摘要:

Proceedings of the Society for Analytical Chemistry Analytical Division Chemical Society Proc. SOC. Analyt. Chem. Vol. 9 No. I Pages 1-24 CONTENTS Specialist Subject Groups . . I Microchemical Methods Group I Annual Meeting of Honorary Secretaries of Groups and Regions .. .. .. 3 Reports of Meetings . . .. 4 SAC Centenary Symbol . . 4 “Modern Analytical Techniques SAC “Belfast” Symposium Glasgow 1972 .. . . 22 Papers Accepted for The Analyst 24 Notice .. .. .. .. 24 Forthcoming Meetings Back Cover for Bio-active Substances” . . 6 January 1972 PAYCAL Vol. 9 No. I PROCEED I N G S January 1972 OF THE SOCIETY FOR ANALYTICAL CHEMISTRY ANALYTICAL DIVISION CHEMICAL SOCIETY Hon. Secretory W. H. C. Shaw Officers of The Society for Analytical Chemistry and the Analytical Division of The Chemical Society C.Whalley President Hon. Treasurcr G. W. C. Milner Secretary Miss P. E. Hutchinszn 9/10 SAVILE ROW LONDON WIX IAF Tolephono 01-734 9864 Editor J. B. A t t r i l l Assistant Editor P. C. Weston Hon. Assistant Secretary D. I. Coomber Proceedings is published by The Society for Analytical Chemistry and distributed t o all members of the Analytical Division and t o subscribers with The Analyst; subscriptions cannot be accepted for Proceedings alone. Single copies can be obtained direct from The Chemical Society Publications Sales Office Blackhorse Road Letchworth Herts. SG6 IHN (NOT through Trade Agents) price 25p. post free. Remittances MUST accompany orders. 0 The Society for Analytical Chemistry INDEXES 1971 The Index to Volume 8 of the Proceedings and the Index to Volume 96 of The Analyst will be distributed to Members and to Subscribers in February. The Index to Volume 21 of Analytical Abstracts covering July to December 1971 is expected to be ready for distribution during April and copies will automatically be sent to Members and Subscribers entitled to receive them.

ISSN:0037-9697    

DOI:10.1039/SA97209FX001

出版商:RSC    

年代:1972

数据来源: RSC

摘要:

SOCIETY FOR ANALYTICAL CHEMISTRY ANALYTICAL DIVISION CHEMICAL SOCIETY Forthcoming Meetings-continued from buck covey Februar y-continued Wednesday 16th WESTERN REGION and EDUCATION AND TRAINING GROUP. BRISTOL N Discussion on “Imaginative Novel Practical Work.” to be introduced by G. Department of Chemistry The University Bristol ; 6.30 p.m. “Inorganic Analysis by Gas Chromatography,” by W. I. Stephen. Haworth Lecture Theatre Haworth Building The University Edgbaston RADIOCHEMICAL METHODS GROUP and JOINT PHARMACEUTICAL ANALYSIS GROUP. “The Philosophy of the Pharmaceutical Control of Short-lived Kadionuclide “The Production of Short-lived Radioactive Substances for Rledical Research,” “The Use of Isotopically Labelled Compounds in Drug Substances and Forniu- Pharmaceutical Society of Great Britain 17 Bloomsbury Square London NORTH WEST REGION AUTOMATIC METHODS GROUP and ELECTROANALYTICAL Speakers Professor D.A. Barker A. Townshend B. Fleet and 11. B. Roodyn. Manchester; 10 a.m. CHROMATOGRAPHY AND ELECTROPHORESIS GROUP. Discussion on “Accuracy and Precision in Quantitative Chromatography,” to be The Leicester Lounge 44 Glasshouse Street London W. 1 ; 6.15 p.m. THERMAL ANALYSIS GROUP on “Kinetic Data from Thermal Analysis.” Nickless B. Bush V. J . Jennings and J . D. R. Thomas. Thursday 17th MIDLANDS REGION. BIRMINGHAM Birmingham 15; 6.30 p.m. Thursday 17th LONDON Preparations,” by J . C. Charlton. L>. J . Silvester. lated Product Stability Studies,” by H. E. Hudson and &I. F. Jones. W.C.l; 2.30 p.m. GROUP on “The Use of Enzymes in Analysis.” Thursday 17th MXNCHESTER Friday 18th LONDON introduced by G.W. Goodman. Tuesday and \Vedncsday Tuesday 22nd 22nd and 23rd Tour of Research Laboratories 3 p.m. ORMSKIRK Wednesday 23rd “Some Observations on the Shapes of TE Curves and the Kinetic Parameters “Interpretation of Data from Programmed Thermogravimetry,” by J . R. “Attempts to Measure Kinetics of Reactions in the Seighbourhood of Hot “Some Problems Encountered in Obtaining Kinetic Data from Thermoanalytical “Kinetic Studies Using Thermal Volatilisation Analysis,” by I. C. McNeill. “Kinetics of Polymerisation,” by K. E. J . Barrett. “Kinetics of Decomposition of Inorganic Solids,” by M. C. Ball. Pilkington Brothers Ltd. Research & Development Laboratories Lathom NORTH WEST REGION jointly with the Liverpool Scction of the CS. “The Chemist a t Sea,” by J.P. Riley. Liverpool; 6.30 p.m. Derived from these Curves,” by M. A. Hughes. MacCallum. Wires,” by R. A. W. Hill. Measurements,” by M. D. Judd. Ormskirk Lancs; 9.30 a.m. Thursday 24th LIVERPOOL SOCIETY FOR ANALYTICAL CHEMISTRY ANALYTICAL DIVISION CHEMICAL SOCIETY Forthcoming Meetings January Friday 21st WESTERN REGION Annual General Meeting. CARD IFF “Fibre Examination Techniques for Identification and Characterisation University of Wales Institute of Science and Technology Cathays Park Cardiff ; Purposes,” by J. E. Ford. 6 p.m. Friday 21st SCOTTISH REGION. GLASGOW “Water for Industry-Production and Quality Control,” by T. D. Rees. Univer- sity of Strathclyde Glasgow ; 4 p.m. Thursday 27th MIDLANDS REGION. BIRMINGHAM “Surface Contaminants on Organic Materials (Practical ilpplications of *Iuger The University Edgbaston Birmingham ; 6.30 p.m.Spectroscopy),” by Jh-. Barber. February Wednesday 2nd LONDON Wednesday 2nd LONDON Tuesday 8th SHEFFIELD Tuesday 8th LONDON Tuesday 15th EDINBURGH ORDINARY MEETING on “Nuclear Magnetic Resonance and Electron Magnetic “Nuclear Magnetic Resonance and its Applications to Structural and Quantitative “Purity Control by NMR,” by I. J. Lawrenson. “Electron Magnetic Resonance as an Analytical Tool,” by B. D. Flockhart. Scientific Societies Lecture Theatre 23 Savile Row London W. 1 ; 6.30 p.m. RADIOCHEMICAL METHODS GROUP. Discussion on “The Implications of Xew Regulations Governing the Transport of Radioactive Materials,” to be introduced by E. J . Wilson. Room T214 Polytechnic of the South Bank Borough Road London S.E.1; 3.30 p.m.NORTH EAST REGION and MICROCHEMICAL METHODS GROUP joiiztlv ZL ith the Sheffield Metallurgical and Engineering Association on “Thc Detei-mination of Fluorine. ’’ Resonance in Analytical Chemistry.” Analysis,” by M. E. A. Cudby. “Why Fluorine Analysis?” by T. R. Farnworth. “Recommending a Standard Method for Fluorine-the Work of the SAC Sub- Committee for Fluorine (Analytical Methods Committee),” by R. J. Hall. “Fluorine and the Organic Elemental Micro-analyst,” by B. T. Saunderson. “The Determination of Fluorine in Non-metallic Materials by Direct Electron “Toxic Properties of Inorganic Fluorine Compounds,” by R. Y . Eagers. Conference Room BISRA The Corporate Laboratories British Steel Corporation SPECIAL TECHNIQUES GROUP on “Mossbauer Nuclear Quadrupole and E.S.R. Speakers to include T. C. Gibb and B. C. Gilbert. London. SCOTTISH REGION jointly with the Edinburgh University Geological Society and the Edinburgh and South East Scotland Section of the CS on “Gcological Prospecting in Scotland.” Speakers S. Bowie and M. T. Gallagher P. Bowden and E. Stevens and RI. J . Russell. Grant Institute of Geology The University Edinburgh; 4 p.m. Excitation,” by M. Corbett. Hoyle Street Sheffield S3 7EY; 3.30 p.m. Analysis-An Up-to-date Assessment of These Techniques.” [continued inside back covey Printed by W Heffer & Sons Ltd Cambridge England

ISSN:0037-9697    

DOI:10.1039/SA97209BX003

出版商:RSC    

年代:1972

数据来源: RSC

摘要:

4 SAC CENTENARY SYMBOL [Proc. SOC. Analyt. Chem. The Society for Analytical Chemistry Centenary 1974 THE Society for Analytical Chemistry's Centenary Committee is offering a prize of L l O for the best design submitted by a member of the Society for a symbol that can be printed on notices etc. connected with the Centenary Celebrations in 1974. To give an idea of what the Committee has in mind the symbols used for the Joint Anglo- Dutch Symposium held in London in April 1970 and the Third SAC Conference held in Durham in July 1971 are reproduced below- O U A H A M 1 9 7 1 w 1. > d. 3. 4. 6. RULES Sketches and suggestions should be submitted t o the Honorary Publicity Officer The Society for .li~alytical Chemistry 9/10 Savilc Row London W1X lAF before February 29th 1972. Copyright in all designs submitted shall belong to the Society for Analytical Chemistry. The Society may make use of any design or of part of any design submitted irrespective of whether it is the prize-winning design or not. l'hc Society is not bound to use the prize-winning design. 'The Committee's decision is final.

