摘要:

Proceedinas wof the Analytical Division ofThe Chemical SocietyCONTENTS283 New Member of Council283 Summaries of Papers283 'Electroanalysis'289 'High-performance LiquidChromatography in Clinical andBiological Chemistry'307 Equipment News31 2 Correspondence312 New British Standards312 Publications Received313 Conferences and Meetings315 Obituary31 6 Analytical Division DiaryVolume 16 No 10 Pages 283-31 6 October 197PADSDZ 16(10) 283-316 (1979)ISSN 0306-1 396PROCEEDINGSOctober 1979OF THEANALYTICAL DIVISION OF THE CHEMICAL SOCIETYOfficers of the Analytical Divisionof The Chemical SocietyPresidentR. BelcherHon. SecretaryP. G. W. CobbHon. Treasurer Hon. Assistant SecretariesJ. K. Foreman D. I. Coomber, O.B.E.; D. C. M.Squirrel1Secretary Hon. Publicity and Public Relations Officer Editor. ProceedingsDr. A. Townshend, Department of Chemistry,University of Birmingham, Birmingham, B15 2TTMiss P. E. Hutchinson P. C. WestonProceedings is published by The Chemical Society.Editorial: The Director of Publications, The Chemical Society, Burlington House, London, W1 V OBN.Telephone 01 -734 9864. Telex 268001.Subscriptions (non-members): The Chemical Society, Distribution Centre, Blackhorse Road,Letchworth, Hens., SG6 1 HN.Non-members can only be supplied with Proceedings as part of a combined subscription with The Analystand Analytical Abstracts.0 The Chemical Society 1979CS ANALYTICAL DIVISIONA Meeting onANALYSIS OF ENDOGENOUS OPIATE PEPTIDESwill be held at 2.30 p.m. onWednesday, December 12,1979atThe Linnean Society, Burlington House, London, W.1Papers presented will be "Biological Roles of Endogenous Opioid Peptides," byProfessor H. W. Kosterlitz and "Peptide Analysis," by Dr. D. G. Smyth.The endogenous opiates are morphine-like substances which occur principallyin the central nervous system and have also been found more recently in a varietyof other tissues in the body. These substances have powerful pain-killing pro-perties but their exact physiological roles are not yet established. There appear tobe three distinct endorphic peptides which react preferentially with their ownreceptors and will be expected to show differing patterns of opiate activity.For further details contact the Secretary, Analytical Division, The ChemicalSociety, Burlington House, London, W1 V OBN

ISSN:0306-1396    

DOI:10.1039/AD97916FX034

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

October, 1979 CONFERENCES AND MEETINGS 315Analytical Division Diary, continuedNovember, continuedThursday, 29th: London Friday, 30th, 5 p.m.: CardiffBiological Methods Group : Annual GeneralMeeting, followed by a meeting on “Bio-logical Considerations in the ImPrOvementof the River Thames and its Catchment.”Speakers: Sir John Hanbury and M. J.Andrews. Burlington House, Piccadilly,London, W.l.Western Region, jointly with the South East“Affinity Chromatography, ” by P. Dean.University of Wales Institute of Science andWales Section of the CS.Technology, CardiffAnalytical Division DiaryNOVEMBERFriday, 2nd, 5 p.m.: GlasgowScottish Region : Annual General Meeting,followed by a discussion meeting.A discussion on “Is Classical Analysis ReallyDead ? ” will be introduced by ProfessorL.S. Bark and R. A. Chalmers.Department of Chemistry, University ofStrathclyde, Cathedral Street, Glasgow.Friday, 9th, 7.30 p.m.: PrestonNorth West Region : Ladies Night, jointly withthe Lancaster and District Section of CSand Preston Polytechnic.The Polytechnic, Preston.Wednesday, 14th, 10.15 a.m. : WarringtonRadiochemical Methods Group : Annual Gen-eral Meeting, and meeting on “ActivationAnalysis in Industry.”“Nuclear Techniques in the Assessment ofCoal Quality,” by S. D. Robertson, S. T.Holding, J. J. Rowson and G. R. Gilmore.Friday, 16th-“Enthalpy Determinations,” by P. G. Laye.“Purity Determinations,” by P. Burroughs.“The Use of Thermal Methods of Analysis forStudying Surface Phenomena,” by E.Patterson.“The Derivation of Kinetic Parameters fromDTA and DSC Data,” by M.I.Pope.“Crystallinity Measurements and Crystal-lization Phenomena,” by J. Haye.“Applications of DTA-Various Fields withParticular Reference to Phase Transitions,”by D. Dollimore.Fulmer Grange, Stoke Poges, Nr. Slough,Berkshire.Tuesday, 20th, 2.30 p.m. : LoughboroughMidlands Region : Annual General Meetingand meeting on “Microprocessors” sup-ported by “Original Papers by YoungerResearch Workers” (Elwell AwardPresentation).Fisons, Loughborough.‘‘Ion Beam Activation Current Applicationsand Future Potential for Thin Layer Wednesday, 21st9 ‘Oa30 a.m*: BristolWestern Region and Atomic SpectroscopyGroup : current awareness Symposium on“Plasma Emission Spectroscopy.”Chemistry Department, The University,Bris to1 .Activation,” by T.W. Conlon.Selby District,’’ by D. E. Green.“Multi-element Particulate Survey in theAnnual General Meeting (2 p.m.>.“Applications of Neutron Activation Analy-sisin ICI,” by J. S. Heslop and D. Green.“A Study of the Potential Market for theUniversity Activation Analysis Service,”by D. C. Jackson.Universities Research Reactor, Risley,Warrington, Cheshire.Thursday and Friday, 15th and 16th:Stoke PogesThermal Methods Group : Annual GeneralMeeting and meeting on “Fundamentalsand Quantitative Applications of DTA andDSC.”Thursday, 15th-“The Similarities of and Differences Between“Criteria for Quantitative DTA and DSC,”“Temperature Calibration,” by E. L. Chars-“Specific Heat Determinations,” by P. G.Annual General Meeting (5.20 p.m.) .DTA and DSC,” by R. C. Mackenzie.by F. W. Wilburn.ley.Laye.Thursday, 22nd : HarlowEast Anglia Region : Annual General Meeting,followed by a meeting on “Analysis ofPolymers.” Speakers : G. Cheeseman andL. H. Ruddle.Revertex, Harlow.Thursday, 22nd, 4 p.m. : GlasgowScottish Region, jointly with the Glasgow andWest of Scotland Section of the CS and theAndersonian Chemical Society.“Optoacoustic Spectroscopy,” by G. F.Kirkbright.Room C133, Chemistry Department, Uni-versity of Strathclyde, Cathedral Street,Glasgow G1 1XL.Wednesday, 28th, 6.30 p.m. : LondonMicrochemical Methods Group.A discussion on “Polarography-Does itStill Work?” introduced by R. C. Rooney.The Savoy Tavern, Savoy Street, London,w.c.2.[continued inside back coverPrinted by Heffers Printers Ltd Cambridge Englan

