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microbial enzymes, in particular, is most promising. Further developments in the immobilization of enzymes will lead to an increased application of 'dip-stick' or bio-electrode type probes or even spot tests for the rapid detection and assay of drugs or metabolites. Such developments can only increase the robustness and range of applications of enzymemediated assay techniques in clinical chemistry. The potential for drug assays based upon microbial enzymes is vast. It is conceivable that, providing a suitable micro-organism can be found, this type of assay could be developed for a great number of the drugs in use today. At present, however, there appear to be few workers actively engaged in this area of research, and, in view of the long development time required for this type of assay, initial progress is likely to be slow. This situation is likely to change as the metabolism of drugs by micro-organisms becomes more widely understood and the demand by clinicians for a greater number of simple, rapid assay procedures rises.

4 McCullough, J. L., Chabner, B. A. and Bertino, J. R. (1971)J. Biol. Chem. 246, 7207 5 Broughall, J. M. and Reeves, D. S. (1975) Antimicrob. Agents Chemother. 8, 222 6 Boeckx, R. L. and Brett, E. M. (1981) Clin. Chem. (Winston-Salem NC) 27, 819 7 Wiener, K. (1978) Ann. Clin. Biochem. 15, 187 8 Price, C. P., Hammond, P. M. and Scawen, M. D. Clin. Chem. (Winston-Salem NC) (in press) 9 Roder, A., Siedel, J., Mollering, H., Seidel, H. and Gauhl, H. (1982) European Patent Application EP 054146 Dr M. D. Scawen obtainedhis B.Sc. (1969)from the University of Shefgqeld and his Ph.D. (1974)from the University of Bristol. In 1976 hejoined the staff of the Microbiological ResearchEstablishment, Porton. On its transfer to the PHLS in 1979 he joined the Microbial Technology Laboratory, Porton Down, Salisbury SP4 0JG, UK wherehe researchesinto the use of enzymesfor drug and metabolite assays and the developmentof novel systemsfor enzyme purification.

References

Dr P. M. Hammond graduatedfrom the Institute of Biology in 1978. After a briefperiod at the Microbiological ResearchEstablishment he was employedby Southampton GeneralHospital in the Department of ChemicalPathology and Human Metabolism. Having obtained his Ph.D. from Southampton University in 1982, he is currently employedby the Cambridge Area Health Authority, and is working on microbial drug assimilation at the Centrefor Applied Microbiology and Research, Porton Down.

1 Sherwood, R. F. and Atkinson, T. (1981) Chem. Ind. (London) 7, 241 2 Kabakoff, D. S. and Greenwood, H. M. (1981) in RecentAdvances in Clinical Biochemistry (Alberti, K. G. M. M. and Price, C. P. eds.), Vol. 3, pp. 1-32, Churchill-Livingstone, Edinburgh 3 Falk, L. C., Clark, D. R., Kalwan, S. M. and Long, T. F. (1976) Clin. Chem. (Winston-Salem NC) 22, 785

Dr C. P. Price obtainedhis Ph.D. in clinical chemistryfrom the Universityof Birmingham in 1973. After beingemployedby Southampton GeneralHospital he becamean Assistant Lecturerin the Departmentof Clinical Biochemistry at Addenbrookes Hospital in Cambridge, UK in July 1980. He researchesinto the adaptation of novel enzyme assays to clinical chemistryand has also worked on the application of turbidometric assays to immunochemicaltechniques.

An introduction to inductively coupled plasma source mass spectrometry Inductively coupled plasma (ICP) source mass spectrometry is a revolutionary new method of analysis, combining the speed and convenience of sample introduction into the ICP with the high sensitivity and isotope ratio capability of atomic mass spectrometry. Alan R. Date London, UK The spectacular development of organic mass spectrometry in the last 30 years has given inorganic mass spectrometry the status of a poor relation. Development in this latter field has been restricted to a few specialized, but very valuable, applications using thermal, spark or secondary ion sources. Radical changes in this situation are expected in the near future with the establishment of inductively coupled plasma (ICP) as an ion source. The idea of sampling ions from a high temperature plasma operating essentially at atmospheric pressure 0165-9936/83/$01.00

