yielded by each species under specific conditions (wavelength, applied potential, etc.).It is interesting to note the possibility of combining any of the above three procedures to increase selectivity even *further [e.g., (a) and (b) in derivatisation chromatographic techniques, (b) and (c) by use of a specific detector for halogenated compounds in gas chromatography or (a) and (c) by formation of soluble complexes of different chromaticity]. In addition, the effect of applying any of the six above-described possibilities can be enhanced thanks to the mathematical discrimination afforded by data treatment (e.g., differential kinetic methods, treatment of overlapping chro- matographic peaks).Kinetics is of great relevance to all procedures intended to increase the selectivity of an analytical process. Such influence is materialised in the establishment of differences in reaction rate (e.g., kinetic-based determinations), in mobility (e.g. , chromatography or electrophoresis) or in the evolution of the response of the analytes with time when they are kinetically excited by instrumental parameters (e.g. , time-resolved flu- onmetry and some electroanalytical techniques). This fundamental analytical feature can be improved by730 ANALYST, JUNE 1987, VOL. 112 suitably dealing with the technical and methodological aspects related to the dynamics of the analytical system. Through representative examples involving different analytical tech- niques and methods, the selectivity levels obtained in situa- tions of increasing dynamism are compared below in areas such as equilibrium and reaction rate determinations, kinetic discrimination in reaction rate methods, manual and auto- matic continuous flow methods and conventional and kinetic- based instrumental alternatives.Equilibrium versus Reaction Rate Methods Reaction rate methods* involve two general aspects which endow them with higher selectivity than equilibrium methods, namely the scarcity of parasitic blank signals and the possibil- ity of using kinetic discrimination. An essential feature of reaction rate methods is the fact that they are based on differential rather than absolute measure- ments (signal increments corresponding to given time inter- vals), so that the parasitic signals yielded by other components of the sample generally have no influence. Thus, photometric analyses of wines by conventional equilibrium procedures involve serious drawbacks arising from the sample colour, which call for the use of separation techniques.If it is the rate of product formation that is measured, the results are not affected by the matrix colour. In this manner the author’s team have determined sulphur dioxide directly by reaction with p-rosaniline and formaldehyde in white, red and rosC wines.9 Equally good results are obtained in the direct kinetic - photometric determination of iron in wines by the stopped-flow technique , using thiocyanate as reagent. 10 The selectivity of reaction rate methods is also a result of the so-called kinetic discrimination, which is based on the different rate at which the analyte and other components of the sample react with the reagent. Table 1 gives two examples showing that measurements carried out under dynamic conditions are subject to less serious interferences than equilibrium methods. The photometric determination of FeIU based on the formation of a coloured complex (km.= 425 nm) with pyridoxal thiosemicarbazone can be accomplished by applying the classical equilibrium method11 or by a reaction rate method based on the fact that the complex formation is slowed by the presence of acetate.12 As can be seen in Table 1, the tolerated level of some ions is much higher in the dynamic method. Trace amounts of copper can be determined photo- metrically with the aid of CuU - 6-methylpicolinaldehyde azine - thiosemicarbazide, which involves reactions of exchange of GN- groups.13 Also, the number of species interfering at a given level is much larger when measuring the final absorbance at equilibrium rather than making initial-rate measurements.Kinetic Discrimination in Reaction Rate Methods As has been demonstrated by comparing reaction rate and equilibrium methods, the shorter the time elapsed between the mixing of the ingredients of the analytical reaction and the measurement, the less is the influence of interferents (side reactions). However, if the same chemical system is used to develop different reaction rate methods, the selectivity also increases as the measurement time approaches the mixing time. Fig. 1 shows three representative examples. * These methods are occasionally incorrectly referred to as “kinetic methods”; in fact, they are a particular type of these. The term “kinetic methods” should be applied to those methods in which signals are measured under physical, chemical or physico-chemical non-equilibrium conditions.Thus, for example, FIA and chemiluminescence methods are proper kinetic methods (they are based on typically transient signals). Table 1. Comparison between the selectivity levels afforded by equilibrium and kinetic methods Photometric determination of iron” 9 1 2 : Tolerated ratio (foreign ion: Fe) Foreign Equilibrium Reaction rate species method method Zn2+ . . . . 0.4 10.0 cd2+ . . . . 8.0 24.0 Pd2+ . . . . 120.0 >lOOo.O CN- . . . . 1 5.0 Photometric determination of copper13: Species interfering at a foreign ion to copper ratio of 25 : 1 Equilibrium method Reaction rate method Ag+, Fez+, Co2+, Ni2+, Zn2+, Hg2+, Pd2+, Pt4+ , V5+, Be2+, Mn2+, Cd2+, Pb2+, Cr3+, As3+, La3+, Ce4+, MOW, WVI Ag+, Fez+, Co2+, Ni2+, Zn2+, Hg2+, Pd2+, Pt4+, v5+ Initial period rate rate time rate Time Fig.1. Comparison of the selectivity levels given b different reaction rate methods. (a) Landolt (1) vs. initial-rate 4). Kinetic determination of iron. Selectivity factors (1 : 2) for foreign species:CoZ+ = 10.1; T+ = 8.4; I- = 3.0; and Be2+, Ba2+, Cr3+, Pb2+, tartrate = >2.0. b) Initial-rate (1) vs. fixed-time 2). Kinetic species: 3-methylhistidine = 10.1; cysteine, Mg = 5.0; and glycine = 2.0. (c) Initial-rate method with (1 and without (2) activator. Kinetic determination of manganese. Se ectivity factors 1 : 2) for forei n species: Ti4+, Pb2+ = 30.0; Co2+ = 15.0; Ca2+, &e4+, 103-; Cr + = 7.5; M 2+, Sn2+, Mow, 104- = 4.0; A + = 3.0; Ni2+, C d 2 + , Hg2+, S2- = 1.f; Cu2+ = 1.0; and Bi3+ = 0.07! determination of histi ine.Selectivity factors (1 : 2) ! or foreign 9 The kinetic - fluorimetric determination of FeIII based on its promoting effect on the oxidation of pyridoxal 2-pyridyl- hydrazone by hydrogen peroxide can be implemented in two manners: (a) by exploiting the Landolt effect (the reciprocal of the induction period is proportional to the analyte concentra- tion) and (b) by applying the initial-rate method.14 The first alternative results in increased selectivity factors [Fig. l(a)]. The fixed-time method is subject to a greater number and more severe interferences than the initial-rate method, as has been shown in the kinetic - fluorimetric determination of histidine based on its accelerating effect on the oxidation of 2-amino-1 ,1,3,-tricyanoprop-l-ene (TRIAP) by hydrogen peroxide, catalysed by trace amounts of copper.15 The initial-rate method results in increased selectivity in this instance [Fig.l(b)]. As a rule, activating effects have been exploited to increase the sensitivity of the kinetic determination of the catalyst, and also to determine the activator itself. There is another, little known, advantage involved in its use: the selectivity increase arising from the fact that the measurement zone of the slope of the kinetic curve is closer to the mixing time in the presence of an activator [Fig. l(c)]. Zn” exerts an activating effect16on the catalytic action of MnII on the oxidation of 2-hydroxybenz-ANALYST, JUNE 1987, VOL.112 731 aldehyde thiosemicarbazone by hydrogen peroxide, which yields a highly fluorescent product.17 The selectivity of the kinetic determination of manganese18 in the presence of the activator is much higher than that achieved in its absence. Differential kinetic methods are the best means of exploit- ing kinetic discrimination as they afford the most important objective of selectivity, i. e. , the simultaneous determination of two or more analytes in the same sample. They are based on the difference in the rate constants of two analytes ( k ~ and k ~ ) reacting with the same reagent, R A + R k P B + R & P* to yield products (P and P*) that give rise to non-discriminated signals under certain instrumental conditions.The concentra- tions of A and B can be determined by different methods (logarithmic extrapolation, proportional equation, single- point, etc.) based on mathematical discrimination.19 A description of simultaneous determinations based on differen- tial chemical kinetics is beyond the scope of this paper. The book by Mark et aZ.20 and a recent review on this subjectzl abound with interesting examples of organic and inorganic determinations, respectively. The mutual dependence of reaction rates in differential kinetics gives rise to the so-called synergistic effect, which results in mutually decreased selectivities, to the point of rendering simultaneous kinetic determinations impossible in some instances. This unwanted effect can best be overcome by using the logarithmic extrapolation method.22 However, this method does not allow the magnitude of the effect to be evaluated.If, in addition to the simultaneous determination, one wishes to derive a mathematical expression describing such mutual influence of the analytes of the monitored signal(s), then one can use a recent modification of the proportional equation method.23 Each of the equations derived includes an additional term that is dependent on the concentration of the analytes and accounts for the synergistic effect mathematically. In this manner, the author’s team have resolved mixtures of various amines reacting very similarly with 2-hydroxybenzaldehyde azine ,23 and also mixtures of histidine and histamine15 or imidazole and 4-methylimid- az0le,2~ which favour the oxidation of TRIAP by hydrogen peroxide, catalysed by trace amounts of copper.When the selectivity of a kinetic procedure allows the direct determina- tion of one of the analytes, but that of the second is subject to synergistic effects, the resolution of the mixture is consider- ably simplified. In this way various mixtures of trace metal ions have been resolved on the basis of reactions of exchange of C=N- groups between 6-methylpicolinaldehyde azine and different amines by use of a combined initial-rate - fixed-time method .25 Catalytic versus Non-catalytic Methods The use of catalysts to accelerate analytical reactions is feasible with both reaction-rate and equilibrium determina- tions. Enzymatic determinations feature high selectivity levels approaching specificity in some instances.They are therefore one of the best examples of the use of a kinetic effect to increase this analytical property. The use of dissolved26>27 or immobilised28J9 enzymes in the routine determination of a large number of analytes in complex matrices (foods, biolog- ical fluids, industrial products, etc.) is the best proof of their valuable contribution in this context. A description of these interesting analytical systems is beyond the scope of this Manual versus Continuous Unsegmented Flow Methods One of the salient features of flow injection analysis (FIA)31 is that, as a rule, neither physical (homogenisation) nor chemical equilibrium has been attained by the time that the sample zone reaches the continuous detector.Thus, FIA methods are doubly kinetic* and hence more selective-although less sensitive-in general than their manual counterparts. This increase in selectivity can be attributed to the kinetics of the process: the short time over which reactants and sample mix favours the develoDment of the analvtical reaction and minimises or even eliminates the effect of interferents, which are troublesome in the manual method on account of the much longer delay time. Below are discussed some examples illustrating this improvement in selectivity. Recently, Pacey et aZ.32 achieved a selectivity enhancement in the determination of ozone based on the decolouration of indigo blue in a straightforward FIA assembly. Equilibrium measurements (batch determination) do not allow one to discriminate between ozone, chlorine and permanganate.However, insofar as the reaction with ozone is much faster, the use of a single-channel FIA manifold with a dye carrier into which water samples are injected results in increased selectivity by factors of about 3 and 2 for chlorine and permanganate, respectively. Copper catalyses the oxidation of 2,2’-dipyridyl ketone hydrazone by dissolved oxygen in a basic medium, and the product yielded is highly fluorescent in an acidic medium. Copper can be determined at the ng ml-1 level by the three methods illustrated in Fig. 2: conventional manual kinetic (initial-rate) ,33 conventional FIA34 and stopped-flow FIA35 methods. In addition to other differences, the selectivity afforded by the three methods is very different.Fig. 3 shows a scheme of the range tolerated for a large number of ions. According to Fig. 3, the conventional FIA method is much less selective than the manual kinetic method, which in turn is less selective than the stopped-flow FIA method. These differ- ences in selectivity can be partly accounted for by considering that the delay time in the stopped-flow FIA method (10 s) is much shorter than the residence time of the peak in the FIA method (30 s) and the delay time in the reaction rate method (30 s). Moreover, the great difference in selectivity between the first and the other two types of methods lies in the fact that the latter use the reaction rate as the parameter related to the analyte concentration, in contrast to the first. The determination of physico-chemical parameters is also subject to interferences arising from side reactions, e.g., in the determination of acid - base constants of easily hydrolysable compounds.The literature abounds with inaccurate acidity constants as a result of not taking into account the possible hydrolytic properties of the compound concerned.36 Insofar as the acid - base reaction is faster than the hydrolysis, if absorbance measurements are made immediately after mixing then the contribution of the extent of reaction of the side process will be virtually negligible. Flow injection assemblies are particu- larly suitable for fast, almost simultaneous rneasurements.37 In such a system (Fig. 4), the sample is injected and mixed with a carrier altering the pH and a glass - calomel flow-through electrode is placed close to the flow cell accommodated in the photometer. The experimental variables of the systems are optimised in order to achieve as short a residence time as possible and efficient mixing, so that absorbance measure- ments are not affected by side reactions, which develop to a minimum or even negligible extent during the short interval paper.According to Werner,sO non-enzymatic catalytic reac- tions are scarcely selective in general. The rational optimisa- tion of the experimental variables involved partly improves the situation, although it is often advantageous to resort to separation techniques as a means of increasing the selectivity of catalytic determinations. * Care should be taken in this sense not to confuse unsegmented continuous flow methods proper with reaction rate FIA methods, the latter being a particular example of the former.732 Water HCI or NaOH solution ANALYST, JUNE 1987, VOL.112 - Photometric detector W - Manual kinetic method DPKH FIA Convention a I Cll’l 1 -- DPKH Pump IV Reactor I N a 0 H *A-, Fluorimeter HCI Re act0 r W Stopped-flow \,Fluorimeter Reactor cu2+ pH = 4.4) Fig. 2. Methodological alternatives to develop the kinetic - fluorimetric determination of trace amounts of copper based on i t s catalytic effect on the oxidation of 2,2‘-dipyridylketone hydrazone (DPKH) 100 90 80 .g 70 I u 6o 5 50 d 40 I- 30 20 10 Cd2+ I Mg2+ I Cr3+ I Pb2+ I Sn2+ I NP+ 1 Mn2+ I Fe3+ Coz+ Ag+ Hg2+ Zn2+ Be2+ AP+ F- =Conventional FIA Methods El Manual reaction-rate QZ FINstopped-flow Fig.3. Tolerated ratios (copper to foreign ion concentration) corresponding to the three methods described in Fig. 2 1 Hydrolysis Manual 2 4 6 8 1 0 1 2 PH Sample HCI or NaOH Dilution solution 4 7 Fig. 4. Determination of the acidity constant of 6-methylpicolinaldehyde azine by FIA and manual methods that elapses between reagent mixing and measurement. Fig. 4 shows the results obtained in the determination of the acidity constant of 6-methylpicolinaldehyde azine by the manual and the FIA methods. As can be seen, the unsegmented continu- ous alternative results in a single inflection point on the absorbance - pH graph, whereas the manual procedure gives rise to two more “jumps” at extreme pH values, which should be attributed to the compound hydrolysis, catalysed by H+ or OH- ions.Kinetics are thus a means of avoiding the incorrect assignment of the three pK values of this acyclic azine by overcoming the perturbation from the side reaction. The use of liquid - liquid extraction in a continuous fashion in an FIA manifold also results in remarkably increased selectivity with respect to the conventional procedure usingANALYST, JUNE 1987, VOL. 112 733 decantation funnels, as has been proved in the indirect atomic absorption determination of various species (perchlorate ,38 nitrite and nitrate39740 and anionic surfactants41) based on the formation of ion pairs with charged metal chelates, which are continuously extracted. A sample stream is mixed with the carrier of a suitable pH containing the metal chelate.This stream then merges with an organic phase in the segmenter. The ion pair is transferred through the interfaces formed between the regular segments of both phases in the so-called extraction coil. A device known as a phase separator allows the loop of an injection valve to be filled with organic phase, completely free from the aqueous phase. The loop contents are inserted into a water carrier and led to the atomic absorption spectrometer flame, yielding a transient signal that is directly proportional to the concentration of extracted chelate and indirectly related to the anion analyte concentra- tion. In Table 2 are listed the selectivity factors afforded by the continuous method, in comparison with those provided by the manual procedure. It is interesting to note the avoidance of serious interferences such as those given by iodide, chloride and sulphite in the determination of nitrate in foodstuffs,39>40 and also that of other surfactants in the determination of lauryl sulphate in waste waters.41 This beneficial effect can be attributed both to the decreased interfacial surface area and to the shorter interval over which the two phases are in contact.Analogous results are obtained when the indirect AAS determination of certain species is carried out by a previous precipitation process.42 The manual procedure is based on the measurement of the excess of cations present in the filtrate. The continuous precipitation is implemented in an FIA manifold by injecting the sample (anionic analyte) into a cation - reagent carrier arriving in a continuous manner at the detector flame.The precipitate is retained on a filter and the anion concentration is determined from the negative FIA peak obtained. In Table 3 are listed the selectivity factors obtained for various anions in the indirect determination of trace amounts of chloride by AAS involving the previous precipitation of this anion with an AgI carrier. As can be seen, the continuous (FIA) method clearly excels over the conven- tional manual counterpart in this regard. Table 2. Increase in selectivity achieved by performing liquid - liquid extraction in an automatic continuous configuration in comparison with the batch procedure Application Selectivity Species factor Determination of nitrate39 I- 3 c1- , co32- 10 S042- 100 surfactants41 .. . . NO3- >200 Triton X-100 50 Phthalic, succinic, Determination of anionic glutamic and benzoic acids 20 C104- 10 I- 2.5 Table 3. Comparison of the selectivity achieved in the indirect AAS determination of chloride by precipitation with AgI in a continuous manner (FIA) or by the conventional procedure42 Selectivity Species factor C104- . . . . . . . . 12.0 Fe(CN6)3- . . . . . . 10.0 CN-, SCN- . . . . . . 8.0 Br- . . . . . . . . 7.0 S042-, Fe(CN)64-, 103- . . 6.0 As043-, Br03 -, As02- . . 5 .O Cr042- . . . . . . . . 4.0 I- . . . . . . . . . . 