ISSN:0306-1396    

DOI:10.1039/AD97916BX036

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Vol. 16 No. 10 October 1979 Electroanalysis The following is a summary of a paper presented at a meeting held on January 15th, 1979, at Chelsea College, University of London. Potentiometric Sensors in On-line Applications Ari lvaska Department of Analytical Chemistry, Abo Akademi, 20500 Abo (Turku) 50, Finland In the last few decades some entirely new fields of application have been developed from analytical chemistry and related subjects.Among these fields are the analysis of compounds in industrial and community wastes and the analysis of substances of biological significance at trace levels in the environment and in body fluids. The same extension of analytical chemis- try can also be seen in the chemical industry, where the successful choice of an analytical method for use in on-line applications requires not only chemical and analytical knowledge, but also an understanding of the engineering problems of the particular process and especially 283284 ELECTROANALYSIS Proc.Analyt. Div. Chem. SOC. problems connected with its control. As far as analytical methodology is concerned, new subjects such as the application of analytical techniques in flow-through systems and the use of minicomputers and microprocessors have been developed.When considering a particular task, the analytical chemist is confronted with the problem of selecting the right method and technique to be used. The needs in the chemical industry for improved product yields, lower costs and higher quality demand improved performance from the measuring and controlling equipment.The monitoring of industrial and natural effluents, reaction mixtures and biological fluids are all good examples of the importance of continuous analytical information. Criteria to be Met by an Automatic On-line Method and Instrument The criteria for an automatic method and instrument in on-line use are different from those for research work. The main demand is that the method should be fast and work in a con- tinuous rather than a batch mode.The signal generated by the instrument should preferably be electrical so as to be transferred easily to control equipment and computers. To minimise the dead time of the whole control loop, the sensors used should have a fast response to load changes. The method of analysis should be reproducible and have a high precision rather than accuracy because mostly only changes and differences are of importance in process control.The methods used should also have the required accuracy and precision over the expected range of measurements. The instruments should be easy to set up and run because on the plant they are normally run by unskilled personnel. The instruments are often used in highly corrosive environments and so should stand extremes of temperature and pressure and be able to run for long periods unattended.These are all criteria that need to be evalu- ated very carefully when choosing a method, and especially in designing an instrument. General Features of Potentiometric Sensors in Flowing Streams Potentiometry is a fast method of analysis and is thus very suitable for on-line use.The dead time of potentiometric sensors is small. In an ideal instance, where the species of interest are measured directly in the stream, the dead time caused by the analysis is the same as the response time of the sensor. One of the advantages of potentiometric sensors is that acti- vities rather than concentrations are measured and ion-selective electrodes respond only to free ions and not to those in complex form.These properties are of interest because activities are important in ionic reactions in aqueous systems and thus ion-selective electrodes provide convenient and necessary information on the chemical composition of the process liquid. The progress of reactions of interest can also conveniently be monitored by potentiometric sensors and they are suitable for many samples with no pre-treatment, such as coloured solutions and slurries, where many other methods fail.In potentiometry changes in e.m.f. can be determined with far better accuracy than their absolute values and thus a potentiometric method is usable in process control, where changes from a set value are of importance.Most potentiometric sensors are easy to handle and physically robust and their relative cheapness allows their replacement more frequently than some other process analyses Poten tiometric measurements are usually improved in streaming solutions for several reasons, e.g., the electrode itself cannot influence the sample concentration because the sample is continuously passing the electrode surface.The response time and the sensitivity of the electrode are normally improved owing to the thin diffusion layer and, by placing the reference electrode downstream, the ionic species flowing out of it do not influence the response of the indicator electrode. The reference electrodes used in continuous measurements should fulfil slightly different criteria and obviously should also have different designs from those electrodes which are used in static situations.One of the most important factors is the steady potential at the liquid junction. If the neutral electrolyte of the electrode is flowing through the junction to the stream, a fresh surface is maintained. A large external reservoir of electrolyte is then needed. This also pressurises the electrode against possible pressure variations in the flowing stream and the use of a constant pressure across the electrode tip prevents a reverse flow.October, 1979 ELECTROANALYSIS 285 There is also a non-flowing reference electrode that requires no liquid exchange between the process stream and the internal electrolyte.It has a plastic body that is a barrier to liquid exchange but not to the passage of external current.When used with pH electrodes this compensates the reading for changes in temperature. A third element in conjunction with the electrode pair is the thermo-compensator. Important Definitions Connected with Potentiometric Sensors Sensitivity is specified by the relationship between concentration of the active species and instrument output and hence by the slope of the electrode response.Sensitivity also specifies the minimum detectable change in concentration, governed by the signal to noise ratio of the instrument. Dead band is the range over which an input to an instrument can be varied without a detectable response. With potentiometric sensors the sensitivity may be affected by physical effects such as temperature and by chemical effects such as ionic strength and interfering species.Accuracy indicates how close a measured value is to a true value, and precision indicates how close different measurements on the same sample are to each other. The difference between the accuracy and precision of a potentiometric sensor is a matter of calibration. High accuracy is normally required of an instrument or sensor when used in quality control or in clinical testing, but high precision is necessary in process control.The selectivity of a chemical sensor is its ability to discriminate between the species of interest and possible interferents. With ion-selective electrodes it is given by the selectivity coefficients. Speed of response is usually defined as the time required for the sensor to reach a specified percentage of the total change observed.The most frequently used terms are the rise time, which is defined as the time between 10% and 90% of the total change, time constant from 0% to 63%, and response time, which is four times the time constant, or from 0% to 98%. The dead time of the measuring device is the time interval during which no change is observed a t the detector after alteration of the parameter being measured.Problems Connected with Potentiometric Sensors in On-line Use Many process streams contain solid particles that have to be removed before a potentio- metric measurement can be made. In the long term the surface of the electrode may become coated by inorganic and organic substances and a cleaning procedure should be employed.Cleaning can be effected either mechanically, by automatic brushing of the surface, or by using an ultrasonic cleaning method. By suitable design of the electrode the coating problems can also be reduced. Nevertheless, the electrodes occasionally have to be moved from the stream for proper cleaning. This can be done most conveniently when the electrodes are calibrated.Special care should be devoted to the physical shielding of the sensors when used in highly corrosive environments. The cables from the sensors to transmitters also require shielding. High-impedance converters may sometimes be necessary on the top of the glass electrode in order to convert the signal to a low-impedance signal for transmission. If high flow-rates and narrow lines are used, streaming potentials may be considered.It is probable that such potentials occur in continuously working flow-through cells and give extra disturbances in the form of potential fluctuations when the flow-rate varies. By correct choice of the pump and design of the flow lines, the effect of streaming potentials can be reduced. By increasing the conductivity of the flowing medium, e.g., by adding a neutral electrolyte to the stream, the effect of streaming potentials can also be reduced. In the continuous determination of fluoride the sample stream is mixed with a stream of TISAB buffer containing 1 M sodium chloride.In some instances, however, large amounts of salt cannot be added owing to possible impurities or when low-selectivity electrodes are used.When liquid ion-exchange electrodes are used, a high pressure of the sample stream affects the membrane and also might contribute to fluctuations in potential. As in static potentiometric measurements, the interfering species should be removed or masked before the stream enters the measuring cell. Redox electrodes, for example, respond286 ELECTROANALYSIS PYOC.Analyt. Div. Chem. SOC. to all oxidisable and reducible species in the stream. If the species of interest are bound, de-complexing is necessary. When the sample stream is extracted from the main stream, the pressure is normally reduced. If the stream contains dissolved gases these may cause gas bubbles, which may create an open circuit in the measuring loop.If the electrode has a plastic body, a static charge may be created on it when the stream moves fast and impinges against the electrode body. This charge should be considered and properly compensated for, otherwise electrical noise is produced. There is always drift in the electrodes, especially in continuous use, and in order to obtain reliable readings they should be calibrated frequently.This is normally done manually by transferring the electrodes from the stream into standard solutions. The calibration may also be effected without removing the electrodes from the stream but by connecting a stream of standard solution, which flows through the measuring cell. This method can be employed when the measurement is made in a side stream extracted from the main stream, eg., to be mixed with a reagent before measurement.For an arrangement with a side stream there is also a novel method of performing con- tinuous calibration of the e1ectrodes.l The potentiometric detector cell is connected to the outlet of a stirred tank containing a solution of the ion of interest. A neutral background electrolyte is pumped into the stirred tank and this causes a continuous change in the potential as a function of time at the outlet of the tank.A linear relationship exists between the potential and the logarithm of the concentration. In this procedure the initial concentration in the tank should be the same as the upper concentration limit at which the electrode is used. Different Set-ups in On-line Measurements As far as the position of the electrodes is concerned there are, in principle, two different ways of performing potentiometric measurements in on-line situations.The simplest set-up, shown in Fig. 1, is the case when the electrodes are immersed directly in the main stream. This modification is easiest to operate and another advantage is the short dead time of the system, depending only on the speed of response of the electrode.This simple system can be used only when an electrode sensitive to the ions of interest is available and chemical and physical interferences are negligible. The other modification is that a side stream is extracted from the main stream, as illus- trated in Fig. 2. The side stream may pass through a filter to remove any solids, so as to prevent the build-up of coatings on the electrode or blockage of the tubes.A reagent stream is then mixed with the sample stream, e.g., to buffer the pH, mask interferences, break up complexes of the ion of interest or give a reaction in which measurable ions are formed. The mixed stream can then be temperature and pressure controlled before entering the measuring cell. n Reagent I mV I Waste Fig.1. Measurement directly in the Fig. 2. Measurement in a side stream. Reagent Filter in region 1 and pressure main stream. is added to the sample. and temperature controls in region 2. There are instances in which the ion of interest is normally not present, e.g., the monitoring The information desired is then the possible In a normal run the ion is not present and the potential of the elec- of the efficiency of an ion-exchange coliimn. appearance of the ion.October, 1979 ELECTROANALYSIS 287 trode will drift or show anomalous potentials owing to interferences.One means of over- coming this problem is to add a small amount of the ion of interest to the sample stream so that stable potentials can be obtained. The background ion can be added by mixing the sample stream with a reagent stream.One drawback of this method is that when the flow- rate of the reagent stream varies, the output of the cell will also vary. To overcome this problem a non-interfering or labelled ion may be added to the reagent stream.2 In the mixed stream the difference in potential between electrodes selective to the ion of interest and to the non-interfering ion is measured.This system provides an effective means of eliminating the possible variations in the rate at which the reagent is added to the sample stream. The electrode pair responds only to the ratio of the activities of the ions. Some Applications For process stream applications, the potentiometric sensors are mostly used as monitors. Glass electrodes have found wide application in measuring devices for process control.Many processes are crucially dependent on pH, and waste waters and other effluents are required to have a certain pH before they can be discharged into lakes and rivers. In power stations, sodium- and chloride-selective electrodes are frequently used to monitor the water circuits. The presence of chloride can be associated with corrosive conditions within the boiler.These electrodes give valuable information on leaks in the system and can also be used to detect failure or saturation of an ion-exchange column. Fluoride electrodes are used to monitor the fluoride concentration in drinking waters and calcium electrodes are used to monitor the water hardness in water-conditioning systems.Cyanide concentrations in metal-plating baths are measured with cyanide electrodes. Inert redox electrodes, such as platinum and gold, are used to monitor chromate in plating effluents and for the control of chlorine during the chlorination of cyanide wastes. Sulphide electrodes have found considerable use in the pulp and paper industry, where the concentration of sulphide ion is of great interest in the circulating streams in the process, in gaseous effluents and in waste waters, and in monitoring the progress of the digestion process.An important and critical variable in the digestion of wood chips is the kappa num- ber, which is a measure of the reducing properties of the pulp. This, in turn, is related to the degree of delignification of the wood chips, which depends on the progress of the reaction taking place in the digester.The kappa number can be related to the concentration of hydroxide ions and to the concentration of free sulphide ions3 If a sodium-selective elec- trode is used together with a glass electrode, the alkali error of the glass electrode can be corrected and a true value of hydroxide-ion concentration can be obtained.A sulphide- selective electrode gives immediately the activity of the sulphide ion. Black liquor from the digester is further used and its chemicals recovered in the process. Sulphide ion activity is an important parameter when monitoring the progress of these operations. By oxidising the dilute black liquor, many of the problems of hydrogen sulphide emission in the later stages of evaporation and in the recovery boiler can be alleviated.Sul- phide-ion activity is a measure of the completion of this oxidation procedure, and it is also monitored in several other stages in the chemical recovery and circulating systems. Sulphide electrodes are also used to monitor hydrogen sulphide concentration in the effluent discharged to the environment from a heavy water plant where D20 is produced by a process employing a bithermal isotopic exchange process between counter-current flows of water and hydrogen sulphide gas.* A suitable gas- scrubbing device ahead of the measuring system provides a means of measuring several compounds in air and stack gas.The liquid effluent from the scrubber is continuously monitored. Systems such as this can be used, for example, to determine fluorides in gaseous effluents from phosphate rock and aluminium refining industries.Other applications are the monitoring of hydrogen sulphide and cyanide emissions. Potentiometric sensors can be used indirectly to measure and control species for which electrodes are not available. The measurement of sulphur dioxide and total oxidant gases as atmospheric pollutants can be performed with an iodide electrode.The gas sample is bubbled into an iodine - iodide solution and the change in iodide concentration is recorded. In the production of chlorine in mercury electrolysis cells the waste water contains toxic levels Gaseous compounds can also be monitored with potentiometric sensors.288 ELECTROANALYSIS Proc.Analyt. Div. Chem. SOC. of mercury. This water is normally treated with sodium hydrogen sulphide to precipite the mercury as insoluble mercury(I1) sulphide. Both pH and pS should be carefully controlled because of the formation of soluble polysulphides a t high pH and the formation of hydrogen sulphide at low pH. The sulphide electrode is needed to monitor the concentration of sul- phide, which should not be too high to discharge into waste waters nor too low to remove mercury efficiently.The control of the chemical composition of a stream with potentiometric sensors always encounters the problem of the logarithmic relationship between the potential and the con- centration of the species of interest. When sodium hydroxide is used to neutralise effluent streams containing strong acids, even small variations in either the flow-rate of the streams or the acid concentration cause significant changes in pH due to the slope of the titration curve at the set-point, as shown in Fig.3. If the stream contains weak acids or bases, the slope of the titration curve becomes more favourable for control purposes, as shown in Fig. 4. To decrease the effect of variations in the flow-rate or concentration, small amounts of, for example, sodium carbonate may be added to the stream before its pH is controlled.I Q Fig. 3. Neutralisation of strong acid with strong base. Fig. 4. Buffering around the set point value decreases the effect of variations in process parameters in the output. Small variations in process parameters cause large varia- tions in the output. Conclusion The use and especially the development of continuous on-line methods require a different approach to that used when considering manual batch methods.As illustrated in this paper, potentiometric sensors have several qualities that make them flexible and useful in a wide variety of on-line applications. It should be emphasised, however, that many of the methods mentioned are still under development and are far from the stage when they could be regarded as accepted standard methods. The rapid development of micro-processors and their appli- cations in chemistry offer new possibilities in the control of automatic on-line instruments and in data evaluation. In employing continuous on-line methods, it should always be remembered that careful applications engineering and a thorough understanding of the chemical background of a problem are required, as is the case with any analytical method or instrument. Finally, it should be emphasised that, as always, an analytical method is useful only when we know its limits, and the result of an analysis is correct only when we know its errors. References 1. 2 . 3. 4. Horvai, G., Thth, K., and Pungor, E., Analytica Chim. A d a , 1976, 82, 45. Riseman, J. H., and Frant, M., U.S. Pat., 3,964,988, 1976. Swarz, J. L., Adv. Instrum., 1975, 30, 608. Gulens, J., Jessome, K., and Macneil, C. K., Analytica Chim. A d a , 1975, 96, 23.