was conceived and first demonstrated by Gray x-s using a capillary arc (DC) plasma. This early work was limited by severe inter-element and matrix effects characteristic of the type of plasma used. In order to overcome these limitations, ICP was substituted by Houk and his co-workers (Iowa State University) and by the Institute of Geological Sciences in the UK (in a project based at the University of Surrey under the direction of Gray). The first reported work from both groups 4"5 was limited by the formation of a cool boundary layer over the small apertures used for plasma sampling in the mass spectrometer. Sampling with apertures large enough to induce continuum flow from the bulk plasma was first applied to the low gas O 1983 Elsevier Science Pubhshers B.V.

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temperature microwave induced plasma (MIP) by a third group, in Canada ~'. Continuum flow sampling from the ICP, first achieved in 1981, was reported by two groups at the second Winter Conference on Plasma Spectrochemistry in January 1982 (Refs 7 and 8). Date and Gray 7 reported further progress at the first biennial U K National Atomic Spectroscopy Symposium in 19829 and a more detailed description of their work has been prepared ~°. The technique described in these papers has now been adopted for production of a commercial instrument H. The performance reported by Douglas et al. s has since been improved and is also now available in a commercial form, announced at the 1983 Pittsburgh Conference and Exposition 12.

Instrument operation The operation of a typical ICP source mass spectrometer may be considered with reference to Fig. 1, reproduced from The Analyst 9. In this example solution samples are introduced into the ICP by conventional pneumatic nebulization, without desolvation. In practice, any technique used for sample introduction into a plasma source may be used in this

new application. The combined experience of the three groups currently known to be investigating this method covers ultrasonic and pneumatic nebulization "-1°, hydride generation 7, electrothermal vaporization 1° and flow injection 13. Laser ablation of solid samples is now under investigation in the UK. Sample dissociation, atomization and ionization take place in the tail flame of the ICP, and a portion of the ionized gas is introduced into the vacuum system of the mass spectrometer. The plasma torch is repositioned horizontally in order to achieve this introduction more easily, but in other respects the ICP used is a standard commercial item. The all-argon plasma is normally operated at 1 200 W (reflected power <5 W), with a support gas flow rate of 12 1 min -1, an auxiliary gas flow rate of 0, and a sample carrier gas flow rate of 0.6 1 min -a. The sample solution uptake rate under these conditions is about 2 ml min -1. The detailed design of the plasma sampling interface, the most significant development in this research, is slightly different for each system, but the basic principles are similar. The instrument developed in the UK uses a three-stage vacuum system. The

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big. 1. Schematic diagram of a typical ICP mass spectrometer. The ICP source, shown to the right, is a standard commercial unit with the torch re-positioned horizontally. Samples are introduced to the ICP by conventionalsolution nebulization. A portion of the ionized gas is extractedfrom the tailflame of the ICP into the mass spectrometer through a small hole (typically 0.5 mm) drilled in a water-cooled copper, nickel or nickel alloy cone. The vacuum system operates in three stages. The extractedgasforms a molecular beam in stage 1 (1 torr; rotarypump), and the centre of the beampasses through a skimmer aperture (1 mm, nickel) into stage 2 (10-3 torr; oil diffusion pump, rotary backing pump). The ions arefocused through a differential pumping aperture into stage 3 (10-6 torr; oil diffusion pump, rotary backing pump) containing the quadrupole massfilter and detector. The quadrupole massfilter scan is synchronized with the sweep of a multi-channel scaling data system, shown lower left, so that the signal from ions of a particular mass to charge ratio (m : z) is recorded in a particular channel or group oJchannels.