ISSN:0037-9697    

DOI:10.1039/SA972090004a

出版商:RSC    

年代:1972

数据来源: RSC

摘要:

6 REPORTS OF MEETINGS [Pvoc. SOC. Annlyt. C7aem. Modern Analytical Techniques for Bio-active Substances The following are summaries of ten of the papers presented at a Joint Meeting of the Microchemical Methods Biological Methods Automatic Methods and Radiochemical Methods Groups on September 16th and 17th 1971 at the University of Surrey Guildford and first reported in the October 1971 issue of Proceedings (p. 203). A Review of Automation in the Laboratory Plenary Lecture BY F. L. MITCHELL ANALYTICAL chemistry covers a very wide area and mechanisation must be invading every branch but in none perhaps so much as clinical chemistry where the necessity of circum- stances has made it imperative that we should mechanise and automate as quickly and completely as possible. In any review of this nature it is necessary to use clinical chemistry as a background as so many of our automatic analysers were developed specifically for hospital work and their peculiarities reflect many of the peculiarities in that work.Since the war our workload has been doubling approximately every four years and of necessity we have had to introduce disciplines that were once strange to analytical chemists- mechanisation data processing and quality control. It is fair to say that the introduction of these three disciplines over the last ten years has revolutionised clinical chemistry and the clinical chemist himself. None of the three could have been satisfactorily introduced on its own. Mechanisation in the laboratory ranges from the application of work simplification to the installation of fast computer-controlled multichannel analysers and it is convenient to start with the simplest and work up to the most complicated.Work simplification itself is little more than the use of common sense and simple aids such as automatic dispensers or the slightly more complicated diluters. If the techniques that are used in large numbers are examined in almost every case it is possible to cut down on the number of steps involved and the number of tubes and pipettes used and with a modest financial outlay to incorporate dispensers and diluters at various stages. Such modifications dramatically increase precision and reduce human error and also increase the speed of operation and minimise boredom. The precision of many of the dispensers and diluters available exceeds that of the most experienced and careful technician who uses pipettes.With most of these instruments however the sample has to be presented to the (Medical Research Council Clinical Research Centre Watford Road Harrow Middlesex H A 1 3 U J ) Mechanisation came first and might be said to be the key to the whole revolution. January 19721 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES 7 machine. A less laborious technique is possible with the Fisons system where bulky mech- anisms and storage bottles are housed under the bench and all operations are controlled by a very compact hand-held module that can be moved from tube to tube. Provided precipita- tion is not involved many complex assays can be carried out entirely by using this system for transfer and volume measurement. Fisons have termed this approach “limited function automation.” The final measurement in many assays is by colorimetry and remarkable evolutionary changes in colorimeters and spectrophotometers have taken place in recent years.The presentation of samples has been mechanised and linearisers and converters have been added for changing the optical density into concentration the values of which can often be directly printed out. The automatic measurement of enzyme kinetics proved to be difficult and only recently have machines specially developed for this purpose become available. In this country the LKB reaction-rate analyser is the most widely used instrument in which the decreasing or increasing absorbance of the sample at 340 nm is continuously recorded for 1 to 9 minutes. The Smith-Kline Instrument Co.have recently introduced their Eskalab system of work simplification for emergency measurements. Nothing is required for an assay except a special kit and a special colorimeter. Reagents are in tablet form and the reaction usually takes place in a precision-made disposable plastics colorimeter cuvette. The Technicon Co. of the U.S.A. with the AutoAnalyzer and its multichannel versions have achieved a great deal towards making our revolution possible and they have exploited to its full potential the system of continuous flow originally developed by Dr. Leonard Skeggs. Their second generation analysers now give recordings with flat-topped instead of rounded peaks showing that reactions now reach the steady state. This mode of operation is also used in their multichannel analysers-the SMA 6/60 and SMA 12/60.Many of the new automatic systems now becoming available however use the discrete system of analysis where the samples are contained in separate vessels during part or all of the analytical process and an advantage therefore is that the mechanisation can imitate more closely the traditional manual procedures. The new discrete analysers may be divided into four categories-( 1) discrete discontinuous where there has to be manual intervention to transfer racks of tubes from one process to another; (2) discrete continuous where the process continues from start to finish without manual intervention ; (3) small multichannel machines ; and (4) large multichannel instru- ments that are electronically or computer controlled. The Joyce-Loebl Mecolab and the Baird and Tatlock Analmatic instruments are examples of the first category.Samples are dealt with in batches and racks of tubes are transferred manually from module to module. As the racks can be placed on a centrifuge precipitation reactions can also be carried out. Several instruments fall into the second category and it is possible to give only a few examples. In the Pye-Unicam AC-60 instrument the sample and reaction tubes travel in inseparable pairs and programming is simply performed with a changeable metal template specially made for each analysis. The speed of operation is 120 samples per hour. The Swedish Linson Autolab analyser functions with two chains of tubes one for specimens and one for the reaction tubes which can be made to traverse a constant- temperature water-bath.An advantage of this system is that the specimen chain may be sent round several times for as many analyses as are required. The instrument processes 240 specimens per hour. In the recently announced Braun-Zeiss SysteMatik analyser the various modules can be demounted and used for limited function automation. Sample rings carry sixty specimens and each has a numbered location that is printed out on the report. Only 1 ml of sample is required for the flow-cell so the machine can operate on micro samples. The Monitor Programachem 1040 is another very sophisticated new instrument in which a programme card selects reagents volumes and timings and determines whether kinetic or ordinary measurements are required. The rate of analysis varies from 1040 to 200 samples per hour. A special very advantageous feature is that it can give “stat” results on a “one- off” basis during the night for instance.This machine is possibly the nearest we have yet seen to “slot-machine” chemistry. 8 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES [Proc. SOC. Analyt. Chem. The third category of small multichannel machines includes the Technicon SMA 12/60 so called because it has twelve channels operating at sixty specimens per hour. Print-out can be in terms of concentration but is normally graphical. The Perkin-Elmer C4 Clinical Analyser has a built-in sample identification system. There are four separate channels and a small computing device converts the results into concentration units that are printed out against the sample number from the identification stub.The Du Pont Automatic Clinical Analyser is unique in that the reagents for each test are contained in a special plastics pack which also serves as the reaction chamber and cuvette for the photometric analysis. A separate pack is used for each determination and assays are therefore relatively costly. The speed of operation is perhaps a little slow at about sixty tests per hour but up to sixty different methods can be handled. Start-up is immediate and the instrument can be virtually a complete automatic chemistry laboratory. In the Hycel Mark 10 sixty samples are placed in numbered locations corresponding to a column of ten switches on the programming panel where any combination of ten tests may be selected for each sample. The computer holds these instructions for all samples and indicates the state of those samples in the process of assay.The last the most interesting and certainly the most expensive group of instruments to be considered are the fast multichannel machines of which there are at present three. The Swedish AGA Auto Chemist is the largest analyser of its type we have experienced so far and costs about E250000. I t has twenty-four channels and is controlled by a unit containing inflexible logic while a computer handles the data produced. It receives speci- mens a t the rate of 134 per hour and has the appearance of a small two-storey building. The Swiss Richterich-Greiner instrument is perhaps the most versatile yet developed. A request card from the hospital ward is punched to indicate in machine-readable form which assays are required. Each specimen is diluted one in five and during a journey round the machine can be assayed by thirty different method programmes.If all thirty channels are used for each specimen only ten samples can be assayed per hour but the speed of sample through-put is increased proportionately as the number of assays required on each specimen is reduced. The Vickers MC-300 is the fastest multichannel machine available accepting specimens at 300 per hour and carrying out up to twenty assays on each. It is controlled by computer and blood samples are taken in special containers on the sides of which the specimen identi- fication can be marked by hand in machine-readable form. Reactions and assays are carried out in the machine on removable twin-channel modules that can be operated singly with a special sampling plate to function as fast twin-channel analysers or two may be placed back- to-back to give a four-channel instrument.With the efficient use of these large multichannel machines the cost efficiency of labora- tories can be considerably improved. For example by using the Vickers machine for one hour per day only the cost per sample is approximately 30p or about l+p per assay. This includes depreciation on the capital cost of the machine operator time reagents etc. By using the machine for six hours per day even this cost is halved. In dealing strictly with bench mechanisation it has not been possible to give a rightful place to data processing the application of which is of equal if not greater importance than mechanisation in our laboratories. At present about twenty-five of the clinical chemistry laboratories in the hospitals of Britain are computer-controlled to some extent.The com- puter is fed with patient information test-request information and on-line and off-line signals from the laboratory instruments. It then