2.0 Conventional versus Fast Molecular Absorption Spectroscopy So far we have shown the positive influence (regarding selectivity) of kinetics on the first few stages of the analytical process.As will be demonstrated below, the instrumental kinetic aspects influencing the signal measurement can also be exploited in order to improve this analytical property. The most significant differences between a conventional UV - visible spectrophotometer and a diode-array detector (DAD) are chiefly of a kinetic nature.43.a On the one hand, the monochromator is normally moving in an ordinary photometer and fixed in an image detector configuration. On the other hand, the use of DADs involves taking several simultaneous absorbance readings (a complete spectrum is scanned in as short a time as 0.1-0.01 s), whereas the conventional procedure requires sequential operation and is thus much slower. The large amount of information offered at a great speed by DADs can only be compiled, processed and presented with the aid of a microprocessor.The use of image detectors in UV - visible spectropho- tometry results in increased selectivity owing to the possibility of collecting absorbance data at various wavelengths in a very fast manner and to the differential spectral features of the analytes and the interferences (selectivity increase based on signal discrimination). Multi-component analysis in UV - visible spectropho- tometry has recently become a practical tool as a result of the emergence of image detectors and powerful laboratory computers. Absorbance measurements are made at n differ- ent wavelengths at which x analytes have different molar absorptivities. The resulting system of n equations in x unknowns (x 2 n) is solved by the computer. Although this methodology can be implemented manually by means of a conventional spectrophotometer , it is impractical for more than two analytes. Factor analysis is a mathematical discrimi- nation method that has promising prospects in the pursuit of increased selectivity levels.45 An interesting overview of selectivity in multi-component determinations and the appli- cation of data reduction schemes for various instrumental techniques was published recently.46 Fast-scan molecular absorption spectroscopy also offers major advantages in kinetic determinations.Thus, it is possible to monitor simultaneously several reactions taking place in the same solution, provided that the spectra of the resulting products do not overlap. The procedure involves recording the absorbance - time profiles corresponding to each species at a suitable wavelength. Milano and Pardue47 carried out the kinetic determination of a mixture of lactate dehydrogenase and alkaline phosphatase in serum by this procedure.Both enzymes catalyse the oxidation of lactic acid in the presence of NAD+ and the hydrolysis of phenol- phthalein monophosphate in the same sample aliquot. In practice, the procedure requires the simultaneous monitoring of NADH (340 nm) and phenolphthalein (550 nm). The study of the hydrolysis of potassium benzyl penicillin is of great practical significance as it reproduces the conditions under which this pharmaceutical acts in the stomach and is an excellent example of the advantages provided by the use of fast detection systems in kinetic determinations.48 The hydrolytic process can directly yield penicillenic acid (I) (which may be responsible for allergic reactions to penicillin), penicillic acid (11) and other cleavage products (IV), plus penamaldic acid (111) indirectly. Insofar as I, ILand I11 can be discriminated by use of three different wavelengths (322,240 and 290 nm, respectively), it is possible to record simul- taneously three independent absorbance - time curves to which the classical procedures for kinetic determinations are applied in order to determine the molar fraction of all the final products (that of IV is obtained by difference). Recently, our team published a review49 showing the734 ANALYST, JUNE 1987, VOL.112 advantages of the use of DADs in hydrodynamic analytical systems such as high-performance liquid chromatography (HPLC) and unsegmented continuous flow methods (FIA), which provide a chromatogram or “FIAgram” per monitored wavelength, from which typical three-dimensional representa- tions can be obtained. These two-fold dynamic configurations also result in increased selectivity. The use of DADs in FIA allows one to associate the lessening of interferences afforded by kinetic discrimination to the signal discrimination allowed by the special detector.In this manner trace amounts of iron and copper have been determined by injecting the sample into a sodium acetate - hydroxylamine carrier, which mixes with a stream containing a mixture of selective chromogenic ligands (o-phenanthroline and neocuproine) , The sample zone pro- vides several absorbance readings at as many wavelengths on passing through the DAD flow cell.50 This procedure has also been advantageously used to resolve binary and ternary mixtures of organic isomers.51 The chromatographic separa- tion on the column itself endows HPLC with higher selectivity than FIA.In addition, the ultra-fast detection afforded facilitates the acquisition of complete spectra at different points along each peak, which in turn allows one conveniently to control the purity of a given chromatographic peak.49 Kinetics in Molecular Fluorescence Techniques Because of their intrinsic characteristics, these techniques are more selective than those based on molecular absorption phenomena. However, conventional molecular fluorimetry is often faced with overlapping emission spectra that are a source of interference or render the determination of several analytes in a single sample impossible.Kinetics has a clear beneficial effect on selectivity in molecular luminescence techniques (Fig. 5). Such an effect can be exerted on the system under study, on the instrument or on both. Below are given representative examples demonstrating how the exploitation of the dynamic aspects in this context substantially increases the selectivity level with respect to the conventional mode. The simplest alternative involves taking advantage of the chemical or physico-chemical kinetics of the system under study. Kinetic - fluorimetric determinations52753 allow both ~ ~~ ~ Conventional fluorimetry Overlapped emission Unm Improvement of selectivity bv kinetic maniDulations Direct i nf I uence of kinetics Kinetic - fluorimetric determinations I Chemi( bio)luminescence On the system under study Synchronous f I uorescence spectroscopy Array detectors (excitation - I emission matrix) On the instrumentation Time-resolved fluorimetry I On both /instrument 1 system Fig.5. Influence of kinetics on the selectivity of fluorescence spectroscopy the reduction of interferences through kinetic discrimination54 and simultaneous determinations to be implemented by differential kinetics.55 It is also worth noting the high selectivity levels attained by chemiluminescence and biolu- minescence techniques56957 also based on the physico-chemical evolution of the system studied. In either instance, the instrumentation used should be adapted to the dynamic situation resulting from the chemical or physico-chemical kinetics, especially if the reaction half-life is short or if typical transient signals such as those obtained in chemi- or bio- luminescence techniques are encountered.The manipulation of the instrumental parameters employed in conventional fluorimetry allows the introduction of interesting instrumental modifications, resulting in increased selectivity in comparison with the analytical procedure based on conventional emission. As a rule, the system concerned is quiescent, i.e., chemical equilibrium has been reached by the time the signal is measured. Synchronous excitation fluor- escence spectroscopy58 is more dynamic instrumentally as the two monochromators used are moved simultaneously at constant wavelength increments, in contrast to the sequential movement of both. The result is a single synchronous spectrum (instead of the traditional excitation and emission spectra), which features a narrower band width.This in turn results in decreased spectral overlap and hence in improved selectivity. In fact, a dual derivatisation of the synchronous signal allows mixtures of analytes with overlapping conven- tional and synchronous spectra to be resolved directly and simultaneously. In this manner binary and ternary mixtures of trace amounts of titanium, zirconium and hafnium have been resolved by the formation of chelates whose emission spectra are only slightly different .59 The interference of bilirubin in the determination of fluorescein has been minimised by using the first derivative of the synchronous signal.60 The applica- tion of derivative synchronous fluorimetry to the determina- tion of analyte mixtures in biological fluids (e.g., pyridoxal, pyridoxal-5-phosphate and pyridoxic acid at the nanomoles per litre level61 and epinephrine and norepinephrine62 in human serum samples) has provided excellent results.The kinetic instrumental aspects are of decisive importance in this fluorimetric alternative. Thus, the rate at which both mono- chromators are scanned simultaneously (nm s-1) has a great influence on the analytical properties: the higher it is, the higher the sensitivity will be. It also results in increased selectivity as a result of the decreased number of bands obtained.As can be seen in Fig. 6, the appropriate selection of the scan rate is decisive in this instrumental alternative. Total excitation - emission matrix (EEM) acquisition is a fluorimetric mode featuring multi-component determination capability.63 The use of conventional instrumentation makes this interesting alternative slow and time consuming. However, the joint utilisation of array detectors and a microprocessor for the ;acq isition and fast processing of a large number of data (ulti IW! tely a kinetic modification) allows the determination of several components in a single sample through three-dimensional (hex. - kern. - If) or contour graphs or indeed other calculation procedures.@ The third way in which kinetics can exert a positive effect on the selectivity of conventional fluorimetric techniques involves kinetic-dependent excitation (generally laser- induced) of the sample (analytes), which results in a phenom- enon (fluorescence decay) that is detected by an ultra-fast data-acquisition system, as such a phenomenon is only accessible in the nano- to millisecond range.The kinetic manipulation of instrumental parameters results in a rapid response from the system under study. Both dynamic aspects contribute substantially to selectivity, to the point of facilitat- ing the resolution of mixtures of analytes with strongly overlapped emission spectra, in addition to the determination of luminescence lifetimes or the study of the decay kinetics of species in excited states.65 There are two classical approachesANALYST, JUNE 1987, VOL.112 8 nm s- 735 90 80 70 60 I ’ nm s-’ I - - - - 460 520 580 Nnrn 50 40 30 20 10 g 40L a, - - - - L 0 E: al .- *.’ - 0) a 60 50 40 30 Fig. 6. 480 520 560 Nnm nm s-’ I 1 I I 440 520 600 Nnm 400480 560 640 Unm Influence of the scan rate on the second-derivative synchro- nous s ctra of trihydroxyindole derivative of epinephrine. The only peak gectly and selectively attributable to the concentration of this analyte is that appearing at 54&550 nm, which is obtained for a scan rate of 2 nm s-1 to time-resolved fluorimetry, as follows. (a) In pulse tech- niques, the system is irradiated with an intense, brief light pulse and the intensity of the resulting fluorescence is recorded as a function of time.% Selectivity in this instance is based on the different rates of fluorescence decay.This is therefore a means of overcoming the interference of a given species by kinetic discrimination: the longer lived species can be measured after the fluorescence contributions from the shorter lived species have decayed to a negligible value.67 Multi-component determinations can be accomplished by deconvolution of multi-exponential decays obtained from mixtures.@-70 Indeed, these determinations, like those de- scribed above, are based on differential kinetics. (b) In modulation and phase-shift techniques ,71 continuous sinu- soidally modulated excitation combined with phase-sensitive detection is used. There are several basic alternatives to multi-component determinations. In the indirect nulling approach, each component can be selectively detected by measuring the solution at the detector phase angle required to cancel the other components.A series of independent simultaneous equations can be generated by performing n measurements at n detector phase angles for x components (n 2 x).72,73 * Kinetics in data collection and handling data reduction 1 Fig. 7. Influence of kinetics on the last step of the analytical process Kinetics in Data Collection and Treatment Time can be a variable of great importance in this last stage of the analytical process, particularly when data are generated in large numbers at high rates. Many of the novel instrumental alternatives such as Fourier transform spectroscopy, stopped- flow methods and image detector modes, would be impractical without the rapid response electronic systems and adequate data-acquisition devices (fast recorders, oscilloscopes and microprocessors) available today.Physico-electronic kinetics plays a major role in these instances and therefore influences the selectivity. The use of powerful computers is a brilliant solution to problems arising from the collection of a large number of data, kinetics imposing no limitation whatsoever in most instances. Data processing is a more complex aspect as the rate of data generation usually exceeds the speed of interpretation by several orders of magnitude. This situation is especially complicated in hyphenated methods,74 based on the on-line coupling of a gas or liquid chromatograph to spectroscopic identification techniques such as infrared spectroscopy, mass spectrometry or those involving the use of diode-array detectors, all of which generate large numbers of data.There are two possible means of circumventing this kinetic delay problem,75 both of which are closely related to the selectivity of the last stages of the analytical process (Fig. 7), as follows. (a) Selective data collection involves collecting fewer data than might be possible in a discriminative manner and treating all the collected data. In this instance, the selectivity is determined by the characteristics of the measurement stage (viz., the use of specific detectors in chromatography, monitoring of the signal at a single instrumental parameter, etc). (b) Non-selective data collection involves acquiring all data generated in the measurement stage in an indiscriminate fashion, taking advantage of the kinetic capability of the computer to collect and store data.There are two ways of processing data in this instance. One involves treating all the collected data (e.g. , automated spectral retrieval, pattern recognition, artificial intelligence), which is time consuming. There are few representative samples of this mode of interpretation in anything like real time.76 In the second alternative, data are chosen selectively (data reduction) in order both to shorten the time required to interpret them and not to occupy too much of the computer’s memory. Kinetics and selectivity are closely related in all these examples. A comprehensive discussion of their advantages and disadvantages has been published.74736 ANALYST, JUNE 1987, VOL.112 Conclusion In its broadest conception, kinetics is a fundamental aspect with a decisive influence on most analytical processes. The aim of this paper has been to demonstrate how the exploitation of the physical, chemical and physico-chemical dynamic aspects involved affords a substantial improvement of the invaluable analytical property of selectivity. In fact, kinetics is, in many instances, the foundation of novel technical or methodological analytical alternatives, although this is rarely acknowledged in the analytical literature. A considerable proportion of the so-called “modern” or “advanced” methods and techniques are based on kinetic modifications or considerations involving either instrumentation, the system concerned or both.This is obviously not an exhaustive review of how kinetics contributes to selectivity. The examples given were aimed to be representative rather than comprehensive. Thus, other instrumental techniques such as electroanalytical methods, mass spectrometry and several modes of thermal methods involve a kinetic component of great relevance to their foundation and applications and which affects not only their selectivity, but also their feasibility. In many separation techniques (especially chromatographic methods), the differ- ent rate of migration is the immediate foundation of the separation and hence of the selectivity, although the remote foundation has a dual nature: thermodynamic and kinetic. Other significant properties such as sensitivity and precision are also decisively affected by the kinetic aspects of the analytical process.Approaches similar to that undertaken in this paper could be of great interest in helping to define in a more comprehensive manner the role played by kinetics in analytical chemistry . 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. References Mottola, H. A., and Hanna, A., Anal. Chim. Acta, 1978, 100, 167. Mottola, H. A., J. Chem. Educ., 1981, 58, 399. Pardue, H. L., andFields, B.,Anal. Chim. Acta, 1981,124,39. Mottola, H. A., Quim. Anal., 1983,II, extra, 3. Pardue, H. L., Quim. Anal., 1983,II, extra, 24. Kaiser, H., Fresenius 2. Anal. Chem., 1972,260,252. Belcher, R., and Betteridge, D., Talanta, 1966, 13, 535.Den Boef, and Hulanicki, A., Pure Appl. Chem., 1983, 55, 553. Lazaro, F., and Luque de Castro, M. D., personal communica- tion. Loriguillo, A., Silva, M., and Perez-Bendito, D., unpublished results. Perez-Bendito, D., and Valcarcel, M., Afinidad, 1980, 366, 123. Ballesteros, L., and Perez-Bendito, D., Analyst, 1983, 108, 443. Rios, A., and Valcarcel, M., Quim. Anal., 1983, I, 227. Rubio, S., Gomez-Hens, A., and Valcarcel, M., Anal. Chem., 1984,56, 1417. Gutierrez, M. C., Gomez-Hens, A., and Valcarcel, M., Anal. Chim. Acta, 1986, 185, 83. Moreno A. , Silva, M. , Perez-Bendito, D. , and Valcarcel, M., Analyst, 1983, 108,85. Moreno, A., Silva, M., Perez-Bendito, D., and Valcarcel, M., Anal. Chim. Acta, 1984, 157,333. Moreno, A., Silva, M., Perez-Bendito, D., and Valcarcel, M., Talanta, 1983,30, 107.Perez-Bendito, D., and Silva, M., “Kinetic Methods in Analytical Chemistry,” Ellis Horwood, Chichester, in the press. Mark, H. B., Reichnitz, G. A., and Grienke, R. A., “Kinetics in Analytical Chemistry,” Willey-Interscience, New York, 1968. Perez-Bendito, D., Analyst, 1984,109,891. Papa, L. J., Patterson, J. H., Mark, H. B., and Reilley, C. N., Anal. Chem., 1963,35, 1889. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32, 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. Rios, A., Silva, M., and Valcarcel, M., Fresenius 2. Anal. Chem., 1985,320, 762. Gutierrez, M. C., Gomez-Hens, A., and Valcarcel, M., Microchem. J., in the press. Rios, A., and Valcarcel, M., Talanta, 1985,32, 851.Bergmeyer, H. V. , “Principles of Enzymatic Analysis,” Verlag Chemie, Weinheim, 1978. Guilbault, G. G., “Handbook of Enzymatic Methods of Analysis,” Marcel Dekker, New York, 1976. Carr, P. W., and Bowers, L. D., “Immobilized Enzymes in Analytical and Clinical Chemistry,” Wiley, New York, 1980. Guilbault, G. G. , “Analytical Uses of Immobilized Enzymes,” Marcel Dekker, New York, 1984. Werner, G., Quim. Anal., 1983,II, extra, 68. Valcarcel, M., and Luque de Castro, M. D., “Flow Injec- tion Analysis. Principles and Applications,” Ellis Horwood, Chichester, 1987. Pacey, G. E., Hollowell, D. A., Miller, K. G., Atraka, M. R., and Gordon, G., Anal. Chim. Acta, 1986, 179,259. Grases, F., Garcia-Sanchez, F., and Valcarcel, M., Anal. Chim. Acta, 1981, 125,21.Lazaro Boza, F., Luque de Castro, M. D., and Valcarcel, Cases, M., Analyst, 1984, 109, 333. Lazaro Boza, F., Luque de Castro, M. D., and Valcarcel, Cases, M., Anal. Chim. Acta, 1984, 165, 177. Luque de Castro, M. D., Silva, M. , and Valcarcel, M. , Analyst, 1984, 109, 1375. Rios, A., Luque de Castro, M. D., and Valcarcel, M., Anal. Chim. Acta, 1985, 171, 303. Gallego, M., and Valcarcel, M., Anal. Chim. Acta, 1985, 169, 161. Gallego, M. , Silva, M. , and Valcarcel, M., Fresenius 2. Anal. Chem., 1986,323,50. Gallego, M., Silva, M., and Valcarcel, M., Anal. Chim. Acta, 1986,179,341. Gallego, M., Silva, M. , and Valcarcel, M., Anal. Chem., 1986, 58, 2265. Martinez, P., Gallego, M., and Valcarcel, M., Anal. Chem., 1987, 59,69. Jones, D. G., Anal. Chem., 1985,57, 1057A and 1207A. Pardue, H.L., in Foreman, J., and Stockwell, P. B., Editors “Topics in Automatic Chemical Analysis,” Ellis Horwood , Chichester, 1979, p. 163. Kowalski, B. R., Anal. Chem., 1980,52, 112R. Otto, M. , and Wegscheider, W. , Anal. Chim. Acta, 1986,180, 445. Milano, M. J., and Pardue, H. L., Clin. Chem., 1975,21,211. McCarrick, T. A., and McLafferty, F. W., J. Chem. Educ., 1984, 61, 463. Lazaro, F., Rios, A., Luque de Castro, M. D., and Valcarcel, M., Analusis, 1986, 14,378. Lazaro, F., Rios, A., Luque de Castro, M. D., and Valcarcel, M., Anal. Chim. Acta, 1986, 179, 279. Bermudez, B., Lazaro, F., Luque de Castro, M. D., and Valcarcel, M. , Analyst, A61225. Ingle, J. D., and Ryan, M. A., in Wehry, E. L., Editor, “Modern Fluorescence Spectroscopy,” Plenum Press, New York, 1981, Volume 3, Chapter 3. Valcarcel, M., and Grases, F., Talanta, 1983, 30, 139. Rubio, S., Gomez-Hens, A., and Valcarcel, M., Analyst, 1984, 109,717. Moreno, A., Silva, M., and Perez-Bendito, D., Anal. Chim. Acta, 1984, 159, 319. Kricka, L. J., and Thorpe, G. H., Analyst, 1983, 108, 1274. Seitz, W. R., CRC Crit. Rev. Anal. Chem., 1981, 13, 1. Rubio, S., Gomez-Hens, A., and Valcarcel, M., Talanta, 1986, 33,633. Rubio, S., Gomez-Hens, A., and Valcarcel, M., Anal. Chem., 1985,57,1101. Brigh, F. V., and McGown, L. B., Analyst, 1986,111,205. Petidier, A., Rubio, S., Gomez-Hens, A. , and Valcarcel, M., Anal. Biochem., 1986,157,212. Valcarcel, M. , Gomez-Hens, A., and Rubio, S., Clin. Chem., 1985,31, 1790. Weber, G., Nature (London), 1961, 190,27. Christian, G. D., Callis, J. B., and Davison, E. R., in Wehry, E. L. , Editor, “Modern Fluorescence Spectroscopy,” Plenum Press, New York, 1981, Volume 4, Chapter 4.ANALYST, JUNE 1987, VOL. 112 737 65. Richardson, J. H., in Wehry, E. L., Editor, “Modern Fluor- escence Spectroscopy,” Plenum Press, New York, 1981, Volume 4, Chapter 1. 66. Hieftje, G. M., and Vogelstein, E. E., in Wehry, E. L., Editor, “Modern Fluorescence Spectroscopy,” Plenum Press, New York, 1981, Volume 4, Chapter 2. 67. Richardson, J. H., George, S. M., and Ando, M. E., Nut. Bur. Stand. (U.S.) Spec. Publ., 1979, No. 519, 691. 68. Knorr, F. J., and Harris, J. M., AnaZ. Chem., 1981, 53, 272. 69. Hiraki, K., Morishige, K., and Nishikawa, Y., Anal. Chim. Acta, 1978, 97, 121. 70. Craven, T. L., and Lytle, F. E., Anal. Chim. Acta, 1979, 107, rlcl? L13. 71. McGown, L. B., and Bright, F. V., Anal. Chem., 1984, 56, 1400A. 72. McGown, L. B., and Bright, F. V., Anal. Chem., 1984, 56, 2195. 73. Bright, F. V., and McGown L. B., Anal. Chem., 1985,57,55. 74. Hirschfeld, T., Anal. Chem., l980,52,297A. 75. Anderegg, R. J., Int. Lab., 1986, March, 62. 76. McLafferty, F. W., Int. J . Mass Spectrom. Ion Phys., 1981,19, 358. Paper A61344 Received September loth, 1986