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ionized gas is extracted through a small aperture (0.4-0.8 ram) drilled in a water-cooled copper, nickel or nickel alloy cone which projects into the plasma tail flame. The extracted gas forms a beam in the first (expansion) stage operating at a pressure of about 1 torr, and the core of this beam passes through a skimmer aperture into the second stage (at about 5 x 1 0 -4 torr). The ions are focused through a differential pumping aperture into the third stage containing the quadrupole mass spectrometer and detector (at about 10-'; torr). The quadrupole mass spectrometer acts as a filter, transmitting only ions with a pre-selected mass:charge ratio (m:z). The quadrupole control system may be set for single ion monitoring or may be varied rapidly for scanning over a range in mass. Transmitted ions are detected with a channel electron multiplier operating in the pulse counting mode, i.e. detecting individual ions. The operational convenience of this system owes a great deal to the application of a multi-channel scaling data system (MCS), designed originally for use in Xand y-ray spectrometry. The system is normally set with a data acquisition memory group of 1 024 channels, a dwell time per channel of 1 ms, and 60 separate sweeps. The quadrupole control is set for the first mass and mass range required and its scan is 14N+ II

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synchronized with each sweep of the MCS so that filtered ions with a particular mass:charge ratio are always recorded in the same channel or group of channels. With the above settings, a complete mass spectrum is integrated and recorded in just over 1 min. Data recorded in the MCS may be transferred to other memory groups, recorded permanently on cassettes or printed out on teletype. Several options for data manipulation are available. The combination of pneumatic nebulizer, minimal memory effects in the sample introduction system, and ease of operation of the mass spectrometer and MCS, provides an instrument in which samples may be processed at a rate of one every few minutes. Spectral characteristics

The spectral characteristics of ICP source mass spectrometry are illustrated in Fig. 2. This shows the spectrum obtained for a solution containing AI, Co, As, Br, Rb, In, Te, I, Cs, La, W, Pb, Bi and U, each at 5/~gm1-1 in 1% v/v HNOs, using a data system memory group of 2 048 channels and covering a mass range of 0-270 m :z. With a dwell time for each channel of 500/~s, and integration for 60 separate sweeps, the complete spectrum was taken in just over 1 min. Each isotope covers approximately seven channels and was

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228 therefore addressed for about 0.2 s. The peak channel integral count in each case may be converted to a count rate equivalent, corresponding to the rate found for single ion monitoring. The peak channel integral count for 133Cs* (2 419) is equivalent to 80 633 counts s --~. The mean non-spectral background is about 2 counts per channel, corresponding to a background count of about 60 counts s -1. More recently, spectra have been obtained giving more than 50 000 counts s --x per /~g m1-1, with a background count rate below 10 counts

trends in analytical chemistry, vol. 2, no. 10, 1983

alBr+, 300), but the isotope ratio of 1.13 is very close to the theoretical value of 1.03, and each channel was addressed for only 0.03 s. Most of the tellurium isotopes are covered by the second expanded group. The smallest visible peak, l"~Te ~ (isotopic abundance 0.87%), represents a concentration of about 40 ngm1-1. L a n t h a n u m , with a very low second ionization energy (11.06 eV), occurs mainly as 139La~ (91%).

S-1.

Analytical performance

The major spectral background peaks, some of which are identified in Fig. 2, are formed from the plasma support gas, its impurities, and the hydrogen, nitrogen and oxygen present in the sample solution. The peculiar shape of some of these peaks results from count saturation in the detector and counting chain. The true relative proportions of the peaks may be seen only with the total ion transmission reduced 7. The few background peaks appearing above 41 m:z (ArH*) include ions from the sampling aperture and skimmer (Cu, Ni) and the argon dimer peak at 80 m :z. Although the spectrum appears fairly complex below 41m:z, most elements have at least one isotope available for analysis. In some cases peaks are only visible in the absence of water vapour, and in this case a dry aerosol with purified argon is essential for analysis. The ionization equilibrium of the plasma, and therefore the spectral characteristics of the method, are controlled largely by the plasma support gas, argon, with a first ionization energy of 15.76 eV. Three elements, helium, neon and fluorine, have ionization energies greater than argon, while nitrogen, with a first ionization energy of 14.53 eV, is only 1% ionized. These four elements, and argon, are therefore excluded from the method when an argon plasma is used. In order to illustrate the spectral characteristics for trace elements, two regions of the complete spectrum in Fig. 2 have been subjected to horizontal (128 channels) and vertical ( x 8 ) scale expansion and superimposed above the complete spectrum. In this way, individual isotopes may be identified and, if necessary, quantified. Rubidium, in the first group, and caesium, in the second, with low first and very high second ionization energies, occur as singly charged ions with very similar total count rates. The isotope for rubidium, using only the m a x i m u m channel integral count in each case (8~Rb*, 1 700: STRb*, 700) is 2.43, which compares favourably with the theoretical value of 2.60. The error, the difference between the two values expressed as a percentage, is 6.5%, while the error expected from counting statistics alone is 4.5%. Arsenic and iodine, like caesium, are mono-isotopic, but have higher ionization energies (Cs = 3.89 eV; As = 9.81 eV; I = 10.45 eV) and, therefore, lower count rates. The arsenic oxide and hydroxide ions found under boundary layer sampling conditions 7 are very small or absent. Bromine has an even higher ionization energy (11.84 eV). M a x i m u m channel integral counts for the two isotopes are very low (;gBr +, 339;