produces work sheets labels for tubes reports quality control information and warnings of breakdowns etc. Years ago simple cheap colorimeters were a status symbol and the most expensive item in laboratories. Over the life-time of many of us these have been replaced by photoelectric colorimeters recording spectrophotometers automatic analysers and now computers. Where we used to think hard over spending L25 we now consider instruments costing a quarter of a million pounds. Although capital outlays may now be large we can take pride that in analytical chemistry our productivity and quality of output have probably improved a t a rate that outstrips that of many productivity-proud industries.January 19721 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES 9 The GeMSAEC Fast Chemical Analyser B Y A. R. THOhfSON (Chemical Engineering Division A tomic Energy Research Establishment Harwell Didcot Berks.) THE past two decades have seen remarkable advances in the biological sciences due in no small part to improvements in analytical techniques. There has also been a great increase in the demand for clinical chemical analysis a trend that is likely to continue if not accelerate owing to current developments in mass metabolic screening for the early diagnosis of disease in paediatric medicine and in the development of national health programmes while the demand for more specific analyses (generally requiring expensive reagents such as enzymes) call for economy in the use of such reagents.These pressures particularly from the clinical laboratories have stimulated the development of a number of mechanised and semi-automated chemical analysis systems now widely used in clinical medicine in research laboratories and in the laboratories of the chemical and pharmaceutical industries. Most of the existing systems analyse samples in series and are inherently slow the time required for a batch of samples being a multiple of the time for each analysis and the interval between samples. The most widely used system the Technicon AutoAnalyzer which has proved itself so valuable over the past 10 to 12 years is however subject to baseline instability and sample carry-over.Data reduction in real time is also uneconomic with all but the largest installations as the rate of data acquisition is much slower than the rate at which the data can be evaluated by computers. Further with series-type analysers error detection and self-correction in real time are difficult as is the use of feed-back control the ultimate require- ment for a fully automated system. The above considerations have led to the development of the GeMSAEC analysis system,l which is designed to carry out chemical analyses rapidly in parallel with reduced amounts of reagents (one-fifth to one-tenth of those required by most conventional systems). In this system discrete samples and reagents are transferred by centrifugal force into a number of optical cuvettes in a specially designed centrifuge rotor so that all reactions begin within a short space of time (approximately 5 s).The centrifugal field also enables air bubbles to be removed and the liquid meniscus to be flattened. The rotor is positioned so that its cuvettes spin between a light source and a photomultiplier. The absorbances of the cuvettes are thus observed in real time and are displayed on an oscilloscope which enables mistakes in reagent make-up and sample dilution to be corrected rapidly. Each cuvette has a built-in siphon so that the mixing caused by transfer is enhanced by sucking air back through the siphons which are also used to empty the cuvettes by the application of air pressure. Vacuum and air pressure are also used to wash empty and dry the rotor in situ.As absorbances are measured while the cuvette rotor is spinning (at approximately 500 r.p.m.) dark current can be measured between each sample the absorbance of which is read every 8 ms. Further standards and reagent blanks can be incorporated with each batch of samples and their absorbances are measured within very short time intervals of those of the samples. It is clear therefore that the data should have high statistical accuracy and that the system has great potential for the measurement of the rate constants of chemical and enzymic reactions. The data are generated in a form and at a rate compatible with their manipulation in digital computers and this will allow both rapid evaluation and potentially feed-back control. As the system uses precisely the same experimental methods for small and large batches of samples analyses of priority samples and change-over between analyses are readily achieved.The system is mechanically very simple in that the only moving part is a centrifuge rotor that spins at relatively slow speeds (up to 2000 r.p.m.). The main features of a prototype GeMSAEC system supplied by M.S.E. Ltd. were described. The rotor and transfer disc have provision for sixteen samples and a tungsten lamp and interference filters are used which enables reactions to be monitored in the visible range and at wavelengths down to 340 nm. Vacuum and air pressure are supplied to enable mixing and dumping to be carried out. At present data reduction is carried out manually by measuring peak heights on photographs of the oscilloscope display.A system for collecting data on punched paper tape is at an advanced stage of development and will be evaluated via a Teletype link to the laboratory IBM 360/75 computer. 10 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES [Proc. Soc. A?zcrZyt. Chenz. To date a number of chemical and enzymic analyses have been carried out with the GeMSAEC system. These include the biuret reaction for proteins and determinations of glucose by the hexokinase glucose-6-phosphate dehydrogenase linked system lactate de- hydrogenase and phosphoglycerate kinase. With the biuret reaction readings taken after 90 s give good reproducibility (normal manual methods take 10 to 30 minutes) while analysis for phosphoglycerate kinase takes only 1 to 3 minutes. The particular usefulness of the system for the scanning of large numbers of fractions obtained from column chromatography and density gradient centrifugation was discussed.Reproducibility is limited so far only by the accuracy of measuring peak heights on the photographs (2 to 4 per cent.). REFERENCE 1. Anderson N. G. Amer. J . Clin. Path. 1970 53 778. Ion-specif ic Electrodes and their Use in Automatic Analysis BY J. R. MANSFIELD (Imperial Chemical Industries Limited Petrocheinicals Division Billinghawz Teesside) THE use of ion-specific electrodes allows a simple approach to automatic analysis. These electrodes measure cationic or anionic activity as the glass electrode-the first ion-specific electrode-measures hydrogen-ion activity (pH). As with the glass electrode in pH measure- ment the use of ion-specific electrodes cannot be considered in isolation from the total measure- ment system.The total system in which ion-specific electrodes are used in automatic analysis consists of a measurement system and a sample system. The measurement system consists of an electrochemical potential transducer a measurement module and a data output. An electrochemical potential transducer of necessity requires two electrodes in this instance an ion-specific electrode and a reference electrode. The ion-specific electrode can be regarded as a membrane electrode in which an ion-conducting membrane separates either two liquid phases or a liquid and a solid phase. The membrane is permeable to specific anions or cations. Ion-specific electrodes can be classified according to the composition of the mem- brane.This membrane can be a glass a solution of a liquid ion exchanger or of a biologically active macromolecule in a water-immiscible solvent supported on a porous medium or a crystal- line solid consisting either of a single crystal or of fine crystals in an inert matrix. The majority of commercially available electrodes consist of a membrane separating two liquid phases-a reference solution and a sample. The reference electrode should be compatible with the ion-specific electrode thus eliminat- ing the complication of the necessity for using a salt-bridge. There are a few systems where a salt-bridge may be preferred e.g. ones in which measurements are made at elevated tempera- tures or where the solvent is non-aqueous. There are available sulphate halide oxide and ion-specific electrodes which can be used as reference electrodes.1 The latter can be used with samples that contain a counter-ion of known and constant activity so giving a constant reference potential.The ion-specific reference electrode must be highly selective in its res- ponse to the counter-ion. The use of a combination of two ion-specific electrodes eliminates liquid junction potentials and is to be preferred when measurements are made to an accuracy of better than a millivolt. The response of an ion-specific electrode-as with the glass pH electrode-can be represented by an equation of the form- where + = ~ and 2 = is the charge on the ion. This equation shows that the potential developed by the electrochemical transducer is proportional to the logarithm of the ionic activity (ax).The value of the proportionality constant is dependent on the charge on the ion (2) and the temperature ( T ) . In practice this constant often deviates from ideality. The deviation can be due to variations in the selectivity constants and the activity coefficients which result from changes in the ionic composition of the solutions. The transducer and sample should be kept at a constant temperature to avoid the need for temperature compensation as the temperature coefficient of a system is often a function of temperature. In many practical situations the total ionic strength of the solution E = constant + + In(a,) RT ZF January 19721 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES 11 may vary considerably. It is essential to know the associated variations in the activity coefficients and selectivity constants.Information available from the literature is limited to a few ideal systems. The empirical approach based on the Harned rule is the most versatile for calculating ionic activity coefficients in mixed electrolyte systems containing 1 1 1 2 and 2 2 electrolytes.2 At concentrations below molar the Guggenheim treatment can be used.3 It is only at very low concentrations that activity coefficients may approximate to unity. The response of the electrochemical transducer can be measured with a pH meter the first ion-specific meter of its kind. Because of the greater accuracy required in analysis a new generation of pH meters has been developed that are capable of measuring to the nearest millivolt or better with digital or analogue read-out.These meters have an input impedance greater than 10l2 Q and should have good common mode rejection. Measurements carried out with meters that have an inadequate specification give erroneous results and should not be incorporated into an automatic system. The specification of the measurement system is downgraded by the use of electrical input leads and connectors that have a lower isolation resistance than that of the meter. Ion-specific electrodes can be used in direct potentiometry or as indicator electrodes in potentiometric titrations. Potentiometric determination of ionic activity may be by direct