ISSN:0306-1396    

DOI:10.1039/AD979160283b

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Octobey, 1979 HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY 289 High-performance Liquid Chromatography in Clinical and Biological Chemistry The following are summaries of six of the papers presented at a meeting of the Scottish Region, the Chromatography and Electrophoresis Group and the Joint Pharmaceutical Analysis Group held on February 23rd, 1979, at the University of Edinburgh. Recent Developments in Post-column Reactions for H PLC R.W. Frei Free University, Department of Analytical Chemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands The pros and cons of post-column derivatisation techniques in HPLC are discussed in this paper. The restriction of this detection mode to reactions with relatively fast kinetics can be relaxed somewhat by resorting to air-segmented streams, which permit actual reaction times of up to 30min without excessive band broadening.This aspect is illustrated by cardiac glycosides, where a fluorigenic derivatisation with concentrated hydrochloric acid is carried out post-column and on-line in a 10-min reaction time. The selectivity and sensitivity thus gained permit easy assaying of low-dosage pharmaceutical formulations and dissolution- rate testing.The segmentation principle can also be adapted to advantage for fast reactions, where an excess of a reagent with similar detection properties usually interferes. Of particular importance in this respect are ion-pair or complexation reactions, which are useful, for example, for tertiary amines that cannot easily be derivatised otherwise.The use of extrac- tion detectors is based on the dynamic micro-extraction principle with chloro- and bromo- pheniramines, using dimet hoxyant hracene sulphonate as a fluorescent counter ion. A two or three orders of magnitude improvement in sensitivity can be achieved with band broaden- ing below 20%; the gain in selectivity permits direct assays of complex samples, e.g., urine samples, without sample preparation steps.The coupling of on-column preconcentration techniques with these types of detector will greatly enhance their value in trace analytical problem solving. Both modes discussed give reproducible signals, the relative standard deviation being 2%, and are therefore suitable for accurate quantitative work. Introduction The introduction of chemical reactions in liquid chromatography as an aid to improving the detection properties and selectivities for certain groups of compounds has become well known in the past few years.The best proof of this popularisation is the rapidly increasing number of publications and the appearance of reviews and The two major lines of methodology are defined as pre-chromatographic and post- chromatographic derivatisation techniques, the latter being the subject of this paper.The development of post-column reactors in liquid chromatography during the past years can be traced to the lack of detectors of sufficient sensitivity and selectivity for certain analytical problems. The pros and cons have been elaborated on earlier,lS2 but certainly the major advantages of the reaction detector are that artefact formation is not critical and that the reaction does not have to go to completion or give well defined derivatives so long as it is reproducible.The most serious disadvantage, on the other hand, is the interdependence between the mobile phase and the reaction medium, and this has probably been one of the major reasons why the development of post-column techniques in HPLC has not been very widespread until now.Obviously the kinetics of a reaction are also very important and determine to a large degree the feasibility of using a particular type of reaction detector. There are three different types of post-column reactor: tubular or capillary reactors; bed reactors; and segmented streams, such as are used in autoanalyzers.covering this area.290 Proc. Analyt. Div. Chem. SOC. Tubular Reactors As with any other post-column reactor, the major problem is to avoid excessive dead volume and consequent band broadening during the reaction. This band broadening can occur in the mixing device(s) during addition of the reagent and in the reaction tract. The optimum design is therefore essential in order to avoid substantial losses of chromatographic resolution. The theoretical aspects of band broadening in tubular reactors have been disc~ssed.~ Based on principles of classical flow dynamics it is relatively simple to predict band broadening in tubular reactors.A typical example of the use of a tubular reactor is the reaction of nonapeptides with Fluram.596 It is now possible to use a much shorter reaction time as at 10 s about 90% of the total fluorescence is generated.The design of the mixing unit is also of utmost importance, particularly when solutions of different densities are mixed and when the flow-rates of the mobile phase and the reagent stream differ considerably. In such instances mixing against the stream of eluting agent causes more turbulence and radial mixing and hence less band broadening.The reproducibility of the chromatographic patterns is good and the reproducibility of peak areas for resolved peaks is such that one can truly speak of a quantitative technique. For a peak with k’ equal to 4, the band broadening due to the mixing device and tubular reactor is of the order of 4% for a reaction time of 10s.Theoretical calculations give a somewhat lower value (3.5%) but do not include the influence of the mixing device. For a 1-min reaction time under otherwise similar conditions a band broadening of more than 20% due to the reaction unit would be found, which for many applications would be unacceptable. HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY A plateau for this rapid reaction is reached in about 50 s.In such an instance one might have to consider the use of a bed reactor. Bed Reactors The use of bed reactors consisting of columns packed with glass beads of various sizes has been pioneered primarily by two group^.*^^ Such a bed reactor can be looked on as a chromatographic column in use under conditions where no retention occurs, and the theory for predicting band-broadening phenomena in a bed reactor is consequently based on para- meters such as packing geometry, average particle size, tortuosity, fluid velocity and diffusion coefficients.A study of these aspects has been made.4y7 Again considering the previous example, where a tubular reactor gives band broadening in excess of 20%, with the equations proposed earlier for bed reactors one would obtain band broadening well below 5%.Therefore, for longer reaction times of 1 min and up to 5 min one would preferably use bed reactors, despite the complication thereby introduced. Segmented-flow Reactors For even longer reaction times one would have to resort to the flow-segmentation principle in order to overcome excessive band broadening. The theoretical aspects of air-segmented flows are the most complex and least understood.A semi-empirical approach to coming to a better understanding of the principles involved has been undertaken by Snyder.8 Band broadening in the reaction tract can be attributed, in part, to leakage between the segments (spaced by air bubbles) as a result of wetting on the capillary wall. This effect is, however, insignificant in comparison with the band broadening effects introduced by mixing T-pieces, de-bubblers or phase separators. Much effort will therefore have to be made and is already being put into producing better designs of these parts or possibly into electronic de-bubbling techniques.A typical example for such a longer reaction involves the dehydration of cardiac glycosides with concentrated hydrochloric acid to form a fluorescent p r o d ~ c t .~ This process is enhanced by adding a mixture of hydrogen peroxide and ascorbic acid; the exact mechanism is un- known. The extrapolated kinetic curve for this reaction shows that a plateau is reached after about half an hour and, following our previous philosophy, an actual reaction time of 10 min was chosen.Use of the air-segmentation principle is a feasible approach, both from the point of view of the reaction time and the aggression of the reagents. An all-glass and PTFE autoanalyzer unit was used. The influence of the segmentation on the resolution of the chromatographic bands is demonstrated in Fig. 1.October, 1979 HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY 291 It was possible with such a system to keep the band broadening due to the autoanalyzer reactor unit below lo%, which is surprising when considering that a mechanical de-bubbler was employed.The approximately 100-fold improvement in detectability and the gain in selectivity compensates fully for the loss in resolution. Extraction Detectors One limitation of post-column reaction detectors in general is the requirement that the reagent, usually added in large excess, should not interfere with the detection process.This condition seriously limits the choice of reaction systems, many of which, from a kinetics point of view, would be entirely suitable for such purposes. It was, therefore, resolved10 to investigate the potential of using on-line post-column extraction principles, well known in conventional autoanalyzer work, for example with fluorescent ion-pair formationll ; these principles have recently been used by Gfeller et al.as a basis for HPLC detection.12 As a model system, two amines of important pharmaceutical interest, viz., chloropheniramine and bromopheniramine, were selected. 9,l0-Dimethoxyanthracene-2-sulphonate, which has also been studied by Westerlund and Borg,13 was chosen as the fluorescent pairing ion. The apparatus used for this work consists again of standard Technicon equipment (Fig.2). The aqueous HPLC effluent containing the amine was mixed with an aqueous solution of the counter ion in an air-segmented stream. The mixture passes through a mixing coil in order to ensure complete ion-pair formation, and is then mixed with an immiscible organic solvent heavier than the aqueous phase. This three-phase mixture passes through a second mixing coil, thus enabling the ion pair to be extracted into the organic phase.The stream is then directed to a phase separator where the air, the aqueous phase and part of the organic phase are removed, while the remaining organic layer passes through the flow-cell of a fluorimeter in which the fluorescent ion-Dair is detected.No air segmentation I t I 50 40 30 20 Time/m in L From HPLC I DAS : Air r L m Fluorimeter Fig. 1. Comparison of the Fig. 2. Autoanalyzer arrangement for post-column ion-pair fluorescence signals obtained formation and extraction. Flow-rates: 1, 0.42 ml min-1; 2, with and without air seg- 0.32 ml min-l; 3, 0.43 ml min-l; 4, 0.34 mlmin-1.Coil 1, 5 mentation. Detection of turns; coil 2, 20 turns (reference 10, reproduced with the per- cardiac glycosides separated by reversed-phase liquid chromatography (reference 9, reproduced with the per- mission of Elsevier, Pub- lishers). mission of Huthig, Publishers). The calculated band broadening caused by the ion-pairing fluorescence system was found This value is acceptable for most analytical applica- A comparison of the relative standard deviations obtained with both ultraviolet and to be about 18% for both compounds.tions.292 PYOC. Analyt. Div. Chem. SOC. fluorescence measurement of four replicate injections of bromopheniramine indicated that the ion-pair system contributed negligibly to the analytical error (both detector systems yielding a relative standard deviation of about 3%).Detection limits for both drugs were about 3 ng (5: 1 signal to noise ratio) at a retention time of 10-15 min. This technique was also applied to urine analysis (see Fig. 3) by injection into the HPLC system of 20 p1 of human urine spiked with 1.0 pg ml-l (1 p.p.m.) of chloropheniramine.In this urine sample, chloropheniramine was easily detected by the fluorescence detector. However, with ultraviolet detection at 225 nm there was no observable difference between sample and blank. HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY Time/rnin Fig. 3. Comparison of ion-pair fluorescence and ultraviolet (225 nm) chromatograms of 20 p1 of urine containing 1.0 p.p.m. of chloropheniramine.Arrow indicates interfering peak in ultraviolet results. Chromatography conditions as described in the text (reference 10, reproduced with the permission of Hiithig, Publishers). Although the direct injection of urine into an HPLC system can be considered to be a severe test, the ion-pair fluorescence detector performed exceptionally well. Conclusion Derivatisation techniques can be a very powerful tool to enhance the effectiveness of modern liquid chromatographic techniques and detectors.In post-column techniques, many groups are still working on the optimisation of technical designs to reduce further band broadening, but as the theoretical aspects for these reactors become better known one can expect significant advances in this area. The possibilities of adapting thermal,' photo~hemical~~ and catalytic15 processes to initiate, accelerate and simplify post-column reactions are being actively pursued by many groups and the field is wide open.Coupling of reaction detectors with step gradients6 and with large volume injections6,16 will greatly enhance their usefulness. Even though the examples discussed in this paper were restricted to fluorescence detection, there remains no doubt that this philosophy can be expanded to other detection modes.In electroanalytical detec- tion this has, in fact, already been achieved4*l7 and further work in this direction is in progress.October, 1979 HPLC IN CLINICAL AND BIOLOGICAL CHEMISTRY References 293 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.16. 17. Frei, R. W., Res. Dev., 1977, February, 42. Lawrence, J. F., and Frei, R. W., “Chemical Derivatization in Liquid Chromatography,” Elsevier, Blau, K., and King, G. S., Editors, “Handbook of Derivatives for Chromatography,” Heyden, Deelder, R. S., Kroll, M. G. F., Beeren, A. J . B., and van den Berg, J. H. M., J . Chromatog., 1978, Frei, R. W., Michel, L., and Santi, W., J .Chromat., 1976, 126, 665. Frei, R. W., Michel, L., and Santi, W., J . Chromat., 1977, 142, 261. Jonker, K. M., Thesis, University of Amsterdam, Amsterdam, 1977. Snyder, L. R., J . Chromat., 1976, 125, 257. Gfeller, J. C., Frey, G., and Frei, R. W., J . Chromat., 1977, 142, 271. Frei, R. W., Lawrence, J. F., Brinkman, U. A. Th., and Honigberg, I., J . High Resolution Chromat., Gfeller, J, C., and Frey, G., 2.Analyt. Chem., 1978, 291, 332. Gfeller, J. C., Frey, G., Huen, J. M., and Thevenin, J. P., J . High Resolution Chromat., 1978, 1, Westerlund, D., and Borg, K. O., Analytica Chirn. Ada, 1973, 67, 89. Twitchett, P. J., Williams, P. L., and Moffat, A. C., J . Chromat., 1978, 149, 683. Nachtmann, F., J . Chromat., 1978, 149, 693. Schauwecker, P., Erni, F., and Frei, R.W., J . Chromat., 1976, 136, 63. Kissinger, P. T., Analyt. Chem., 1977, 49, 447A. Amsterdam, 1976. London, 1977. 