ISSN:0003-2654    

DOI:10.1039/AN9871200729

出版商:RSC    

年代:1987

数据来源: RSC

摘要:

ANALYST, JUNE 1987, VOL. 112 739 Catalytic Titrations* Plenary Lecture Ferenc F. Gaal Institute of Chemistry, Faculty of Sciences, University of Novi Sad, V. VlahoviCa 2, YU-21000 Novi Sad, Yugoslavia The application of catalysed reactions to the detection of end-points in titrimetric analysis has led in the last twenty years to the development of numerous methods of titration. After a brief historical overview and a consideration of terminology, the theoretical and experimental foundations of catalytic titrations and their a ppl icat io ns, I i m itat ions and selectivity a re discussed. Keywords : Tit rime tric an a 1 ysis; ca ta 1 ytic tit ra ti0 ns; ca ta 1 ytic en d-p o in t indication The indication of the end-point of titrations has always been an important area of research in titrimetric analysis.’-3 Problems have appeared in different forms, but the aim has always been the same: to improve the possibilities of solving particular analytical tasks.The application of catalysis in analytical chemistry has led, amongst other things, to the possibility of determining very small amounts of substances. Although the use of some catalytic effects in titrimetry has been known for a relatively long time, only a quarter of a century has elapsed since the appearance of the first publica- tion of a method for the determination of titrimetric end- points based on a catalytic reaction. These so-called catalytic titrations are founded on the following principle: Titration reaction- A + K A Y AK . . (1) Titrated component Titrant Reaction product (inhibitor) reagent of titration (catalyst) (catalytically inactive) Indicator reaction- K X + ( Y ) z L + ( O ) .. . . . . A small excess of the titrant catalyses a side-reaction (the “indicator reaction”) and this reaction is used to indicate the end-point of the titration. Concentrations of the indicator reaction components may be often much higher than the concentration of the component to be determined (the inhibitor of the process). As the catalyst is regenerated by the catalytic reaction, very small amounts of catalyst can catalyse the formation of relatively large amounts of the indicator reaction product, leading to marked changes in the chosen physico-chemical property of the titrated solution, which can then be easily measured. In this way it is possible to detect the moment at which a small excess of the titrant has been added and to determine small amounts of the titrated component.Typical idealised curves of catalytic titrations are shown in Fig. 1. Two vertical lines divide the graph into three portions: pre-period, titration period and after-period - excess titrant period. Depending on possible changes in the parameter measured, the curves can have either an ascending or descending course. Segment AB corresponds to the pre- period (no titrant added). Point B represents the beginning of the addition of the titrant, whereas C and C’, respectively, denote the equivalence point a n c t h e beginning of the after-period (titrant added). Slopes BC and BC’, correspond- ing to the occurrence of either a slow spontaneous or a poorly Calysed indicator reaction, are incomparably smaller than CD and C“, which are characteristic of a fast catalytic * Presented at the 2nd International Symposium on Kinetics in Analytical Chemistry, Preveza, Greece, 9-12 September, 1986.reaction. After point D (i.e., D’) has been reached, i.e., from the moment when one of the indicator reaction components has been exhausted, the final segment can have different slopes (depending on a number of factors), but in all instances it is different from that observed during the preceding period. In choosing the system titrated component - titrating reagent - indicator reaction, it is necessary to bear in mind the following requirements. Reaction (1) ought to proceed rapidly and in a stoicheiometric ratio in the direction of the formation of the catalytically inactive reaction product AK.Use has been made of complexometric, precipitation, redox and neutralisation reactions. The titrating reagent should fulfil the requirements for the titrant in titrimetric analysis. The properties of AK are also of great importance. Thus, for example, the complex AK should have a large stability constant, so that the complexation reaction is efficient even at low concentrations of the ligand. Similarly, in the case of the formation of a sparingly soluble compound, its solubility product should be very low. All these requirements are necessary in order to ensure a low concentration of the catalyst before the equivalence point. As for the indicator reaction, it should be sufficiently sensitive so that a small excess of the titrating reagent can induce a marked increase in its rate and, of course, it should proceed under the same conditions as reaction (1).The rates of possible side-reactions between the titrant (K) or titrand (A) with either the indicator reaction components (X, Y) or products (L, 0) should be negligible. Redox and ligand-exchange reactions, and also poly- merisation, acylation and dehydration, have been mainly employed as indicator reactions. ‘i I I I I C J A T = \ > Equivalence point I\ I I Pre-period I Titration period I After period Degree of titration Fig. 1. Idealised forms of catalytic titration curvesANALYST, JUNE 1987, VOL. 112 As the end-point of catalytic titrations is determined on the basis of the parameter changes caused, not by the competition of the titration reaction, but by the catalytic action of the titrant in a small excess, it is possible to improve the sensitivity of some universal, but rather rough procedures (e.g., ther- mometric method) of indicating the end-point.The number of publications concerning the determination of titration end-points by catalytic indicator reactions (Fig. 2) show an increasing interest in the application of this method.4-14 Biennial fundamental reviews on kinetic determinations and some kinetic aspects of analytical chemistry in AnaZyticaZ Chemistry have devoted a special chapter to this method since 1980.15-18 As seen from Fig. 2, after some pioneering works on catalytic analytical methods,19JO there was a normal, but not easily discernable, period of incubation before the appearance of papers on the determination of titration end-points employing catalytic reactions.Erdey and BuzBs,21 in 1960, were the first to employ a visual chemiluminescent indicator reaction (H202 + luminol and H202 + lucigenin) in the titration of EDTA with Cu2+. They were probably influenced by the work of Szebelledy, who, almost twenty years before, supervised some research on the application of chemiluminescence in analytical chemistry.22 In 1962, Yatsimirskii and Fedorova23>24 described a method for the determination of microgram amounts of silver and iodide ions in which I- served as a catalyst of the indicator reaction CeIV- As111 in acidic medium. They performed a would-be titration by following the course of the reaction in samples containing equal amounts of silver and increasing excesses of titrant (I-). By plotting log (absorbance) vs.time (log A - t) straight lines were obtained with slopes proportional to the iodide concentration, indicating that the reaction was first order with respect to iodide. Titration curves were then constructed within the coordinates of the volume of KI solution added and the slope of the straight line log A - t . The equivalance point was determined as the intersection of the straight line with the horizontal, corresponding to the solubility of AgI in water. The authors especially stressed the importance of the catalytic effect in the detection of the titration end-point and proposed the term “catalymetric titrations”.Almost at the same time, Bognhr and SBrosi25 described a similar, but simpler, method. Their “simultaneous indication” of step-wise titration was announced as a new variant of the catalymetric titration. To equal volumes of the solution of Ag+ or Hg2+ they added different volumes of a standard solution of iodide, covering both sides of the equivalence point with several measurements. After adding all the necessary components (including ferroin as the indicator) except CeIV, each solution was diluted to the same volume. By a simultaneous addition of the CeIV solution, the reaction CeIV- As”’- ferroin was enabled to start. In each instance, the 14 I l 3 12 1 I , I I Year Fig. 2. Annual survey of publications on catalytic titrimetry time, t , needed to reach the d o u r change of the indicator, was measured and the quantity llt plotted against the volume of titrant added.The titration end-point was determined as the break on the titration curve constructed in this manner. The possibility of the catalytic determination of the end- point of continuous thermometric titrations of bases with perchloric acid in acetic acid as solvent was pointed out by Keily and Hume26 in 1964. They employed the heat effect of the reaction of acetic anhydride with trace amounts of water present in the titrated solution, the reaction being catalysed by the excess of perchloric acid. However, they did not make much use of the effect that they observed. In 1965, Margerum and Steinhaus27 published a paper on the determination of metal ions using the ligand-exchange reaction of triethylenetetraamine - nickel(I1) and (ethylenedinitri1o)tetraacetate - cuprate(II), catalysed by EDTA.The rate of the indicator reaction was monitored by a spectrophotometric method at different stages of the titration. The rate constant of the ligand-exchange reaction plotted against the volume of the titrating agent resulted in a titration curve with an easily detectable end-point. In the same year, Vaughan and Swithenbank28 published a valuable contribution in which they reported the use of acetone as both the solvent and indicator in the continuous enthalpimetric titrations of acids. They wrote, “The enthalpimetric titration of acidity with non-aqueous alkali often results in a curve that shows only a slight change in slope at the end-point, but, if acetone is used as a solvent for the acid, it has been found to act as an ‘indicator.’ At the neutralisation point a rapid heat rise occurs owing to the formation of diacetone alcohol from the reaction of acetone with the excess of non-aqueous alkali.The conditions under which this reaction occurs have been studied and a method incorporating the best conditions is described.” In a paper by Weisz and Muschelknautz,29 which appeared a year later, it was said that, “The application of catalytic reactions for the end-point indication in volumetric analysis in the classical sense is described: the catalyst itself serves as the titrimetric reagent; substances can be titrated which are able to distinctly diminish the catalytic action (by precipitation or by complex formation).The first drop in excess of the titrant catalyses the ‘indicator reagent mixture.”’ In the beginning of 1965 I began working in this field of research, supervised by Professor Vajgand at the University of Belgrade. The same year we published our first paper30 and, subsequently, my MSc31 and PhD32 thesis on the same subject . In the latter half of the 1960s, several new research centres appeared and extensive research on methodological and experimental problems of catalytic titrations was begun. A chronological survey of some important events in the development of this field is presented in Table 1. These contributions have come from centres all over the world, as can be seen from Fig. 3. A great impact on the development of these methods, especially the development of their theory, was the applica- tion of computers. Taking into account the equilibrium concentration of the catalyst during the titration it is possible to simulate the corresponding catalytic titration. Thus, in some instances, theoretical preparations preceding the experi- ment may be useful in choosing the optimum working conditions, i.e., for obtaining the best experimental results. Different workers have used a variety of terminology in this subject. Some workers have apparently not been aware of the importance of catalysis, which is reflected in the way in which they named the method, it either being called after the titrant, e.g., chelatometric titrations,57 or, after the monitor- ing mode, e.g., enthalpimetric titrations.28 The term “kinetic titration”27 seems to be too broad.Different cambined terms have also appeared, such as cataly-thermometric titra- tion,30931 thennocatalymetric titrations8 and thermocatalyticANALYST, JUNE 1987, VOL. 112 741 Table 1. Some important contributions to the development of catalytic titrimetry Year 1960 1962 1963 1965 1966 1967 1968 1969 1970 1971 1972 1973 1975 1976 1977 1980 1983 1984 1984185 1986 1986 Event Complexometric titration. Chemiluminescent reactions: luminol - Hz02 and lucigenin - H202 catalysed by CuI1 Catalymetric titration of Ag+ by I- with CeIV - As111 reaction as indicator reaction “Simultaneous indication.” Reaction of CeIV - As111 - ferroin in determination of Ag+ and Hg2+ with I- Publication of the first monograph on kinetic methods of analysis Enthalpimetric method.Acetone used both as solvent and “indicator” Application of some catalytic reactions for visual titration Catalymetric thermometric titration of organic bases with HC104 in The term “catalytic thermometric titration” was proposed Potentiometric indication of the catalytic reaction CeTV - Asrx1 catalysed by I- in titrimetry Catalymetric titration of CN-. Cu2+ as catalyst (autoxidation of L-ascorbic acid) and titrant. Spectrophotometric indication Catalytic thermometric titration by coulometric generation of titrant First review dedicated to catalytic titrants and catalytic indication Titrimetric method for the determination of bases with HC104 by in the titrations of organic acids end-point indication the presence of small amounts of H20 (2%) and (CH3C0)z0 (8%) of end-points thermometric indication patented. Reactions: (CH3C0)*0 - ROH catalysed by H+ Substitution titration with catalytic end-point detection First theoretical considerations Differential and derivative catalytic thermometric titrations Contribution to the theory of continuous catalymetric and thermocatalytic titration processes Switching circuit for automatic recording of the time elapsed to reach the “end-point’’ in catalytic spectrophotometric indication Catalysed reactions for indication in titrimetry followed biamperometrically Cationic polymerisation of styrene and alkyl vinyl ethers as a means of end-point indication in thermometric iodimetry Determination of weak acids in mineral insulating oils by catalytic thermometric titration Review on catalytic titrations Evaluation of a perchlorate-selective electrode for catalytic Olfactory indication in catalytic titrations Catalytic potentiometric titrations ( I = constant) Review on catalytic thermometric titrimetry Gaseous catalysts for end-point indication in titrimetric analysis First International Symposium on Kinetics in Analytical Chemistry (Cordoba, Spain, September 27-30,1983) Catalytic refractometric titrations Fluorimetric catalytic titrations Complete mathematical description of catalytic titrations Catalytic conductimetric titrations Second International Symposium on Kinetics in Analytical Chemistry titrations involving periodate indicator reactions (Preveza, Greece, September 9-12,1986) Author(s) Erdey and Buzhs Yatsimirskii and Fedorova BognAr and Sdrosi Yatsimirskii Vaughan and Swithenbank Weisz and Muschelknautz Vajgand and Gahl Vajgand and Gad Weisz and Klockow Mottola and Freiser Vajgand etal.Mottola Goizman Klockow and Garcia Beltran Mottola Vajgand and Gall Goizman Mottola Weisz and Pantel Greenhow Castle and Greenhow Hadj iioannou Hadjiioannou et al. Abe et al. Gad et al. Greenhow Weisz and Schlipf Gall and Topalov Moreno et al. GaAl et al. Gad et al. Reference 21 23 25 33 28 29 30 34 35 36 37 4 38 39 40 41 42 43 44 45 46 9 47 48 49 12 50 51 52 53-55 56 Fig. 3. year a contribution from a given city has been published and the total number of publications originating from the given place Survey of cities in which the investigations on catalytic titrimetry were performed up to 1986.Numbers in parentheses indicate first742 ANALYST, JUNE 1987, VOL. 112 titration42>59 but these are not widespread. Similarly, the term “catalymetric titration”2*25,30,33,36,43,60-73 has gained only relatively poor acceptance. Finally, the term “catalytic titration” with reference to the mode of its monitoring (e.g., thermometric, spectrophotometric and ampero- metric methods (analysis) with catalytic end-point indica- tion”l7.18 and “catalytic titrimetry” have been used most frequently. Certainly, some more consistency in the terminol- ogy would contribute to a better understanding among the people working in this interesting field. metric9,11-14,32,34,37;41,4~9,51-55,64,74-147) together with ‘‘titri- Theory As with other analytical procedures, in the development of catalytic titrations the methodological and experimental foundations were laid before the corresponding theoretical relations were derived.The experimental conditions have been primarily chosen on the basis of extensive experimenta- tion, which is a rather tedious job. In catalytic titrations the titrant can be added either continuously or discontinuously, but in both instances the titration curve is obtained by plotting the change in some physical parameter (which is a function of an indicator reaction rate, i.e., the catalytic activity of a component of the solution to be titrated) against the amount of titrant added. As methods with a continuous addition of the titrant are of greater importance, and experimentally much simpler, their mathematical interpretation has attracted more attention.In 1970 Mottola40 gave the first mathematical treatment of catalytic titration curves, starting from some general expres- sions, but without particular solutions for the titration curves. Goizman42 in 1971 derived equations for catalytic thermo- metric curves, but his work was restricted to the volumetric addition of the titrant, and the volume change of the solution during titration was neglected. Simpson148in 1973 was the first to simulate catalytic spectrophotometric titration curves using a computer, introducing a new quality into these investiga- tions, but he, also, neglected the solution volume changes during titration. G a W in 1977 formulated some general mathematical equations as a basis for the interpretation of the shape of catalytic titration curves, obtained by different techniques, for both coulometric and volumetric addition of the titrant.However, all these workers have assumed that the catalyst concentration before the equivalence point can be neglected. This approximation is acceptable provided that the reaction product formed in reaction (1) is, for example, a sparingly soluble compound with a very low solubility product, or a complex with a very high stability constant. This is not always the case, however. For this reason AbramoviC149 formulated a mathematical description of catalytic titration curves, taking into account the equilibrium concentration of the catalyst in the course of the titration. The theory was developed for catalytic complexometric,~3 precipitation,54 redox54 and neutralisation55J50J51 titrations.To simplify the derivation of the mathematical expressions, it is advisable to introduce several approximations, namely, (1) the titrant is added to an ideally stirred solution; (2) temperature changes during titration do not affect the thermodynamics and kinetics of the reactions; (3) the indica- tor reaction has an incubation period short enough to be neglected; (4) the rate constant of the titration reaction is high, and need not to be taken into account; and (5) changes caused in the measured parameter by the titration reaction are negligibly small compared with the changes due to the indicator reaction. The procedure for obtaining expressions for the simulation of catalytic titrations consists of deriving an equation for the catalytst concentration during the titration.The expression obtained is then introduced into that for the rate of the indicator reaction and this expression is integrated for the limiting conditions. In deriving the equation for the catalyst concentration in the course of, e.g., complexometric titrations, we start from the expression for the instability constant KN of the catalyst - inhibitor complex (assumed to have a 1 : 1 stoi- cheiome try) where c(K) is the molar concentration of catalyst K, c’(A) that of inhibitor A and c(AK) that of the complex AK. After the introduction of the appropriate expressions for c’(A) and c(AK) into expression (3), a relationship is obtained for the equilibrium concentration of the catalyst during the titration.In volumetric complexometric catalytic titrations, c’(A) and c(AK) during the titration are c’(A)= ’*) -c(AK) . . . . (4) V, + jt and c(AK) = - jtc(T) - c(K) . . . . . Vr + jt where c(A) is the initial molar concentration of the inhibitor, c(T) the concentration of the titrant, V, the initial volume of the titrated solution (1), j the rate of addition of titrant (1 s-1) and t the time (s). Introducing these expressions into equation (3) gives the following relation for the catalyst concentration during the titration: where E k = {Vr[C(A) + KN]}~; F k = 2Vrj(K~[c(A) + KN] - c(T)[c(A) - KN]}; and Gk = /’[C(T) KN]’. If the indicator reaction is of first- or pseudo-first-order with respect to the indicator reaction component X, then its rate can be expressed as * * (7) c(X> i --- dc(X) - [k; + ki, c(K)] c(X) + - dt Vr + It where k; is the rate constant of the first-order spontaneous reaction (s-I), ki, the rate constant of the first-order catalysed reaction (1 mol-1 s-1) and c(X) the molar concentration of the indicator reaction component. The last term in equation (7) describes the change in the rate of the indicator reaction due to the increase in volume of the reaction mixture during the titration; obviously it is equal to zero in the coulometric mode of the titrant addition.If the catalyst concentration is expressed by equation (6) and equation (7) is integrated for the limiting conditions c(X) = co(X) at t = 0, an expression is obtained for describing the concentrationof componentxat time t: c(X) = C,(X) - vr exp(-Mk) .. . . V, + jt where c,(X) is the initial molar concentration of the indicator reaction component andANALYST, JUNE 1987, VOL. 112 743 in which where Ak = {Vr[C(A) + c(T)]}~; Bk = 2Vr{KN[c(A) - c(T)] - c(T)[c(A) + c(T)]); and Ck = [c(T) + K N ] ~ . As the concentrations of the reaction products are often measured during the titration, it may be suitable to express the reaction rates as a function of these concentrations. If product L is formed from component X in equation ( 2 ) in a 1 : 1 ratio, then the concentration of X remaining at time t will be: - c(L) . . . . (11) c(X) = C,(X) -- Vr Vr + jt where c(L) is the molar concentration of the indicator- reaction product. On the basis of equations (8) and (11) it follows that the concentration of L at time t is Vr Vr +lt c(L) = C,(X) - [ 1 - exp( -&)I .. (12) In a similar way, it is possible to derive the relations for second-order indicator reactions. In the example of coulometric complexometric catalytic titrations the concentrations of the inhibitor, c'(A), and of the complex, c(AK), are c'(A) = c(A) - c(AK) . . . . . . (13) It c(AK)=--c(K) . . . . . nFV, where I is the generating current (A), F the Faraday constant (C mol-I), and n the number of electrons in the half-reaction for the electrochemical generation of the titrant. The intro- duction of these parameters into expression (3) leads to an equation for the catalyst concentration It - (EL)& + [EL + Fit + (It)2]& c(K) = . . (15) 2nWr where EL = {nn/,[c(A) + KN]}2 and FL = 2nFVr I[KN - c(A)].Further steps in deriving the expressions for the titration curves are similar to those in the preceding procedure. By employing the above approach it is also possible to derive a mathematical expression describing the blank titra- tions. In this instance, the catalyst concentration, c(K), is a linear function of time as there is no titrand in the reaction vessel. Here also, the two modes of titrant addition were considered.54 As the mathematical expressions obtained in this way are rather complex, the conditions under which the equilibrium concentration of catalyst can be neglected have also been determined.5~-5S~150." By employing the above equations, i.e., by simulating the catalytic titration curves, it is possible to choose, in a relatively simple way, the optimum experimental conditions.The effect of a number of factors on the shape of simulated titration curves is illustrated in Figs. 4-8. From Fig. 4, lines A-D, it can be seen that the titration end-point determination is difficult when the complex instabil- ity constant, KN, is >lO-7 (line B) under the given working conditions. However, if the rate constant of the catalysed reaction is lower, it may be possible to determine the end-point even for values of the complex instability constant >lo-7. Fig. 4 also shows the catalytic titration curves obtained on the basis of the approximative expressions derived by Goiz- man42 and Simpson148 (line E) and GaaP2 (line F). It should be mentioned again that under certain working conditions (for example, at a small value of the instability constant and at a 0 1 2 3 4 5 6 7 8 9 1 0 Concentration of indicator reaction p r ~ d u c t / l O - ~ M Fig.4. Simulated curves for catalytic complexometric titrations obtained with volumetric addition of titrant and a first or pseudo-first order indicator reaction, under the following conditions: V, = 3 x 10-2 1. c (X = 10-3 M; k: = 10-3 s -1; k; = 103 1 mol-l s-l; c(T) = 5 x lo-; $; c[A) = 5 x 10-4 ~ ; j = 10-5 1 s-1. Curves A-D, equilibrium catalyst concentration taken into account [according to equation (12)]; Ekzobtained on the basis of the expressions obtained by Golzman and Simpson148; F, obtained according to the expressions obtained by GaBl.32 Values for KN are: A, 10-6; B, C, and D, 10-10.Equivalence point, 3 .OO mF3 -Concen&ation of indicator reaction product/l c4 M Fig. 5. Effect of rate constant for the second-order catalysed reaction, kk, on the shape of the simulated curves in volumetric redox catalytic titrations obtained under the followin conditions: V, = 3 X M; c(A) = 5 'x" id-. M; j = 10-5 1 s-1; K, = 105. Values for kk (12 mol-2 s-1) are: A, 107; B, 106; C, 105; D, 104; and E, 18. Equivalence point, 3.00 ml54 1. c (X = 10-3 M; k, = 10-3 1 mol-1 s-1; c$T) = 5 X744 ANALYST, JUNE 1987, VOL. 112 O.O I Concentration of indicator reaction pr~duct/lO-~ M Fig. 6. Effect of generating current, I, and concentration of titrand, c(A), on the shape of simulated coulometric precipitation catalytic titration curves employing a first-order indicator reaction, under the following conditions: V, = 3 x 10-2 1; co(X) = 10-3 M; k: = 10-5 s-1; k; = 103 1 mol-1 s-1; n = 1; K = 10-13.Values for I and c(A) are: A, 4.82 X 10-2 A and 5 x lo-;&; B, 4.82 x 10-3 A and 5 X M; C, 4.82 x 10-4 A and 5 x 10-5 M; D, 4.82 x 10-5 A and 5 x 10-6 M; E, 4.82 x 10-6 A and 5 x 10-7 M. Equivalence point, 300.3 ss4 0.0 0.5 1 .o 1.5 v) 2.0 z 5 2.5 E i= r 4 3.0 3.5 t 4.5 4 . 