measurement or by the methods of standard addition or subtraction. In direct potentio- metric measurements the system must be calibrated which requires the use of two standard solutions to determine the slope and the offset.The alternative method of known increments (addition4,5 and s ~ b t r a c t i o n ~ ~ ~ ) is more accurate. A method described by Durst7 and developed by Brand and Reclinitzs enables greater accuracy to be obtained by the use of multiple addition and a successive approximation computation. In the last two methods the ionic activity is calculated without a knowledge of the slope or the offset. Several ionic parameters of a system can be determined by using a series array of ion- specific electrodes for example pH and pC1 can be determined by using the appropriate electrode and referring it to a common reference electrode. As the form of the equation for the response of these electrodes is simple automatic computation of ionic activity from the data from a multiple electrode system is not complex.In an automatic system the analyser may handle discrete samples in a continuous or discontinuous manner or may pump the sample in a continuous flow through the cell. The metering of discrete samples and the controlling of flow must be carried out accurately. This is particularly critical when samples of 1 to 10 p1 volume are dispensed or a flow of less than 1 1 h-l is maintained. Microtip electrodes and flow-through cells are used when small samples are dispensed. The ion-specific electrode is simple device. Its response can be measured relatively easily and evaluated with a minimum of mathematical manipulation. This makes it an ideal transducer for automatic analysis. However care must be taken in selecting the correct type of ion-specific electrode and the associated reference electrode.The results must be inter- preted with due regard to the properties of the electrolyte system. REFERENCES 1. 2. 3. 4. 5. Ibid. 1970 2 5. 6. Ibid. 1969 1 25. 7. 8. Ives D. I. G. and Janz G. J. “Reference Electrodes,” Academic Press London 1961. Leyendekkers J . V. and Whitfield M. Analyt. Chern. 1971 43 322. Guggenheim E. A. Phil. Mag. 1935 19 588. Ovion Reseavch IHC. Newsletter 1969 1 9. Durst R. A. Micvochim. Acta 1969 3 611. Brand M. J. D. and Rechnitz G. A. Analyt. Chern. 1970 42 1172. High Pressure Liquid Chromatography BY D. RI. HEINEKEY (Imperial Chernical Industries Ltd. Oygaizics Division Hexagon House Blackley Manclzester) ADSORPTION chromatography in columns was until about 1967 characterised largely by slow separations and low column efficiencies.However at about this time theoretical studies indicated that all forms of chromatography were governed by essentially the same parameters 12 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES [Proc. Soc. Analyt. Clzewz. and that if the correct conditions were chosen then the speed and efficiency of liquid chromato- graphy could approach that of gas chromatography. The principal suggestions were that liquid chromatography would require long narrow columns small particle sizes and high pressures for high speed separations. Small values of the plate height H are obviously desirable for maximum resolution and efficiency although large values may be compensated for by an increase in the column length. Of fundamental importance therefore is the dependence of H on experimental conditions and especially the variation of H with solvent velocity v particle diameter d, and the bed packing structure.H versus SOLVENT VELOCITY- Early experimental work by Snyder1 and Stewart Amos and Perry2 suggested that the dependence of H on v in adsorption chromatography could be described by the Van Deemter equation with the longitudinal term set to zero i.e. H = A + Cv. This seemed to be applic- able to conditions under which v was varied over a small range. When the range of v was increased it was found that this simple equation became inadequate. Although a modified and more complex form of the Van Deemter equation can be used to describe the results an alternative simple empirical relationship has been ~bserved,~ namely H = Dv0s4.Here D is a constant for a given set of experimental conditions. The general shape of a graph of H against v therefore shows a levelling-off at higher velocities. A consequence of this is that if it is required to double the efficiency in a fixed time it is necessary to increase the column length by a factor of three. This means that the pressure required has to be increased by a factor of nine. On the other hand if the column length is kept constant the efficiency can be doubled by reducing the solvent velocity by a factor of two-thirds thereby increasing the analysis time by a factor of three. VARIATION OF H WITH PARTICLE SIZE AND BED STRUCTURE- The dependences of H on d and bed structure are interrelated. Theoretically for a bed of fixed structure i.e. where all the particles retain the same relative positions and con- figurations as the particle size is reduced a single graph of reduced plate height (12 = H/d,) against reduced velocity (V = ZJ d,/D, where D is the diffusion coefficient of the solute in the moving phase) should describe the various H veyszhs v graphs for a given chromatographic system as d is varied.In practice however it is found that it is more difficult to pack particles into regular even beds as the particle size is reduced so that a deviation from the curve obtained at higher particle size is found on the graph of h against V . The point at which this deviation occurs varies with the technique used to pack the column. However provided that regular packing occurs then a decrease in particle size of 50 per cent. results in a similar decrease in H .Therefore a reduction in particle size of 50 per cent. will double the efficiency of the column if the column length is kept constant,with an increase in pressureof a factor of four. Various packing techniques have been used such as the addition of the packing in small amounts with consolidation of each addition by tapping and bouncing the column but these techniques usually show some deviation from regular packing if the particle size is reduced to below about 100 pm. However the dry method first described by Sie and van den Hoed4 has been found to give regular beds with a particle size of 50 pm. The method was originally used with columns of 10-mm diameter but it is also applicable to columns of 3-mm diameter. Other workers have attempted to pack finer silica particles into columns but until very recently only limited success was achieved.OTHER VARIABLES- Pore structure-In so far as the type of pores present can affect the rate of diffusion in the stationary phase one of the requirements of the type of silica used is that it should have an open pore structure without long narrow pores. This means that the pore diameter should be as large as possible bearing in mind that increasing pore diameter leads to a smaller surface area and hence to lower adsorptive properties. A useful compromise is a type of silica such as Davison 952 which has an average pore diameter of 15 nm and a surface area of about 300 m2. This type of silica shows significantly lower plate heights than does the usual type of silica used in adsorption chromatography which have pore diameters of about 3 nm.Column packing techniques have therefore received considerable attention. January 19721 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES 13 Column diameter-An increase in the column diameter tends to lead to increased plate heights and it now seems generally accepted that the most useful column diameter is 3 to 4 mm which is similar to that used in gas chromatography. Water content-The water content of the adsorbent is important in that fully activated adsorbents show much larger values of H than do the same adsorbents with 4 to 20 per cent. of water added to the fully activated material. This is probably due to the blocking of very active sites that have slow adsorption - desorption kinetics or to the filling with added water of small diameter pores in which mass transfer is slow.Viscosity of the solvent-The viscosity of the solvent affects separation through its effect on D and column permeability. As a rough guide an increase in the viscosity of the solvent by a factor of two will result in some loss in separation efficiency at constant time. Vis- cosities of solvents should therefore be kept as low as possible in fast separations. OTHER SUPPORTS- The previous discussion has been concerned principally with the optimisation of con- ditions for the packing of silica-gel columns. Because of the packing problems encountered with fine silica particles other types of supports and adsorbents have been developed. These are typified by porous glass beads and the controlled porosity supports that have a hard impenetrable core and a thin surface of porous materials5-’ These have the advantage of reduced mass-transfer distances with the porous material which should lead to lower plate heights.These bead-type materials also have the advantage of smoothness and high density both factors contributing to ease of packing and hence to regular packing. Because this type of material has greatly reduced surface area there is a significant loss in adsorptive capacity and therefore they are more suitable for liquid - liquid chromatography than for adsorption chromatography . For practical purposes it has been found that a column 75 cm long 3.5 mm i.d. packed with Davison 952 of particle size 47 to 53 pm gives H = 1.0 mm at a flow-rate of 1 cm s-l and H = 0.4 mm at a flow-rate of 0.3 cm s-l. The pressure required for the higher flow-rate is 300 p.s.i.g.which means that glass columns can be used with safety. Such a column is capable of separating up to six components in 20 minutes. KEFE REXCES 1. 2. 3. 4. 5. G . 7. Snyder L. R. Analyt. Chem. 1967 39 698. Stewart H. N. M. Amos R. and Perry S. G. J . Chromat. 