149, 669. 1979, 2, 11. 213. HPLC Analysis of Antidepressants on Silica Gel 1. D. Watson Department of Biochemistry, Royal Infirmary, Glasgow, G4 OSF Tricyclic antidepressant drugs are widely used in the amelioration of depression. Of this class of drugs amitriptyline and imipramine, which are derived from the tranquilliser chlor- promazine, are the most commonly used.All three drugs are metabolised mainly by ring hydroxylation and mono- and didesmethyla- tion of the aminopropyl side chain. The major metabolites of chlorpromazine, amitriptyline and imipramine are the 5-sulphoxide, 10-hydroxy and 2-hydroxy compounds, respectively ; however, the pharmacologically active metabolite for each of these drugs are 7-hydroxy- chlorpromazine, nortriptyline (desmethylamitriptyline) and desmethylimipramine.1-3 Plasma levels for each of the parent psychosedatives and their pharmacologically active metabolites have been demonstrated as having a significant correlation with assessed clinical response.ls495 As individuals exhibit genetically determined variation in their handling of such drugs the response of each to his dosage schedule should be assessed by monitoring plasma levels.Variations in drug metabolism are also reflected in the urinary profile; such monitoring requires a simple, reliable quantitative method. Chlorpromazine, amitriptyline and imipramine have all been determined by use of gas - liquid chromat~graphy~s~~~ but had one or more of the following requirements : lengthy purification procedures, derivative formation to improve the chromatography of the meta- bolites and specific detectors. These requirements resulted in lengthy complex assays, with which it was difficult to achieve reproducible results.Thin-layer chromatography with fluorimetry of the eluted zone8 or isotope dilutiong have also been tried.In an attempt to simplify the assay of these drugs, high-performance liquid chromato- graphy (HPLC) has been used, and several procedures have been published employing ion exchange,lO silica geW12 and reverse phase ion pair.13 As silica gel provides, in effect, normal-phase elution, the least polar compound elutes first. The drugs are less polar than their metabolites, therefore the sensitivity for the parent drug is enhanced.Initially, the separation of amitriptyline and its metabolites was studied. Although silica gel would not normally have been the chosen chromatographic mode, as the molecules are organic, ionic and water soluble, it was hoped to resolve isomeric metabolites. A preliminary separation of amitriptyline and nortriptyline using a methanol - ammonia (100: 1.5) solvent, with protriptyline as internal standard, was achieved (Fig.1). The sensitivity and linearity were found to be such that they would be compatible with the determination of plasma levels.14 Other workers have used this as the basis of a method for chloripramine and its desmethylated294 Proc. Analyt.Div. Ckem. SOC. metab01ite.l~ An unidentified metabolite peak and nortriptyline could not be resolved by use of this solvent system. Recourse to other thin-layer chromatographic solvents indicated that a benzene - dioxan - ethanol - ammonia solvent mixture could provide satisfactory resolution ; however, this solvent is opaque to ultraviolet light. Proportional calculatj on of the Hildebrand solubility parameters of ultraviolet-transparent solvents can provide an alternative solvent of comparable polarity; a medium polarity solvent such as dichloro- methane - propan-2-01 was indicated.Propan-2-01 acts as a co-solvent for ammonia and serves to de-activate the silica gel reproducibly. Ammonia is required in the solvent in order to reduce peak tailing.A systematic investigation of solvent composition indicated that the ratios of the alcohol and base had profound effects on the chromatography of amitriptyline and its metabolites. A general ratio of dichloromethane - propan-2-01 - ammonia (1OO:x:y) was considered and x or y varied only when the other was maintained at the ratio of the original solvent; variations in x from 1-50 and y from 0.05-1.0 were employed.Solvent selectivity will vary more than solvent strength as alcohols and bases are known to have selective properties.16 The efficiency, retention and resolution of the different solvent combinations were examined for the two main metabolites, nortriptyline and 10-hydroxyamitript yline. HPLC IN CLINICAL AND BIOLOGICAL CHEMISTRY A$ a U t m - 0.5 % 2 1 .o .c 0.8 2 C 0.6 E E Q: 0.4 + m .- 0 d 2.0 1.0 Retention volume/ml Fig.1. HPLC of ami- triptyline, nortriptyline and protriptyline. A, Amitrip- tyline, N, nortriptyline, P, protriptyline. Solvent, meth- anol - ammonia (100 + 1.5); flow-rate, 0.5 ml min-I; col- umn, 5 ,urn silica, 25 cm x 2 mm i.d. ; wavelength, 240 nm. 0 0.4 0.8 1.2 1.6 Rs Fig. 2. Effect of changes in am- monia and propan-2-01 ratios on the resolution of 10-hydroxyamitriptyline and nortriptyline.RS = Resolution function. A, Base changes; B, propan- 2-01 changes. Retention decreased exponentially with increase in the solvent component ; however, there was a reversal of the relative positions of the two peaks. In Fig. 2 R, = 0 is the point of no resolution. Propan-2-01 ratios of 1.75 and 10 gave the best efficiencies and asharp peak of efficiency was obtained for ammonia at 0.2.Base line resolution is achieved a t R, >, 1.5 (Fig. 2). From these results it is possible to arrive at an optimum solvent compo- sition for the separation of amitriptyline and its polar metabolites; the chosen solvent was in the ratio (100: 10: 0.2). Fig. 3 shows the resulting chromatogram of an extract from the urine of a patient on amitriptyline.By using “infinite” diameter columns with this solvent, efficiency was sufficiently improved to resolve amitriptyline completely from the solvent front. Alternatively, reduction of the propan-2-01 content to 1.75 resulted in greater retention of all the peaks. This was compatible with the requirements for retention, resolution and efficiency.The methods are as previously publishedl1,l2 and have been applied to other tricyclic structured drugs, i.e., plasma and urinary imipramine and desmethylimipramine (Figs. 4 and 5 ) and plasma chlorpromazine (Fig. 6). The silica is probably protected from the deleterious effects of the base as columns have remained effective for as long as 2 years; possibly the alcohol or water contained in theOctober, 1979 HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY I 10-0HN 10-OHA 1 I I I I 16 12 8 4 Retention volurne/rnI 1.5 (u C 1.0 m -2 s1 a 2 0.5 D Fig.3. HPLC of urinary amitripty- line and metabolites. A, Amitriptyline ; DI fi I 20 10 Retention volurnehl 295 I 10-OHA, 10-hydroxyamitriptfiine ; N, HPLC of plasma imipramine and metabo- nortriptyline; IS, internal standard lites.I, Imipramine; DDI, didesmethylimipramine ; (protriptyline) ; 1 0-OHN, 1 O-hydroxy- IS, internal standard (nortriptyline) ; DI, desmethyl- nortriptyline; U/D, unidentified. Sol- imipramine ; 2-OHI, 2-hydroxyimipramine. Solvent, vent, dichloromethane - propan-2-01 - dichloromethane - propan-2-01 - ammonia (100 + 75 + ammonia (100 + 10 + 0.2); flow-rate 0.2); wavelength, 251 nm.Other conditions as in 1 ml min-l; column, 5 pm silica, 25 Fig. 3. cm x 2 mm i d . ; wavelength, 240 nm; chart speed, 25 cm h-l. solvents is forming a layer on the silanol groups with hydrogen bonding, effectively liquid - liquid chromatography. It is unlikely that such a large proportion of alcohol (10%) is acting entirely in this manner, and the majority will be part of the mobile phase; yet there is a clear optimum concentration.The mechanism of separation does not appear to be readily identifiable. Propan-2-01 and water contained in the solvents have a de-activating effect on silica1'; this is probably achieved by solvation of the acidic silica. The sharp peak of efficiency found with ammonia content changes may reflect a neutralisation of the acidity of silica; thus silanol groups may become ionic ammonium silanol groupings.The relative attraction between this postulated surface effect and the mobile phase provides a mechanism for separation. The dependence of efficiency on solvent proportions and the requirement of a lengthy initial period of equilibration to achieve reproducible column Fig.4. I 32 24 16 8 Retention volume/ml 0 1 .o 0) C 0.5 4 Q 8 II Fig. 5. HPLC of urinary imipramine and metabolites. Other abbreviations as in Fig. 4. Wavelength, 251 nm. 10-OHI, 10-Hydroxyimipramine; 10-OHDI, 10-hydroxydesmethylimipramine; 2-OHDI, 2-hydroxydesmethylimipramine. Con- ditions as in Fig. 3.296 Proc. Analyt. Div. Chem. SOC. performance suggest that a modification of the silica is taking place.Another apparent anomaly, possibly related to the separation mechanism, is the order of elution of the amine metabolites of the tricyclic drugs, viz., tertiary, primary, secondary; this order may relate to the relative contributions of the inductive and steric basicity of the aminopropyl side chain. Alcoholic basic eluting agents on silica gel columns can be optimised for the separa- tion of other weak bases, e.g., nicotine,l* colchicinelg and nitrazepam.20 Such normal phase systems can be devised by utilising the influence of the secondary solvents.They provide an alternative to reverse phase where, in drug metabolism studies at least, the compound of greatest interest is the parent compound, which is usually non-polar and present in relatively low concentrations. HPLC IN CLINICAL AND BIOLOGICAL CHEMISTRY 1.5 1 .o 01 C m f2 4 2 0.5 20 10 0 Retention volume/ml Fig.6. HPLC of plasma chlorpromazine and metabolites. C, Chlorpromazine : IS, in- ternal standard (amitriptyline) ; DDC, dides- methylchlorpromazine : DC, desmethylchlor- promazine ; 7-OHC, 7-hydroxychlorpromazine ; CSO, chlorpromazine 5-sulphoxide.Wave- length, 258 nm. Conditions as in Fig. 4. A further potential of normal-phase systems is in the direct linking of liquid chromato- graphs to mass spectrometers. A problem of such linking is the excess of solvent molecules and attendant impurities that may swamp the mass spectrometer. The answer is probably to evaporate the solvent prior to feeding the eluted analyte to the mass spectrometer on a moving belt. Solvents employed in normal-phase chromatography can be chosen such that low boiling-point solvents are used ; reverse-phase solvents may include a preponderance of water, requiring higher temperatures.However, excessive heat can lead to thermal degradation of the analyte, thereby nullifying one of the major advantages of HPLC as an analytical system.It is possible, therefore, that normal-phase separations may be the mode of choice in direct mass spectrometric - liquid chromatographic interfacing. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. References Mackay, A. V. P., Healey, A. F., and Baker, J., BY. J. Clin. Pharmac., 1974, 1, 425. Hucker, H. B., and Porter, C. C., Fedn Proc. Fedn Am. Socs Exp. Biol., 1961, 20, 172.Hermann, B., Schindler, W., and Pulver, R., Med. Exp., 1959, 1, 381. Braithwaite, R. A., and Widdop, B., Clin. Chim. Acta, 1971, 35, 461. Gram, L. F., Reisby, N., Ibsen, I.. Nagy, A., Dencker, S., Bech, P., Peterson, G. O., and Curry, S. H., Analyt. Chem., 1968, 40, 1251. Cooper, T. B., Allen, D., and Simpson, G. M., Psychopharmac. Commun., 1975, 1, 445. Faber, D. B., Mulder, C., and Man in't Veld, W.A., J . Chromat., 1974, 100, 55. Maguire, K. P.. Burrows, G. D., Coghlan, J. P., and Scoggins. B. A., Clin. Chem., 1976, 22, 761. Smith, D. J., Spectra-Phys. Chromat. Rev., 1975, 1, 1. Watson, I. D., and Stewart, M. J., J . Chromat., 1977, 132, 155. Watson, I. D., and Stewart, M. J., J . Chromat., 1977, 134, 182. Christiansen, J.. Clin. Pharmacol.Ther., 1976, 19, 318.October, 1979 HPLC IN CLINICAL AND BIOLOGICAL CHEMISTRY 297 13. 14. 15. 16. 17. 18. 19. 20. Knox, J. H., in Dixon, P. F., Gray, C. H., Lim, C. K., and Stoll, M. S., Editors, “High Pressure Watson, I. D., and Stewart, M. J., J . Chromat., 1975, 110, 389. Moyes, R. B., and Moyes, I. C. A., Postgrad. Med. J . , 1977, 53, 117. Bristow, P. A., “L.C. in Practice,’’ HETP, Wilmslow, 1976, p.149. Snyder, L. R., and Kirkland, J . J., “Introduction to Modern Liquid Chromatography,” John Wiley Watson, I. D., J . Chromat., 1977, 143, 203. Jarvie, D., Park, J., and Stewart, M. J., Clin. Toxicol., in the press. Pye Unicam Ltd., Application note, 7042.33.1506.1 1, Pye Unicam, Cambridge, 1976. Liquid Chromatography in Clinical Chemistry,” Academic Press, London, 1976, p.7. and Sons, New York, 1974, p. 254. The Role of HPLC in the Investigation of the Poisoned Patient M. J. Stewart* Toxicology Unit, Department of Clinical Chemistry, Royal Infirmary, Edinburgh, EH3 9 Y W The range of analyses required by clinicians faced with poisoned patients is theoretically very wide, but at present the instrumentation and expertise available in all but a few hospital laboratories has placed severe constraints on the number of drugs that can be determined routinely, especially when rapid analyses are required out-of-hours.The great majority of poisoned patients are treated by conservative clinical techniques without the need for analysis of the drug or poison concerned. There are, however, a few circumstances in which a quantitative determination of the plasma concentration of the substance concerned may be of value for the prognosis or in order to decide whether to institute a specific form of treatment.This small group is shown in Table I. TABLE I DRUGS/POISONS FOR WHICH A QUANTITATIVE DETERMINATION IS REQUIRED AS AN EMERGENCY Salicylate, Paracetamol, Paraquat, Iron, Lithium, Quinine, Phenobarbitone Simple, acceptable quantitative techniques for this group of compounds should be available in every hospital laboratory that serves an acute admissions unit.The methods currently in use consist of a variety of colorimetric and spectrophotometric techniques, with fluori- metry for quinine. (Lithium is determined by absorption or emission spectrophotometry.) Some larger laboratories employ gas - liquid chromatography, but the technique is not widespread for emergency use.Some of the methods employed may include the determination of inactive metabolites. The use of such methods has, in the past, led to failure to appreciate the true extent of poisoning and to errors in treatment. This is particularly so with paracetamoll and the barbiturates,2 where failure to measure the true excretion of unchanged barbiturate in the urine led to a 10-year controversy over the use of forced alkaline diuresis.There is now a greater awareness in clinical laboratories of the need for specific methods of drug analyses and HPLC theoretically offers the answer to some of these problems. Emergency Methods Of the five compounds in Table I that are amenable to HPLC analysis, methods have been produced for all but paraquat.Published methods for paracetamo13 and salicylate4 employ protein precipitation with perchloric acid as the only sample preparation, after which a sample of the supernatant solution is injected on to the column. Quantitative determination of the unchanged drug and its major metabolites is obtained in each instance.The paracetamol method has been refined by us to produce a method that can provide a quantitative result in less than 5 min. The method for quinine and its metabolites requires an initial extraction procedure, but is more specific than some of the fluorimetric methods * Present address : Department of Biochemistry, Royal Infimary, Glasgow, G4 OSF.298 Proc.Analyt. Div. Chem. SOC. currently in use.5 Some of the metabolites of quinine may be pharmacologically active and their separate determination may be clinically useful. The barbiturates show their highest absorbance at pH 10, which is too alkaline to be used with silica columns. There is, fortunately, adequate sensitivity at wavelengths less than the optimum. More important are the differences in molar absorptivity between the different barbiturates, which, unlike gas - liquid chromatography with flame ionisation detection, requires that each barbiturate should be read from a separate calibration graph.Phenobarbitone is the species most commonly required to be analysed quantitatively in order to determine the need for forced alkaline diuresis to enhance excretion. Several methods have been published for the analysis of phenobarbitone by HPLC, often along with other anti-convulsants which may well be taken concurrently in cases of self-p~isoning.