Although ICP source mass spectrometry is now available in a selection of commercial instrumentation u'12, no comprehensive account of its analytical performance has been published. In assessing such performance, the following features should be considered: power of detection, sensitivity, selectivity, element coverage, dynamic range, precision, freedom from matrix effects, accuracy, ease of operation, speed of analysis, ease of automation, isotope ratio capability and cost effectiveness. The spectrum reproduced in Fig. 2 illustrates the potential of the method for both rapid multi-element trace analysis and for isotope ratio determination. Most elements ~are detected as singly charged monatomic ions. The exceptions are those elements T A B L E I. C o m p a r i s o n of detection limits (2o- blank, ng m F 1) Element Lithium Boron Magnesium Aluminium Titanium Vanadium Chromium Manganese Cobalt Zinc Germanium Arsenic Selenium Rubidium Silver Cadmium Indium Tellurium Caesium Barium Lanthanum Cerium Tungsten Gold Mercury Lead Bismuth Thorium Uranium

ICPSMS 3 1 0.5 0.6 0.3 0.4 0.2 0.8 0.5 3 1 7 15 0.3 0.2 0.5 0.1 0.5 0.1 0.3 c 0.2 c 0.2 c 0.5 0.2 0.4 0.3 0.2 0.2 c 0.4

ICPAES a 1.9 3.2 0.1 t5 2.5 3.3 4.1 0.9 4 1.2 1 35 30 4.7 1.7 42 27 0.9 6.7 32 20 11 17 28 23 43 170

FAAS a 2 l 000 0.2 20 50 20 3 3 5 0.6 50 100 100 2 2 1 30 70 15 20 t 600 500 I0 200 20 40 7 000

Boumans, P. W . J . M . (1980)Line Coincidence Tablesfor Inductively Coupled Plasma Emission Spectrometry Pergamon Press, Oxford and New York. u I n s t r u m e n t a t i o n Laboratory, Data Sheet 091/11/79 (1979) c Doubly charged ion. a

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forming very strongly bound oxides, for which very small oxide peaks may also be present [e.g. thorium oxide at about 4% (Ref. 9)], and elements with low second ionization energies which will be present partly as doubly ionized species (e.g. the rare earth elements) x°. The absence of stray ion signals (c.f. stray light in atomic emission spectrometry) results in spectra offering excellent selectivity. Good detection and sensitivity are also apparent in the example given above. State-of-the-art detection limits (20- blank) for a series of elements are compared in Table I with those of two well-established methods of analysis, ICP atomic emission spectrometry (ICPAES) x~ and flame atomic absorption spectrophotometry (FAAS) is. While FAAS provides good detection for a limited number of elements, ICPAES has excellent detection for a large number of elements, but is particularly good for light elements. Inductively coupled plasma source mass spectrometry (ICPSMS), however, provides excellent detection over a wide range from lithium to uranium. In many other respects, its analytical performance is similar to ICPAES. A guide to other aspects of performance may be gained from the limited number of publications available in the field. Calibration curves extending over four orders of magnitude in concentration for a few elements have been reported by one group 8, and over six orders of magnitude for one element by another group 9. The presentation of calibration curves implies a certain degree of short term precision, which is expected to be on a par with ICPAES. Data for long term precision are unavailable. In this respect, the copper sampling cones used by Gray and Date erode gradually under the influence of the very high temperature plasma flame, but have lifetimes in excess of 50 h. The nickel alloy cones used by Douglas et al. have much longer lifetimes, and nickel is not recorded in the spectra. Salt condensation over the sampling aperture, reported in the early boundary layer sampling work 4'~, is not a serious problem with continuum sampling, and solutions containing up to 5 000/xg ml -~ total salt concentration may be run routinely. In terms of freedom from matrix effects, performance is again similar to ICPAES. Douglas et al. 8 show that the addition of up to 1 000/xg ml -~ P04 (phosphate) or aluminium has no effect on calcium signals. Gray and Date 1° report some signal suppression for cobalt and bismuth in the presence of up to 1 000/xg ml -x sodium, but the exact mechanism of this suppression is unknown. Date and Gray 9 show the signal obtained for lead and bismuth in a multi-element solution to be unchanged in the presence of a synthetic geological matrix (at 500/xg ml-~). Douglaset a l . 8 present data for several trace elements in NBS SRM 362 (Low Alloy Steel) at 1 000/xg ml -~ in solution, and report that matrix matching was unnecessary. Accuracy, of course, depends on several factors including freedom from matrix effects, but should be acceptable. Ease of operation and speed of analysis are self-evident in the above discussion of instrument