0 1 d.V 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Concentration of indicator reaction pr~duct/lO-~ M Fig. 7. Simulated curves for neutralisation catalytic titrations of acids and bases obtained with coulometric addition of titrant (strong base - acid) and a first-order indicator reaction, under the following conditions: V, = 3 x 10-2 1; c,(X) = 10-3 M; k: = 10-3 s-1; k; = 105 1 mol-1 s-1; I = 4.82 x 10-4 A; c(A) = 5 x 10-5 M; KHs = 10-14; n = 1.Curves A-F, degree of acid dissociation taken into account55; G, obtained on the basis of the mathematical expression derived for titrations of strong acids and bases; H, obtained on the basis of the approximate expression. Values for acidity constant: A, 10-6; B, 10-5; C, 10-4; D, 10-3; E, 0.1; F, 10. Equivalence point, 300.3 s150 0 1 2 3 4 5 6 7 Time/lO* s Fig. 8. Effect of autoprotolysis constant, KHs, on the shape of the simulated coulometric neutralisation catalytic titration curves for a diprotic acid - base, with a first or pseudo-first order indicator reaction, under the following conditions: V, = 3 X 1; c,(X) = 1 M; k: = 10-5 s -1; k; = 103 1 mol-1 s-1; I = 4.82 X 10-1 A; c(H2C), c(C) = 5 x 10-2 M; n = 1; Kl = 5 x 10-4; K2 = 5 x 10-8.Values for KHs are: A, 10-10; B, 10-12; C, C’, C 10-14; D, 10-16. Equivalence points: 300.3 and 600.6 s151 high ratio of the concentration of titrant to that of titrand) satisfactory agreement is obtained between all simulated catalytic titration curves. As can be seen from Fig. 7, lines D-F, if the acidity constant is greater than 10-3, it is possible to employ the equation derived for titrations of strong acids and bases (as lines D-F are identical with line G). The effect of the solvent autoprotolysis constant on the shape of the titration curve of a diprotic acid or base is illustrated in Fig. 8. It is evident that, by choosing a suitable solvent, it is possible to determine either only the first (lines A and B) or the second (line D) equivalence point, or both (lines C, C’ and C ) .Lines C, C’ and C were obtained under the same working conditions, apart from the ordinate scale. Lines C and C are the most suitable for the determination of the first and the second equivalence points, respectively. On the other hand, the parameter changes in line C’ are not very convenient for determination of either the first or the second equivalence point. Therefore, by choosing a measuring instrument with a satisfactory sensitivity the determination of both equivalence points can be made possible. The effect of the following parameters has been studied: the rate constant of the spontaneous and catalysed indicator reaction; the initial concentrations of the indicator reaction components; the rate of titrant addition; and the number of electrons involved in the reaction of electrochemical genera- tion of the titrant. By taking all of these into account, and on the basis of the kinetic data of a potential indicator reaction, it is possible to estimate whether the reaction considered would be suitable for the indication of the titration end-point in the determination of particular analytes.In addition, after an experimental check, simulated catalytic titration curves might also be used for the determination or revision of the indicator reaction kinetics, and for the determination of all other parameters influencing the shape of the catalytic titration curves (Fig. 9). All these results are very important, especially in the development of new determination procedures and in decid- ing the optimum experimental conditions.Had the present- day theories of catalytic titrations been known twenty years ago, a lot of experimental work would have been saved.ANALYST, JUNE 1987, VOL. 112 745 > E -280 . 0“ v;? 01 I -260 m I + -240 E lLi - I I I I I I J 0 0.50 1.00 1.50 2.00 2.50 3.00 Volume of 2.0 x lo-* rnol dm-3 of MnS04/cm3 Fig. 9. Ex erimental (A) and simulated (A’-E’) catalytic poten- tiometric PO4-/(Hg - Hg2SO4)] titration curves of 18.25 mg of EDTA wit 2.0 X 10-2 M MnS04 in the presence of periodate (1.3 X 10-4 M), TEA (9.7 x 10-3 M) and phosphate buffer, pH 6.84. Curves A-E, were obtained for the following values of k; (1 mol-1 s-l); A’, 700; B’, 500; C’, 400; D’, 350; E’, 184142 Practical Aspects The actual task in determining the optimum experimental conditions is to measure the rate of the indicator reaction during titration, i.e., to register changes in the concentration of the “monitored species,’’ which is one of the components of the indicator reaction.Practically, it will usually suffice to register changes in the measured parameter (temperature, absorbance, electrode potential, etc.) as a function of the amount of the titrant added; the obtained (graphical) data permit the determination of the titration end-point. As far as the necessary apparatus is concerned, catalytic titrations can be performed by means of any of the set-ups employed in titrimetric analysis, and consist of the following parts: (i) a titrant system for the addition or generation of the titrant; (ii) a titration vessel with or without a sensing device (detector system) and stirrer; and (iii) (in instrumental end-point detections) the device for measuring and/or recording the measured parameter.Closed systems should always be used, in order to protect both the titrated and titrating solution from the influence of some external agents, such as atmospheric moisture or C02. Mode of Titrant Addition The titrating reagent in catalytic titrations can be added in either a discontinuous or continuous way. The former mode has been employed in two kinds of titrimetric determinations: the standard series and the method based on the titration of one aliquot only. In the first instance, to equal volumes of the titrated solution, containing all but one component of the indicator reaction, increasing volumes (some of them exceed- ing the equivalence point) of a standard solution of the titrating reagent are added.The solutions are then diluted to the same volume, and equal volumes of the missing indicator reaction component solution are poured in simultaneously. The time interval required to reach a relevant change in the system, corresponding to the exhaustion of the limiting reaction component, is then measured. The time ti is a function of the amount of the catalyst (titrating reagent) present in the solution.25@>62,79 A graph of llri against the volume of the titrating reagent added permits the determina- tion of the titration end-point as the break on the resulting curve.A similar procedure has been employed in spectrophoto- metric ,23,24,61,@ 967-71 oscillopolarograp hic ,63 t hermome tric79 and amperometric methods with two identical electrodes.79 In these instances the logarithm of the measured parameter is plotted as a function of time for each stage of the titration. The slopes of the resulting straight lines are proportional to the concentration of the titrating reagent in the solution. The titration curve is finally constructed by plotting the values of the slopes obtained against the volume of titrating reagent. The titration end-point is determined as the intersection of two straight lines, one of which may be horizontal, corre- sponding to the solubility product in precipitation titra- tions.23*24961~@ In some instances the titration curve is con- structed from the total apparent rate constant of the indicator reaction as a function of the amount of titrant added.27 A common characteristic of these procedures is that they require a lot of time.A simpler discontinuous method, based on the titration of one aliquot, has been used more frequently. In these instances, the solution containing the indicator reaction components is titrated with a standard solution of the catalyst. After the addition of each volume increment of the catalyst solution, the parameter of interest is measured, usually in equal time intervals, and the values obtained are presented graphically as a function of the amount of catalyst. This procedure has been employed in catalytic titrations with visual, thermometric and potentiometric indication of the end-point .The continuous mode of titrant addition has been used more frequently, and in recent years it has become ubiquitous. The titrating reagent is added continuously either by a volumetric or coulometric (constant current) method, the latter having two variants, i.e., either internal or external generation of titrant. The external generation of titrant81782 allows some of the possible reactions of the indicator reaction components at the electrodes to be avoided. According to the literature, the volumetric method has been used more frequently than coulometry. (Coulometric generation of the titrant has been found in 15 papers of experimental theoretical nature5%-55J5O9151 and in some instances the coulometric catalytic titrations have been used as a suitable comparative method.107,108,111,112~153) For the coulometric generation of the titrant, either commercially available coulostats or laboratory-made electrical circuits can be used.For the volumetric addition of the titrating solution, either macro, micro or ultra-micro motor-driven micrometer-syringe burettes can be used. It is my opinion that the possibilities offered by the coulometric method have not been sufficiently employed, and that there is a lot to be achieved in this field. character ,31,32,37,41 ,51,56,74,80-84,133,147,152 in five papers of a Titration Vessel The choice of the titration vessel depends on the mode of titrant addition and, partly, on the method of the titration end-point indication. An ordinary beaker can be used in visual and some other determinations.Instrumental methods of monitoring the titration course require special vessels or cells. Hence, photometric titrations have been carried out in rectangular or cylindrical cells, made either of glass or quartz. These may be of substantial volume,1@J54 when the solution is stirred with a magnetic stirrer, whereas in flow-through systems their volume is smaller. In the latter instance, mixing and circulation of the solutions from the reaction tube to the photometric flow-through cell is accomplished by means of a variable speed tubing pump.40 Modern phototitrators fur- nished with an immersion photo-probe consisting of a double bundle of glass fibres appear to be very practical.143 Refrac- tometric titrations have also been performed in a flow-through cell connected to a peristaltic pump.51 The cell was construc- ted with PTFE outlets enabling aggressive chemicals to be handled.Special closed vessels have been constructed to work with gases5OJ52J55 in the catalytic thermometric end-point indication. Owing to very pronounced signals, Dewar vessels appear to be unnecessary. Coulometric titrations require different cells for the interna180@ (most frequently H-type) and external titrant generation.81782 In external generation, the titration is carried out in a beaker and the titrating reagent is generated in a special cell.746 ANALYST, JUNE 1987, VOL. 112 A local excess of titrant must be avoided as it imitates the irreversible indicator reaction and may cause the appearance of a premature end-point.Efficient stirring of the titration solution with a magnetic stirrer or rotary-paddle stirrer is therefore required. Titration Procedures and Methods of End-point Detection With respect to the actual procedure, several types of catalytic titrations can be distinguished. In direct titrations, the concentration of the inhibitor is determined by its titration with a standard solution of the catalyst. If, however, a catalyst of an unknown concentration serves as the titrant, whereas a standard solution of the corresponding inhibitor is the titrand, then the titration is referred to as a reverse titration. The catalyst content can be also determined by back-titrations; an excess of the standard solution of the inhibitor is titrated with a standard solution of the catalyst.This method can also be employed in the determination of components having neither a catalytic nor an inhibitory effect on the indicator reaction, provided that they form, for example, a more stable complex or a less soluble precipitate than that of the catalyst. A common feature of all the above techniques is that the catalyst serves as the titrating reagent. However, it is possible to add the catalyst in its inactive form (complex or precipitate) to the solution containing the sample (inhibitor) and the indicator reaction components. The solution of a substance that forms a more stable complex with the inhibitor, i. e., a less soluble precipitate, than that of the inhibitor + catalyst, is used as titrating reagent.For all the time that the inhibitor is present in its free form, the catalyst remains inactive. At the moment at which the inhibitor has completely reacted, the excess of titrant will displace the catalyst from the complex or precipitate and the rate of the indicator reaction will be markedly enhanced. These are so-called substitution catalytic titrations.39.162 They can also be performed either as direct, reverse or back-titrations. In those instances when the above titration techniques do not work, an indirect method may be tried.89,N The procedure requires either the precipitation of the substance to be determined or its decomposition. As a result, one of the components obtained might be determinable by one of the procedures above. A survey of techniques used for monitoring catalytic titrations is given in Table 2.The thermometric method has been used most frequently as it can easily be employed to follow the rate of a whole range of catalytic reactions. From the use of this we have witnessed a rapid development of thermometry. Potentiometric and amperometric methods, owing to their high sensitivity, are suitable for the determina- tion of micro-amounts of substances. It is to be expected that the number of papers on catalytic titrations, especially those based on spectrophotometric and luminescent methods, will show a substantial increase, due primarily to the appearance of suitable equipment. Finally, a better understanding of the subject will be of help to those workers who, when good signals have been obtained at the equivalence point, have wrongly ascribed these to some other effects (e.g., to the electrode material), ignoring the importance of possible Generally, the end-point of catalytic titrations can be determined either by visual, graphic or automatic methods. The simplest way is, of course, the visual observation of changes occurring in the titration solution; an excess of titrant triggers the indicator reaction, which is observed as a change of colour of the solution.In instrumental methods the end-point is mainly deter- mined graphically from plots (directly or after first or second derivation) of measured parameters versus the amount of titrant added. Changes at the equivalence point are not instantaneous, and, consequently, graphical extrapolation is necessary for both linear and non-linear titration curves.The end-point of catalytic titrations can be determined by the automatic stopping of the titration (i.e., of titrant addition) on the basis of the signal obtained by the second-derivative technique, either by means of commercially available titrators or by laboratory-constructed derivator units.9~40.41,43,77,78,129,154 However, the effect of the concen- tration of the indicator reaction components, intensity of stirring of the solution during titration and of a spontaneous occurrence of the indicator reaction can crucially affect the values of the measured parameter and, thus, the location of the end-point. Such errors are more probable in automatic catalytic titrations compared with the non-catalytic titrations.catalytic action involved. 58,83,111,112,153,163 I 164 Table 2. Methods of end-point detection in catalytic titrimetry Lowest determined concentration of compounds/mol 1-3 Method non-instrumental methods: I. Visual and other Colour change Liberation of gas Olfactory 11. Instrumental methods: Optical methods (Spectro)photometric Chemiluminescent Fluorimetric Refractometric Interferometric Conductimetric High-frequency conductimetric Voltammetric - amperometric Voltammetric - oscillopolarographic Potentiometric ( I = 0) Potentiometric (I = constant) Other methods Thermometric Manometric Electroanalytical methods Inorganic 3 x 10-7 9 x 10-3 6 x 10-4 3 x 10-10 5 x 10-6 5 x 10-5 - - - - 6 x 10-6 5 x 10-6 2.5 x 10-8 6 x 10-6 6 x 10-8 - Organic 2 x 10-3 2 x 10-4 - 1 x 10-7 5 x 10-4 5 x 10-5 1 x 10-3 2 x 10-4 5 x 10-4 - - 2 x 10-2 - 4 x 10-8 2 x 10-6 6 x 10-4 Important references 29,50,76,156 29,74 48,76 36,40,132,154 21,157,158 52 51 161 56 56 44,49,97,100,113,157 63 35,78,97,128 49,97,100,113 28,34,80,82,87,159,160 32,152ANALYST, JUNE 1987, VOL.112 747 This might be the reason why semi-automatic techniques have been used so frequently. In these titrations, the titrant is added continuously and the titration curve is registered by means of a recorder. Finally, in so-called manual catalytic titrations, the titrant is added discontinuously and changes in the system are followed either by visual observation or by measuring discontinuously a characteristic parameter of the limiting component of the indicator reaction, which is often unreliable.The construction of the corresponding titration curve is rather tedious and time consuming and this may be the reason why this method is now rarely used. In order to eliminate possible methodological errors, blank titrations are usually performed in all of these determinations. Applications Neutralisation Catalytic Titrations A substantial number of neutralisation catalytic titrations have been developed.26,28,30,34,37,38,41,46,51,55,56,58,59,66,73-75,80, 81,8~88,90,92-95,101-104,106,109,11~119,133,1~,141,144,147,15~152,1~,161, 165-173 Use has been made of polymerisation, acylation, dehydration, Claisen condensation and various redox reac- tions for the indicator reaction. As some polymerisation reactions are catalysed by acids and some by bases, these reactions have been employed as indicators in the determina- tion of both organic acids and bases of different strength and in the determination of some inorganic acids.Acetic or pro- pionic anhydride has often been used as one component of the acylation reaction, whereas compounds containing one or more hydroxy groups (in addition to water) have served as the other. The acylation reactions catalysed by strong acids have been used in the determination of organic bases of different strength. The catalysed Claisen reaction,75 dehydration reac- tions74J52 and some redox reactions have not been used extensively. The thermometric method has been used almost exclusively for monitoring the course of neutralisation catalytic titrations.In some instances, visual ,41,74 manometric,l52 interfero- metric,lbl conductimetric,56 high-frequency conductimetric56 and refractometric51 methods have been employed. The titrant addition has been carried out by both volumetric and coulometric (either with internal or external generation) methods. Both manual and semi-automatic techniques have been employed and the direct titration procedure has been used almost exclusively. Complexometric Catalytic Titrations There have been a great number of papers on complexometric catalytic titrations. 