19G8 38 209. Snyder L. R. J . Chromat. Sci. 1969 7 352. Sie S. T. and van den Hoed N. Ibid. 1969 7 257. Kirkland J. J. Ibid. 1969 7 7. Horvath C. and Lipsky S. R. Ibzd. 1969 7 109. Kirkland J. J. Ibid. 1969 7 361. Some New Developments in Thin-layer Chromatographic Apparatus BY J. P. LEPPARD AND A. D. R. HARRISON ( Wolfson Bioanalytical Centre University of Surrey Guildford Surrey) THE first half of this lecture was devoted to a brief survey of some of the apparatus that has appeared in the literature or on the market in the past two or three years.The categories considered were General Equipment Sample Applicators Plate Scanners and New Concepts and particular attention was paid to novel approaches adopted in each piece of apparatus mentioned. Mention was made of an evaluation study of a machine not yet on the market. This is a flame-ionisation detector for the detection and quantification of thin-layer chromato- graphic “spots” on quartz rods coated with silica gel. This gave good results with sample loadings of 1 to 10 pg the main difficulties encountered being the difficulty of sample applica- tion and inadvertent retention of the developing solvent on the adsorbent coating when it was supposedly dried in preparation for the next run. The second half of the lecture was devoted to apparatus that has been designed or evaluated at the Wolfson Bioanalytical Centre.Unsuccessful ideas were considered first and included the use of capacitance and conductivity measurements for detection and quantifica- tion of thin-layer chromatographic spots and a perforated (rear-loading) plate designed to 14 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES [Proc. SOC. Analyt. Clzem. facilitate sample loading. Mention was made of a novel spot-scanning device which has a promising future. Application of the sample to a thin-layer chromtographic plate is a very tedious and at the same time very important stage in the thin-layer chromatographic process. As a module in what could eventually be an integrated automatic thin-layer chromatographic system the Wolfson Centre has designed and is evaluating a sample applicator which seems likely to fulfil all the requirements of such a machine within a precision level of 10 per cent.or possibly 5 per cent. It consists of a reciprocating drive which moves mounted pick-up arms into a row of sample cups and then on to the plate. Up to twelve samples can be spotted simul- taneously and up to ninety-nine aliquots of a given sample can be spotted without interven- tion by the operator. Timers allow for drainage of the sample on to the plate and for drying of the spots. Solvents as volatile as chloroform have been shown to give good results. Radioimmunoassa y BY B. A. L. HURN I Wellcorne Research Laboratories L a ragley Couvt Beckenham Ken/) RADIOIMMUNOASSAY is based on the antigenic competition between the substance that is to be determined and a radioactively labelled analogue of that substance for a limited number of binding sites provided by a suitable antibody; the more unlabelled substance is present the less radioactive analogue will be able to bind to the antibody.The technique therefore has similarities to other types of isotope dilution procedures and more especially to competitive binding assays. The main feature that distinguishes radioimmunoassay from these other techniques is the fact that it is based on the reaction of an antigen with an antibody. This type of reaction normally has a high association constant (k the antibody has a high “avidity”) which is essential for stoicheiometry in the competitive binding process and it has the great advantage that a very wide range of types of compound will stimulate the production of antibodies (i.e.they are said to be “immunogenic”). The range of substances that have so far been measured by radioimmunoassay includes protein and polypeptide hormones (down to eight amino-acids in size) steroids enzymes prostaglandins iodothyro- nines viruses and tumour-specific substances. Many compounds that are not immunogenic in their native state may be made so by conjugation to other substances such as proteins. Once an antibody of sufficient avidity has been produced the major technical difficulty in radioimmunoassay is concerned with the separation of antibody-bound radioactivity from the remainder of the reaction mixture. A very wide range of physical chemical and immuno- logical techniques have been proposed for this purpose over the years.Those in most com- mon use a t the present time include immunological methods (the “double-antibody’’ method SO called because a second antibody is used to precipitate the complex of first antibody with antigen) differential physical absorption on t o substances such as charcoal covalent binding of the antibody (before its use in the assay) to a solid support such as cellulose and physical adsorption of the antibody to a solid support such as polystyrene. The variety of methods available indicates that none is totally satisfactory; for this reason (and others) the proper validation of a radioimmunoassay system is a matter to which much critical attention should be given prior to its routine use. Although immunospecificity of antisera is not essential in radioimmunoassay systeiiis the over-all specificity of the test is most important.This is normally determined by the purity of the labelled antigen as only those substances that can compete with the binding of this antigen will register in the test. On some occasions it is desirable that the assay should be insensitive to species variation in a protein hormone (that is to say hormone-specific but species-nonspecific as in the assay of hzwzan parathyroid hormone using antiserum raised against the bovine hormone with labelled bovine hormone as the tracer). On the other hand there are occasions when the maximum possible specificity is essential as when individual steroids are being measured in the presence of others of the same family. While appropriate chemical modification of the antigens used for immunising the animals can sometimes be of assistance in the control of specificity the most important contribution to the problem is usually made by painstaking selection of the most suitable reagent from a large number of antisera.There are various problems of specificity. January 19721 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES 15 Once these various problems have been solved radioimmunoassay allows the routine measurement of a wide variety of substances with a high degree of sensitivity and specificity and most important of all to the clinical chemist it can usually be applied directly to bio- logical fluids in all their complexity without prior extraction and purification. Some Recent Applications of Neutron-activation Analysis BY G.C. GOODE (United Kingdonz Atotnic Energy A uthority A tonzic Weapons Research Establishment Alderwmsioii Reading Berks. RG7 4PR) NEUTRON-ACTIVATION techniques are among the most sensitive for a large proportion of elements in the periodic table and offer some advantages over alternative methods when extremely low concentrations are being measured. In particular the techniques can be non- destructive and avoid some of the contamination problems that often prevent the use of alternative analytical methods at the limit of their sensitivity. In biological systems the concentrations of many trace elements are extremely low and neutron-activation methods have been applied extensively to the analysis of body fluids and tissue in studies concerned with extending knowledge of the rble of trace elements in metabolic processes.Much of the published literature concerned with establishing the normal levels of trace elements in materials of biomedical interest appears to be erroneous probably mainly as a result of problems of contamination and of the limitation of some of the analytical methods used. This is well illustrated by the large variations in the reported concentration of the least abundant element cobalt in whole blood for which values ranging from 0.12 to 0.0005 p.p.m. have been recorded. The importance of obtaining comparative data from different analytical methods and of establishing reliable biological standards in this area of work cannot be overstressed. REVIEW OF TECHNIQUES The methods used in neutron-activation analysis were briefly reviewed with particular reference to y-spectrometry and radiochemical separation techniques.Various aspects of automation in this field were outlined and the economics of these techniques for multi- element survey analysis were considered. ~-SPECTROMETRY- The introduction of Ge(Li) detectors with greatly improved resolution over the NaI type has made a considerable impact in the bio-analytical field and greatly extends the possibility of non-destructive multi-element analysis for a number of materials. For the maximum utilisation of the large amount of spectral data produced by these systems com- puter methods need to be introduced and a number of “peak search and fit” routines are becoming available for the precise allocation of peak energies and measurement of peak areas. NaI detectors although of inferior resolution offer a far greater sensitivity and again the introduction of computer methods using least-squares fitting has greatly extended the amount of reliable analytical data that can be obtained.RADIOCHEMICAL SEPARATION TECHNIQUES- Although instrumental non-destructive neutron activation offers an attractive method of analysis its application in the biomedical field is often limited by the large amounts of activity from 15-0-hour sodium-24 14-5-day phosphorus-32 and 35.9-hour bromine-82 that are produced. Removal of major intevfering activity-This can be achieved in several ways; a recent powerful technique involves the selective retention of interfering ions on a suitable column of an insoluble inorganic material. The properties of a variety of such materials have been investigated by Girardi Pietra and Sabbioni,l several of which appear to offer extremely useful analytical possibilities.Some examples including hydrated antimony( V) oxide for removal of sodium-24 and acidic aluminium oxide for removal of phosphorus-32 were discussed. Radiochemical group se9avation-A useful compromise is provided by the group separation approach in which groups of nuclides are removed by a suitable separation technique from Several approaches to radiochemical separation are possible as follows. 16 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES [Proc. Soc. AnaZyt. Clzem. the irradiated material after dissolution and individual y-spectra for each group are resolved appropriately. This method has been applied primarily because of developments in automa- tion for example in ion exchange2 and solvent e ~ t r a c t i o n .~ Separation of individual elements-For elements of particularly low abundance e:g. chromium and cobalt specific separation procedures are sometimes necessary to provide meaningful data and classical radiochemical methods involving the measurement of the over-all chemical yield are applied. EXAMPLES OF APPLICATIONS ANALYSIS OF BONE- Neutron-activation methods have been used a t A.W.R.E. in collaborative studies with several hospitals for following changes in the composition of bone from patients undergoing various treatments for renal disorders. 14-MeV neutron activation is applied to small biopsy samples of bone for the determination of fluorine by using the reaction I91;(n 2n)l*F followed by thermal neutron activation for the determination of calcium magnesium and sodium.Data for a number of trace elements present are also available from the y-spectra recorded including aluminium which is of current interest in studies on the toxic effects of hyperaluminaemia. RfULTI-ELEMENT ANALYSIS O F BLOOD AND TISSUE- There is considerable interest in obtaining reliable multi-element data for a variety cf materials of biomedical interest including blood serum and tissue in relation to known pathological conditions and neutron-activation methods are playing an important r61e in such studies. Elements that can be determined by a combination of non-destructive and radiochemical methods in blood for example include bromine chlorine cobalt chromium copper iron mercury potassium manganese sodium phosphorus rubidium selenium and zinc and various analytical approaches to their determination including the application of selective ion-retention media and automated solvent extraction were discussed.DETERMINATION OF COBALT AND CHROMIUM- Radiochemical separation schemes for the specific separation of cobalt and chromium from blood and urine involving ion-exchange and solvent-extraction techniques were des- cribed. Analytical data for these low-abundance elements is being obtained to study the corrosion of metallic implants used in orthopaedic surgery. REFEREKCES 1. 