Measurement of isotope ratios by Doppler-free laser spectroscopy

Measurement of 7Li/6Li isotope ratios by resonant Doppler-free two-photon diode laser atomic absorption spectroscopy in a low-pressure graphite furnac...
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Anal. Chem.

1987, 5 9 , 1232-1236

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Figure 4. Reconstructed total ion electropherogram of five quaternary ammonium salts, at lo-' M (14-17fmol injection) concentration, obtained by CZE-MS: (A) tetramethylammonium bromide; (B) trimethylphenylammonium iodide; (C) tetraethylammoniumperchlorate; (D) tetrapropylammoniwn hydroxide; (E) tetrabutylammonium hydroxide.

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limitation of the technique but only as the first medium used in CZE-MS. Of course, other buffers may be more desirable for particular CZE applications. Nonvolatile buffers, for example, should pose few problems based on the work of Fenn and co-workers (9). The ability to combine CZE with mass spectrometry is not as surprising as it might appear a t first glance. The cathode need not be in a buffer reservoir, but only biased negative with respect to the anode. Thus, a metalized segment of capillary tubing or other electrical contact with the buffer provides the essential control of the electric field. This approach (necessary for mass spectrometric interfacing) does not alter the electroosmotic flow, at least to an extent that is detectable with fluorescence detection just prior to the electrospray. The success of this approach is further supported by the high efficiency separations presented in this communication. On the basis of these initial results, electrospray ionization appears to provide an ideal interface for the marriage of a highly efficient separation technique, capillary zone electrophoresis, with the sensitive and highly specific detector provided in the mass spectrometer. Future work will aim at obtaining enhanced sensitivity and separation efficiencies and exploring the role of various instrumental parameters relevant to CZE separations and MS detection. LITERATURE CITED Mikkers, D. W. P.; Everaerts, F. M.; Verhegge, Th. P. E. J . Chromatogr. 1979, 169, 11. Jorgenson, J. W.; Lukacs, K. D. Science 1984, 222, 266. Lauer, H. H.; McManigllL D. Anal. Chem. 1988, 58, 166. David, P. A.; Pellechia, P. J.; Manning, D. L.; Maskarlsha, M. P. Report ORNL/TM-9141, April 1984. Pretorius, V.; Hopkins, 9. J.; Schieke, J. D. J . Chromatogf. 1974, 9 9 , 23. Arpino, P. J.; Beamgrand, C. Int. J . Mass Spectrom. Ion Processes 1985, 6 4 , 275. Bruins, A. P. J . Chromafogr. 1985, 323, 99. Mack, L. L.; Kralik. P.; Rhonde, A,; Dole, M. J . Chem. Phys. 1970, 52, 4977. Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. 9. Anal. ChSm. 1985, 57, 675.

Figwe 5. Reconstructed total ion electropherogram of five quaternary ammonium salts, at lo-' M (0.7-0.9 fmol injection) concentration, obtained by CZE-MS: tetramethylammonium bromide (rn /I 74);trimethylphenylarnmonium iodide (rn/ z 136);tetraethylammonium perchlorate (rn/z 130);tetrapropylammoniumhydroxide (rnl z 186);tetrabutylammonium hydroxide (rn / z 242).

0.7-0.9 fmol injection, obtained by decreasing Vi to 20000 V and C to lo-' M. Though the separation efficiencies in Figure 4 vary from 26 000 and 100 000 theoretical plates, they are increased to between 35000 and 140000 theoretical plates in Figure 5. Such increases in efficiency with decrease in sample concentration and size suggest further improvement may be possible with higher buffer ionic strength (2). The water-methanol solution used in this preliminary work should not be construed as a

Jos6 A. Olivares Nhung T. Nguyen Clement R. Yonker Richard D. Smith* Chemical Methods and Separations Group Chemical Sciences Department Pacific Northwest Laboratory P.O. Box 999 Richland, Washington 99352 RECEIVED for review October 31, 1986. Accepted December 18,1986. We thank the U.S. Department of Energy through Contract DE-AC06-76RLO-1830and the U.S. Army Medical Research Institute of Infectious Diseases for support of this work. Pacific Northwest Laboratory is operated by Battelle Memorial Institute.