21,27,29,36,39,40,44,47-49,52,53,57,63,65,68-70,76,91, 98-100,120-127,131,132,134,136,139,142,143,154,15~158,162,17~179 Differ- ent redox reactions have been used as indicator reactions, whereas ligand exchange has been used only in two instances.27263 The decomposition of hydrogen peroxide, and oxidations by the same agent, have been frequently used as redox indicator reactions, whereas reactions involving iodate, periodate, chlorate, perborate and oxygen have received much less attention.Various metal ions, such as Co2+, Cu2+, Mn2+, Pb2+, Fez+ and Ni2+, have served as catalysts in these reactions. By employing solutions of these cations as titrants, different complexones (EDTA, DCTA, DTPA, NTA, etc.) have been determined by the direct titration method. In addition, reverse, back-titration and substitution methods have been used to determine a number of cations. The ligand-exchange reaction using EDTA as a catalyst appeared to be suitable for the indication of the end-point in the direct catalytic titrations of a limited number of cations.The course of complexometric catalytic titrations has been followed by visual, photometric, spectrophotometric, amperometric, oscillopolarographic, potentiometric ( I = 0, I = constant), thermometric, manometric, chemiluminescent and olfactory methods. The volumetric mode of titrant addition only has been employed. The manual and semi- automatic procedures have been the most frequently used, although there are papers on the use of automatic methods.43.154 Precipitation Catalytic Titrations These titrations have also received considerable attention although somewhat less compared with those of the two groups already di~~~~~ed.2~25,29,35,44,50,54,60-62,64,67,71,77-79,81, 82,89,91, %,97,113,128,12~131,137,145,154,157,159,178,1~,181 Only redox reactions have been used for indication, and those involving CeIV as the oxidising agent have appeared most frequently.Iodate, permanganate, persulphate, hydrogen peroxide, iodine and Mn"' have been used to a lesser extent. Iodide, molybdate, Th4+, Ag+ andH2S have served as catalystsin these indicator reactions. The use of an iodide solution as the titrant has allowed the direct determination of Ag+ , Hg2+, Pd2+ and Au3+, whereas a number of anions (Cl-, Br-, I-, SCN-, CN-, etc.) and cations (Ag+, Pd2+, Hg2+, Au3+) have been determined by reverse, back-titration and substitution titra- tions. An indirect method has allowed the determination of some secondary amines. Solutions of molybdate, Th4+ and Ag+ have been used as titrants in direct titrationsof Pb2+ and of some anions (F- , SiF62-, C1- , Br- , I- and SCN-).Several metal ions have been determined by direct, back-titration and the indirect method using sodium sulphide solution as the titrant. The course of these titrations has been followed by visual, photometric, spectrophotometric, amperometric, poten- tiometric ( I = 0, I = constant) and thermometric methods. The addition of titrant was either volumetric or coulometric with external generation. The manual and semi-automatic procedures have been most frequently used, but there are papers on the use of automatic meth0ds.77~129~154 Redox Catalytic Titrations This group of catalytic titrations has been least investi- gated.45,54,105,110,135,155,182,183 Both redox and polymerisation reactions have been used as the indicator reaction.Several cations have been determined (direct and back-titration method), and some organic substances, permanganate and water (direct method). The course of the titration has been monitored by visual, photometric and thermometric methods with volumetric addition of the titrant. Both manual and semi-automatic techniques have been used. The above methods have been widely used in the analysis of different complex materials. Catalytic thermometric titrations (nominal molarity 6.001 M)/ml (1 &vision = 0.2 rnl) Fig. 10. Effect of adding tetraethylthiuram disulphide on the catalytic thermometric titration of insulating oils.& A, Oil no. 18: B, oil no. 18 + 0.02% TET; C, oil no. 12; D, oil no. 12 + 0.02% TET748 ANALYST, JUNE 1987, VOL.112 have been successfully used for the determination of the phenol and resorcinol components of resins,94,95J71 phenolic hydroxy groups in ~081,165 acids in mineral insulating 0ils32~46 (Fig. lo), petroleum bitumens104 and in formaldehyde,a dithiocarbamates and phosphorodithiolates in oils105 and for the analysis of sulphonamides (Fig. 11). 103,147 The same technique has also been employed in the analysis of the L-dopa contents of tablets and capsules,86 alkaloids and related basic compounds, extracted from crude drug formulations or natural materials88 or in pharmaceutical products.30~34~37~80 Further, the method has also been successfully employed in studies of molecular structure and reactivity,lC@ the mechan- ism of condensation and rearrangement reactions of mono- and di-functional carbonyl compounds,138 the interaction of dipolar aprotic solvents with water146 and the reaction mechanism of iodimetric end-point indication and an evalua- tion of a copolymerisation indicator reaction.183 Catalytic potentiometric titrations78 (Fig. 12) have been used for the determination of the iodide content in table salts.136 Further, a catalytic spectrophotometric method for the determination of Ca2+ and Mg2+ (Fig. 13) was successfully employed for their determination in milk.98 The fluoride content in technical phosphoric acid was determined after the removal of phosphate. 184 The Fe, Cu, Ni and Mn content in metallurgical samples was determined. 143 The silver content of pharmaceutical prepara- tions of silver sulphadiazone was also determined.72 A selective determination of some acidic substances present in mixtures (e.g., thiols, carboxylic acids, polyhydroxylic phenols) can be achieved by using different combinations of titrant, solvent and indicator.Hence, for example, the difference in the results obtained by employing perchloric acid and boron trifluoride as titrating reagents for bases, and potassium hydroxide and tetrabutylammonium hydroxide as titrating reagents for acids, can be explained by the structural properties of the molecules titrated. 12 The catalytic thermometric method of titration end-point determination has also found a significant application as a comparative method in the development of some other determination procedures. 106108,111,112,153,185 Some very clever and useful analytical solutions can be found among established methods leading to a wider use of catalysis in titrimetric analysis.Limitations and Selectivity The indication of titration end-points by catalytic reactions is an example of a novel application of irreversible indicators in titrimetric analysis. Therefore, if sufficient care is not exer- cised, the known shortcomings of irreversible indicators will affect all these determinations. However, owing to their sensitivity, catalytic titrations are suitable for the determina- tion of low concentrations of substances (Table 2). The limit of their applicability depends on the value of characteristic constants, such as solubility product, acidity - basicity con- stant, stability constant, etc., depending on the kind of titration reaction employed.In addition, properties of the indicator reaction and solvent, and temperature, play a significant role. In order to improve the possibility of the application of catalytic titrations to complex materials analysis, it would be necessary to increase the selectivity of the method. It is often true that in direct titrations only one end-point is registered. However, by choosing a proper combination of the solvent, indicator reaction and titrating reagent, it is possible to determine several components in mixtures. For example, in contrast to carboxylic acids, many thiols cannot be determined using acrylonitrile as an indicator. However, they can be titrated if acrylonitrile is replaced by acetone or cyclohex- anone.92 Thus, a selective determination of thiols and - 1 Titranthl(1 division = 1 ml) Fig.11. Catalytic thermometric titration curves for sulphon- amides. 103 2, Phthalylsulphathiazole; 4, succinylsulphathiazole; 8 and 9, sulphadiazine; 13, sulphamerazine; 14, sulphamethizole; 15,17 and 19, sulpha yridine; 20, sulphaquinoxaline; 21-26 and 28, sulpha- thiazole; 29, sulphaurea. t , End point, curve 21 Volume of Kl/ml Fig. 12. Recorded curves for the semi-automatic spectro hoto- metric (A) and potentiometric (B) titration78 of mercury(II7 with iodide. Al and B,: 20 ml of 1.2 x 10-4 M HgII titrated with 6 X 10-3 M KI. A2 and B2: 20 ml of 1.2 x 10-5 M HgII titrated with 6 x 10-4 M KI I 1 I A B C D I I Volume of Mn"/ml Fig. 13. Recorded curves for the semi-automatic indirect catalytic titration98 of alkaline earth ions.A, 50.0 pg of Ca."; B, 50.0 pg of Mg"; C, 64.7 pg of Sr"; and D, 60.0 pg of BaII. Conditions: 5 ml of 1.01 X 10-3 M EDTA solution, 10 ml of 0.429 M ammonia solution titrated with 9.1 X 10-3 M MnII solution. Theoretical end-points: 4.18, 3.29, 4.74 and 5.07 ml, respectively carboxylic acids in a mixture of the two can be accomplished by performing two titrations (one for each kind of indication). A similar approach has been employed in the selective determination of resorcinol and pheno1,93 and of resorcinol and phenol melts in novolac,94 in resols95 and in their corresponding mixtures. The necessary selectivity has been achieved by employing a solvent mixture in which resorcinol can be titrated either as monoprotic or diprotic acid, depend- ing on the solvent composition.Phenols in solution and their melts in resins have been titrated only as monoprotic acids and the mixture composition has been determined from a calibra- tion graph, constructed on the basis of the results obtained by using two corresponding solvent systems.ANALYST, JUNE 1987, VOL. 112 749 In the titrations of some polyfunctional organic acids85J51 and bases32J51 some inter-inflections appear on the titration curve. It has been found that these correspond to the neutralisation of stronger acidic or basic groups. The appear- ance of the inflections can be ascribed to the inability of the weaker acidic groups to hinder the polymerisation - acylation reaction. As a result, two or more inflections appear. These represent the limits of the interval within which the polymerisation - acylation reaction has commenced, but its rate is low, as an insufficient amount of catalyst is present in the solution.Conclusions On the basis of the above it can be concluded that catalytic titrations allow the end-point indication of a great number of titrations. In addition, the method has already obtained a sound theoretical basis. As catalytic titrations show, in certain instances, an advantage over non-catalytic titrations in respect of sensitivity, their significant development in the future is to be expected. The application of pre-concentration and separa- tion methods might induce some new research interest in the field. Also, some novel indicator reactions, together with new kinetic data on the existing reaction, in conjunction with the use of modem computerised titration systems, will certainly open some new possibilities.As with other kinetic methods of analysis, catalytic titrations can play a significant role in chemical education because they require an interdisciplinary approach to chemical problems using modern teaching means (electronic measuring instruments and computers). Finally, standardisation of current terminology would allow better scientific communication. I express my sincere gratitude to Professor Vilim Vajgand, my teacher, and to Dr. Biljana AbramoviC, my pupil, who, for many years, have worked with me in this field. 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. References Szabadviry, F., “History of Analytical Chemistry,” Pergamon Press, Oxford, 1966. SzabadvBry, F., Acta Chim. Hung., 1959,20,253. BBnyai, E., “KCmiai IndikBtorok,” Miiszaki Konyvkiad6, Budapest, 1961. Mottola, H. A., Talanta, 1969, 16, 1267. Weisz, H., Allg. Prakt. Chem., 1971, 22, 98. Gary, A.-M., and Schwing, J.-P., Bull. SOC. Chim. Fr., 1972, 3657. Weisz, H., and Pantel, S . , Fresenius Z. Anal. Chem., 1973, 264,389. Mottola, H. A., CRC Crit. Rev. Anal. Chem., 1975,4,229. Hadjiioannou, T. P., Rev. Anal. Chem., 1976,3, 82. Weisz, H., Angew. Chem. Znt. Ed. Engl., 1976, 15, 150. Parry-Jones, R. L., Educ. Chem., 1976, 13,76. Greenhow, E. J., Chem. Rev., 1977,77,835. Rugu, Y., Chin. J. Pharm. Anal., 1981, 1,54. Kiss, T. F. A., Talanta, 1983,30,771.Mottola, H. A., and Mark, H. B., Jr., Anal. Chem., 1980,52, 34R. Mottola, H. A., and Mark, H. B., Jr., Anal. Chem., 1982, 54, 70R. Mottola, H. A., and Mark, H. B., Jr., Anal. Chem., 1984,56, 101R. Mottola, H. A., and Mark, H. B., Jr., Anal. Chem., 1986,58, 269R. Sandell, E. B., and Kolthoff, I. M., J. Am. Chem. SOC., 1934, 56, 1426. SzebellCdy, L., and BBrtfay, M., Z. Anal. Chem., 1936, 106, 408. Erdey, L., and Buzhs, I., Anal. Chim. Acta, 1960, 22, 524. Moldvai, R., PhD Thesis, University of Budapest, 1942. Yatsimirskii, K. B., and Fedorova, T. I., Dokl. Akad. Nauk SSSR, 1962, 143, 143. Yatsimirskii, K. B., and Fedorova, T. I., Zh. Anal. Khim., 1963, 18, 1300. BognBr, J., and SArosi, Sz., Mikrochim. Acta (Wien), 1963, 1072. 26. 27. 28. 29. 30.31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. Keily, H. J., and Hume, D. N., Anal. Chem., 1964,36,543. Margerum, D. W., and Steinhaus, R. K., Anal. Chem., 1965, 37,222. Vaughan, G. A., and Swithenbank, J. J., Analyst, 1965, 90, 594. Weisz, H., and Muschelknautz, U. , Fresenius Z. Anal. Chem., 1966,215,17. Vajgand, V. J., and GaPl, F. F., Glas. Hem. Drus. Beograd, 1966,31, 103. Gad, F. F., MSc Thesis, University of Belgrade, 1968. GaBl, F. F., PhD Thesh, University of Belgrade, 1977. Yatsimirskii, K. B., “Kineticheskie Metody Analiza,” Goskhi- mizdat, Moscow, 1963. Vajgand, V. J., and Gad, F. F., Talanta, 1967, 14,345. Weisz, H., and Klockow, D., Fresenius Z.Anal. Chem., 1967, 232,321. Mottola, H. A., and Freiser, H,, Anal. Chem., 1968,40,1266. Vajgand, V. J., Kiss, T. A., GaB1, F. F., and Zsigrai, I. J., Talanta, 1968, 15, 699. Goizman, M. S., Avt. Svid., No. 231 192, 1968t1969: Byull. Zzobret., No. 35, 1969. Klockow, D., and Garcia Beltrin, L., Fresenius Z. Anal. Chem., 1970,249,304. Mottola, H. A., Anal. Chem., 1970, 42, 630. Vajgand, V., and Gas, F., “Proceedings of the 2nd Confer- ence on Applied Physical Chemistry, VeszprCm,” Volume 1, AkadCmiai Kiad6, Budapest, 1971, p. 683. Goizman, M. S . , Zavod. Lab., 1971,10, 1164. Mottola, H. A,, MPZAppl. Notes, 1971,6, 17. Weisz, H., and Pantel, S . , Anal. Chim. Acta, 1972, 62, 361. Greenhow, E. J., Chem. Znd., 1973,697. Castle, D. A., and Greenhow, E. J., “The Determination of Weak Acids in Mineral Insulating Oils by Catalytic Thermo- metric Titration,” IP 75-15, Institute of Petroleum, London, 1975, p.1. Hadjiioannou, T. P., Koupparis, M. A., andEfstathiou, C. E., Anal. Chim. Acta, 1977, 88, 281. Abe, Sh. , Kon, Sh., and Yamagata, A. , Asahi Garasu Kogyo Gijutsu Shareikai Kenkyu Hokoku, 1977,3Q, 225. Gad, F. F., AbramoviC, B. F., Szebenyi, F. B., and CaniC, V. D., Fresenius Z. Anal. Chem., 1977,286,222. Weisz, H., and Schlipf, J., Anal. Chim. Acta, 1980, 121, 257. GaB1, F. F., and Topalov, A. S., Jenaer Rundsch., 1984, 29, 184. Moreno, A., Silva, M., PCrez-Bendito, D., and ValcBrel, M., Analyst, 1984, 109, 249. GaB1, F. F., and AbramoviC, B. F., Talanta, 1984, 31, 987. AbramoviC, B. F., Gad, F. F., and PauniC, Dj. z., Talanta, 1985,32, 549.GaB1, F. F., and AbramoviC, B. F., Talanta, 1985,32,559. Gad, F. F., AbramoviC, B. F., and Cserven&k, R. I., Microchem. J., 1986,34,295. Weisz, H., and Kiss, T., Fresenius Z. Anal. Chem., 1970,249, 302. Vajgand, V., Pastor, T., Todorovski, T., GaB1, F., and TodoroviC, M., “Proceedings of the Analytical Chemical Conference, Budapest,” Volume 1, Hungarian Chemical Society, Budapest, 1966, p. 152. Goizman, M. S., Dokl. Akad. NaukSSSR, 1969,184,599. Bognir, J., and SBrosi, Sz., Mikrochim. Acta (Wien), 1966, 534. Fedorova, T. I., and Yatsimirskii, K. B., Zh. Anal. Khim., 1967,22,283. BognBr, J., and SBrosi, Sz., Mikrochim. Acta (Wien), 1969, 463. Polyanskaya, A. A., Sb. Aspir. Rub. Kazan. Univ., Estetstv. Nauki, Khim. Geogr. Geol. Kazan, 1970,51. Vajgand, V.J., StojanoviC, D. Dj., and JeleniC, Dj. B., Glas. Hem. Drus. Beograd, 1970,35,223. Reznik, B. E., Chuiko, V. T., and Vershinin, V. I., Zh. Anal. Khim., 1972,27,395. Bark, L. S., and Ladipo, O., Analyst, 1976, 101,203. Fedorova, T. I., Yatsimirskii, K. B., Schvedova, L. V., and Ermolaeva, T. G., Zh. Anal. Khim., 1975,30,59. Vanni, A., and Amico, P., Ann. Chim., 1975,65,347. Kreingold, S . U., and Antonov, V. I., Tr. Vses. Nauchno Zssled. Znst. Khim. Reakt. Osobo Chist. Khim. Veshchestv, 1975,37, 174.750 ANALYST, JUNE 1987, VOL. 112 70. 71. 72. 73. 74. 75. 76. 77 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. Vanni, A., Amico, P., and Roletto, E., Ann. Chim., 1976,66, 213.Fedorova, T. I., Yatsimirskii, K. B., Vasil’eva, E. V., and Markichev, V. G., Zh. Anal. Khim., 1977,32, 1951. Sriraman, K., Sarma, B. S. R., and Kalidas, K., Indian Drugs, 1985,22,544. Greenhow, E. J., and Ladipo, O., Fresenius 2. Anal. Chem., 1985,321, 485. Vajgand, V., Gad, F., ZrniC, Lj., Brusion, S., and Velimi- roviC, D., “Proceedings of the 3rd Analytical Chemical Conference, August 24-29, 1970,” Volume 11, AkadCmiai Kiad6, Budapest, 1970,443. Gail, F. F., AvramoviC, B. Dj., and Vajgand, V. J., Microchem. J., 1982, 27, 231. Abe, Sh., Kon, Sh., and Matsuo, T., Anal. Chim. Acta, 1978, 96,429. Hadjiioannou, T. P., Piperaki, E. A., and Papastathopoulos, D. S., Anal. Chim. Acta, 1974, 68, 447. Hadjiioannou, T. P., and Piperaki, E.A., Anal. Chim. Acta, 1977,90,329. Gail, F. F., Soros, V. I., and CaniC, V. D., Mikrochim. Acta (Wien), 1975,II, 689. Vajgand, V. J., GaA1, F. F., and Brusin, S. S., Talanta, 1970, 17,415. Vajgand, V. J., Gall, F. F., ZmiC-Zeremski, Lj. P., and Soros, V. I., “Thermal Analysis, Proceedings of the Third ICTA, Davos,” Volume 2, Birkhauser Verlag, Basle, 1972, p. 437. Gad, F., and Bjelica, L., Chem. Anal. (Warsaw), 1976, 21, 227. Gail, F. F., KuzmiC, D. Lj., GaSi, K. M., and MiljkoviC, D. A., Microchem. J., 1984,29,7. Gahl, F. F., and AvramoviC, B. Dj., J . Thermal Anal., 1983, 26,285. Greenhow, E. J., and Shafi, A. A., Analyst, 1976, 101,421. Greenhow, E. J., and Spencer, L. E., Analyst, 1973,98,485. Greenhow, E. J., and Spencer, L. E., Analyst, 1973, 98, 90.Greenhow, E. J., and Spencer, L. E., Analyst, 1973,98, 98. Kiba, N., Suzuki, Y., and Furusawa, M., Talanta, 1981, 28, 691. Kiba, N., Sawada, Y., and Furusawa, M., Talanta, 1982, 29, 416. Kiss, T. F. A., “Thermal Analysis, Proceedings of the Third ICTA, Davos,” Volume 2, Birkhauser Verlag, Basle, 1972, p. 209. Greenhow, E. J., and Loo, L. H., Analyst, 1974, 99, 360. Greenhow, E. J., and Hargitt, R., Proc. SOC. Anal. Chem., 1973, 10,276. Greenhow, E. J., Hargitt, R., and Shafi, A. A., Angew. Makromol. Chem., 1975,48,55. Greenhow, E. J., and Shafi, A. A., Angew. Makromol. Chem., 1976,53, 187. Kiba, N., and Furosawa, M., Anal. Chim. Acta, 1978,98,343. Gail, F. F., and AbramoviC, B. F., Talanta, 1980,27,733. Ternero, M., PCrez-Bendito, D., and Valdrcel, M., Micro- chem.J., 1981, 26, 61. Piperaki, E. A., and Hadjiioannou, T. P., Chim. Chron., 1977, 6, 375. GaAl, F. F., AbramoviC, B. F., and CaniC, V. D., Zb. Rad. Prirod.-mat. Fak. Novi Sad, 1978, 8, 199. Greenhow, E. J., Analyst, 1977, 102, 584. Greenhow, E. J., and Shafi, A. A., Talanta, 1976, 23, 73. Greenhow, E. J., and Spencer, L. E., Anal. Chem., 1975,47, 1384. Greenhow, E. J., and Nadjafi, A., Anal. Chim. Acfu, 1979, 109, 129. Greenhow, E. J., and Spencer, L. E., Analyst, 1976,101,777. JovanoviC, M. S., Ga61, F. F., and Bjelica, L. J., Fresenius 2. Anal. Chem., 1971,255,277. Gad, F. F., SiriSki, J. S. , JovanoviC, M. S., and Branovatki, B. Dj., Fresenius 2. Anal. Chem., 1972,260,361. Gail, F. F., Leovac, V. M., and AvramoviC, B. Dj., Fresenius Z. Anal. Chem., 1973,266,355.Greenhow, E. J., J. Chem. SOC., 1979, 1248. Greenhow, E. J., and Spencer, L. E., Analyst, 1975,100,747. Bjelica, L. J., Vajgand, V. J., and VelimiroviC, D. Lj., Mikrochim. Acta (Wen), 1976,11, 241. Gail, F. F., KuzmiC, D. Lj., and Horvat, R. I., Microchem. J., 1978,23,417. 113. Gail, F. F., AbramoviC, B. F., and CaniC, V. D., Talanta, 1978,25, 113. 114. Greenhow, E. J., and Spencer, L. E., Talanta, 1977,24,201. 115. Dajer de Torrijos, L., and Greenhow, E. J., Proc. Anal. Div. Chem. SOC., 1979, 16,7. 116. Greenhow, E. J., and Shafi, A. A., Proc. Anal. Div. Chem. SOC., 1975, 12, 286. 117. Greenhow, E. J., Nadjafi, A., and Dajer de Torrijos, L., Analyst, 1978, 103,411. 118. Parry-Jones, R., Chem. Znd., 1974,770. 119. Ga& F. F., MiljkoviC, D. A., GaSi, K. M., and KuzmiC, D.Lj., FreseniusZ. Anal: Chem., 1982,312,618. 120. Gail, F. F., CsBnyi, M. J., and AbramoviC, B. F., Zb. Rad. Prirod.-mat. Fak. Novi Sad, 1979, 9, 387. 121. Abe, Sh., and Matsuo, T., Bunseki Kagaku, 1971,20, 1168. 122. Abe, Sh., and Takahashi, K., Bunscki Kagaku, 1974,23,1326. 123. Hotta, N., Toyama Kogyo Koto Semmon Gakko Kiyo, 1976, 10, 1. 124. Abe, Sh., and Kon, Sh., Bunseki Kagaku, 1976,25, 846. 125. Abe, Sh., Takahashi, K., and Matsuo, T., Nippon Kagaku Kakhi, 1973,5,963. 126. Gomez Hens, A., Temero, M., PCrez-Bendito, D., and Valdrcel, M., Mikrochim. Acta (Wien), 1979, I, 375. 127. Ternero, M., Pino, P., PCrez-Bendito, D., and ValcBrcel, M., Anal. Chim. Acta, 1979, 109, 401. 128. Hadjiioannou, T. P., and Timotheou, M. M., Mikrochim. Acta (Wien), 1977, I, 61.129. AbramoviC, B. F., Gad, F. F., CservenAk, R. I., and Varga, A. Gy., Microchem. J., 1984,30, 162. 130. GaB1, F. F., and AbramoviC, B. F., Mikrochim. Acta (Wien), 1982, I, 465. 131. Hotta, N., Toyama Kogyo Koto Semmon Gakko Kiyo, 1977, 11, 83. 132. Abe, Sh., Nakamura, N., and Matsuo, T., Bunseki Kagaku, 1981,30, 809. 133. GaA1, F. F., and Topalov, A. S., Zb. Rad. Prirod.-mat. Fak. Novi Sad, 1982,12, 5. 134. Timotheou-Potamia, M. M., Koupparis, M. A,, and Hadjiio- annou, T. P., Mikrochim. Acta (Wien), 1982,II, 433. 135. Greenhow, E. J., and Jeyaraj, G. L., Anal. Proc., 1982, 19, 326. 136. Timotheou-Potamia, M. M., and Hadjiioannou, T. P., Mikro- chim. Acta (Wien), 1983,II, 59. 137. Timotheou-Potamia, M. M., Koupparis, M. A., and Hadjii- oannou, T.P., Microchem. J., 1983,28, 392. 138. Marrero-Ardila, D., and Greenhow, E. J., Anal. Proc., 1983, 20, 130. 139. Raya-Saro, T., and Perez-Bendito, D., Analyst, 1983,108,857. 140. Qiao, W., Quinghua, Y., and Rugu, Y., Nanjing Yaoxueyuan Xuebao, 1984, 15, 8. 141. Greenhow, E. J., and Viiias, P., Talanta, 1984, 31,611. 142. AbramoviC, B. F., and GaAl, F. F., Microchem. J., 1985, 32, 226 143. Raya Saro, T., and PCrez-Bendito, D., Anal. Chim. Acta, 1985, 172,273. 144. Greenhow, E. J., and Ortuiio, J. A., Analyst, 1985, 110,713. 145. Sanchez-Pedreiio, C., Hernandez Cordoba, M., and Viiias, P., Talanta, 1985, 32, 218. 146. Godhino, 0. E. S., and Greenhow, E. J., Anal. Chem., 1985, 57, 1725. 147. Gail, F. F., Topalov, A. S., and VitCz, Zs. J., Microchem. J., 1986,33, 71. 148. Simpson, B. E., MSc Thesis, Oklahoma State University, 1973. 149. AbramoviC, B. F., PhD Thesis, University of Novi Sad, 1982. 150. AbramoviC, B. F., Gaal, F. F., and AbramoviC, B. K., J. Serb. Chem. SOC., 1986,51,265. 151. AbramoviC, B. F., Gahl, F. F., PauniC, Dj. z., and AbramoviC, B. K., paper presented at the 10th International Symposium on Microchemical Techniques, Antwerp, 25-29th August, 1986, abstract no. 176; to be published. 152. Vajgand, V., Bjelica, L., and Gadl, F., “Euroanalysis 11,” AkadCmia Kiad6, Budapest, 1975, Abstract 11-97. 153. Bjelica, L. J., Vajgand, V. J., Pastor, T. J., and PaliC, D. V., Glas. Hem. Drus. Beograd, 1976,41, 195. 154. Weisz, H., Pantel, S., and Ludwig, H., Fresenius Z . Anal. Chem., 1972,262,269. 155. Weisz, M., and Schlipf, J., Anal. Chim. Acta, 1982, 134,349. 156. Weisz, H., and JanjiC, T., Fresenius 2. Anal. Chem., 1967,227, 1.ANALYST, JUNE 1987, VOL. 112 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. Pantel, S., and Weisz, H., Fresenius 2. Anal. Chem., 1976, 281,211. Erdey, L., Weber, O., and BuzBs, I., Talanta, 1970,17, 1221. Weisz, H., Kiss, T., and Klockow, D., Fresenius 2. Anal. Chem., 1969, 247,248. Greenhow, E. J., and Spencer, L. E., Analyst, 1973,98, 81. Vajgand, V. J., and Todorovski, T. J., Glas. Hem. Drus. Beograd, 1966,31, 153. Kiss, T., Fresenius 2. Anal. Chem., 1970,252, 12. Vajgand, V., and Pastor, T., Glas. Hem. Drus. Beograd, 1962, 27,263. Bjelica, L. J., Vajgand, V. J., Pastor, T. J., and PaliC, D. V., Fresenius 2. Anal. Chem., 1976,280,382. Vaughan, G. A., and Swithenbank, J. J., Analyst, 1970, 95, 890. Greenhow, E. J., Chem. Ind., 1972,422. Greenhow, E. J., Chem. Znd., 1972, 466. Greenhow, E. J., and Spencer, L. E., Analyst, 1974,99, 82. Greenhow, E. J., Chem. Znd., 1974,456. Greenhow, E. J., and Dajer de Torrijos, L. A., Analyst, 1979, 104,801. Ga91, O., MSc Thesis, University of Skopje, 1982. Godinho, 0. E. S., Pasquini, C., Urzedo de Queiroz, R. R., and Aleixo, L. M., Anal. Lett., 1984, 17, 135. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 751 Greenhow, E. J., and Kashanipour, M., Analyst, 1985, 110, 1209. Kiss, T. F. A., Mikrochim. Acta, 1972,420. Kiss, T. F. A., Mikrochim. Acta, 1973,847. Kiss, T. F. A., Mikrochim. Acta, 1975,II, 471. Duewer, D. L., and Christian, G. D., Chem. Biomed., Environ. Instrum., 1979,9,373. Weisz, H., and Schlipf, J., Anal. Chim. Acta, 1983, 147, 247. Salinas Lopez, F., Berzas Nevado, J. J., and Espinosa Mansilla, Y. A., Anal. Quim., 1984, 80,125. Burton, K. C., and Irving, H. M. N. H., Anal. Chim. Acta, 1970,52, 441. Kiss, T. F. A., Zb. Rad. Prirod.-mat. Fak. Novi Sad, 1971, 1, 139. Pantel, S., and Weisz, H., Anal. Chim. Acta, 1980, 116, 421. Greenhow, E. J., and Jeyaraj, G. L,, Analyst, 1983,108,991. AbramoviC, B. F., MSc Thesis, University of Novi Sad, 1979. Greenhow, E. J., and Kashanipur, M., Analyst, 1984,109,931. Paper A61346 Received September loth, 1986