2. 3. Girarcli F. Pietra R. and Sabbioni E. Eztraiom Rep. 1969 EUR 42S7e. Samsahl K. Nucleonics 1966 8 252. Goode. G. C. Baker C. W. and Brooke N. M. Aizalysl 10G9 94 72s. Polarograph y BY D. F. MUGGLETON (May and Baker Ltd.Dagenhavn Essex) POLAROGRAPHY is an electroanalytical technique devised by Heyrovsky some fifty years ago that can be used with advantage to investigate qualitatively and quantitatively a wide range of organic and inorganic systems. It is concerned with the current - potential relation- ship set up when oxidisable or reducible substances are electrolysed under defined conditions and a dropping mercury electrode is used to obtain a renewable and reproducible interface at which to study electrode processes. The basic circuit for direct current polarography consists of a dropping mercury electrode (a glass capillary internal diameter approximately 0.06 0.03 mm connected to a reservoir containing pure mercury which emerges from the electrode tip as a regular series of drops) and a reference electrode (mercury pool silver - silver chloride calomel etc.) both of which are immersed in the solution under investigation.A battery and rheostat (or equivalent arrangement) enables the potential difference (+0.4 to -2.6 V) between the dropping mercury electrode and reference electrodes to be varied in a uniform and predetermined manner. A calibrated galvanometer (or potentiometer recorder) measures the current (in microamperes) January 19721 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES 17 flowing through the system. Much useful work can be carried out with relatively simple apparatus although more sophisticated equipment will of course greatly extend the range of operations possible. Solutions for polarography may be aqueous or non-aqueous wholly chemical or partly biological but they must contain in addition to the polarographically active species an excess of an indifferent salt that does not participate in the electrode reaction.This so- called supporting electrolyte carries nearly all of the current that flows through the solution and thereby ensures that the contribution of the polarographically active species can be attributed exclusively to diffusion processes. Polarographic reductions necessitate the prior removal of oxygen and this is generally achieved by bubbling an inert gas e.g. nitrogen through the solution. If biological media e.g. blood serum froth badly then rapid specific and quantitative de-oxygenation can be achieved by the addition of glucose glucose oxidase and catalase Polarograms (S-shaped current - potential curves) provide qualitative and quantitative information.The position of the polarographic wave on the potential axis (conventionally indicated by the half-wave potential E+) is characteristic of the test substance whilst the height of the wave [the difference between the residual (i.e. initial) and limiting (i.e. final) currents is designated the diffusion current] is proportional to concentration. With mixtures multi-branched polarograms are obtained each component generating a wave with a par- ticular half-wave potential and diffusion current. Oscillations of the current recorder arising from natural growth and decay of the mercury drops may become unacceptably large with very dilute solutions that necessitate high- sensitivity measurements Electrical damping will overcome this difficulty but with the possibility of consequent distortion of wave-shape and to a lesser extent loss of resolution.Alternatively and preferably current-sampled (or Tast) polarography can be used and the current is measured over only a limited and pre-selected part of the drop life. Direct-current polarography involves measurements with many mercury drops to cover the required potential range. Cathode-ray polarography is a variation of the technique that is particularly useful to analysts. The range of potential over which the electrode reaction occurs is then traversed during the life-time of a single drop. A rapid sweep of potential is applied at the end of the drop life and the current - potential relationship observed on a cathode-ray screen (with optional photographic recording).Cathode-ray polarography offers the advantages of speed greater sensitivity and the use of differential and derivative techniques to resolve waves having only small differences of Et. Environ- mental studies are now of considerable importance and metals such as lead and cadmium present in samples of chemical or biological origin are conveniently determined polarographi- cally. Some anions e.g. chlorate and bromate similarly generate cathodic waves whilst others e.g. halides and cyanide can be studied anodically. Oxygen (commonly removed as already described as a necessary preliminary step to the measurement of other compounds) exhibits two waves and is consequently amenable to polarographic determination. The sensitivity of detection attainable by polarographic methods will depend somewhat on the nature of the compound being measured but will in general extend to to M.A very wide range of organic compounds can be studied polarographically under cathodic i.e. reducing conditions including unsaturated hydrocarbons organic halogen compounds carbonyl-containing compounds quinones organic acids and their derivatives nitro com- pounds and many heterocyclic compounds. Some compounds that are not polarographically active themselves may become amenable to study at the dropping mercury electrode after formation of an appropriate derivative. Thus chlorpromazine on treatment with bromine gives a well formed cathodic wave that can be used for analysing mixtures of chlorpromazine (polarographically inactive). Similarly nitration of aromatic hydrocarbons nitrosation of certain alkaloids and hormones and treatment of steroids with Girard’s reagent have all proved useful in obtaining derivatives suitable for polarographic estimation.Veterinary feed additives especially those containing a nitro group e.g. dimetridazole and furazolidone can often with advantage be determined polarographically while the past few years have witnessed an expansion in the use of polarographic techniques for pesticide analysis as they may render superfluous difficult and time-consuming extraction and clean-up procedures. Polarography of inorganic materials has been used extensively in many fields. 18 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES [Proc. Soc. AnaZyt. Chem. Polarography can often be carried out directly in a wide range of biological fluids e.g.plasma urine bile and saliva so that drugs and their metabolites may be rapidly and simultaneously determined in microgram or sub-microgram amounts by using samples of no more than 0-5 to 1.0 ml. Special techniques exist whereby measurements can be made on samples as small as 0.05 ml. Response at the dropping mercury electrode depends on diffusion processes and polaro- graphy offers the possibility of studying protein-binding phenomena without prior separation of free and bound drugs. Disturbance of equilibria is thus avoided. Drug bound to protein (high molecular weight) will diffuse only slowly and hence make no significant contribution to the observed diffusion current which will be due entirely to the free drug (low molecular weight). From the known total drug concentration free and bound components can be calculated from measurements of diffusion current.Polarographic procedures are worthy of consideration for a wide range of analytical problems. They frequently permit the simultaneous qualitative and quantitative deter- mination of several compounds on a limited amount of sample with an accuracy comparable with that attainable by other methods. Satisfactory results are obtained even at the parts- per-million level and as the final assays rarely take more than a few minutes polarography is particularly valuable in busy laboratories handling large numbers of samples. Problems in the Use of Enzymes in Bio-analysis BY B. J. GOULD AND V. MARKS (Department of Biochemistry University of Surrey Guildford Surrey) BECAUSE enzymes used for bio-analysis must be pure they are also expensive.Fortunately they are usually very effective and specific catalysts which accounts for their increasing use in analytical chemistry. Enzyme-catalysed reactions can be followed by the formation of product or by the removal of substrate. After a certain time the reaction will go to equilibrium or completion. This total change can be estimated and with the aid of a standard curve or some known property e.g. molar extinction coefficient of one of the substrates or products (frequently NADH reduced nicotinamide adenine dinucleotide) the amount of an unknown substance can be determined. The extent of this change must be determined under carefully controlled conditions as it may be affected by enzyme concentration [El substrate concentration [S] cofactors inhibitors ionic strength temperature pH and time of incubation.A fairly typical development of a biochemical assay can be seen in the successive methods used for the estimation of bile acids.l Initially the bile acids were extracted modified chemi- cally and then separated by gas - liquid chromatography. Subsequently they were estimated by the more sensitive technique of fluorimetry following prolonged extraction and purification. Later the direct enzymic method using spectrophotometric estimation was used and this allowed direct estimation as the enzyme used was specific enough to ensure that only a limited range of substrates were estimated. The latest development has been the use of the enzymic method with the more sensitive fluorimetric analyses. The enzymic reaction involved 1s- 3-E-h ydroxysteroid deh ydrogenase Steroid alcohol + NAD + -+ steroid ketone + NADH + H+ where NAD represents nicotinamide adenine dinucleotide a coenzyme that is readily detected both spectrophotometrically and fluorimetrically.The steroid ketone formed was trapped with hydrazine thus increasing the sensitivity of the method. One of the first applications of enzymes to clinical biochemistry by relatively inexperi- enced workers was for the detection of glucose in urine by means of a stick impregnated with glucose oxidase - peroxidase - chromogen. It has the advantages that it is specific for glucose sensitive easy and rapid to perform. One disadvantage of the method is its very specificity because several other sugars of clinical importance that can occur in urine are n6t detected although they are detected by the older and less specific chemical methods.Other disadvantages of the stick method are that the enzymes are susceptible to inhibitors and interfering substances it is not quantitative and the sticks are liable to deteriorate especially January 19721 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES 19 if they are stored incorrectly. This latter point could be easily checked if a negative result was obtained by dipping the stick into a solution known to contain glucose and showing that it reacted normally. In practice this would be too cumbersome a procedure for routine use. How- ever this enzyme also catalyses reactions involving several substances other than galactose and this lack of specificity should not be ignored.2 Thus enzymes can be too specific or not specific enough depending upon their application.When using an enzyme to measure the concentration of its substrate one commonly uses a low concentration of substrate and a relatively high concentration of enzyme to ensure that the reaction is quickly completed. When enzymes are used to estimate other enzymes then one must have a relatively low concentration of the enzyme being measured and some- thing like a 100-fold excess of the second or indicator e n ~ y m e . ~ Then the initial velocities observed should be close to the true value for the enzyme being measured. This use of a large excess of an enzyme may make these assays very expensive. For instance in a method for the estimation of 5’-nucleotidase in which two other enzymes were used the amount of the final enzyme in the system glutamate dehydrogenase is theoretically sub-optimal and this appears to have been partly due to the cost of the m e t h ~ d .