~~~ A compromise wavelength of 200-220 nm is used for detection.Paraquat at present remains to be satisfactorily analysed by HPLC, although it can be resolved on ion-exchange columns8 and therefore could be run as an ion pair on reversed-phase silica gel following extraction as an ion pair from ~ l a s m a .~ HPLC IN CLINICAL AND BIOLOGICAL CHEMISTRY Barbiturate analysis is less satisfactory at present for two reasons. Screening Screening techniques are greatly over-used and should only be employed where the clinician genuinely has no information relating to the substance ingested. Bad screening techniques are dangerous and may also lead to legal problems when controlled drugs are involved.Traditionally, gas - liquid chromatography (using 2 columns) or thin-layer chromatography have been used. The latter is more popular where identification rather than quantification is required and where the number of samples handled is large. HPLC offers the possibility of providing a fuller screening programme if used in conjunction with gas - liquid chromatography. HPLC should be able to provide the best separation and quantification but currently suffers from the lack of a universal detector.The area in which gas - liquid chromatography is liable to maintain its position as the technique of first choice is that of volatile compounds such as alcohols and amphetamines, but despite the theoretical advantages, few results have yet emerged concerning HPLC screening methods.This may be a reflection on the flexibility of the technique, which enables an optimised system to be developed for any single drug or group of drugs. For this reason, universal solvent systems covering a large range of drugs are less popular.The few studies which have been reported indicate that normal-phase silica gel is acceptable for the resolution of drugs of abuselo and for a variety of basic drugs, such as tricylic anti-depressants and phenothiazinesll and q~inidine.~ The systems used were based on the methanol - ammonia system that is used widely for thin-layer chromato- graphy, but for HPLC careful control of the concentrations of secondary solvents is necessary for reproducible determinations.The advantages and disadvantages of the three techniques are shown in Table 11. TABLE I1 COMPARISON OF TLC, GLC AND HPLC AS SCREENING TECHNIQUES TLC GLC HPLC Range of analytes . . . . No limits Sample preparation. . . . Rapid Method of identification . . Relative retention + Quantitation .. .. . . Moderate Metabolite analysis . . . . Possible Recovery of sample. . . . Possible Speed .. . . Slow specific sprays Volatile sample Variable Rapid Relative retention required Good Usually not possible Not possible Ultraviolet absorbance required Rapid Rapid Relative retention + Good Possible UV scanning Possible Reversed-phase silica gel columns have been used for a large number of pure drugs by Twitchett and Moffat12 and found to be more successful for acidic than basic compounds, which give poor peak shape and resolution.Twitchett et al. ,13 using cation-exchange columns, were able to demonstrate good separa- tion of a wide range of basic compounds when using the eluting agent at pH 3 and methanolOctober, 1979 HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY 299 concentrations of 60%.This procedure is now increasingly being replaced by ion-pair chromatography on reversed-phase silica gel, on which elegant separations are being achieved, such as that for the tricyclic anti-depressants and their metabolites reported by Mellstrom and Braithwaite.14 HPLC screening will continue to expand as complementary to gas - liquid chromatography, but a comprehensive system is still some way from development. The use of normal- and reversed-phase columns in parallel would seem to be indicated in order to provide rapid identification of unknown peaks, with stop-flow scanning as a useful additional indicator of structure.HPLC for Analysis of New Drugs and Metabolites Despite the development of many derivative techniques, gas - liquid chromatography has never been the most satisfactory method for the determination of water-soluble compounds.HPLC opens up the prospect of quantitative analyses of a range of highly polar drugs, which have been neglected up to now. In particular, the antibiotics and anti-cancer drugs are now amenable to investigation, and unusual clinical problems, such as overdose with colchine,l5 can now be fully investigated.The ability to measure metabolites along with the parent drug already allows a clearer understanding of the metabolism and excretion of some species of drugs. Sensitivicv The majority of HPLC systems at present in use employ ultraviolet detectors, the sensitivity of which has been questioned. Fortunately, the great majority of drugs exhibit acceptable ultraviolet absorption, especially when wavelengths of the order of 200 nm are employed.For this reason, reversed-phase systems, using solvents that are optically clear at this wavelength, are increasingly being used. For those drugs with a high absorption at a higher wavelength, the sensitivity of HPLC can approach that of the electron-capture detector.Fig. 1 shows the trace obtained from the analysis of a plasma sample containing 50 mg each of diazepam and its major metabolites. The limit of detection of these benzodiazepines in that system is 10 pg 1-1 using a 1-ml sample of plasma. 0.004 - 0.003 Ec (D m c! 0.002 C m e a Q 0.001 a I I I I I Elution volurne/rnl 4 Fig. 1. Separation of diazepam and its metabolites by HPLC plasma extract.Conditions: column, 10 mm x 4 mm i.d. stainless steel; packing, Hypersil-ODS (Shandon) ; solvent, acetonitrile with 0.0 1 M sodium acetate buffer (pH 4.6), 30 + 70; pump rate, 2 ml min-l; wavelength, 240 nm (0.01 AUFS); volume injected, 5 pl. Extract from a spiked plasma containing 50 ng each of diazepam (D), desmethyldiazepam (DM), tem- azepam (T) and oxazepam (0) in 1 ml.Internal standard, prazepam (100 ng), added before extraction. It is, perhaps, too early to attempt to review the impact of HPLC upon toxicological The flexibility, rapidity and operator acceptability of currently available tech- analysis.300 Proc. Analyt. Div. Chem. SOC. niques are leading to a rapid increase in the standard of analyses which the laboratory can offer to the ward.HPLC IN CLINICAL AND BIOLOGICAL CHEMISTRY 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. References Stewart, M. J., Adriaenssens, P. I., Jarvie, D. R., and Prescott, L. F., Ann. Clin. Biochem., 1979, 16, 89. Bloomer, H. A., and Maddock, R. K., in Matthews, H., Editor, “Acute Barbiturate Poisoning,” Excerpta Medica, Amsterdam, 1971, pp. 233-253. Adriaenssens P.I., and Prescott, L. F., J . Pharm. Pharmac., 1978, 6, 87. Prescott, L. F., King, I. S., Brown, L., Balali, M., and Adriaenssens, P. I., Proc. Analyt. Div. Chem. Guentert, T. W., Coates, P. E., Upton, R. A., Combs, D. L., and Riegelman, S., J . Chromat., 1979, Soldin, S. J., and Hill, J. G., Clin. Chem., 1976, 22, 856. Kabra, P. K., Stafford, B. E., and Marton, L. J., Clin. Chem., 1977, 23, 1284.Pryde, A., and Darby, F. J., J . Chromat., 1975, 139, 311. Jarvie, D. R., and Stewart, M. J., Clinica Chim. Acta, 1971, 94, 241. Jane, I., J . Chromat.. 1975, 111, 227. Watson, I. D., and Stewart, M. J., J . Chromat., 1977, 134, 182. Twitchett, P. J., and Moffat, A. C., J . Chromat., 1975, 111, 147. Twitchett, P. J., Gorvin, A. F. P., and Moffat, A. C., J . Chromat., 1976, 120, 359.Mellstrom, B., and Braithwaite, R., J . Chromat., 1978, 157, 379. Jarvie, D. R., Park, J., and Stewart, M. J., Clin. Toxicol., 1979, 14, 375. Soc., 1979, 16, 300. 162, 59. HPLC in Clinical Pharmacological Studies of Analgesic Drugs L. F. Prescott, I. S. King, Lindsey Brown, M. Balali and P. I. Adriaenssens University Department of Therapeutics and Clinical Pharmacology, The Royal Infirmary , Edinburgh, EH3 9YW Most of the methods used for the assay of analgesics such as aspirin and paracetamol in biological fluids are non-specific and there are major difficulties with the determination of their metabolites.Gas - liquid chromatographic methods overcome the problems of specificity, but with therapeutic concentrations of these drugs derivatisation is necessary because of their polar nature.The introduction of HPLC has been a great advance and it is now possible to separate and measure many polar drugs and metabolites by using simple procedures. We have developed simple, rapid and specific HPLC assays for aspirin, salicylic acid, paracetamol, antipyrine, diflunisal, phenylbutazone and mefenamic acid in plasma and urine.With aspirin and paracetamol, metabolites can be measured simultaneously. No solvent extraction is required and the whole procedure can usually be completed within a few minutes. The general method for the determination of these drugs in plasma consists of the addition of a suitable internal standard, precipitation of plasma proteins with perchloric acid or acetone, centrifugation and direct injection of 5-10 pl aliquots of the clear supernatant into the HPLC column. The aqueous internal standard is added and an aliquot of the mixture injected into the column.The columns were internally polished stainless steel, 100-150 x 4.5 mm i.d., packed with 5 pm C,,-bonded spherical silica (Hypersil-ODS or Spherisorb-ODs) and fitted with septum injectors. An Orlita pump (Model AE 104) was used with a Cecil Model 212, Pye Model LC3 or Waters Model 440 ultraviolet detector, a strip-chart recorder and an integrator.The solvent was aqueous propan-2-01 containing combinations of formic or acetic acid, potassium nitrate, potassium dihydrogen phosphate and modifiers such as ethyl acetate. Details of the assays of these analgesics in plasma are summarised in Table I.Minor modifications to the solvent composition were occasionally required for optimum separation of the drugs and metabolites from potentially interfering peaks in urine. Using the ratios of the peak area or peak height of the drugs to those of internal standards, the calibration graphs were linear over a wide range of concentrations and passed through the origin.The limits of measurement are less than 1 pgml-l and the standard deviation of the methods over the therapeutic range of plasma concentrations is 3-5%. The methods for urine are even simpler.October, 1979 G AS HPLC IN CLINICAL AND BIOLOGICAL CHEMISTRY TABLE I DETAILS OF HPLC ASSAYS OF ASPIRIN AND METABOLITES, PARACETAMOL AND METABOLITES, DIFLUNISAL, ANTIPYRINE, PHENYLBUTAZONE AND MEFENAMIC ACID IN PLASMA Drug Paracetamol Aspirin Diflunisal Antipyrine Plasma protein Metabolites precipitant Sulphate, glucuronide, Perchloric cysteine and mercap- acid turic acid conjugates Salicyluric, gentisic, Perchloric and salicylic acids acid - Acetone - Perchloric acid Phenylbutazone Oxyphenbutazone Acetone Mefenamic acid - Acetone Solvents * 0.1 M KH,P04 - (100: 0.1 : 1.7) 0.02 M KNO, in (100: 12:4) 0.16 M KNO, in (55 : 25 : 20) (200 : 20: 0.14) 0.16 M KNO, in FA - IPA 2% AA - IPA - EA 2% AA - IPA - E A H20 - IPA - TE 2% AA - IPA - EA (55 : 25 : 20) 0.08 M KNO, in (50 : 45 : 5) 2% AA - IPA - EA Internal standard N-Propionyl- p-aminophenol Acet-fi-toluidide, benzoic acid Flufenamic acid Amidopyrine Flufenamic acid Niflumic acid 301 Reten- tion time/ min? 6.5 4.0 4.2 3.5 6 6 * FA = 98% formic acid.AA = acetic acid, IPA = propan-2-01, EA = ethyl acetate, TE = triethylamine. t Primary drug. SA BZ 10 20 Time/m in I Minutes Fig. 1. HPLC of plasma from a patient with aspirin overdosage. SU = Salicyl- uric acid, AS = acetylsali- cylic acid, AT = acet-p- toluidide, BZ = benzoic acid, SA = salicylic acid. Fig. 2.HPLC of blank urine (left) and urine from a patient with aspirin overdosage (right). G = Gentisic acid, SU = sali- cyluric acid, AS = acetyl- salicvlic acid, AT = acet- P-toluidide internal stan- dard, BZ = benzoic acid internal standard and SA = salicylic acid. - - - P Fig. 3. HPLCof plasma from a patient who took 18 mg kg-1 of oral antipyrine (AP) for determination of the plasma anti- pyrine half-life.AM = Amidopy- rine internal stan- dard.302 HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY Proc. Analyt. Div. Chem. SOC. Details of the methods for the determination of paracetamol and its sulphate, glucuronide, cysteine and mercapturic acid conjugates have been published.lY2 Chromatograms obtained during routine assays of aspirin, antipyrine and diflunisal in plasma are shown in Figs.1 4 . I " 5 1 Mi nu tes Fig. 4. HPLC of blank plasma (left) and plasma following a therapeutic dose of diflunisal (DF) (right). FA = Flufen- amic acid internal stan- dard. These methods have been used for clinical pharmacological and pharmacokinetic studies in healthy subjects and hospital patients. No serious problems have been encountered with interference from endogenous compounds or other drugs.Extensive and detailed studies have been carried out on the metabolism of paracetamol following overdosage and the effects of treatment with sulphydryl compounds, such as N-acetylcysteine. In addition, the effects of different diets, other drugs, liver disease and gastrointestinal disease on paracetamol absorption and metabolism have been studied. This work simply could not have been done in the time without simple, rapid methods for the determination of paracetamol and its metabolites.The methods could readily be scaled down for plasma samples of 100 pl and virtually all currently available, acidic, anti-inflammatory analgesics can be chromatographed under these conditions. References 1. 