operation. The data system used with the instrument described above may be interfaced with both an automatic sample changer and a micro-computer. Automation of the system is therefore relatively simple. It is perhaps fair to state that the necessity for sampling cone replacement leads to a little more operator intervention than is necessary with ICPAES, but considerably less than with the established methods of analysis in atomic mass spectrometry. A major advantage of this new application of the inductively coupled plasma is the ability of the developed system to provide isotope ratio information very rapidly. Data presented for the multi-element spectrum reproduced above suggests that the error for isotope ratios might approximate that expected from counting statistics alone. The rapid scanning facility of the quadrupole mass spectrometer and multi-channel scaling data system may be used to limit the influence of fluctuations in the sample introduction system. By limiting the mass range and increasing scan speed, sufficient counts may be accumulated for each isotope of interest to reduce error to within 0.1-1%. This error is acceptable for a wide range of applications, including rapid stable tracer studies in biochemistry, isotope dilution analysis, and pollution monitoring. Date and Gray 7'1'~report the rapid determination of lead isotope ratios in galena (natural lead sulphide) samples, and zinc isotope ratios in dithizone extracts from blood plasma and faeces 1'~. Douglas and Quan 12 use the method to determine copper in NBS SRM 1571 (Orchard Leaves) by isotope dilution. This new method of analysis will undoubtedly find application in a number of fields in the near future. The cost of a developed instrument is not expected to exceed that of a multi-channel direct-reader in ICPAES (about £100 000), and compares very favourably with the cost of instrumentation for analysis by classical atomic mass spectrometry.

Acknowledgement The work carried out in the UK was supported by the Institute of Geological Sciences and the Commission of the European Communities (DG XII). This paper is published with the approval of the Director, Institute of Geological Sciences (NERC).

References 1 Gray, A. L. (1974) Anal. Proc. 11, 182 2 Gray, A. L. (1975)Analyst 100, 289 3 Gray, A. L. (1978) inDynamic Mass Spectrometry Vol. 5 (Price, D. and Todd, J. F. J., eds), Ch. 8, Heyden, London 4 Houk, R. S., Fassel, V. A., Svec, I-I.J., Gray, A. L. and Taylor, C. E. (1980) Anal. Chem. 52, 2283 5 Date, A. R. and Gray, A. L. (1981 ) Analyst 106, 1255 6 Douglas, D.J. and French, J. B. (1981/Anal. Chem. 53, 37 7 Date, A. R. and Gray, A. L. (1983) Spectrochim. Acta Part B Atomic Spectr. 38, 29 8 Douglas, D. J., Quan, E. S. K. and Smith, R. G. (1983) Spectrochim. Acta Part B Atomic Spectr. 38, 39 9 Date, A. R. and Gray, A. L. (1983) Analyst 108, 159 10 Gray, A. L. and Date, A. R. Analyst (in press)