~ Another enzyme that is frequently measured by using a coupled enzyme system is aspartate aminotransferase. In the original method of Karmen Wroblewski and LaDue,5 sub-optimal values of the substrates were used. This has been investigated by several workers but it is also worth noting that one of the substrates or-oxoglutarate inhibits the enzyme at relatively low concentrations. Another assay method for aspartate aminotransferase measures the rate of formation of the enol form of oxaloacetate. Unfortunately the enzyme produces the keto form and it has been shown6 that the keto - enol transformation may be the rat e-limiting step under certain conditions.The previously mentioned glucose oxidase - peroxidase - chromogen system can also be used in solution to measure glucose concentrations. A recently developed (non-carcinogenic) chromogen “Perid” is generally satisfactory but if serum is dialysed to remove protein in an automated method it has been shown that the uric acid also present in the serum can convert the normal coloured product to a colourless one. Another interfering substance this time with o-dianisidine as the chromogen is ascorbic acid. Once again a colourless product is formed. This can create a problem in the estimation of the urinary glucose of pregnant women who have consumed large quantities of vitamin pills. Several naturally occurring compounds ranging from inorganic phosphate to low- molecular-weight peptides are known to inhibit urinary enzyme^.^ The inhibitors can usually be removed at least partially by dialysis or Sephadex gel filtration.A recent development in the use of enzymes for bio-analysis has been to insolubilise the enzyme by binding it to an inert carrier. This allows the enzyme to be used more economically. It can be used either in batch techniques in which it can be recovered after use by filtration or centrifugation or it can be used continuously. Other advantages of bound enzymes are that they are usually more stable and it may be possible to change the optimum pH for enzyme activity. The main disadvantage is that the bound enzymes are usually less active but this may be overcome by using more enzyme and higher temperatures. The bound enzymes may also be more difficult to assay.Unfortunately they are also very expensive so it is consider- ably cheaper to insolubilise one’s own particular enzyme. Having mentioned some of the problems that can arise from the use of enzymes in bio- analysis it is worth pointing out that provided suficient care is taken it is possible to measure amounts of NADH as small as 10-19 mol.* The measurement of galactose with galactose oxidase is becoming more popular. 1. 2. 3. 4. 5. 6. 7. 8. REFERENCES Murphy G. M. Billing B. A. and Baron D. N. J . Clin. Path. 1970 23 594. Avigad G. Amaral D. Asensio C. and Horecker B. L. J . Biol. Chem. 1962 237 2736. Bergmeyer H. U. “Methods of Enzymatic Analysis,” Academic Press London 1963 p. 10. Ellis G. Belfield A. and Goldberg D. M. Proc. 7th I n t .Congr. Clin. Chem. GenevalErian 1970 Karmen A, Wroblewski F. and LaDue J. S. J . Clin. Invest. 1955 34 126. Banks B. E. C. Lawrence A. J. Thain E. M. andVernon C. A. J . Chem. SOC. 1963 5799. Werner M. Maruhn D. and Atoba M. J . Chromat. 1969 40 254. Lowry 0. H. Passonneau J. V. Shulz D. W. and Rock M. K. J . Biol. Chem. 1961 236 2746. 2 95. 20 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES [Proc. SOC. Analyt. Chem. Pharmaceutical Aspects of Proton Magnetic Resonance Spectroscopy BY D. M. RACKHAM (Lilly Research Centre Limited Erl Wood Manor Windlesham Surrey) PROTON magnetic resonance spectroscopy (P.M.R.) is now in the middle of its third decade and has moved a long way from its origins in theoretical physics. Recent developments in the design of P.M.R. apparatus techniques and associated chemical reagents have permitted the widespread use of the method for problems in chemical biochemical pharmacological and pharmaceutical analysis as witnessed by the explosion in the literature of nuclear mag- netic resonance (N.M.R.) (more than 3000 papers during 1970).P.M.R. experiments involve the observation of the absorption of radio-wave (VHF) energy as a proton placed in a high magnetic field is raised from a low to a high energy state. Because the amount of energy involved in the transition is so small kcal mol-I) no chemical degradation of molecules is induced (cf. ultraviolet absorption). An important reason for the high cost of present-day N.M.R. spectrometers (which can be in excess of fTl00 000) is the need for highly stable magnetic fields and frequency sources.The perform- ance of today’s spectrometers has been humorously (and accurately) compared with an optical telescope capable of resolving two cats sitting a few inches apart on the surface of the moon. Fortunately the instrument manufacturers have kept in mind the requirements and financial resources of the analyst in industry. Spectrometers of moderate performance have recently been announced costing less than i6000 (Perkin-Elmer 60 MHz) or fT3000 (Varian 30 MHz). Some of the most useful developments in accessories and techniques include- (i) resonance of other nuclei e.g. carbon-13 fluorine-19 phosphorus-31 and boron-1 1 ; (ii) Fourier transform equipment and CATS ; (iii) Spin decoupling techniques; (iv) INDOR ENDOR and CIDNP; (v) Lanthanide shift reagents.Four parameters are relevant to the interpretation of a P.M.R. experiment- (i) The chemical shift -This refers to the position on the spectrometer chart with respect to a standard reference material (usually taken as tetramethylsilane at the scale position 6 = 0.00). Most protons fall within the range S = 0 to 10 but some may fall a t abnormally low fields (e.g. enol protons at S = 20) or at abnormally high fields (e.g. transition-metal hydrides at 6 = -50). Large shifts in a 6 value can be induced in either direction by addition of an appropriate lanthanide reagent. (ii) The coupling constant (J)-This is a valuable guide to molecular substitution and geometry . (iii) The signal area-This is usually measured by electronic summation of the area under a peak or group of peaks. For most conditions encountered these areas are a direct quantitative measure of the number of protons causing the signal(s).(iv) Exchange eflects-Addition of a few drops of deuterium oxide to the P.M.R. sample can cause removal of signals for labile protons (attached to N S or 0). Four areas of application of P.M.R. to pharmaceutical analysis warrant particular attention as follows. USES I N QUALITATIVE ANALYSIS- For the identification of products and intermediates in synthesis and metabolism P.M.R. is best used in conjunction with other physical techniques e.g. infrared (I.R.) and mass spectrometric measurements. The pharmaceutically important conversion of penicillins to cephalosporins can be totally rationalised in terms of changes in the P.M.R. and I.R. spectra. The method also serves as a complement to I.R.for studies of hydrogen bonding and tauto- merism. Information regarding the site of protonation of amine and alcohol species is often uniquely determinable by P.M.R. January 19721 MODERN ANALYTICAL TECHNIQUES FOR BIO-ACTIVE SUBSTANCES 21 PROBLEMS OF STEREOCHEMISTRY AND OPTICAL ISOMERISM- Measurement of the coupling constant and use of the Karplus equation relating its magnitude to inter-proton angles have proved valuable in determining the stereochemistry of biologically active molecules e g . nicotine methadone and prodine. When optical isomerism oceurs and one enantiomer only may possess pharmacological activity P.M.R. spectroscopy has been less successful. Whitesides and Lewis1 have recently reported an optically active europium complex that can form diastereoisomers of markedly different chemical shifts with a pair of enantiomers.This seems to hold great potential for previously intract able problems. APPLICATIONS TO BIOMOLECULES AND DRUG - RECEPTOR INTERACTIONS- P.M.R. offers the advantage over solid-state X-ray crystallographic structure analysis that it can be applied to solutions (e.g. in deuterium oxide). Studies on enzymes have proved particularly rewarding. In some remarkable experiments staphylococcal nuclease has been prepared with all except one type of amino-acid residue deuterated. The resultant simplifica- tion of the P.M.R. spectrum allows deductions to be made regarding the effects of substrates and inhibitors. The enzyme pancreatic ribonuclease reveals several very small absorptions in the aromatic region which can be assigned to the individual imidazole protons of the histidine residues at positions 12,48 105 and 119 in the peptide chain.By following the shifts of these protons in buffers and after addition of enzyme inhibitors the ionisation of the imidazole rings and the site of enzyme inhibitor interaction were deduced. Much information can be gleaned from the observation of proton relaxation times regard- ing the sites of interaction of small molecules with proteins (e.g. penicillin or ephedrine with bovine serum albumin). QUANTITATIVE ANALYSIS- Modern electronic integrators on P.M.R. instruments have an accuracy of i-2 per cent. and are therefore adequate for use in quantitative analysis by peak area measurements. Samples need not be completely pure provided impurity peaks do not seriously interfere with those of the assayed compound.A further advantage sometimes not appreciated is that the integral curves have a higher intrinsic signa1:noise ratio than a simple qualitative scan. The preferred method of quantitative analysis of mixtures refers all integrals to those of an added internal standard which should have a sharp peak well separated from the assayed component with which it should not react. This procedure has found wide acceptance eg. the assay2 of hypoglycaemic agents using maleic acid as added standard. Further information regarding P.M.R. for quantitative analysis in pharmaceutical chemistry can be found in a recent re vie^.^ CONCLUSION- The next few years will undoubtedly see further progress with regard to instrumental developments in P.M.R. (e.g. Fourier transform systems). By virtue of this and the dramatic growth of interest in carbon-13 magnetic resonance and the use of lanthanide shift reagents the pharmaceutical analyst can look forward to a rich harvest of exciting and challenging work. REFERENCES 1. 2. 3. Whitesides G. M. and Lewis D. W. J . Amer. Chem. SOL 1970 92 6979. Rehse K. 2. anal. Chem. 1969 246 22. Rackham D. M. Tdanta 1970 17 895.