2 . Howie, D., Adriaenssens, P.I., and Prescott, L. F., J. Pharm. Pharmac., 1977, 29, 235. Adriaenssens, P. I., and Prescott, L. F., BY. J. Clin. Pharmacol., 1978, 6, 87. Clinical Analysis of Steroids by HPLC P. F. Dixon, P. Lukha and N. R. Scott Pathology Depadment, Bromley Hospital, Bromley, Kent Most of the physiologically active steroids possess some specific ultraviolet absorbance, e.g., the A4-3-0~0 configuration of corticosteroids at about 240nm and the aromatic A-ring of oestrogens at about 280 nm, and can thus be detected directly in HPLC systems.However, the sensitivity is low and theory predicts that steroids measurable in plasma are at present limited to the most abundant corticosteroid, cortisol, its precursors if cortisol biosynthesis is blocked by disease or pharmacological agents, administered steroids and, during late pregnancy, oestriol and progesterone.In urine, most steroids have been reduced to non-October, 1979 HPLC 17S CLINICAL AND BIOLOGICAL CHEMISTRY 303 ultraviolet absorbing metabolites and direct analysis is likely to be confined to unconjugated cortisol and cortisone and, in pregnancy, oestrogens. Trefz et aZ.l and Van den Berg et a1.2 described normal-phase systems for measuring plasma cortisol.We have investigated the reversed-phase system ODS silica with aqueous methanol, which is likely to be available in any laboratory practising HPLC because of its inherent simplicity and versatility. It separates cortisol from most other relevant steroids, but not from prednisolone (Table I). A l-ml sample of plasma is conveniently extracted after adding alkali and 200 ng of prednisone internal standard by vortex mixing with 7 ml of dichloromethane, 5 ml of which are evaporated and reconstituted in 100 p1 of the eluting agent (45% aqueous methanol); 72 pl are introduced via a sample loop.Fig. 1 shows a chromatogram of a plasma extract and gives the working conditions. Standards need to be prepared in a protein matrix (3% BSA) as the recovery (86%) is substantially greater from water.Good correlations have been obtained with a standard fluorimetric technique3 in uncontaminated samples, and with a competitive protein binding method (Radiochemical Centre Cortipac) in drug contaminated samples. The lowest measurable concentration is about 30 pmo11-l.The method has been used successfully for the rapid determination of plasma cortisol in samples from patients when drug interference, particularly from spirono- lactone, has caused the results by fluorimetry to be suspect. However, as a batch of samples can be prepared for chromatography in about 20min and each chromatogram runs for about 20 min the method is suitable for a small routine workload.In normal practice the inability of the system to resolve cortisol and prednisolone scarcely imposes any limitations on its use. TABLE I STEROID RETENTION DATA Conditions as in Fig. 1. K’ Retention relative Compound capacity factor t o cortisol Prednisone . . .. .. 7.7 0.72 Cortisol . . .. . . 10.7 1.00 Prednisolone . . . . 11.0 1.03 Corticosterone . . . . 19.7 1.85 Dexamethasone .. . . 22.0 2.06 1 l-Deoxycortisol . . . . 23.7 2.23 17-OH-Progesterone . . 68.0 6.10 Spironolactone . . . . 46.0 4.35 A similar system has recently been described by Reardon et al.,4 for measuring cortisol and its immediate precursor, 1 l-deoxycortisol, in plasma after administration of the 1 l-hydroxylase blocker Metyrapone as a test of pituitary function. Suitably modified systems should theoretically be capable of measuring abnormal concentrations of the earlier cortisol pre- cursor, 17-hydroxyprogesterone, in plasma from patients with congenital adrenal hyper- plasia.The separation and measurement of administered synthetic steroids needs the superior resolving power of normal-phase systems, e.g., silica with the mobile phase 5% ethanol in dichloromethane, saturated with water.By using suitable internal standards therapeutic concentrations of prednisolone, 6-methylprednisolone, prednisone and dexamethasone can be measured. The sensitivity of oestrogen determination is considerably improved at short wavelengths, e.g., about 20-fold for oestriol at 200 nm compared with 278 nm. This gain is undoubtedly associated with loss of specificity.Plasma oestriol can be measured during the last trimester of pregnancy after a tedious hydrolysis and extraction procedure and chromatography in a normal-phase system with detection at 217 nm.5 Alternatively, by using the electro-oxidative property of the phenolic A-ring a sensitivity at least as good as that in optical systems a t 200 nm can be obtained by potentiostatic amperometry, and with considerably enhanced specificity.The direct determination of oestriol conjugates in urine during pregnancy has been investigated. Van der Wal and Huber6 have reported extensively on separations of oestrogen304 HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY Proc. Analyt. Div. Chem. SOC. 15 10 5 0 Time/min Chromatogram of plasma extract: 1, prednisone; 2, cortisol. Mobile phase methanol - water 45 : 55; stationary phase, ODS Hypersil in 125 x 4.5 mm column with 25-mm guard column; elution rate, 1 ml min-I.Fig. 1. Detection, ultraviolet a t 239 nm. conjugates and concluded that the reversed-phase ion-paired partition system ODs silica, with a mobile phase of methanol - phosphate buffer containing cetyltrimethylammonium bromide, was amongst the best for oestriol conjugates, although they and other workers could not resolve the two major metabolites oestriol-16-glucosiduronate and oestriol-17- glucosiduronate in any system.We have studied the above system in detail with regard to the four conjugates oestriol-3-glucosiduronate, -16-glucosiduronate, -17-glucosiduronate and -3-sulphate. In general, retention is increased by reducing the concentration of methanol and, to a lesser extent, by reducing the pH and reducing the ionic strength of the buffer.The ion-pairing agent greatly increases retention, particularly that of the sulphate relative to the glucosiduronates. The amberlite resin XAD-2 extracts all of the oestrogens measured by a routine total oestrogen procedure and these can be eluted with methanol in virtually lOOyo yield.’ How- ever, such eluates are too complex for successful isocratic HPLC analysis.Stepwise elution of XAD-2 with increasing concentrations of methanol in water showed that more than 85% of the glucosiduronates are eluted between 20y0 and 60% aqueous methanol. A prepara- tive schedule was used as follows: 5 ml of urine were applied to a Pasteur pipette column of XAD-2, washed with 3ml of 20% methanol in pH 8 phosphate buffer and eluted with 5 ml of 60% methanol in phosphate buffer.A chromatogram of 50 pl of the eluate is shown in Fig. 2, which also gives the working conditions. Only the peaks corresponding to oestriol-3-glucosiduronate and the combined oestriol-l6/17-glucosiduronates regularly appear in pregnancy urine and are absent in non-pregnancy urine.At 278 nm the sensitivity is low and maximum scale expansion is necessary at these concentrations. The electrochemical detector gives a sensitive and more specific response for the D-ring glucosiduronates (Fig. 3). Following hydrolysis by /3-glucuronidase the 16/ 17-glucosiduronate peak disappears com- pletely when monitored at 278 nm or by the electrochemical detector, but at 220 nrn a small amount of ultraviolet-absorbing material is still detected, confirming the lack of specificity at this wavelength.In a small series the concentrations of oestriol-16/17- glucosiduronates were about half of the total oestrogen concentrations with a very close correlation. Electrochemical measurement yielded concentrations significantly lower and a correlation slightly better than those obtained by ultraviolet absorption at 220 nm, with the results obtained at 278 nm intermediate between the two.This preliminary work could thus be made the basis of a rapid and specific determination of the combined oestriol-16- and -17-glucosiduronates in pregnancy urine. The 16- and 17-glucosiduronates remain unresolved.October, 1979 HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY n n 0.005 AU Fig.2. Chromatogram of preg- nancy urine: 1, oestriol-3-gluco- siduronate (purity undetermined) ; 2, combined oestriol-16/17-gluco- siduronates. Mobile phase meth- anol - phosphate buffer pH 8,0.05 M - cetyltrimethylammonium bromide 50 : 50 : 0.1 ; stationary phase, ODS Hypersil in 125 x 4.5 mm column; elution rate, 1 ml min-l.Detec- tion, ultraviolet a t 220 nm. 12 4 -1 H 2 min Fig. 3. Chromatogram of pregnancy urine. As in Fig. 2 except: elution rate, 2 ml min-l; detection, potentiostatic amperometry at + 1.2 V. 305 References 1. 2. 3. 4. 5. 6. 7. Trefz, F. K., Byrd, D. J., and Kochen, W., J . Chromat., 1975, 107, 181. Van den Berg, J. H. M., Mol, C.H. R., and Thijssen, J. H. H., Clinica Chim. Acta, 1977, 78, 165. Mattingly, D., J . Clin. Pathol., 1962, 15, 374. Reardon, G. E., Calderella, A. M., and Canalis, E., Clin. Chem., 1979, 25, 122. Paul, J., and Dixon, P. F., unpublished work, Van Der Wal. Sj., and Huber, J. F. K., J . Chromat., 1978, 149, 431. Stoll, M. S., and Dixon, P. F., unpublished work. Development of HPLC Methods for the Determination of Enzymes of the Haem Biosynthetic Pathway C.K. Lim Divison of Clinical Chemistry, Clinical Research Centre, Harrow, Middlesex, H A 1 3 U J The enzymes of the haem biosynthetic pathway are commonly determined in research and routine clinical laboratories as they are important in the study of abnormal porphyrin and haem biosynthesis and their associated diseases.The methods currently used are compli- cated, time consuming and relatively insensitive. HPLC, with its high resolving power, its ability to analyse compounds insufficiently stable for gas - liquid chromatography without the need for derivatisation and with its simple sample clean-up procedures, is well suited for the development of assay methods for enzymes. It is possible, by using suitable precursors, to isolate products or substrates by HPLC from the incubates.This practice eliminates the need for intermediate reactions and complicated extraction procedures. It should therefore be possible to develop sensitive assay systems for the individual enzymes. The general approach should also be applicable to the analysis of other enzyme systems.With the determination of 6-aminolaevulinic acid synthetase (ALA-S), 6-aminolaevulinic acid dehydratase (ALA-D) and uroporphyrinogen I synthetase (URO I-S) as examples this paper discusses the possibility of potentials of HPLC in enzyme analysis. Determination of ALA-S the condensation of glycine and succinyl-CoA to form ALA. ALA-S is the rate-limiting enzyme in the haem biosynthetic p a t h ~ a y .l - ~ It catalyses This enzyme is elevated in the306 Proc. AnaZyt. Div. Chem. SOC. liver of patients with hepatic p0rphyria.~3 Conventional spectrophotometric methods cannot be used to measure ALA-S in bone marrow because of its low activity. It has there- fore been assayed by means of radioactivity counting, using a labelled precursor, for example, 14C-a-ketogl~tarate.~ In this method the 14C-ALA produced was isolated in a semi-pure form by ion-exchange chromatography for conversion into 14C-ALA pyrrole by reaction with ethyl acetoacetate.The 14C-ALA pyrrole was then further purified by use of paper chromatography for radioisotope counting. The whole procedure is both complicated and time consuming. ALA was well separated from a-ketoglutarate on an octadecylsilane (ODS) column with 0.005 M heptanesulphonic acid - acetonitrile (95 : 5) as the solvent system.Unlabelled ALA was used to mark the position of the peak for convenient peak collection. Complete purification of ALA was not necessary in this instance provided that the sample isolated was not con- taminated with radioactivity from 14C-a-ketoglutarate. HPLC I N CLINICAL AND BIOLOGICAL CHEMISTRY By using HPLC, however, it can be considerably simplified.Determination of ALA-D Erythrocyte ALA-D is commonly assayed according to the method of Bonsignore et aZ.7 and its modification^.^^^ These methods involved the use of ALA as the enzyme substrate and the determination of the porphobilinogen (PBG) formed in the incubation mixture. However, some of the PBG formed is converted into porphyrinslO and the enzyme activity calculated from the amount of PBG in the incubate is therefore inaccurate. A methodll based on measuring the amount of ALA consumed in the incubation mixture is more accurate but PBG interferes with the determination and an effective way of separating ALA from PBG is therefore essential.ALA and PBG can be separated by reversed-phase ion-pair chromatography on an ODS column with 0.005 M heptanesulphonic acid - acetonitrile (95: 5 V/V) as the eluting agent at a flow-rate of 1 ml min-l. The separation can also be achieved on a Hypersil APS column using acetate buffer (pH 4.6). The sample preparation and incubation procedures for ALA-D were as described by Tomokuni.ll The amount of ALA utilised and/or PBG formed could be measured directly by means of HPLC and the enzyme activity could therefore be calculated. Determination of URO I-S URO I-S together with URO III-cosynthetase are the two enzymes that catalysed the conversion of PBG into uroporphyrinogen 111. URO I-S deficiency is indicative of acute intermittent porphyria.12 I t is assayed by extracting and measuring the uroporphyrin formed in the incubation mixture with PBG as the substrate.13 Although this method is simple it is non-specific as other porphyrins can be co-extracted. The methyl esters of the porphyrins can be separated on a Hypersil APS column by using methyl acetate - heptane (40:60 Y/Y) as the eluting agent. Thus, by converting the extracted porphyrins from the incubation mixture into their methyl esters and measuring the uroporphyrin concentration by means of HPLC the enzyme activity can be calculated. Conclusion This paper summarises three different approaches to enzyme determination, colorimetry, By using HPLC these methods can easily The principle is radioisotope counting and spectrophotometry. be modified to provide a rapid and specific method of enzyme analysis. also applicable to other enzyme systems. 1. 2. 3. 4. 5. 6. 7. 8. References Granick, S., and Urate, G. J. Biol. Chem., 1963, 238, 821. Tschudy, D. P., Welland, F. H., Collins, A., and Hunter, G., Jr., Metabolism, 1964, 13, 396. Marver, H. S., Collins, A., Tschudy, D. P., and Rechcigel, M., Jr.. J. Biol. Chem., 1966, 241, 4323. Nakao, K., Wada, O., Kitamura, T., Uono, M., and Urate, G., Nature Lond., 1966, 201, 838. Tschudy, D. P., Perlroth M. G., Marver, H. S., Collins, A., Hunter, G., Jr., and Rechciger, M., Jr., Takaku, F., and Nakao, K., Life Sci., 1970, 10, 721. Bonsignore, D., Calissano, P., and Cartasangna, C., Med. Lav., 1965, 56, 199. Burch, H. B., and Siegel, A. L., Clin. Chem., 1971, 17, 1038. Proc. Natn. Acad. Sci. U.S.A., 1965, 58, 841.October, 1979 EQUIPMENT NEWS 9. 10. 11. 12. 13. Weissberg, J. B., Lipschutz, F., and Oski, F. A., New Engl. J . Med., 1971, 284, 565. Granick, S., and Mauzerall, D., J . Biol. Chem., 1958, 232, 1119. Tomokuni, K., Clin. Chem., 1974, 20, 1287. Batlle, A. M. Del C., and Rossetti, M. V., Int. J . Biochem., 1977, 8, 259. Batlle, A. M. Del C., Xifra, E. A. W., and Stella, A. M., Int. J . Biochem., 1978, 9, 871. 307