ISSN:0003-2654    

DOI:10.1039/AN9871200739

出版商:RSC    

年代:1987

数据来源: RSC

摘要:

ANALYST, JUNE 1987, VOL. 112 753 Determination of Kinetic Parameters for 3a-Hvdroxysteroid Dehydrogenase Using the Five Major Bile Acids and their Conjugates as Substrates and Correlation with their Structure and Solubility* Evriklia S. Lianidou and Panayiolis A. Siskost laboratory of Analytical Chemistry, University of Athens, 104 Solonos Street, 106 80 Athens, Greece Kinetic parameters for 3a-hydroxysteroid dehydrogenase (3a-HSD) using the five major free bile acids and their glycine and taurine conjugates as substrates and P-nicotinamide adenine dinucleotide (P-NAD+) as coenzyme were determined from initial rate measurements. Four different mathematical methods were used for the evaluation of the results: (a) the double reciprocal plot, (b) the direct linear plot, (c) the Woolf plot and (d) the Scatchard plot methods.The determined kinetic parameters were correlated with the structure of the substrates (number of free hydroxy groups) and their absolute aqueous solubilities. Keywords: Fluorimetric method; kinetics; enzymes; bile acids; 3a-h ydroxysteroid deh ydrogenase In recent years there has been increasing interest in the bile acids (BA) field.1 The determination of the three main bile acids [cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)] in biological fluids has medical significance for the diagnosis of hepatobiliary diseases2 CDCA and ursodeoxycholic acid (UDCA) are used clinically for gallstone dissolution as an alternative to surgery.3 Litho- cholic acid (LCA) is the main bile salt constituent of gallstones and studies concerning its properties and determination have increased recently because of its presence in urinary pre- cipitates and its hepatoxicity.4 Hence there is increasing interest in studying the physicochemical properties and in developing analytical methodology for BA.1 ~ 5 The kinetics of BA with 3whydroxysteroid dehydrogenase (3a-HSD) have been partially studied .6,7 Similar kinetic studies with 7a-HSD have led to the simultaneous determina- tion of primary BA based on the differences in their kinetic parameters.8 This paper describes a more systematic study on the kinetics of the enzyme-catalysed oxidation of the five major BA and their glycine (GCA, GCDCA, GUDCA, GDCA, GLCA) and taurine (TCA, TCDCA, TUDCA, TDCA, TLCA) conjugates.We have determined fluorimetrically the kinetic parameters for 3a-HSD using BA as substrates and P-NAD+ as coenzyme. The determined kinetic parameters were calculated using four different mathematical methods: the double reciprocal plot,9 the direct linear plot,10 the Woolf plot and the Scatchard plot methods.llJ2 The results show that the number and positions of free hydroxy groups play a significant role in the affinity of the substrate for the enzyme and that there is a good correlation between their Michaelis - Menten constants, Km, and the absolute aqueous solubilities of the BA. The relationship between the physicochemical properties of BA and their biological properties may explain many of the effects of BA.5 The reported kinetic differences may be used for the development of differential kinetic methods for the determina- tion of bile acids in aqueous mixtures and real samples.Experimental Apparatus All measurements were performed using a Perkin-Elmer M 512 fluorescence spectrophotometer with a 1 .OOO-cm path length with continuous stirring and at constant temperature (25.0 k 0.1 "C). The NADH fluorescence was monitored * Presented at the 2nd International Conference on Kinetics in t To whom correspondence should be addressed. Analytical Chemistry, Preveza, Greece, 9-12 September, 1986. using an emission wavelength of 455 nm and an excitation wavelength of 340 nm. The fluorescence signals were recorded and the initial slope, AFlAt, was taken as a relative measure of the reaction rate.8 The instrument was calibrated against NADH standard solutions under conditions similar to those of the reaction and the initial reaction rate was calculated as A[NADH]/At (1 mol-1 s-1).Reagents All solutions were prepared in doubly distilled, de-ionised water from analytical-reagent grade materials. 3a-HSD. 3a-HSD (E.C. 1.1.1.50). Obtained from Milli- pore as a powder from Pseudomonas testosteroni with an activity of about 0.6 U mg-1. A stock solution of 0.6 U ml-1 was prepared in 0.020 M Tris - 12.0 mM EDTA buffer (pH 7.2). This enzyme was stored at -10 "C and was stable for 1 week. Working enzyme solutions (0.03 U ml-1) were pre- pared fresh daily by appropriate dilution of the stock solution with the Tris buffer and kept in an ice-bath when in use. P-NAD+. A 55 mM solution was prepared by dissolving 0.0796 g of B-NAD+ (Sigma) (ca.98.7% pure enzymatically) in 2.00 ml of water. BA solutions. BA and their glycine and taurine conjugates were obtained from Calbiochem. Stock solutions of BA (1 mM) were prepared by dissolving appropriate amounts of their sodium salts in water. The stock solutions of LCA and its conjugates (50 p ~ ) were prepared in buffers of pH 9.5, because of the insolubilitv of this bile acid in water.13314 The stock solutions were standardised enzymatically using stan- dard NADH solution in the same buffer (Calbiochem, A grade, 101.78% pure enzymatically, disodium salt tetra- hydrate). Working BA solutions were prepared by appro- priate dilution of the stock solutions in water and at concentrations depending on the Km of the BA.The working concentration must be in the range 0.3-2 Km.9 Buffer solutions. Pyrophosphate, glycine and ethanolamine buffer solutions, each 0.1 M and pH 9.5, containing 5 mM EDTA were used for the determination of kinetic parameters. Procedure A 2.00-ml volume of buffer solution, 0.100 ml of enzyme solution and 0.100 ml of P-NAD+ solution were injected into the cell. The stirrer was started and, after the fluorescence signal of the mixture had stabilised, the reaction was initiated by injecting 0.100 ml of BA solution. The signal was recorded for about 2 min and the initial reaction rate was calculated from the slope of the initial linear part of the reaction curve (AFlAt). Absolute reaction rates, A[NADH]IAt (1 mol-1 s-l), were calculated using NADH standards.754 ANALYST, JUNE 1987, VOL.112 Results and Discussion General considerations concerning optimum pH, tempera- ture, effect of P-NAD+ concentration on the reaction rate, NADH monitoring and further details of the reaction mechan- ism were similar to those reported previou~ly.6~7J5J6 Further, the reaction of bile acids with the 3a-HSD was also studied using the commercially available NAD analogue thionico- tinamide-DPN, 3-acetylpyridinedeamino-DPN and 3-acetyl- pyridine adenine dinucleotide (APAD) (Sigma), but none of them reacted as 3a-HSD coenzyme. Determination of K,,, Values for 3a-HSD The Km values of LCA, TLCA and GLCA were determined in the three buffers (pyrophosphate, glycine and ethanolamine) each 0.100 M and pH 9.5. Table 1 shows that the nature of the buffer does not have a significant effect on the initial reaction rate and on the kinetic parameters of GLCA.However, pyrophosphate buffer was chosen for all subsequent studies because of its low background fluorescence. An increase in the concentration of the buffer, and hence an increase in the ionic strength, causes a decrease in Km.7 Four different mathematical methods were used to deter- mine the kinetic parameters: (a) the double reciprocal plot,g (b) the direct linear plot,lO (c) the Woolf plot and (d) the Scatchard plot11 methods. The double reciprocal plot is severely affected by experimental errors, whereas the proce- dure for obtaining estimates of Km and Vmm. by direct linear plots is based on distribution-free statistics and is much less dependent on assumptions than the least-squares approach to data fitting.17 The Woolf plot performs well on both well behaved data and scattered data; it is preferable to the double reciprocal plot12 and is clearly superior to the Scatchard plot for well behaved data.11 Table 2 shows the Km values of the fifteen bile acids calculated using these four methods.The calculations were carried out using suitable computer programs written in Fortran. In Table 3, K, values obtained in this work are compared with those cited in the literature.6JJ6 Even though the conditions (temperature, buffer, ionic strength and measuring techniques) were very different, many of our Km values are of the same order of magnitude as those published previously. The differences that do occur may be due to the different experimental conditions used.K, values for LCA and UDCA and their conjugates are given here for the first time. Kinetic Parameters of 3a-HSD in Relation to Structure and Solubility of BA Fig. 1 shows the stereo-formula of DCA18 and the structure of the five bile acids. The hydrophobic and hydrophilic character of each bile acid molecule and its aqueous solubility depend mainly on the number and position of free hydroxy groups. An increase in the number of hydroxy groups results in an increase in hydrophilic character. 19 According to our data the number and position of hydroxy groups in the bile acid molecule influence the kinetic parameters of 3a-HSD. Similar observations concerning both 3a-HSD and 7a-HSD have been reported previously.6920.21 This is the first systematic work to attempt to correlate the structure of the substrate with the kinetic parameters of 3a-HSD.The K, values increase as the number of hydroxy groups increases and a linear increase from LCA through CDCA to CA is observed (y = 22.6~ - 25.8; r = 0.91, n = 8). LCA, which is a monohydroxy bile acid and the most hydrophobic and least soluble of all, shows the greatest affinity as substrate to the enzyme. When the Km values of the dihydroxy bile acids are compared, it is apparent that DCA, in which the second hydroxy group (12a-) is far from the 3a-position, has the lowest K, value and the greatest affinity Table 1. Effect of buffer solutions (0.100 M) on the kinetic parameters from initial rate measurements of GLCA at pH 9.50. [P-NAD+] = 2.39 m; 25 "C Background K,/ vmax./ fluorescence, Buffer p , ~ 1-1 nmol s-1 Kob$s-l relative units Pyrophosphate .. 1.28 8.36 0.653 2.0 Glycine . . . . 1.75 8.54 0.488 17.0 Ethanolamine . . 1.22 8.99 0.737 10.0 Table 2. K, values (v) for the 3a-HSD using the five major bile acids and their glycine and taurine conjugates as substrates in 0.1 M pyrophosphate buffer, pH 9.50,25 "C Michaelis - Menten constant, K,,,/~M Bile acids LCA DCA Free . . . . . . Glycine conjugates Taurine conjugates 0.55* 1.92 0.39t 1.79 0.40$ 1.82 0.640 1.85 1.28 1.37 0.97 1.81 0.87 1.75 1.29 1.64 0.60 2.53 0.58 2.25 0.59 1.73 1.04 2.24 * Double reciprocal plot method. t Direct linear plot method. 5 Scatchard plot. $ Woolf plot. UDCA CDCA 4.45 7.7 4.53 9.5 4.79 16.9 4.90 12.7 10.2 23.9 10.9 19.2 10.1 19.2 10.3 20.4 4.76 6.4 4.60 6.5 3.46 8.0 4.03 7.9 CA 40.5 56.3 54.2 54.9 153 154 171 180 38.2 39.3 36.9 38.9 Table 3.Comparison of K, values (w) for 3a-HSD using bile acids as substrates with published values Bile acid This work* Ref. 6* Ref. 7* Ref. 16* LCA . . GLCA . . TLCA . . DCA . . GDCA .. TDCA . . UDCA . . GUDCA .. TUDCA . . CDCA . . GCDCA . . TCDCA . . CA . . . . GCA . . TCA . . 0.55 1.28 0.60 1.92 1.37 2.53 4.45 10.2 4.76 7.7 23.9 6.4 40.5 38.2 153 <<5 4.0 4.2 14 36 22 30 36 208 89 251 205 63 * Determined by the double reciprocal plot method. DCA 0 acid Position and orientation of OH groups LCA 3ff DCA 3aIl2a! U DCA 3ar.7p CDCA 3a,7ff CA 3a,7a11 2ar Fig. 1. Stereo-formula of DCA and position of hydroxy groups of the five major bile acids in manANALYST, JUNE 1987, VOL.112 755 60 . 5, g 5O A *-’ m 40 - s C 3 0 - / 0 100 200 300 400 500 Absolute aqueous soiubility/pM Fig. 2. Michaelis - Menten constants (K,) of the ma’or free bile acids with 3ar-HSD and NAD as a function of their absoiute aqueous solubilities. Linear function: y = 0.127~ - 6.43; r = 0.92, n = 5 to the enzyme. In UDCA the C7 hydroxy group is P-oriented and the two hydroxy groups lie 8 8, apart with the 7-OH function oriented towards the opposite side of the hydrocar- bon ring system, whereas in its epimer CDCA the two hydroxy groups are a-oriented, lying about 5 8, apart.19 Therefore, UDCA reacts three times faster that CDCA. The lowest affinity as substrate to 3a-HSD is shown by CA, which is the most hydrophilic of all, having three hydroxy groups (Fig.1). No substantial kinetic difference was observed among the free glyco and tauro conjugates of the same bile acid, as they have the same number of hydroxy groups in the same position. The only exception is GCA, which according to our experimental results gives a very high K, value and was not included in the calculation of the linear function. Fig. 2 shows that a reasonable correlation ( r = 0.92) exists between the K, values and the absolute aqueous solubilities of the free bile acids,l9 which depends mainly on the hydrophilic and hydrophobic properties of the molecules. LCA, which is the most hydrophobic of all, is the least soluble and the best substrate for the enzyme, whereas CA, which is the most hydrophilic, is the most soluble and has the smallest affinity to the enzyme.Solubility data are not available for all the conjugated bile acids. Our findings suggest that the number and position of hydroxy groups in BA (polarity) are the major factors that differentiate the kinetic parameters of 3a-HSD with BA as substrates and play a significant role in their affinity to the enzyme. Similarly, correlation of the polarity of BA with their affinity constants for albumin has been reported recently.22 Differences in kinetic parameters have already been used for the development of differential kinetic methods for the determination of primary BA using 7a-HSD .*3 However, 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. the kinetic determination of one BA in the presence of others with the available purity of 3a-HSD does not seem so easy, although LCA reacts about 100 times faster than CA, because CA exists in serum in higher concentrations than LCA.The authors express their thanks to Dr. A. Papanastasiou- Diamandi for valuable suggestions and constructive criticism and Mr. Thanos Tsekouras for writing the computer pro- grams. The work was supported partially by a research grant from the University of Athens. References Street, J. M., Trafford, D. J. H., and Makin, H. L. J., J. Lipid Res., 1983, 24, 491. Pennington, C. R., Ross, P. E., and Bouchier, I. A. D., Gut, 1977, 18, 903. Park, Y.-H., Igimi, H., and Carey, M. C., Gastroenterology, 1984, 87, 150. Yanagisawa, J., Ichimiva, H., Nagai, M., and Nakavama, F., J.Lipid Res., 1984, 25, 750. Hofman, A. F., and Roda, A,, J. Lipid Res., 1984, 25, 1477. Siskos, P. A., Tzouwara, S. M., and Philianos, S. M., Anal. Lett., 1980, 13, 1589. Bolt, M. G., and Boyer, J. L., in Paumgartner, G., and Stiehl, A., Editors, “Bile Acids Metabolism in Health and Disease,” Falk Symposium 24, MTP Press, Lancaster, UK, 1976, p. 285. Papanastasiou-Diamandi, A., Siskos, P. A., Hadjiioannou, T. P., and Triantafilides, I. K., Anal. Chim. Acra, 1984, 160, 243. Segel, I. H., “Biochemical Calculations,” Second Edition, Wiley, New York, 1976, p. 234. Cornish-Bowden, A,, and Eisenthal, R., Biochim. Biophys. Acta, 1978, 523,268. Cressie, N. A. C., and Keightley, D. D., Biometrics, 1981,37, 235. Wilkinson, G . N., Biochem. J., 1961, 80, 324. Small, D. M., and Admirand, W., Nature (London), 1969,221, 265. Matschiner, J. T., in Nair, P., and Kritchevsky, D., Editors, “Bile Acids,” Volume I, Plenum Press, New York, 1971, p. 16. Bergmeyer, H. U., Editors, “Prinicples of Enzymatic Analy- sis,” Verlag Chemie, New York, 1978, p. 19. Skalhegg, B. A., Eur. J. Biochem., 1975, 50, 603. Adams, K. A. H., Storer, A. C., and Cornish-Bowden A., J. Chem. Educ., 1984,61,527. Herndon, W. C., J. Chem. Educ., 1967,44,724. Igimi, H., and Carey, M., J. Lipid Res., 1980, 21, 72. Steensland, H., J. Clin. Lab. Invest., 1978, 38, 447. Skalhegg, B. A., andFausa, O., Scand. J. Gastroenterol., 1977, 12,433. Roda, A., Cappelleri, G., Aldini, R., Roda, E., and Barbara, L., J. Lipid Res., 1982, 23, 490. Papanastasiou-Diamandi, A., Diamandis, E. P., and Siskos, P. A., Clin. Chim. Acta, 1983, 134, 17. Paper A61336 Received September 1 Oth, 1986 Accepted October 28th, 1986

ISSN:0003-2654    

DOI:10.1039/AN9871200753

出版商:RSC    

年代:1987

数据来源: RSC

摘要:

ANALYST, JUNE 1987, VOL. 112 757 Kinetic = Potentiometric Determination of Amino Acids Based on Monitoring their Reaction with Dinitrofluorobenzene Using a Fluoride-selective Electrode* Eleni Athanasiou-Malaki and Michael A. Koupparist Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 104 Solonos Street, Athens 10680, Greece A kinetic - potentiometric method is described for the determination of amino acids, dopa and aspartame, based on monitoring their reaction with 2,4-dinitrofluorobenzene using a fluoride-selective electrode at pH 9.0 and 25 "C. Initial-slope and fixed-time (60 s) methods were used to construct calibration graphs, in most instances in the range 1 x 10-4-5 x 1 0 - 3 ~ . Binary mixtures of cysteine - cystine can be determined at pH 8 and 9.Dopa and aspartame were determined in commercial formulations with a precision and accuracy of 2-3% without interference from excipients or coloured or turbid solutions. The method is very simple and rapid. Reaction constants and orders of reaction were experimentally determined for various amino acids. Keywords: Kinetic determination; fluoride-selective electrode; amino acids; 2,4-dinitrofluorobenzene; aspartame and dopa The combination of the selectivity, sensitivity and simplicity of kinetic methods of analysis with the inherent advantages of ion-selective electrodes, i. e. , high selectivity, sensitivity and freedom from optical interferences, produces an excellent and versatile technique. 1 The fluoride-selective electrode (a solid-state membrane electrode with an LaF3 crystal as the electroactive material) has proved to be one of the most successful ion-selective electrodes.It has a low detection limit (1 x 10-6~) and, apart from high concentrations of OH-, is free from interferences (potentiometric selectivity coefficient @?,OH- = 0.1). Despite the extensive use of this electrode in the direct potentiometric determination of fluoride in a great variety of samples (some of the developed methods have been adopted as official methods by various associations), and of metal ions which form complexes with fluoride, the utilisation of this electrode in kinetic analysis is limited because of the low reactivity of fluoride. Recently, a sensitive potentiometric reaction-rate method for the determination of peroxidase and peroxidase-coupled reactions (glucose and cholesterol) has been proposed for clinical applications, based on the monitor- ing of the peroxidase-catalysed rupture of the covalent C-F bond in certain organofluoro compounds (4-fluoroaniline, 4-fluorophenol, etc.) in the presence of H202.2 A similar method has been proposed and evaluated for the kinetic determination of peroxidase in coloured and turbid extracts of plants, where the titrimetric and other available spectro- photometric methods are unsuitable.3 In this paper, an extension of the application of fluoride- selective electrodes to kinetic analysis is described by the development and evaluation of a kinetic method for the determination of amino acids based on monitoring their reaction with 2,4-dinitrofluorobenzene (DNFB).This so- called Sanger reagent has been proposed as a label for the terminal amino acid group in the determination of the amino acid sequence of proteins,4 for active site labelling of enzymes and for studying protein tertiary structures.5 Apart from its use in structural analysis, DNFB has been employed for the spectrophotometric determination of amino acids and primary and secondary amines,Cg amino acid nitrogen in plasma and urine (Goodwing>lo), the gravimetric determination of mor- phine (a phenolic a1kaloid)ll and other phenols,Q the * Presented at the 2nd International Symposium on Kinetics in t To whom correspondence should be addressed. Analytical Chemistry, Preveza, Greece, 9-12 September, 1986. spectrophotometric determination of isoniazidl3 and various antibiotics14 and as a derivative reagent in GLC for phenols and aminesl5 and in HPLC for amines.16 The above spectrophotometric methods have inherent disadvantages in that a long time is required for the completion of the reaction and that heating is required to speed up the reaction.Additional steps are also required for the hydrolysis of the excess DNFB and the extraction of the DNB - amino product for measurement, and the methods cannot be applied to the determination of coloured or turbid samples. There is also an absence of any kind of selectivity between the various amino acids in the spectrophotometric methods. All these drawbacks can be eliminated, with a slight decrease of the sensitivity, by using a kinetic - potentiometric method.The reaction between DNFB and various amines and amino acids has been extensively studied by spectrophotometry in organic and aqueous organic solvents.17J~ The reaction is a two-stage process involving nucleophilic addition followed by the loss of the fluoride ion. Although in some systems this second step is slow as it is subject to base catalysis,lg it is rapid in polar solvents containing a hydroxy group.20 Kinetic studies in aqueous solutions showed an increased reaction rate and the reactions were found to be accelerated by micellar catalysis, which was used to speed up spectrophotometric determinations.21 Hydroxide ions also react with DNFB ,22723 but the pH dependence of the rates of reactions between DNFB and amino acids is entirely accounted for by the effect of pH on the state of ionisation of the amino acids, and there is no evidence for base catalysis.24 Experimental Apparatus The system for potentiometric rate measurements consists of a combination fluoride electrode (Orion Model 96-49) and a conventional analogue electrometer (Corning Model 12 research pH meter) with 0.1 mV resolution connected to a multi-speed variable-span recorder.All of the measurements were carried out in a thermostated (rfiO.2"C) plastic double- wall reaction cell with continuous magnetic stirring. The electrode was stored in a 1 x 10-3 M fluoride solution between measurements and overnight. Reagents All reagents were of analytical-reagent grade and de-ionised, distilled water was used throughout.758 ANALYST, JUNE 1987, VOL.112 Amino acid and drug solutions. Working standard solutions in the range 1 X 10-5-5 x 1 0 - 2 ~ were prepared daily from 0.1000 M stock solutions made from analytical-reagent grade amino acids (Merck or Fluka), with the addition of 1 M sodium hydroxide or hydrochloric acid if required for dissolution. A stock solution (0.0100 M) of dihydroxyphenyl-L-alanine (L- dopa) was prepared daily by dissolving the appropriate amount of the drug (Fluka) in 5 ml of 0.1 M hydrochloric acid and diluting with water to 100 ml. Working standard solutions of aspartame (L-aspartyl-L-phenylalanine methyl ester) in the range 1 x 10-4-5 x l o - 3 ~ were prepared daily from a 0.1000 M stock solution made from the pure substance (Fluka). DNFB working solution, 4.0% mlV (0.215 M) in acetone.A 1 .OO-g mass of dinitrofluorobenzene (Sigma) was dissolved in 25 .O ml of acetone. This solution was stored in a sealed amber glass vial in the refrigerator and was opened only when used. It was stable for at least one month. This reagent should be carefully handled as it is vesicatory. Mixed borate buffer solution, 0.0300 M, p H 9.0. Contains 3.0 X l o - 5 ~ NaF and 5.0 x l o - 3 ~ trans-1,2- diaminocyclohexane-N,N,N',N'-tetraacetic acid (DCTA). Prepared by dissolving 11.4 g of Na2B407. 10H20 and 1.73 g of DCTA in 800 ml of water and adding 300 1.11 of 1 .OOO M NaF solution. The pH was adjusted to 9.0 by the dropwise addition of 5 . 0 ~ NaOH using a pH meter and diluting to 1 1. Other mixed buffers of similar composition which were used in the kinetic and method development study were made from acetic acid (pH 3 - 9 , NaH2P04 (pH 6-8.5) and borate (pH Standard fluoride solutions. Working standard solutions were prepared from a stock 1.0000~ NaF solution with appropriate dilution.All fluoride solutions were stored in polyethylene bottles. 9-10). Measurement Procedure Pipette 10.00 ml of a working standard or sample solution of the analyte and 5.00 ml of the pH 9.0 mixed borate buffer into the thermostated (25.0 "C) reaction cell. Start the stirrer and after the potential has stabilised (about 20 s) adjust the recorder pen to the higher potential side of the chart recorder, set to 20 mV full scale, and start recording. Initiate the reaction by the injection of 100 pl of DNFB working solution with a Hamilton micro-syringe and record the reaction curve for about 2-3 min.Evacuate the cell, wash twice with water and proceed to the next sample. A blank (H20) should be included for each calibration graph. Estimate graphically the initial slope AElAt (mV s-1) or the potential change AE (mV) for a 60 s time interval. Using the standard solutions of the analyte, construct a calibration graph of AElAt vs. concentration (initial slope method) or 10AE'S - 1 vs. concentration (fixed time method). AE for the blank should be subtracted from every AE measured for the standards or the sample. The slope of the electrode response ( S ) (also required in the kinetic study) is periodically determined by successive additions of 100 pl of 1.5 X 10-3 and 1.5 x 1 0 - 2 ~ NaF standard solutions in 10.0 ml of H20 mixed with 5.00 ml of buffer, and measuring the potential with the electrometer.Sample Preparation The sample solutions should be approximately neutralised, if required, using a drop of phenolphthalein indicator. For the determination of L-dopa and aspartame in capsules or tablets, not less than 20 tablets or the contents of not less than 20 capsules are weighed and finely powdered or mixed. An accurately weighed portion of the powder containing 0.014.1 mmol of L-dopa or 0.01-0.5 mmol of aspartame is transferred into a 100-ml calibrated flask, dissolved with water using a Vortex mixer and diluted. Portions of the clear supernatant are determined as under Measurement Procedure. Results and Discussion Study of the Electrode Characteristics In order to evaluate the operational characteristics of the fluoride-selective electrode at pH higher than the optimum (5-5.5), calibration graphs were constructed in the concentra- tion range 1 X 10-6-1 x l o - 4 ~ at pH 6, 7, 8, 9 and 10 at temperatures of 35, 45, 55 and 65°C.The slope of the electrode response (S) increased with temperature up to 55 "C and then stabilised at 65 "C at each pH studied. For the pH of interest, pH 9.0 (borate buffer), the slope was -63.7, -67.4, -72.2 and -70.6 mV decade-1, respectively, for the four temperatures studied. The slightly super-Nernstian behaviour (104-111%) and the stabilisation of the slope at 65 "C can be explained by the temperature dependence of the hydroxide interference on the fluoride-selective electrode, the variation of the solution temperature coefficient (the effect of tempera- ture on the ion activity and the expansion of the solution) and also by the variation of the temperature gradient between the internal reference solution of the fluoride combination elec- trode and the thermostated test solution.In a recent study25 this electrode was found to show a significant thermal hysteresis (a broad E - Tpattern). The lower linear concentration limit (LLCL) was practically constant in the temperature range studied but increased with an increase in pH. At pH 9, the optimum for the determina- tion, the LLCL was 1 x l o - 5 ~ instead of 1 x 1 0 - 6 ~ as at pH 5, and the calibration graph was linear up to 1 X 10-1 M. The pH 6-8 phosphate buffers and the pH 10 borate buffer gave a higher linear limit (2 X 10-5 M) because of the slight interference of phosphate and hydroxide ions.The dynamic response time was also studied by rapidly changing the fluoride concentration and measuring the time for 95% stabilisation of the new potential. The dynamic response time measured for a concentration change of 5 X 10-5 M, at an initial concentration of 1 x 10-5 M (25 "C) was 24 s in the pH 9 borate buffer and 12 s in the pH 5 acetate buffer. The electrode was 3 years old at the time of study; a faster response is expected for a new electrode.26 From the approximate exponential equation of E - t curves for step-wise concentration changes1 AE, = AEm [1-exp(-t/T)] . . . . (1) where AE, is the potential change after time t,AE, is the potential change at equilibrium state and T is the time constant of the ion-selective electrode for the particular step change, a time constant of 8 s was calculated for the concentration change of 1 x 10-5 to 5 x 10-5 M at pH 9.0 and 25 "C.If linear E - t recordings are expected during a reaction producing fluoride ions, the actual recordings will also be linear, after the initial reaction, and shifted in time by T.l The slopes AElAt, from which kinetic information is extracted, are in error by a factor of 1-exp( -tlT). It can be easily calculated that the error introduced by the response time limitations of the electrode for slope measurements obtained after time 4.6 T (i.e., 37 s) will be equal to 1% of the correct value. From the above study, it was concluded that the fluoride- selective electrode can be used for kinetic work in weak alkaline solutions (pH 9.0) if a fluoride concentration, equal to its LLCL (1 x 10-5 M), was present before the start of the reaction and all measurements were obtained after a time of 30 s.The fluoride concentration of the mixed buffer serves to establish this initial concentration. The DCTA was added to the mixed buffer to mask the possible interferences of Fe3+ and AP+. Kinetic Study of Amino Acid - DNFB Reactions The reaction of amino acids with DNFB is a well knownANALYST, JUNE 1987, VOL. 112 759 example of a nucleophilic aromatic substitution reaction, with the formation of an intermediate complex, and can be depicted by the following scheme27: kl k-1 m H 2 + (N02)2Ca3F (N02)2Ca3(F)h2R k2.(N02)2C&NHR + H+ + F- . . (2) Assuming a steady state, the rate of fluoride formation is described by the equation klk2 k-1 + k2 d[F-]/dt =- [RNH2],[DNFB],=K[RNH2],[DNFB], (3) where K is the over-all second-order rate constant. By differentiation of the Nernst equation for the fluoride elec- trode with respect to time, we have dE/dt = S’(l/[F-I) (d[F-]/dt) . . . . (4) where S’ is the slope of the E vs. In C calibration graph. Equation (4) is valid in the log - linear part of the electrode response graph, i.e., for [F-] > 1 x 10-5 M. Combining equations (3) and (4), at the start of the reaction where the initial slope is measured (with the precautions discussed previously), we have (AE/At)o = S’(l/[F-]o)K [RNH2]0 [DNFBIo . . (5) As the pH seriously affects the concentration of the reactive non-protonated amino gr0up,24 the stoicheiometric concen- tration C0,RNH2 of the amino acid can be used in equation ( 9 , with substitution of the reaction rate K by the experimental (pH 9) stoicheiometric reaction rate Kst (AHAt), = S’(l/[F-]o)Ks‘ G,RNH~[DNFB]o .. (6) The value of K can be easily calculated from Kst using the Table 1. Kinetic parameters of the reaction of amino acids with DNFB. Conditions: pH 9.0; 25°C; [DNFB],, = 1.43 X 1 0 - 3 ~ Kst ( f SD) */ K( f SD)t/ 1 mol-1 1 mol-1 Reaction order$ Amino acid s-1 x lo2 s-1 x 102 (f SD) Glycine . . Alanine . . Valine . . Leucine . . Isoleucine Serine . . Threonine Cysteine . . Cystine . . Methionine Phen ylalanine Tyrosine . . L-Dopa . . Tryptophan Aspartic acid Asparagine Glutamic acid Glutamine Lysine . .Histidine . . Arginine . . Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.77 f 0.04 0.312 f 0.001 0.33 f 0.01 0.093 f 0.004 0.1660 f 0.0008 1.57 f 0.06 0.91 f 0.02 75.52 f 0.01 4.14 f 0.03 0.212 f 0.006 2.27 f 0.06 11.5 f 0.4 0.100 f 0.002 2.61 f 0.04 0.54 f 0.06 0.147 f 0.006 0.096 f 0.002 0.120 4 0.004 0.1196 f 0.0008 0.212 f 0.002 0.234 f 0.002 0.3547 f 0.0001 9.6 f 0.3 1.577 f 0.006 1.61 f 0.06 0.47 f 0.02 0.944 f 0.005 4.0 f 0.1 33.8 f 0.8 - - 0.52 f 0.01 5.4 f 0.1 - - - 3.1 f 0.3 0.25 f 0.01 0.62 f 0.01 0.259 f 0.008 - - - 11.48 f 0.01 1.05 f 0.04 0.91 f 0.01 0.96 f 0.05 1.01 f 0.04 0.99 f 0.01 1.06 f 0.06 1.09 f 0.04 1.05 f 0.04 0.97 f 0.04 0.93 f 0.09 1.02 2 0.03 1.06 f 0.03 0.89 f 0.05 1.1 * 0.1 0.9 f 0.1 1.06 f 0.08 1.07 f 0.04 0.90 f 0.05 0.91 f 0.03 1.01 f 0.03 1.02 f 0.03 0.94 f 0.01 * Calculated by least-squares regression of equation (6) for five different concentrations of amino acid in the range shown in Table 3.t K = Kst/ai, where ai = fraction of free amino group. $ Calculated by least-squares regression of the logarithmic form of equation (6) for five different concentrations of amino acid. equation K = Kst/ui, where ui is the fraction of amino acid species in the form of RNH2 at pH 9.0, provided that the amino group is the only group which reacts with DNFB. As DNFB is also subject, to a small extent, to hydrolysis at pH 9.0 (with a rate constant of 0.12 1 mol-1 s-1 at 25 OC),Z the initial rate (AE/At)o for the reaction of DNFB with amino acids must be corrected for this.A blank of (AE/At)o, hydrolyis can be easily measured or calculated from the plots of From expenments with various concentrations of the amino acid and a constant concentration of DNFB, the reaction order with respect to the amino acid and Kst (and thus K ) can be obtained. From similar experiments, in which the DNFB concentration was varied in the range 1.8 X 10-4-7.2 X 10-4 M with constant amino acid concentration (glycine, 6.7 X l o - 4 ~ ) the reaction order for DNFB was found to be 1.02 k 0.03, which agrees with previous spectrophotometric results .21.24 Table 1 shows the results of this kinetic study at 25°C. The reaction is seen to be first order with respect to all the amino acids.Values of Ksf are given for all the amino acids tested, but the K values were calculated only for those amino acids with a single amino group and no other reactive functional group. Literature kinetic data for the reactions of DNFB with amino acids were found using spectrophotometry (monitoring the intermediate complex and the final product), at pseudo- first-order conditions and usually without correction for DNFB hydrolysis. Hence there are differences between these results and the potentiometric data reported here, which are based on the rate of fluoride liberation in the final step of the reaction. Bunton and RobinsonB reported values of K = 0.19 1 mol-1 for glycine and 0.122 1 mol-1 for phenylalanine, which (AEIAt)o VS. C R M 2 . Time Fig. 1 . Effect of pH on the glycine - DNFB reaction at 35°C.Glycme, 1 x l o - 4 ~ ; DNFB, 1.43 x l o - 3 ~ Time Fig. 2. Effect of temperature on the glycine - DNFB reaction at pH 9.0. Glycine, 5 x l o - 4 ~ ; DNFB, 1.43 x 10-3141760 ANALYST, JUNE 1987, VOL. 112 are about twice our potentiometric values. Wong and Con- ners21 reported half-lives of the reactions, under pseudo-first- order conditions and at 25 "C, from which Kst (1 mol-1 s-1) at pH 9.2 can be calculated (Kst 0.0917 for glycine, 0.0379 for alanine, 0.103 for phenylalanine, 0.150 for tyrosine, 0.367 for tryptophan and 0.0458 for glutamic acid). These values, in which the hydrolysis of DNFB is also included and which are based on the intermediate complex formation, were compared with our potentiometrically determined Kst values (at pH 9.0).The two sets of results were about the same for tyrosine, but greater for phenylalanine (4 times), glycine (5 times), alanine (12 times), tryptophan (14 times) and glutamic acid (47 times). Cysteine has the greatest Kst value because of the reactivity of the sulphydryl group, followed by tyrosine which possesses a phenolic group which reacts rapidly with DNFB . Figs. 1 and 2 show the effect of pH and temperature, respectively, on the reaction. Both variables increase the reaction rate, hence the reaction mixture has to be both buffered and thermostated. From experiments at various temperatures in the range 25-65 "C, Kst values were calculated for the glycine - DNFB reaction and using Arrhenius plots E , values of 18.5 k 0.4 kcal mol-1 for pH 9.0 and 18.4 k 0.6 kcal mol-1 for pH 10.0 were found.Wong and Connors21 reported a AH value of 16.1 k 0.5 kcal mol-1 for the alanine - DNFB reaction at pH 9.2 using spectrophotometric measurements. The hydrolysis of DNFB was also studied by the poten- tiometric procedure. The values of Kh (first-order reaction rate constant for hydrolysis) found are 6 (41) X10-6 s-l (35"C), 2.6 (k0.5) xlO-5 s-1 (45°C) and 7.6 (k1.6) XlO-5 s-1 (55°C). For the reaction with hydroxide ions, second- order rate constants, KOH (1 mol-1 s -I), of 0.05 4 0.01 (35 "C), 0.24 f 0.08 (45 "C) and 0.8 k 0.3 (55 "C) were found. Using mixed experimental hydrolysis constants from different Time Fig. 3. Typical reaction curves of the proline - DNFB reaction for calibration raph at pH 9.0 and 25 "C.DNFB, 1.43 X l o - 4 ~ . Proline: (a) blank; (f ) 1 x l o - 4 ~ ; (c) 3 x 10-4 M; (d) 5 x 10-4 M; and (e) 10 X 10-4 M Table 2. Experimental data for the construction of calibration graphs for the determination of isoleucine with initial-slope and fixed-time procedures. Initial slope: (AE/At),, = 6.42 (f0.16) X + 3.62 (f0.02)C, r = 0.99992. Fixed time: 10AEIS - 1 = 0.043 f 0.003 + 208 f 2C, r = 0.9997 Concentration/ (AElAt),-J RSD, YO RSD, Yo 10-4 M 103mVs-1 (n = 3) 10*"S -1 (n = 3) 1 .OO 1.01 1.0 0.067 0.8 3.00 1.71 0.102 5.00 2.46 0.139 8.00 3.56 0.215 10.00 4.25 0.255 30.00 - 0.666 temperatures the E , of the hydrolysis of DNFB was found to be 27.5 4 0.9 kcal mol-1. Barends et aZ.23 reported values of 5 X 10-6 s-1 for Kh and 0.12 1 mol-1 s-1 for KOH- at 40 "C using HPLC, whereas Bunton and Robinson22 reported a KOH- of 0.12 1 mol-1 s-1 at 25 "C by spectrophotometry at high (0.01 M) hydroxide concentrations.Determination of Amino Acids, L-Dopa and Aspartame As is concluded from equation (6), the initial slope of the reaction curve is linearly related to the amino acid concentra- tion as the reaction is first-order with respect to amino acids (Table 1). pH 9.0 (borate buffer) and 25 "C were selected as optimum conditions for routine determinations as a compromise of their effects on the rate of the amino acid and hydrolysis-reactions and on the operational characteristics of the fluoride-selective electrode. Amino acids with an increased reaction rate at pH 9.0, such as cysteine and tyrosine can also be determined at pH 8.0 with a decrease in the sensitivity of the determination.Fig. 3 shows typical reaction curves used for the calibration graph of an amino acid (proline). Two kinetic methods can be Table 3. Analytical characteristics of the determination of amino acids in aqueous solutions using the initial-slope method Linear range1 Amino acid 10-4 M Glycine . . . . 2-50 Alanine . . 5-100 Valine . . . . 5-100 Leucine . . 5-100 Isoleucine . . 5-100 Serine . . . . 2-50 Threonine . . 5-100 Cysteine . . 0.05-0.8 Cystine . . . . 0.5-15 Methionine . . 5-100 Phenylalanine 1-20 Tyrosine . . 0.2-20 Tryptophan . . 5-100 Asparticacid . . 2-50 Asparagine . . 1CL100 Glutamic acid 20-200 Glutamine . . 5-100 Lysine . . . . 5-100 Histidine . . 5-100 Arginine . . 5-100 Proline .. . . 1-20 L-Dopa . . . . 1-20 Aspartame . . 5-50 Slope ( f S.D .)I mV s-1 1 mol-1 25.6 f 0.5 9.92 f 0.2 8.13 f 0.2 1.76 f 0.06 3.62 f 0.02 21.6 f 0.06 9.30 f 0.09 874 f 7 104 f 1 6.98 f 0.6 40.6 f 0.5 155 f 1 8.39 k 0.09 30.5 f 0.9 2.01 f 0.07 1.10 2 0.05 4.61 f 0.04 5.14 f 0.09 4.03 f 0.09 4.12 2 0.09 11.5 2 0.2 4.61 f 0.3 48.4 2 0.9 Detection RSD, % limit/ (n=3) 1 0 - 4 ~ 0.999 0.9 0.5 0.999 1.2 1 0.997 1.2 1 0.998 2.7 3 0.99993 1.0 3 0.999 1.0 0.5 0.999 1.3 1 0.998 2.0 0.01 0.998 2.0 0.1 0.998 2.0 2 0.9997 2.1 0.3 0.99991 1.7 0.07 0.998 2.1 1 0.9990 2.5 0.4 0.998 2.7 5 0.998 1.2 10 0.999 2.0 2 0.999 1.6 2 0.9991 2.0 2 0.9994 0.5 2 0.9990 2.0 0.5 0.999 1.5 0.2 0.999 1.8 0.2 Table 4. Comparison of results obtained by kinetic - potentiometric and established methods for the determination of dopa and aspartame Amount found 2 SDImg (n = 3) Nominal Present Reference content/mg method method* Dopa: Madopar capsules (Roche).. . . . . 50 52.7 f 0.8 52.0 f 0.7 100 105 f 2 107 k 3 200 204 k 3 207 f 4 Aspartame: Canderel tablets (Searle) . . . . . . 18 18.5 f 0.5 19.0 f 0.7 * USP spectrophotometric method for dopa, HPLC for asp art ame .29ANALYST, JUNE 1987, VOL. 112 761 Table 5. Recovery of aspartame (1.00 x l o - 3 ~ ) from synthetic mixtures with various excipients (100 mg ml-l) Excipient NaCl . . . . . . Carbobo14000* . . Magnesium stearate Talc . . . . . . Lactose . . . . Mannitol . . . . Galactose . . . . Sorbitol . . . . Sugar . . . . . . * Carboxypolymethylene. Recovery, % . . 101.0 . . 98.9 . . 97.1 . . 98.4 . .102.5 . . 98.7 . . 100.7 . . 98.3 . . 102.1 Mean 100.2 Table 6. Determination of cysteine - cystine binary mixtures. Slopes of 54.09; Kc,,,, = 991.1; and Kc,,,, = 93.37 calibration graphs (mV s-1 1 mol-1): KcysH, 8 = 109.6; KCYS,, 8 - - Amino acid taken/M Amino acid found*/M Cysteine Cystine Cysteine Cystine 1.00 x 10-5 5.00 x 10-3 9.20 x 10-6 5.14 X 10-3 2.00 x 10-5 1.00 x 10-3 1.91 x 10-5 1.03 x 10-3 3.00 x 10-5 5.00 x 10-4 2.90 x 10-5 5.07 x 10-4 * Average of three determinations (initial-slope method). used for such graphs, the initial-slope or the fixed-time method. It was found that a 60-s time interval for AE reading gave the most linear calibration graphs and that the initial- slope method is more precise. Table 2 shows typical examples of calibration graphs for L-isoleucine obtained using the two methods; excellent linearity and reproducibility were obtained.Data obtained for the amino acids tested are shown in Table 3, which summarises the useful analytical ranges, slopes of the calibration graphs and standard deviations, precision (obtained by triplicate measurements of a medium standard) and the detection limit (the concentration giving an initial slope three times the slope of the blank). Exact information about the relative reactivity of the various amino acids cannot be concluded from the slopes of the calibration graphs as the expegments were run over a 2-year period and the slope of the electrode response was gradually decreased. The proposed method was applied to the determination of L-dopa (amino acid) and aspartame (a-dipeptide) in phar- maceutical formulations.The latter is a very promising sweetener. The results of the analysis of some commercial formulations and comparisons with official or established methods are shown in Table 4. A recovery study performed on synthetic mixtures of aspartame with various excipients gave a mean recovery of 100.2% (range 98.4-102.5%) (Table 5). Attempts to determine aspartame in cola drinks failed because of the high concentration of phosphoric acid present. Phos- phate ions in high concentrations interfere with the response of the fluoride-selective electrode and an unstable potential base line was obtained. Determination of Amino Acids in Binary Mixtures Pairs of amino acids with different reaction rates at different pH can be determined in binary mixtures using the method of proportional equations.30 Cysteine (CYSH) and cystine (CYS2) in binary mixtures were successfully determined by measuring the initial slope of the reaction ( R ) for the mixture at pH 8 and 9.According to the method of proportional equations where KcY~H and Kcys2 are the slopes of the calibration graphs of cysteine and cystine, respectively, at pH 8 or 9. Table 6 shows the results obtained. Conclusions The proposed method is a very simple and rapid way of determining amino acids, either individually or in some instances in binary mixtures. Dopa and aspartame can be determined in pharmaceutical formulations without interfer- ence from the excipients in turbid and coloured solutions. Amines, hydrazides, mercaptans and some phenols interfere.The work also shows the usefulness of ion-selective electrodes in kinetic studies. 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. References Efstathiou, C. E., Koupparis, M. A., and Hadjiioannou, T. P., Ion-Sel. Electrode Rev., 1985, 7, 203. Siddigi, I. W., Clin. Chem., 1982, 28, 1962. Skaltsa, H. D., Koupparis, M. A., and Philianos, S. M., J. Assoc. Off. Anal. Chem., 1986, 69, 1006. Sanger, F., Biochem. J., 1945,39,507. Singer, S. J., Adv. Protein Chem., 1967, 22, 1. McIntire, F. C., Clements, L. M., and Sprouli, M., Anal. Chem., 1953, 25, 1757. Kolbezen, M. J., Eckert, J. N. E., and Brefschneider, B. F., Anal. Chem., 1962,34,583. Dubin, D. T., J. Biol. Chem., 1960, 235, 783. Goodwin, J. F., Clin. Chim. Acta, 1968, 21, 231. Goodwin, J. F., Clin. Chem., 1968, 14, 1080. Garratt, D. C., Johnson, C. A., and Lloyd, C. J., J. Pharm. Pharmacol., 1957,9, 914. Zahn, H., and Wurz, A., Z. Anal. Chem., 1951, 134, 183. Poole, N. F., and Meyer, A. E., Proc. SOC. Exp. Biol. Med., 1958,98,375. Ryan, J. A., J. Pharm. Sci., 1984, 73, 1301. Timbrell, J. A., Wright, J. M., and Smith, C. M., J. Chromatogr., 1977, 138, 165. Lawrence, J. F., and Frei, R. W., “Chemical Derivatization in Liquid Chromatography,” Elsevier, Amsterdam, 1976. Bunnett, J. F., and Garst, R. H., J. Am. Chem. SOC., 1965,87, 3875. Forlani, L., J. Chem. Res. ( S ) , 1984, 260. Bunnett, J. F., and Randall, J. J., J. Am. Chem. SOC., 1958,80, 6020. Bunton, C. A., and Robinson, L., J. Org. Chem., 1970, 35, 733. Wong, M. P., and Connors, K. A., J. Pharm. Sci., 1983, 72, 146. Bunton, C. A. and Robinson, L., J. Org. Chem., 1969,34,780. Barends, D. M., Blauw, J. S., Mijnsbergen, C. W., Govers, C. J. L. R., and Hulshoff, A., J. Chromatogr., 1985,322, 321. Bunnett, J. F., and Hermann, D. H., Biochemistry, 1970, 9, 816. Efstathiou, C. E., Pentari, J. G., and Hadjiioannou, T. P., Anal. Chem., 1986, 58, 233. Mertens, J., Van den Winkel, P., and Massart, D. L., Anal. Chem., 1976,48,272. Connors, K. A., “Reaction Mechanisms in Organic Analytical Chemistry,” Wiley, New York, 1972, p. 274. Bunton, C. A., and Robinson, L., J. Am. Chem. SOC., 1970, 92, 356. “United States Pharmacopeia,” 20th Revision, Mack, Easton, PA, 1900, p. 441. Garmos, R. G., and Reilley, C. N., Anal. Chem., 1962, 34, 600. Paper A61343 Received September loth, I986 Accepted February 9th, 1987