ISSN:0037-9697    

DOI:10.1039/SA9720900006

出版商:RSC    

年代:1972

数据来源: RSC

摘要:

22 SAC “BELFAST” SYMPOSIUM GLASGOW [PYOC. soc. Analyt. Chem. SAC “Belfast” Symposium Glasgow March 23rd and 24th 1972 SCIENTIFIC PROGRAMME THE scientific programme of the SAC Symposium originally announced for Belfast and now to be held a t the University of Glasgow in March 1972 has now been fixed. This Symposium is being organised in honour of Cecil L. Wilson Professor of Analytical Chemistry in The Queen’s University in Belfast in recognition of his long and varied services to the teaching and practice of analytical chemistry and in celebration of Professor Wilson’s 60th birthday. The Society had originally intended to hold this Symposium at The Queen’s University but in view of the present political disturbances in Belfast and elsewhere in Northern Ireland the location of the Symposium was subsequently changed to the University of Glasgow.A full social programme is being arranged both for participants and for non-scientific guests and on Saturday March 25th there will be an all-day excursion to local places of interest. Accommodation will be in Queen Margaret Hall. I t is expected that an exhibition of modern analytical instruments will be mounted during the period of the Symposium. Intending delegates who have not yet completed and returned the preliminary application form attached either to the original notice or to the revised notice circulated with the October issue of the Journal should at once notify the Secretary Society for Analytical Chemistry 9/10 Savile Row London W1X lAF England of their intention to attend as registration forms are now being distributed and must be completed and re turned b y Friday March 3rd.PROGRAMME PLENARY LECTURI~S by-Professor R. Belclier PA IT RS- Thursday March 23rd Stream A- Professor H. Weisz. “The Dissolution of Materials in Fluoride Media,” by J. B. Heatlriclge Ilcpartnient of Chemistry The LTniversity Sheffield S3 7HF. “Special Decomposition Methods in I:ltramicro arid Extreme Trace -4nalysis of Elc.ments,” by G. ’I‘?)lg -1/ax-Planck-Institut fur Metallforschung Schwabisch Gmuntl Ccrmany. “Microwave Spectroscopy in Analytical Chemistry,” by J . Cuthbcrt Cambridge Scientific Instruments Ltd. Chesterton Road Cambridge CB4 3AW. “Cse of Cyclotrons for A4ctivation Analysis with Fast Neutrons,” by V. Krivan Max-Planck-Institut fur Metallforschung Schwabisch Gmund Germany and H. Miinzel 1 nstitiit fur Iiadiochcmie Kernforschungszentrum Karlshruhe Germany.“The Determination of Trace Elcments in Close Compositional antl Textural .4nalogucs of 1,unar Rocks by Xeutron Activation Analysis,” by H. I. Drever Dept. of Gcology l’ni\.cmity o f St. .\iitlrcws and J . E. Whitley Scottish Research Reactor Centre East Kilbridc. “Thc Determination of Chromium and Molybdenum in Steel by A4toniic-absorption Spectrometry using an Air - Acetylene Flame,” by J. M. Ottawav and N. K. Pradhan L’nivcrsity of Strathclydc Cathedral Street Glasgow C. 1. “Submicrotrace Analysis of Metal Surfaces by Filament Atom Iieser\-oir Spectroinctry ’ I by I . I,. Shresta and T. S. Wcst Tmperial College of Science antl Technology Loiidon S.lV.7. “The Determination of Impurities in Selenium by Atomic-absorption Spectrophotometry,” by 1C.c. Rooney and C. G. Pratt Rooney & lliard Ltd. Blackwater Station Estate Cainberley Surrey. Friday March 24th Stream A- “A Novel High Sensitivity Thermobalancc,” by E. 1,. Charsky .A. C. F. T\“imp and J. P. Retlfc~rn Stanton Redcroft Ltd. Copper Mill Lane London SlVI 7 OBN. “The Determination of Uranium in High Molecular LVeight ;Imine Extracts Obtained from .\queous Sulphate Solution,” by S. J . Lyle I. Salvadori and A. Stcvcnwn T h e Chemic‘tl T.aboratorv Tiniversity of Kent at Canterbury Canterbury Kent. January 19721 SAC “BELFAST” SYMPOSIUM GLASGOW 23 “The Analytical Chemistry of Some Chelating Ion-exchange Resins Based on Pyridine,” by 1,. S. Bark and J. P. Martin University of Salford Salford 5 Lancs. “The Determination of Nitrogen Dioxide and Nitric Oxide with Saltzman-type Reagents,” by M.P. Mendoza National Coal Board Coal Research Establishment Stoke Orchard Nr. Cheltenham Glouccstershire. “lmproved Determination of Manganese in Water by Catalytic Oxidation of NN-Diethylaniline by Periodate,” by G. Ghersini and G. Pampurini CISE Segrate Milano Italy. “The A4nalytical Properties of Alizarin Methyl Sarcosine,” by I<. A1-Ani M. A. Leonard and G. T. Murray Department of Analytical Chemistry Queen’s University Belfast. “Sew Analytical Methods Involving the Masking of Periodate with Molj.l)date,” by A. Townshend Chemistry Department Birmingham 7 Jniversity Birmingham. ‘<The II’ork of the Metallic Impurities in Organic Matter Sub-Committee of the ;Inalytical Methods Committee-With Particular Reference to the Determination o€ Arscnic and Selenium,” by L.E. C,oles Glamorgan County Council. County Public Hcalth Laboratory Institute of Preventive Medicine The Parade Cardiff CF2 317 J. Thursday March 23rd Stream K- “ A New High Iicsolution Fast Scanning Infrared Interferometer,” by J . Cuthbert Cambridge Scientific Instruments Ltd. Chesterton Road Canilridge CB4 3AW. “Purity Determinations 1 7sing a Yew Quantitative DTX System,” by H. Staub and W. Pcrron Mettler 1 nstrumcnte A4.G. CH - 8606 Greifcnsee Switzerland. “Maiiua! and Automated Methods for Determining Cephalosporin C,” by I . J . Hotlgson T. Maine and J . R. Slater Glaxo Laboratories Ltd. Barnard Castle C o . Durham. “Luminescence Analysis of Oxylm-biturates,” by L. A. Gifford \V. 1’. Iiayes Jd. .I. King J . N. Miller ant1 L). Thorburn Burns Loughlmrough Lnivcrsity of lechnology Leics.and J. \V. Bridges University of Surrey Guildford. “The Direct Therrnomctric Determination of Barbiturates in Dosage Form,” by ,2. E. Beezer and W. F. Smyth Chelsea College of Science and Technology Manresa Road London S.W.3. “The Determination of Moisture in Materials Containing Fats or Oils [:sing a Deuterium Oxide Dilution Technique,” by P. B. Mansfield Newport Instruments Ltd. Ncwport Pagnell Bucks. “Some Data on the Analytical Chemistry of the 17-Side-chain of Steroids of the Pregnane Series,” by S. Goriig Chemical Works G. Richter Budapest Hungary. “The Determination of Prophylactic Additives in Animal Feeds (The Work of the Prophylactics in Animal Feeds Sub-committee of the Analytical Methods Committee) ,” by R. E. \Yeston. Laboratory of the Government Chemist Cornwall House Stamford Street London S.E.1. (former member of the Sub-committee). Friday March 24th Stream B- “F’olarographic Determination of Trace Amounts of Cyanide,” by L. S. Bark and €3. S. Lim Department of Chemistry and Applied Chemistry University of Salford Salford 5 Lancs. “Polarographic Studies on Phenylarsenoxide,” by A. Watson and G. Svehla Department of Analytical Chemistry The Queen’s University Belfast. ”Bromide and Todide Selective Elcctrodes for Following Oscillating Chemical Reactions,” by E. IGrds arid M. Burger Institute of Inorganic and Analytical Chemistry L. Eotvos University Budapest Hungary. “Oxidation - Rcduction Titrations in ArN-Dimethylformamide,” by R. D. Braun and J . T. Stock Department of Chemistry University of Connecticut Storrs.Conn. 06268 U.S.A4. “Automation of Electrochemical Techniques,” by J. K. Foreman Laboratory of the Government Chemist Cornwall House Stamford Street London S.E. 1. “Electrogeneration of Metal Ions from Liquid Amalgams,” by Th. J . M. POUW G. den Boef and U. Hannema Laboratory for Analytical Chemistry University of Amsterdam The Netherlands. “Coulometric Titrations Using a Vitreous Carbon Working Electrode,” by V. J . Jennings and A. Dodson Lanchester Polytechnic Coventry. “The Determination of Carbon in Steel by a Coulometric Method,” by B. Metters €3. G. Cooksey and J . M. Ottamfay Chemistry Department University of Strathclyde Cathedral Street Glasgow C. 1. Further details and registration forms can be obtained from the Secretary Society for Analytical Chemistry 9/10 Savile Row London W1X 1AF. These must be completed and returned by March 3rd.

ISSN:0037-9697    

DOI:10.1039/SA9720900022

出版商:RSC    

年代:1972

数据来源: RSC

摘要:

24 PAPERS ACCEPTED FOR THE ANALYST [Proc. SOC. Analyt. Chem. Notice SECOND INTERNATIONAL SYMPOSIUM ON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY JULY 3RD TO 7TH 1972 GUILDFORD THE Symposium organised by the Nuclear Magnetic Resonance Spectroscopy Discussion Group of The Chemical Society will take place at the University of Surrey Guildford. Invited speakers are G. Binsch F. A. Bovey K. R. Ernst H. Fischer C. W. Haigh and E. W. Randall. Short contributed papers can be accepted for inclusion in the programme and intending speakers should submit a title and 250-word synopsis to Dr. L. H. Sutcliffe Donfian Laboratories Grove Street P.O. Box 147 Liverpool L69 3BX not later than February 1st. Further information can be obtained from Dr. J. F. Gibson The Chemical Society Burlington House London W1V OBN.

ISSN:0037-9697    

DOI:10.1039/SA972090024b

出版商:RSC    

年代:1972

数据来源: RSC