Measurement of Isotope Ratios by Doppler-Free Laser Spectroscopy Applying Semiconductor Diode Lasers and Thermionic Diode Detection Sir: In a recent correspondence (1)we pointed out why the application of tunable lasers in analytical spectroscopy is not common outside research laboratories. Although laser-based techniques like laser-induced flourescence (LIF), laser-enhanced ionization (LEI), and resonance ionization spectros-

copy (RIS) have already shown their potential and their extreme detection sensitivities (1-7), the complexity, the high cost, and the limited wavelength ranges of the most commonly used dye lasers are the reasons for the slow establishment of analytical laser spectroscopy. However, we are convinced that

0003-2700/87/0359-1232$01.50/0 0 1987 American Chemical Society

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Figure 1. Experimental arrangement for isotope ratio measurements by resonant Doppler-free two-photon laser spectroscopy applying thermionic diode detection.

the acceptance of methods based on laser spectroscopy will grow in the near future. The increasing interest will be due to the present rapid development of semiconductor diode lasers which can replace the dye lasers. Diode lasers are small-size and low-cost devices. They do not need to be aligned and have very stable output power; they are very efficient and have a very long life. The easiest way to tune a diode laser in wavelength is to change its temperature, which causes a slight change in the effective band gap in the semiconductor due to a change of the average energy of the current carriers. At present, continuous wave (CW) single longitudinal mode diode lasers are already available commercially for lasing wavelengths down to about 735 nm. There are companies that will offer diode lasers with wavelengths of about 680 nm a t room temperature in the near future. Research groups have already reported CW single mode laser operation in the yellow spectral range (8). Another way of extending the wavelength range to the blue or even the UV region is given by the development of highpower diode array lasers. Generation of 5.4-W CW power at 850 nm has been obtained recently (9) by these devices. Because the laser radiation of diode lasers can be focused nicely to a few micrometer beam waists (IO),efficient second harmonic generation can be performed in nonlinear crystals. In particular, in the wavelength range 840-940 nm, very efficient frequency doubling by KNb03 crystals has been demonstrated (11). Very recently the second harmonic generation of 420 nm in a LiNb03 waveguide attached to a diode laser was reported (12). It is expected to obtain a power of 1-5 mW with 50 mW of fundamental power from the semiconductor diode laser. The complete setup is not larger than a paper clip. In this correspondence, where preliminary results of isotope ratio measurements in Ba are given, we will demonstrate the simple but powerful application of diode lasers in isotopeselective trace element detection and isotope dilution techniques using LEI Doppler-free two-photon spectroscopy and thermionic diode detection (13),which may become a n easy to operate and low-cost alternative to mass spectrometry. EXPERIMENTAL SECTION Experimental Setup. The experimental arrangement for isotope-selectivetrace element measurement has been described in a recent publication in this journal (I). It applies the technique of resonant Doppler-free two-photon laser spectroscopy with thermionic diode detection. The light of two tunable CW single mode lasers is passed through two thermionic diodes in counter-