ISSN:0306-1396    

DOI:10.1039/AD9791600289

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

October, 1979 EQUIPMENT NEWS 307 Equipment News Atomic-absorption Spectrophotometers Model 280 is a new microcomputer-controlled, single-beam atomic-absorption spectrophoto- meter. The optical system employs a high- dispersion grating monochromator and features quartz-coated reflecting optics. The built-in microcomputer permits automatic calibration of the display with one or two standards. Accessories include an accessory lamp turret and power supply for a second hollow-cathode lamp, a deuterium arc background corrector and a chopper for the optimum use of electrodeless discharge lamps.The Model 380 is a double-beam, micro- computer-controlled atomic-absorption spectro- photometer with the same high-energy optical system as the Model 560, including a dual- blazed grating. This Model has an automatic gain control and an optional double-beam deuterium-arc background corrector with auto- matic intensity control.Microcomputer gas controls featuring push-button selection of two oxidants and one fuel are optionally available. The instrument couples directly to the PRS-10 printer sequencer, or to the TR-2 teletypewriter readout after installation of a printed-circuit board.A communication interface providing EIA RS-232 C output is also available. Perkin-Elmer Ltd., l’ost Office Lane, Beacons- field, Buckinghamshire, HP9 1QA. Autosampler for Furnace Atomic Absorp- tion A microcomputer-controlled Model AS-40 auto- sampler for furnace atomic absorption is now available, designed for use with the HGA-500 and the new HGA-400 heated graphite furnaces.The AS-40 is offered in two versions, one especi- ally designed for the completely automated Model 5000 instrument, and the other for use with all other Perkin-Elmer microcomputer- controlled atomic-absorption instruments. Automatic calibration of the Models 5000, 703, 603, 560 or 450 is built in. Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 1QA.Liquid Chromatograph The Series 3B Liquid Chromatograph is a dual-pump, microprocessor-controlled, solvent- delivery module offering virtually unlimited gradient capabilities and using five segments, each with a variety of concave, convex or linear solvent programmes. The Series 3B also offers the capability of a computer-orientated linkage to data manipulation and reduction apparatus.Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 1QA. Processor for Liquid Chromatography An automated methods processor for liquid chromatography enables the user to conduct a complete liquid chromatographic analysis from injection to processed and printed answer com- pletely automatically. The processor program- mes and controls the analysis of up to 42 con- secutive samples from a cassette tape pro- gramme.Other programmes can be generated from the SIGMA 10 keyboard. Analytical control features include pump method, data processor method, sample selection, sample sequence and detector wavelength change. Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 1QA. Automated High-performance Liquid Chromatography The LDC Chromatography Control Module (CCM) connects to the present range of LDC liquid chromatographs and is available in two forms.The control module can automate and govern the HPLC - integrator system, or a more versatile unit is available, which incorporates the control module, integrator and printer in the one instrument. A hard copy of the para- meters can be produced.The VDU screen on both versions displays instructions in language rather than codes. Harrison Carloss Ellis, Church House, 14 King Street, Newcastle-under-Lyme, Staffordshire, ST5 1EJ. Liquid Chromatograph Columns A range of analytical liquid chromatographic columns comprised of eight columns is available, each with a guaranteed efficiency and a very long working life.308 EQUIPMENT NEWS Proc.Analyt. Div. Chem. SOC. Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 1QA. Spectrophotometric Detector for Liquid Chromatography The LC-75 is a variable wavelength ultraviolet detector and when used with the LC-75 auto- control unit i t enables operations such as automatic stop flow scanning, automatic absorb- ance ratioing, wavelength optimisation, auto- zeroing and base-line correction for gradient runs to be carried out.Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckingharnshire, HP9 1QA. Syringes Syringes for use with the new Carlo Erba on- column injector for capillary columns are available, having a 0.23 mm 0.d. needle to inject the sample directly into the 0.3 mm i d .capillary column. Scientific Glass Engineering (UK) Ltd., 657 North Circular Road, London, NW2 7AY. Infrared Spectrophotometer The microprocessor-controlled Model 98 series of infrared spectrophotometers consists of three instruments : the Model 598, scanning from 4 000 to 200 cm-I, the Model 398, scanning from 4 000 to 400 cm-l and the Model 298, which covers the range 4000 to 600cm-l. All of these instru- ments are available in the choice of three slit programmes and six scan times.Standard features include repeat scan facilities, wave- number limits, a choice of chart formats and rapid indexing of both chart and monochromator to 4 000 cm-l. When used with the interface accessory, the 98 series can be connected directly to the infrared data station.Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 IQA. Amino Acid Analyser The LKB 440 enables complete protein hydroly- sate analysis to be carried out in a little over 1 h and can detect less than 100 pmol of amino acids. LKB Instruments Ltd., LKB House, 232 Addington Road, Selsdon, South Croydon, Surrey, CR2 9PX. Rubidium Photocathode A special rubidium photocathode gives en- hanced sensitivity in the 400-650 nm spectral range, giving typical improvements of 50% in green and 20% in blue sensitivity compared with standard bialkali photomultipliers.The Series 9900 photomultipliers incorporate this rubidium photocathode. The increased “Corn- ing Blue” figure will lead to improved pulse- height resolution, while the improved response in the green region will enable higher counting efficiencies to be achieved in high-energy physics appIications in which BBQ wavelength shifters are used.EM1 Electron Tubes, Bury Street, Ruislip, Middlesex. Hydride Generation System for Plasma Emission Spectroscopy A hydride-generation system enables the SMI Spectrospan range of plasma emission spectro- meters to be used for the determination of ultra-trace levels of arsenic, antimony, selenium and other hydride-producing elements, which are difficult to detect by conventional atomic- absorption techniques.The system provides a 10-100 fold increase in sensitivity over direct nebulisation for arsenic and selenium. It can also be used for mercury-vapour production for low-level mercury determination. Techmation Ltd., 58 Edgware Way, Edgware, Middlesex, HA8 8HP.Ion Source An ion source design for providing very stable, high-intensity beams of metallic ions, which is also compatible with ultra-high vacuum installa- tions, consists of a source of liquid gallium and a stabilised high voltage control unit that provide a stable source of gallium ions at currents ranging from 5 to 100 pA and at energies from 0.1 to 10 keV.Dubilier Scientific Ltd., Chaucer Trading Estate, Launton Road, Bicester, Oxfordshire, OXG OTU. Pre-heater for Flame Photometers A modification in the design of Model 435 and 455 flame photometers has eliminated the need for a water separator by providing small heaters to warm and dry the air from the instrument’s compressor, thus eliminating the detrimental effects of condensation even under extreme temperature and humidity conditions.Corning Medical, Corning Ltd., Halstead, Essex, CO9 2DX. Fabry - Perot Etalons and Interferometers Several types of Fabry- Perot ktalons and interferometers are offered for teaching and research purposes. With the simplest type,October, 1979 EQUIPMENT NEWS 309 which is used extensively in teaching labora- tories, the spacing between the plates can be continuously adjusted from 0.05 to 5 mm.Research-grade models include facilities for pressure-scanning Fabry - Perot spectroscopy, oscillating Fabry - Perot interferometry and other high-resolution spectroscopic techniques. Glen Creston Instruments Ltd., 16 Carlisle Road, London, NW9 OHL. Process Control System An integrated process control system, named Spectrum, introduces two new microprocessor- based elements, a control unit and a high speed communications link, and can incorporate such established electronic equipment as Spec 200, Videospec and the Fox 3 and Fox 1A computers.In shared control using the Microspec unit, one digital processor performs the monitoring and control functions for a number of process- control loops.Distributed control using the Foxnet process communications link enables control sub-systems to be installed at appro- priate locations throughout a plant with the ability to intercommunicate with each other and with centralised equipment. Foxboro-Yoxall Ltd., Redhill, Surrey, RH1 2HL. Process Control System Microprocessor-based industrial and process control equipment, GEM 80, falls into two series, GEM 80/100 and GEM 80/200.Initially, only the “200” series will be available. The GEM 80/200 series is based on a sub-rack central unit housing double high printed circuit Euro- cards containing the processor, memory and power supply, to which input - output termina- tion panels or sub-racks can be added. Products in this series include a high-performance pro- grammable logic controller (GEM 80/210), a process micro-controller with digital and analo- gue capability (GEM 80/220) and a more advanced micro-controller with video display capability (GEM 80/230).GEC Electrical Projects Ltd., Broughton Road, Rugby, Warwickshire. Digital Image Analysis System A simple quantitative analysis system, called MOP (Manual Optical Picture analysis) uses the inter-relation of the operator’s discriminat- ing judgement with the high-speed calculation ability and memory of its microcomputer in a semi-automatic mode of operation.Three different models of the MOP system are offered in modular design. The images to be analysed can be in the form of photographs, including X- rays, micrographs, projections and drawings, or they can be directly evaluated through a micro- scope.Reichert- Jung UK, 820 Yeovil Road, Slough, Berkshire, SL1 4JB. Isoelectric Focusing Equipment The Model 1415 horizontal electrophoresis cell is designed for electrofocusing applications, im- munoelectrophoresis, DNA electrophoresis and zone electrophoresis. The cooling platform is 43 cm long to accommodate two 20 cm gel trays.A series of small holes in the lid make possible checks on the pH along the length of the gel without interrupting the current. Analytical electrofocusing is improved by the use of photo- polymerisation of the acrylamide gel, rather than chemical polymerisation, which introduces substances that may contain ions that will disturb the stability of the pH gradient.Bio-Rad Laboratories Ltd., Caxton Way, Holywell Industrial Estate, Watford, Hert- fordshire, WD 1 8RP. Swing-out Ball Valves Whitey swing-out ball valves in the 3 to 1 in size range are now available with a variety of end-connection combinations, including integral Swagelok, female pipe, tube socket weld or pipe socket weld connections. The mixed-end design reduces the number of potential leak points, by eliminating the need for additional adapter fittings.Removal of a single bolt allows the centre body section to swing out for ease of seat maintenance. The full flow shut-off valves are standard in brass and 316 stainless steel, with service ratings to 2 500 lb in-2 and 450 O F . Spring-return and double-acting pneumatic actuators are available for remote operation.Techmation Ltd., 58 Edgware Way, Edgware, Middleses. Digital RTD - Platinum Thermometer The Wahl Digital RTD - Platinum heat prober thermometer is a pocket-sized, high-accuracy thermometer for instant digital readings to 0.1 “C for surfaces, liquids, powders and gases. A selection of 15 interchangeable RTD - platinum probes are available for every type of plant, processing or laboratory application over temperature ranges of - 50 to + 500 “C.Wahl International Ltd., c/o Beam Com- munications Ltd., 117 Piccadilly, London, WIV 9FJ.310 EQUIPMENT NEWS Proc. Analyt. Div. Chem. SOC. Temperature Alarm Monitor The Tempalarm monitor incorporates six individually programmable channels adjustable to high and low limit over the full operating range of 0-99.9 "C with a minimum separation of 10.2 "C.Audible alarms and individual channel fault lights indicate out-of-range temperatures. A large digital temperature display with 0.1 "C resolution allows the temperature to be checked a t each station by means of its own channel- selector switch. Baker Hosegood Associates Ltd., 64 South Street, Epsom, Surrey, KT18 7PH.Disposable Inert Filter Elements A disposable filter element called Fluorobond, composed of borosilicate glass fibres bonded with fluorocarbon resin for optimum inertness, makes possible ultra-high efficiency gas filtra- tion with minimum absorption. Five grades of filters give in-liquid filtration from 25 to 0.3 pm retention and in-gas filtration from 90 to 99.999 9+ yo of 0.6 pm particles (the two finer grades are gas-sterilising filters).The flow-rates are as follows: one 51 mm diameter by 230 mrn long tube can filter up to 175 1 min-1 (39 g.p.m.) of water, or up to 3 240 Nm3 h-l (1 910 s.c.f.m.) of air. The Fluorobond elements offer tempera- ture resistance to 150 "C in dry gas, resistance to virtually all chemicals except hydrofluoric acid, and are unaffected by water or oil.Balston Ltd., Springfield Mill, Maidstone, Kent. Filters In-line filters in corrosion-resistant Hastelloy C are now available for use in applications such as corrosive fluid handling, acid handling and alkaline systems. The filters are available with %-in female NPT end connections. All parts are manufactured in Hastelloy C , including the filter elements, which are available in 2, 7, 15, 60 and 90 pm sizes.Maximum operating pressure at room temperature is 3 000 lb in-2, with a maximum differential pressure across the element of 1000 lb in-2. The maximum operating temperature of the filters is 500 "C. Techmation Ltd., 58 Edgware Way, Edgware, Middlesex, HAS SJP. Magnetic Stirrer Now available are two hot-plate magnetic stirrers, the MS7 and MS8, both having a wipe- clean hot-plate and simple on - off press-button operation.The MS3, a magnetic stirrer with- out hot-plate, provides efficient stirring facilities for volumes up to 10 1. Scientific and Educational Aids Ltd., Vale Road, Windsor, Berkshire, SL4 5 JL. Angle- head Centrifuge A compact angle-head centrifuge has a maxi- mum capacity of 120ml and the eight-plate angle head will accept tube sizes up to 15 ml.A solid-state power controller adjusts the speed of the centrifuge. The top speed is 5 000 rev min-1 and the maximum RCF is 2 500 g. The self balancing action of the mounting makes critical balancing of tubes unnecessary. A. Gallenkamp and Co. Ltd., P.O. Box 290, Technic0 House, Christopher Street, London, EC2P 2ER.Hot - plates A range of laboratory hot-plates is announced, consisting of the Model PC35 hot-plate, the Model PC35 1 hot-plate with integral magnetic stirrer and the Model PC353 magnetic stirrer only, made with a Pyroceram top plate. Pyroceram is pre-treated for resistance to thermal shock, and offers advantages over iron, aluminium and steel-topped models, including resistance to corrosion, excellent uniform thermal conductivity for even heat-up, strength, chemical inertness, resistance to scratching and an easy to clean surface.The surface area of all models is 225 cm2 (35in2), ample to accommodate a 2-1 beaker, and temperature settings are variable up to a maximum of 510 "C. Corning Laboratory Products, 1 Princes Street, Richmond, Surrey. Liquid Handling Equipment A range of micro-syringes for general and specialised purposes, in sizes from 1 to 1 000 pl, and high-pressure models for HPLC, designed specifically for pressures up to 340 atm, are now available.Designed for pipetting liquids in volumes from 1 to 1000 p1, the Eppendorf single- and triple-volume pipettes have built-in tip ejection. The range of Fortuna Optifix bottle-top dispensers, providing volumes from 0-100 ml, have a PTFE-coated piston, giving trouble-free dispensing of all media including concentrated mineral acids and metal alkali solutions.Eppendorf automatic dispensers will deliver 10-1 000 p1 volumes with an accuracy and repeatability of better than 0.5%. Anderman & Company Ltd., Central Avenue, East Molesey, Surrey, KTS OQZ.New Products A glucose- enzymatic assay for use in an ultraviolet micro-procedure is specific for theOctober, 1979 EQUIPMENT NEWS 31 1 quantitative determination of glucose in serum, plasma, cerebrospinal fluid and urine. The procedure is based on the coupled enzyme method developed by Slein, and utilises hexakinase (HK) and glucose-6-phosphate de- hydrogenase (G-6-PDH).The assay is rapid, sensitive and accurate. The Quantimune radioimmunoassay system for oestriol measures unconjugated oestriol in serum. The system is based on the solid- state support technique using Immuno-beads as the support. The sensitivity and precision of the system is excellent throughout the range. Bio-Rad Laboratories Ltd., Caxton Way, Holywell Industrial Estate, Watford, Hertford- shire, WDl 8RP.The carcinoenibryonic antigen (CEA) control serum (human), a stable, lyophilised control serum containing no preservatives, stabilisers or other catalytic or non-reactive ingredients, is now available. Manufactured from pooled human serum with two levels of the antigen, it provides CEA representative of the high normal range (2.5-5.0ng ml-l) and a higher level, which represents the range clinically significant for malignancies (10-20 ng ml-l).Elevated lipid control serum (human) is designed specifically for monitoring levels of cholesterol, triglycerides and high-density lipoprotein (HDL) cholesterol in serum. The lyophilised material offers constituent values for a number of different manufacturers kits. A stable, lyophilised pooled human urine control, with constituent values that include a number of parameters embracing areas of endocrinology, toxicology and biochemistry, is also now available.Bio-Rad Laboratories Ltd., Caxton Way, Holywell Industrial Estate, Watford, Hertford- shire, WDl 8RP. Literature A range of discharge lamps manufactured by Ushio Electric, Japan, including xenon short-arc lamps, super-high pressure mercury arc lamps, low pressure mercury lamps and xenon long- arc lamps, are detailed in a catalogue now available.Spek-Glass, 22 Upton Close, Henley- on-Thames, Oxfordshire. A 12-page colour brochure describing the Model 98 series of infrared spectrophotometers, viz., Models 298, 398 and 598, is available. Perkin- Elmer Ltd., Post Office Lane, Beaconsfield, Buckinghamshire, HP9 1QA.An 1 %page, full-colour brochure, describing the 5000 atomic-absorption spectrophotometer is now available. The brochure contains a description of the control panel, an illustration of the set up procedure for a cadmium deter- mination and a discussion of automatic wave- length selection. Automated operation is discussed with refcrence to sequential multi-element analysis.Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 1QA. The Spring 1979 (Volume 1, No. 1) edition of Peak includes information on the HPlO8lB + HP3350 combination for automated HPLC, the use of gas chromatography - mass spectrometry with negative chemical ionisation and automa- tion and the routine application of liquid chromatography.Hewlett-Packard, Central Mailing Department, P.O. Box 529, Amstelveen 1134, The Netherlands. A brochure on Model 1081B isocratic liquid chromatograph with automatic sampling and integrated microprocessor control is available. Hewlett-Packard, P.O. Box CH-1217, Meyrin 2, Geneva, Switzerland. The March/April 1979 edition of Measurement Computation News gives information on the HP8566A spectrum analyser as well as news of components and software support materials.Hewlett-Packard, Winnersh, Wokingham, Berkshire, RGll 5AR. The March/April 1979 edition of Internationat Laboratory contains articles on a microcomputer- controlled multi-sample rotating-disc colori- meter, a method for converting polarographic units of enzyme activity into International Units, determination of immunoglobulins with an automatic discrete analyser, ion-selective electrodes in titrations using tetraphenylborate, evaluation of a nitrate ion-selective electrode, measurement and application of the oxygen index test, processing data from an inductively coupled plasma emission spectrometer using APL, and the application of discrete wave- length HPLC at 214 nm. International Scien- tific Communications Inc., 808 Kings Highway, Fairfield, Conn. 06430, USA. Industrial and Scienti3c Instruments is a postal factfinder service, covering instrumentation and equipment in the fields of analysis, measurement simulation and testing, etc. It is published monthly and copies are available free-of-charge on application. IS1 Factfinder Service, Han- over Press Ltd., P.O. Box 67, London, N6 5BR.312 CORRESPONDENCE Proc. Analyt. Div. Chem. SOC. International Laboratory Direct Information national Scientific Communications, c/o Inter- Sevvice, published quarterly, is a source of national Laboratory Inquiry, Sycamore House, information on the latest available laboratory Woodside Road, Amersham, Buckinghamshire, instruments and equipment, with tear-off reply HP6 GAA. postcards. Copies can be obtained from Inter-