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11 Plasmaquad Data Sheet No. 02.511 (1983), VG (Isotopes) Limited, Winsford, Cheshire, UK. 12 Douglas, D. J. and Quan, E. S. K. (1983) Elan 250 ICP/MS Paper 402, 1983 Pittsburgh Conference, Sciex Inc., Thornhill, Ontario, Canada 13 Houk, R. S. and Thompson, J.J. (1983)Biomed.Mass Spectrom. 10, 107 14 Boumans, P. W.J.M. (1980)Line Coincidence Tablesfor Inductively

Coupled Plasma Atomic Emission Spectrometry Pergamon Press,

Oxford and New York 15 Instrumentation Laboratory (1979), Data Sheet 091/ 11/79 16 Date, A. R. and Gray, A. L. (1983)Int.J. MassSpectrom. Ion Phys.

48, 357 Alan R. Date is at the Institute of Geological Sciences, 64/78 Gray's Inn Road, London WC1X 8NG, UK.

Kinetic analysis of microemulsions using microprocessor controlled UV/visible -spectrometry Microprocessor controlled spectrometers greatly extend the possibilities for kinetic analysis of complex thermal or photochemical reactions. As a result, more sophisticated evaluation procedures, such as derivative spectroscopy or dynamic multi-component analysis become possible. GQnter Gauglitz TDbingen, FRG For the examination of the photochemical behaviour of diphenylpolyenes in microemulsions it is necessary to obtain a large n u m b e r of high-precision digitized spectra, taken at different times during the reaction 1'2. The dependence of photochemical q u a n t u m yield and rate constants on solvent, type of microemulsion and temperature also has to be determined. In order to examine these photoisomerization reactions it is necessary to be able to detect small changes in absorbance with a high accuracy, to overcome disturbances due to stray light and background absorption, and to record the spectrum at a n u m b e r of optimal reaction times. These problems can be solved by the use of a microprocessor which controls the recording of spectra, data acquisition, data storage, and the opening and closing of photo-shutters. The use of a microprocessor thus enables extensive kinetic analysis, even in turbid solutions.

Methods of kinetic analysis Fig. 1 represents a 'reaction spectrum '3, i.e. a set of spectra, recorded at predetermined times during a reaction. Different reaction domains (time intervals in the course of a reaction during which one step of the whole mechanism dominates) are symbolized by another form of graphical representation. Each spectrum is digitized at an appropriate wavelength resolution and is stored on a tape or disk for further evaluation. Even though the digitized absorbances at a few wavelengths are sufficient to determine the n u m b e r of steps in the reaction sequence and the kinetic constants, using the digital information as a whole will allow the determination of an improved signal-to-noise 0165-9936/83/$01.00

ratio, better multi-component analysis, and the calculation of derivative spectra. Whereas isosbestic points in the reaction spectrum provide rather inadequate information about the complexity of the reaction 4, 'absorbance diagrams '3'4 permit the determination of the n u m b e r of linear, independent steps 3"4 in the sequence of reactants, spectroscopically observable intermediates and final products. To construct these diagrams, 'characteristic wavelengths' are selected from the reaction spectrum which are characterized either by an obvious change in the absorbance during the reaction or by characteristic spectroscopic behaviour with respect to different steps in the reaction. At each observed reaction time the absorbances of two wavelengths are plotted against each other to yield the absorbance diagram, the aim being to linearize the relation between the absorbance changes at different wavelengths. The form of the resulting diagram demonstrates the complexity of the reaction. Diagrams of higher 'rank' are constructed by more complex combinations of the changing absorbances 3'~. The rank of the diagram corresponds to the n u m b e r of linear independent steps in the reaction. Wavelengths which fall within the absorption range of all reaction products should be selected for use in these diagrams to avoid loss of information. The time at which the reaction is observed must be the same for all the wavelengths of a spectrum. This can be easily achieved in the case ofphotoreactions, where a photo-shutter simply interrupts irradiation during the recording of the spectrum. However, in the case of 'fast' thermal reactions (which go to completion in a period comparable to the recording time) the correct absorbances have to be obtained by interpolation of absorbance-time curves, since the absorbances measured at different wavelengths belong to different ~) 1983 Elsevier Science Pubhshers B.~,.