ISSN:0003-2654    

DOI:10.1039/AN9871200757

出版商:RSC    

年代:1987

数据来源: RSC

摘要:

ANALYST, JUNE 1987, VOL. 112 763 Kinetic - Potentiometric Determination of Monosodium Glutamate in Soups and Soup Bases and of Glutamic Dehydrogenase" Dimitrios P. Nikolelis Chemistry Department, Laboratory of Analytical Chemistry, University of Athens, 104 Solonos Street, 106 80 Athens, Greece A simple and selective procedure has been developed for the determination of glutamic acid and glutamic dehydrogenase by using an ammonia gas-sensing electrode. Glutamic acid is deaminated by bacterial glutamic dehydrogenase in the presence of P-NAD+. A linear relationship exists between the initial rate of ammonia release and the substrate concentration or the enzyme activity. Optimum conditions for the determinations were established. Glutamic acid in the range 1 .O X 10-4-1 .O x 10-3 M and enzyme in the range 0.0500-0.750 U can be determined with relative errors of about 2%.A method is given for determining monosodium glutamate in soups and soup bases. The method was compared with the official AOAC method; satisfactory agreement was achieved. Keywords: Ammonia gas-sensing electrode; kinetic - potentiometric determination; i-glutamic acid determination; glutamic deh ydrogenase determination; soup and soup bases analysis Monosodium glutamate (MSG), a widely used food additive, has been claimed to be responsible for the Chinese restaurant syndromelJ and a controversy exists around a report that it produces brain lesions in the neonate m0use.3~4 Therefore, there is a need for a rapid, simple and sensitive method for the determination of this compound. Non-enzymatic methods for the determination of MSG have been based mainly on chromatographic techniques,s-7 but most of the proposed procedures are time consuming, cumbersome and, in some instances, irreproducible.On the other hand, potentiometric sensors for the steady-state measurement of MSG have been developed,g79 but these methods have not been applied to the determination of this compound in foods. In this paper, an initial-rate kinetic method for the determination of L-glutamic acid is described. The method is based on the use of glutamate dehydrogenase to deaminate the acid. Deamination is followed by potentiometric measure- ment of the initial rate of ammonia production. Optimum operating conditions have been established to ensure short measurement times and maximum sensitivity for glutamic acid in the range 1.0 x 10-4-l.0 x 10-3 M.The method is suitable for the determination of MSG in soups and soup bases. The method has also been used to determine glutamate dehy- drogenase in aqueous solutions by adding a constant concen- tration of the substrate to the sample and measuring the initial rate of the potential change. Experimental Apparatus The apparatus and the electrode assembly used were essen- tially identical with those described previously. 10 An Orion Model 95-10 ammonia gas-sensing electrode was used and potentials were recorded with a Heath - Schlumberger Model SR-255 potentiometric recorder. A Corning Model 12 research pH meter, acting as a voltage monitor, was inserted between the electrode and the recorder to match the high output impedance of the electrode with the relatively low impedance of the recorder.All measurements were made with a thermostated cell at 37 k 0.2 "C. When not in use, the electrode was kept in 0.10 M citric acid buffer (pH 43.11 * Presented at the 2nd International Symposium on Kinetics in Analytical Chemistry, Preveza, Greece, 9-12 September, 1986. Reagents All solutions were prepared with de-ionised, distilled water from analytical-reagent grade materials and were stored in a refrigerator when not in use. Tris - HCl buffer, 0.10 M, pH8.5. Dissolve 12.1 g of Tris in water, adjust the pH to 8.5 with 6 M hydrochloric acid solution and dilute to 1 1 with water. P-Nicotinamide adenine dinucleotide (P-NA D+) solution, 0.0060 M.Dissolve 0.400 g of P-NAD+ in water and dilute to 100 ml. Adenosine 5'-diphosphate (ADP) solution, 0.50 mM. Dis- solve 21.4 mg of ADP in water and dilute to 100 ml. L-Glutamic dehydrogenase stock solution, 25 .O U ml-1. Dilute 0.050 ml of enzyme suspension [L-glutamic dehydro- genase, E.C. 1.4.1.3, from bovine liver (Sigma Chemical), solution in 50% glycerol, 37 U mg-1 of protein, defined and measured as recommended by the supplier] with 0.50 mM ADP solution to 10.0 ml. Prepare working standard solutions containing 0.500,2.50 and 7.50 U ml-1 of enzyme from the 25 U ml-1 solution by dilution with 0.50 mM ADP solution (prepare just before use). L-Glutamic acid stock solution, 0.0100 M. Dissolve 0.0735 g of glutamic acid in 1.5 ml of 1 M hydrochloric acid and dilute to 50.0 ml with water.Prepare working standard solutions of concentrations 1.00 x lO-4,3.00 x lO-4,6.00 x lo-4and 1.00 X 10-3 M from the stock solution by dilution with water (prepare just before use). Procedure Preparation of samples For products in the dry form, pulverise them uniformly in a mortar and weigh 0.80 g of sample. For undiluted, concen- trated soups, homogenise the entire undiluted contents of a can in a blender and weigh 4.0 g of sample. For consommk (clear, condensed) soup, weigh 0.45 g of sample. Dilute the samples to about 35 ml with water at room temperature and mix until all water-soluble substances are in solution. Quanti- tatively transfer the sample into a 50-ml calibrated flask, dilute to volume with water and mix (solution A). Transfer 1.00 ml of solution A into a 10-ml calibrated flask, dilute to volume with water and mix.Determination of MSG in soups Pipette 4.00 ml of 0.1 M Tris - HCl buffer (pH 8.5) and 1.00 ml of 6.00 X 10-3 M P-NAD+ into a thermostated cell and injectANALYST, JUNE 1987, VOL. 112 into this solution 0.200 ml of glutamic dehydrogenase stock solution (about 5 U). Immerse the ammonia electrode in the solution and start the stirrer; when the electrode potential has stabilised (1-2 min), start the recorder and pipette 1.00 ml of soup sample into the reaction cell. Record the potential, which decreases during the reaction, for 4-6 min. Determination of the blank Treat standard aqueous solutions containing 1.00 X lO-4,5.00 x 10-4 and 10.0 x 10-4 M glutamic acid by the above procedure, and plot the initial reaction rates against the concentration of the standards.The intercept of the linear plot on the abscissa gives the blank value. Determination of glutamic dehydrogenase Pipette 4.00 ml of 0.1 M Tris - HCl buffer (pH 8.5), 1.00 ml of 6.0 x 10-3 M P-NAD+ and 1.00 ml of 0.0100 M glutamic acid into the thermostated cell. Immerse the ammonia electrode in the solution and proceed as in the determination of MSG. Start the reaction by injecting 0.100 ml of glutamic dehydro- genase standard solution or sample solution into the cell with a 0.1-ml Hamilton microsyringe. Calculations Calibration is carried out by plotting the initial slope (AE min-1) against the molar concentration of glutamic acid or glutamic dehydrogenase activity (U).Results and Discussion L-Glutamic acid is specifically deaminated in the presence of glutamic dehydrogenase according to the equation L-glutamate + NAD+ + H20 - 2-oxoglutarate + When the glutamic acid sample solution is pipetted into the buffered system containing the enzyme and B-NAD+, the potential of the ammonia gas-sensing electrode decreases with time and the initial rate of potential change is directly related to the concentration of substrate added. The same reaction can be used to determine the enzyme activity. The effects of buffer composition and pH were studied in order to establish conditions that provide good sensitivity and short measurement times. The initial rates of ammonia production in different buffers and at various pH values were monitored at 37 "C, as shown in Fig.1. Borate and glycine buffers (pH 8.0) were found to inactivate the enzyme NADH + NH4+ 40 30 > lu & 20 1c 0 1 2 3 Timeimi n 4 Fig. 1. Recorded graphs of potential vs. time for the glutamic dehydrogenase catalysed hydrolysis of glutamic acid, in the presence of NAD+ at (A) pH 9.5 (carbonate buffer); (B) pH 8.0 ( hosphate buffer ; (C) H 8.0 (imidazole buffer); (D) pH 7.5 (fris - HC1 buffer ; (E) p!I 9.0 (Tris - HC1 buffer); F) H 8.0 (triethanolamine buffer ; (G) pH 8.0 (Tris - HCl bufferf; &) pH 8.5 (Tris - HCI buffer I . Enzyme activity 0.625 U; other conditions as in procedure for glutamic dehydrogenase determination completely, whereas phosphate buffers (pH 8.0) inhibited the enzyme. The enzyme acitivity in imidazole buffers (pH 8.0) was relatively low, and Tris - HCl and triethanolamine buffers seemed the most satisfactory.With Tris - HCl buffer, there was an increase in the initial rate with increase in pH. Reaction rates were measured at pH 8.5, however, to ensure a better precision and larger buffer capacity. The effect of variations in the NAD+ concentration on the sensitivity of the enzyme determination was studied. The sensitivity of the method increased (whereas the blank remained almost constant) as the concentration of NAD+ was increased up to 6.0 x 10-3 M , which was chosen for the recommended procedure. Similar results were obtained for substrate determinations. The sensitivity of the method increases with increase in the amount of enzyme used up to 5 U of glutamic dehydrogenase, which was used in the recom- mended procedure.Variations in the glutamic acid concentration used in the enzyme determination showed that the slope of the calibration graph increased with increasing concentration of substrate used, up to 0.010 M glutamic acid, whereas the blank remained almost constant; a further increase in substrate concentration did not improve the sensitivity or the blank. A study of the effect of temperature in the range 2637 "C showed that the initial rate of potential change and the sensitivity of the method increased sharply with increasing temperature. A temperature of 37 "C is recommended. The selectivity of the method is good. Although glutamic dehydrogenase has been reported to catalyse oxidations with hydroxy acids (in the presence of phenylhydrazine), only L-glutamic acid reacts in the glutarnic dehydrogenase system if a Tris buffer is used and there are no interferences from D-isocitric, L-rnalic, gluconic, oxalic, L-lactic and DL-citric acids.12 The interference from other amino acids (e.g., DL-norvaline , L-valine , L-methionine , L-a-aminobutyrate , DL- norleucine, L-alanine, L-leucine and L-isoleucine) is negli- gible, as the oxidation of L-glutamate is stimulated by ADP.The oxidation of the other amino acids, in contrast, is inhibited by ADP.13 It was also found that when starch, lactose and glucose are present with glutamic acid in a 10 : 1 excess, no effect on the glutamic acid determination is observed. The stability of the reagents used has been discussed previously.14 It is well known that glutamic dehydrogenase in solution and the absence of protective agents is rapidly inactivated.15.16 Therefore, the stability of the enzyme in the presence of various stabilisers was examined.It was found that a 200-fold dilution of the enzyme suspension with a solution containing ADP at concentrations higher than 0.50 mM stabilised the enzyme for at least 1 d. It was further found that the initial reaction rate was a maximum for this ADP concentration, as the deamination of glutamate is stimulated by ADP.13715 The addition of bovine serum albumin and human serum is also said to stabilise the enzyme.17 A 200-fold dilution of the enzyme suspension with human serum stabil- Table 1. Determination of glutamic acid in aqueous solutions Amount taken*/ Amount found?/ 1 .oo 1.02 3.00 3.07 5.00 4.91 6.00 5.91 8.00 8.12 9.00 9.21 10.0 9.80 10-4 M 10-4 M Relative error, Yo +2.0 +2.3 -1.8 -1.5 +1.5 +2.3 -2.0 * Initial concentrations. t Single runs; the regression equation is AE min-1 = 2.57 X 104 [glutamic acid] + 3.32; correlation coefficient = 0.9997.ANALYST, JUNE 1987, VOL.112 765 Table 2. Determination of glutamic dehydrogenase (GDH) in aqueous solutions Amount taken*/ Amount found?/ Relative error, U U O/O 0.0500 0.0495 -1.0 0.0750 0.0759 +1.2 0.125 0.122 -2.4 0.250 0.254 +1.6 0.375 0.368 -1.9 0.500 0.508 +1.6 0.675 0.611 -2.2 0.750 0.766 +2.1 * Activities in 0.1 ml of sample. t Single runs; the regression equation is AE min-1 = 30.3(GDH activity) + 2.10; correlation coefficient = 0.9996. Table 3.Determination of monosodium glutamate in soups and soup bases MSG found, % m/m Product Proposed method* AOAC methc Beef bouillon cubes (A) . . Beef bouillon cubes (B) . . Chicken bouillon cubes (A) . . Chicken bouillon cubes (B) . . Minestronesoupmix . . . . Beefnoodlesoupmix . . . . Vegetablesoupmix . . . . Tomatocream . . . : . . Condensed chicken broth . . Condensedbeefbroth . . . . 9.83 9.86 9.76 9.72 2.38 4.22 10.62 3.92 1.24 0.76 * Double runs; standard additions method. 9.70 9.80 9.70 9.95 2.30 4.40 10.57 4.00 1.28 0.74 ised the enzyme, for at least 90 min. The use of 5% bovine serum albumin for dilution resulted in stability of the enzyme preparation for about 60 min. The activity of these prepara- tions. however, decreased by 22 and 34% in 90 and 120 min, respectively.When bovine serum albumin was used for dilution, the activity of the resulting preparation was about 66% of the initial value. Immobilisation of the enzyme on the electrode was further attempted in an effort to reduce the cost per analysis and simplify the procedure. When an enzyme suspension contain- ing 0.50 mM ADP was placed directly on the electrode surface, the enzyme activity was lost in 1 h. Other procedures were investigated for the immobilisation of the enzyme,l8J9 such as cross-linking with glutaraldehyde on albumin or on activated glass beads (in both instances an amount of 0.50 mM ADP was added). These procedures stabilised the enzyme, but the activities of these preparations were about 10% of the initial enzyme activity in solution.Applications Under the optimum conditions described, there was a linear relationship between the initial rate of evolution of ammonia and glutamic acid concentration up to 1 X 10-3 M. Results for the determination of glutamic acid in aqueous solutions are given in Table 1. Glutamic acid in the range 1.00 X 10-4-1.00 X 10-3 M was determined with an average error of 1.9%. The relative standard deviation was 2.0% for a 5.00 X 10-4 M glutamic acid sample (six results). Similarly, there was a linear relationship between the initial rate of evolution of ammonia and enzyme activity. With aqueous solutions, glutamic dehydrogenase was determined in the range 0.0500-0.750 U in 0.1 ml of sample with an average error of 1.8% (Table 2). The relative standard deviation for 0.375 U of glutamic dehydrogenase was 2.2% (six results).Application of the procedure to commercially available soups and soup bases gave the results in Table 3. Monosodium glutamate was determined by both the pro- posed method and the official AOAC method.20 Because of the cost of preparing calibration graphs and because a reference soup without MSG is not available for calibration, a standard additions procedure was used to determine MSG in soups by the proposed method. In general, there was satisfactory agreement between the results obtained by the two methods. The proposed method is faster than chromato- graphic procedures (4-6 min compared with more than 2 h using chromatography) and there is no need for separation steps. The accuracy of the proposed method was checked indirectly by means of recovery experiments carried out with three representative soup samples in which MSG was added to the sample.Subtraction of the MSG content of the soup showed that the recovery of added MSG (1.00 x 10-4-7.00 X 10-4 M) was satisfactory (97.3-102.9%, mean 100.5%). The author is indebted to the State Chemical Laboratory (Athens, Greece) for the analysis of the soups and soup bases. This work was supported by research grants from the University of Athens. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. References Schaumburg, H. H., andByck, R., N. Engl. J. Med., 1968,279, 105. Schaumburg, H. H., Byck, R., Gerstl, R., and Mashman, J. H., Science, 1969, 163, 826. Olney, J. W., Science, 1969, 164,719. Heywood, R., James, R.W., and Worden, A. W., Toxicol. Lett., 1977, 1, 151. Fernadez-Flores, E., Johnson, A. R., and Blomquist, V. H., J. Assoc. Off. Anal. Chem., 1969, 52, 744. Bailey, B. W., and Swift, H. L., J. Assoc. Off. Anal. Chem., 1970, 53, 1268. Coppola, E. D., Christie, S. N., and Hanna, J. G., J. Assoc. Off. Anal. Chem., 1975,58,58. Hikuma, M., Obana, H., Yasuda, T., Karube, I., and Suzuki, S., Anal. Chim. Acta, 1980, 116, 61. Yanushyavichyute, R., Cheskis, B. I., Paulyukoyis, A. B., and Kazlauskas, D. A., Zh. Anal. Khim., 1983,38, 498. Efstathiou, C. E., Nikolelis, D. P., and Hadjiioannou, T. P., Anal. Lett., 1982, 15A, 1179. Keelye, D. F., and Walters, F. H., Anal. Lett., 1983, 16A, 1581. Guilbault, G. G., Sadar, S. H., and McQueen, R., Anal. Chim. Acta, 1969,45, 1. Tomkins, G. M., Yielding, K. L., Curran, J. F., Summers, M. R., and Bitensky, M. W., J. Biol. Chem., 1965,240,3793. Nikolelis, D. P., Anal. Chim. Acta, 1985, 167, 381. Frieden, C., J. Biol. Chem., 1959, 234, 815. Di Prisco, G., and Strecher, H. J., Biochim. Biophys. Acta, 1966, 122, 413. Ellis, G., and Goldberg, D. M., Clin. Chim. Acta, 1972, 39, 472. Brown, G., Thomas, D., Gelff, G., Domurato, D., Berjonneu, A. M., and Guille, C., Biotechnol. Bioeng., 1973, 15, 359. Marshall, D. L., Biotechnol. Bioeng., 1973, 15, 447. “Official Methods of Analysis of the Association of Official Analytical Chemists,” Twelfth Edition, AOAC, Washington, DC, 1975, Sections 20.149-20.151. Paper A61329 Received September loth, 1986 Accepted December 8th, 1986

ISSN:0003-2654    

DOI:10.1039/AN9871200763

出版商:RSC    

年代:1987

数据来源: RSC