ISSN:0306-1396    

DOI:10.1039/AD9791600307

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

312 CORRESPONDENCE Proc. Analyt. Div. Chem. SOC. Publications Received Chemistry. A Conceptual Approach. York, Cincinnati, Toronto, London and Mel- Fourth Edition. bourne: Van Nostrand. 1979. Price f114.20 Charles E. Mortimer. Pp. xix + 815. New (hardback); f17.50 (softback).October, 1979 PUBLICATIONS RECEIVED 313 New Applications of Lasers to Chemistry. Based on a symposium sponsored by the ACS Division of Analytical Chemistry a t the 175th Meeting of the American Chemical Society, Anaheim, California, March 1P15, 1978.Edited by Gary M. Hieftje. ACS Symposiztm Series 85. Pp. x + 244. Washington, D.C.: American Chemical Society. 1978. Price $23.50. Carbon- 13 NMR in Polymer Science. Based on a symposium sponsored by the Macromolecular Science Division at the 61st Conference of The Chemical Institute of Canada in Winnipeg, Manitoba, June 4-7, 1979.Edited by Wallace M. Pasika. ACS Sympo- siunz Series 103. Pp. x + 334. Washington, D.C. : American Chemical Society. 1979. Price $29.75. Analytical Chemistry of Liquid Fuel Sources. Tar Sands, Oil Shale, Coal, and Petroleum. Based on a symposium co- sponsored by the Divisions of Petroleum Chemistry and Analytical Chemistry at the 173rd Meeting of the American Chemical Society, New Orleans, Louisiana, March 21- 25, 1977.Edited by Peter C. Uden, Sidney Siggia and Howard B. Jensen. Advances in Chemistry Series 170. Pp. x + 341. Washington, D.C.: American Chemical Society. 1978. Price $32. Ultratrace Metal Analysis in Biological Sciences and Environment. A symposium sponsored by the Division of Analytical Chemistry a t the 174th Meeting of the American Chemical Society, Chicago, Illi- nois, August 29-30, 1977.Edited by Terence H. Risby. Advances in Chemistry Series 172. Pp. viii + 263. Wash- ington, D.C. : American Chemical Society. 1979. Price $36.50. Coulometric Analysis. Edited by E. Pungor and I. BuzAs. Conference held at Mdtrafiired, Hungary, 17-19 October, 1978. Pp.x + 302. Budapest: Akadkmiai Kiad6. 1979. Price $27. Organochlorine Insecticides and Poly- chlorinated Biphenyls in Waters 1978. Tentative Method. Department of the Environmen t/National Water Council. Methods for the Examination of Water and Associated Materials. Pp. 28. London: HM Stationery Office. 1979. Price L1.25. Microanalyse et Microscopie Electronique a Balayage.Edited by F. Maurice, L. Meny and R. Tixier. Ecole d'Ete' de St-Martin-d'Hdres 11-16 Sep- tembre, 1978. Pp. xxx + 534. Orsay, Les gditions de Physique. 1978. Wilson and Wilson's Comprehensive Ana- lytical Chemistry. Volume IX. Ultra- violet Photoelectron and Photoion Spec- troscopy, Auger Electron Spectroscopy, Plasma Excitation in Spectrochemical Analysis. Edited by G. Svehla. Pp. xvi + 305. Amsterdam, Oxford and New York : Elsevier. 1979. Price $73.25; Dfl150. Sugar Analysis. Official and Tentative Methods Recommended by the Inter- national Commission for Uniform Methods of Sugar Analysis (ICUMSA). Edited by Ferdinand Schneider. Pp. xiv + 268. Peterborough: ICUMSA. 1979. Price

ISSN:0306-1396    

DOI:10.1039/AD979160312c

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

October, 1979 PUBLICATIONS RECEIVED 313 Conferences and Meetings Fechem Conference on Education in Analy - tical Chemistry in a Changing World December 10-12, 1979, Baden Bei Wien, Austria This Conference, organised by the Working Party on Analytical Chemistry of the Federation of European Chemical Societies, will be held a t the Hotel Gutenbrunn. The format will be a series of Plenary Lectures in the mornings and evenings, separated by group discussions (6-8 people to each group).Among the lecturers will be H. Malissa, E. Pungor, D. Betteridge, L. Massart, D. Klockow, W. M. Terhyi and J. T. Clerk. The address of the Conference Secretariat is Verein Osterreichischer Chemilter, Eschen- bachgasse 9, 1010 Vienna, Austria. Workshop on Specific Ion Electrodes December 18, 1979, London This workshop will be held at King’s College and organised by the Mineralogical Society.The use of specific ion electrodes in geochemistry is a rapidly expanding field, which offers unique opportunities for the in situ analysis of aqueous cationic and anionic species. Participants will314 CONFERENCES AND MEETINGS Proc. Analyt. Div. Chem. SOC. initially be instructed in theoretical aspects of electrode behaviour, providing the necessary background for workers in this field to discuss problems in the measurement of pH and cationic and anionic species in natural waters.The range of available equipment and its handling characteristics will be dealt with by a scientist from Orion. The Workshop will include a large element of practical work using a range of specific ion electrodes, and will therefore be restricted to approximately 25 participants.A small fee will be charged to cover expenses. Application forms and further details can be obtained from Dr. R. W. Raiswell, School of Environmental Sciences, University of East Anglia, Norwich, NR4 7TJ. Specific Element Chromatographic Detec- tion by Plasma Emission Spectroscopy January 7-11, 1980, Sun Juan, Puerto R ~ C O As a part of the programme for the International Winter Conference on Developments in Atomic Plasma Spectrochemical Analysis there will be held a special symposium on the design and application of plasma spectroscopic detectors for specific element detection in gas chromato- graphy and in high-performance liquid chroma- tography, chaired by P.C. Uden of the Univer- sity of Massachusetts. It is planned that papers will be delivered by international experts in this rapidly developing field to cover the interfacing of inductively-coupled plasma (ICP) , microwave (MIP, MWP) and d.c. plasma (DCP) discharges to chromatographic systems. For further information on the Conference contact International Winter Conference, 1980, ICP Information Newsletter, Chemistry Depart- ment, GRC Tower I, University of Massachu- setts, Amherst, Mass.01003, USA. Environmental Mineralogy January 11, 1980, London This full-day session will be held at the Geo- logical Society in Burlington House as part of the 1980 Anniversary Meeting of the Minera- logical Society. Papers are invited on toxic, radioactive and biologically active minerals, morphological, chemical and other features of hazardous minerals, identification and assess- ment techniques and control measures.Persons interested in contributing to the meeting are asked to contact the convenors: Dr. A. A. Hodgson, Cape Asbestos Fibres Ltd., Iver Lane, Cowley, Uxbridge, Middlesex, UB8 2JQ and Dr. D. C. Goldring, British Steel Corporation, Teesside Laboratories, Ladgate Lane, Middles- brough, Cleveland, TS8 9EG.Biological Fluidised Bed Treatment of Water and Wastewater Afiril 14-17, 1980, Manchester The Water Research Centre (WRC), in collabo- ration with the University of Manchester Institute of Science and Technology (UMIST), is organising an international conference on this important new treatment process to be held in April 1980.Contributions have been invited from leading experts in Britain, the USA, Canada, Australia and South Africa. The latest work a t WRC and UMIST will be described and there will be an opportunity to visit one or two large pilot plants. Facilities for poster papers will be provided at the conference. The conference programme will include papers on: the removal of nitrate and ammonia from water; treatment of sewage in fluidised beds; workshop on basic principles of fluidised beds ; process engineering of fluidised beds ; treatment of industrial wastewater ; process economics ; and de-nitrification of concentrated industrial wastes.For further information please contact the Conference Organiser, Water Research Centre, Medmenham Laboratory, Henley Road, Med- menham, P.O.Box 16, Marlow, Buclungham- shire, SL7 2HD. VIth International Histochemistry and Cytochemistry Congress August 17-22, 1980, Brighton This Congress will be organised on behalf of the International Federation of Societies for Histo- chemistry and Cytochemistry by the Royal Microscopical Society. Held in association with Micro 80, it will include a large trade exhibition of light and electron microscopes and related scientific equipment and materials.The Scientific programme includes symposia and topics on the following subjects: innovation and validation of methods for enzymes; tumour histochemistry ; autoradiography ; botanical histochemistry ; muscle pathology; zoological histochemistry ; electron histochemistry ; min- eralised tissues; nuclear histochemistry ; muco- substances ; liver pathology; cytofluorimetry ; analytical cytology ; pharmacology and toxi- cology; neurohistochemistry ; quantification ; membranes; cell uptake and transport; haema- tology ; histochemistry of phagocytic cells; staining mechanisms ; immunocytochemistry ; developmental processes ; cell injury and death; tropical pathology ; and neuroendocrine histo- chemistry.The programme will be arranged so that the participants can visit the poster sessions and trade exhibition during the day.October, 1979 CONFERENCES AND MEETINGS 315 A social programme for delegates and accom- panying guests is being arranged. For further details apply to the Secretariat, VIth International Histochemistry and Cyto- chemistry Congress, Royal Microscopical Society 37/38 St Clements, Oxford, OX4 1A J .International Workshop on Trace Element Analytical Chemistry April 27-29, 1980, Neuherberg, Federal Republic of Germany This Workshop, which is to be held in the GSF in Neuherberg, near Munich, will be organised by the GSF (Gesellschaft fur Strahlen- und Um- weltforschung mbH) and the Working Group on Trace Elements in the Life-Sciences of the AGF (Arbeitsgemeinschaft der Grossforschungsein- richtungen) in co-operation with the Inter- national Atomic Energy Agency and the European Communities.It will consist of a series of invited papers on specific problem areas, followed by an extended discussion period in which all participants will be invited to take part.Short contributed papers (about 10 min) are also solicited for the Workshop, but abstracts of them must be submitted in tripli- cate (in English) by November 30, 1979. The Workshop is to consider biomedical and biochemical functions of trace elements, pro- blems of sampling and handling of biomedical samples, specific trace element analytical methods and analytical quality control (possi- bilities for systematic errors). All corre- spondence concerning the Workshop should be addressed to Dr.P. Schramel, Gesellschaft fur Strahlen- und Umweltforschung, Physikalisch - Technische Abteilung, Ingolstadter Land- strasse 1 , D-8042 Neuherberg, Federal Republic of Germany. Semlab '81 June 2-5, 1981, London The UK's major laboratory diagnostics and medical instrumentation exhibition, previously entitled Labex, will in future return to a London venue and have a new title, Semlab, to reflect a considerable enlargement of its scope.The first exhibition in the new series, Semlab '81, the International Scientific, Educational and Medical Laboratory Equipment Exhibition, will be held in the Grand Hall, Olympia. This new exhibition will cover comprehensively all services for the laboratory, equipment, instru- ments and consumables.The show will also include medical equipment, such as diagnostic, patient care and monitoring devices, and will also feature analytical and process control instruments. The British Laboratory Ware Association have already agreed to act as sponsors for Semlab '81 and additional sponsorship and support are expected shortly from the Scientific Instruments Manufacturers Association of Great Britain. Further information is already avail- able from: Mr. Tony Lee, Semlab '81 Sales Manager, Industrial and Trade Fairs Limited, Radcliffe House, Blenheim Court, Solihull, West Midlands, B91 2BG. Obituary Mr. A. W. Hartley We deeply regret to announce the death of Mr. A. W. Hartley, formerly Chairman of the East Anglia Region and Group Chief Analyst of Spillers Ltd.

ISSN:0306-1396    

DOI:10.1039/AD9791600313

出版商:RSC    

年代:1979

数据来源: RSC