or copropagating direction to achieve Doppler-free spectra. While one thermionic diode, filled with the element under investigation (Ba), serves to lock the lasers properly to the Doppler-free isotopic component and to control the frequency and power drifts of the lasers,solid samples, introduced by a probe to the other thermionic diode (analytical diode), are atomized electrothermally from a Re band with a shallow depression. To activate the cathode filament of the analytical thermionic diode, Sr vapor was used. Both thermionic diodes were filled with 100 mtorr He buffer gas. The setup is shown schematically in Figure 1. During the evaporation process of the sample, one of the lasers is switched in frequency between the isotopic components of interest and, if necessary, the background spectrum, which is due to velocity changing collisions. The switching is performed and controlled by a PC computer via a digital-to-analogconverter. The signals of the thermionic calibration diode A with, e.g., a natural isotopic composition, and of the analytical diode B with the unknown isotopic ratio are amplified and stored by the computer. Because of their easy handling and excellent performance, we are using diode lasers for switching between the isotopic components. In our particular experiment, where isotope ratios in Ba were measured, semiconductor diode lasers (Hitachi HL 7801) induced the first step of the two-photon transition 6s' 'S0-6s6p 3P,-6s6d TIz at = 791.35 nm or (Hitachi HLP 1400) the second step of the transition 6s2 'S0-6s6p IPP,-6s6dIDz at A,, = 821.25 nm. In both cases the other step was induced by the light of a commercial, frequency-stabilized,single-mode CW dye ring laser (Spectra Physics 380 D; dye, Rhodamin 560) at A, = 552.06 and 553.7 nm, respectively. To avoid saturation broadening of the Doppler-freecomponents,the beams of the semiconductordiode laser and of the dye laser were expanded to cross sections of about 3 cm2 and attenuated by neutral density filters. Typical laser intensities of 1-3 mW/cm2 were used for the dipole transitions. A higher intensity could be used only for the 6' 'S0-6s6p 3P1 transition, where the transition probability is lower. Operation of the Diode Lasers. The diode lasers we use produce CW single longitudinal mode radiation in the range 760-805 nm (HL 7801) and 810-860 nm (HLP 1400) with maximum powers of 5 and 15 mW, respectively. The rough tuning of the wavelength is done by temperature while finer tuning can be obtained by changing the current of the diode. A typical tuning capability is 0.2-0.3 nm/OC. For this purpose the laser diode is placed in a small copper heat sink, which in turn is mounted on a Peltier element. The temperature is tuned in a typical interval -20 to +60 O C and controlled by a temperature sensor. With a simple electronic loop the temperature can be kept stable to about 1/200 O C . For stabilization to about 1 MHz we apply a highfinesse confocal Fabry-Perot interferometer (fsr = 2 GHz) where we are locking the frequency of the diode laser to the slope of the transmission line (see, e.g., ref 14). We achieved line widths of the diode lasers that were typically about 23 MHz.

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Flgure 2. Resonant Doppler-free two-photon spectrum inducing the 6s2 1S,-6s6p 'P1-6s6d ID, transition in barium by counterpropagating laser beams.

In a second electronic loop the reference signal from the Fabry-Perot setup acts on the laser diode current. The frequency of the locked diode laser can be changed by applying a voltage to the piezo element of the Fabry-Perot. If the isotope ratio of an element has to be determined, both lasers are tuned to the isotope resonance of the two-photon transition. While one laser, in our case the dye laser, is fixed in frequency, the second laser, the diode laser, is driven by different bias values from a computer via a digital-to-analog(DA) converter from one isotopic component to the other. The background signal can also be measured by this procedure. Relevance of the Thermionic Calibration Diode. One important task of thermionic diode A, as discussed in our earlier publication, is to serve as a reference for a proper locking of the lasers to the Doppler-free resonances. The other function is to give the signal ratio of a normalized sample (e.g., the ratios of the natural abundances). Here one has to remember that the intensities of the lines in resonant Doppler-free two-photon spectroscopy depend on the position of the laser frequency within the Doppler profile of the components. For example, if we want to measure the ratio of a very rare isotope to a prominent one, we have to tune the first laser to the center of the Doppler profile of the rare isotope which is only in the wing of the prominent isotope. Thus, the line intensity of the rare isotope is enhanced compared with the intensity of the more prominent one. The enhancement can be very large and depends on the magnitude of the isotope shift of the first transition. If the gas temperatures in diode A and B are comparable, the measured signal ratio of diode A can be used as a standard for the unknown isotope ratio in the evaporated sample of diode B. To find the right laser intensities, one has always to make a compromise between the demand of high signal to noise ratio (large intensities) and small saturation effects of the induced transitions (low intensities). Saturation broadening reduces the isotopic selectivity of the method considerably. On the other hand, larger laser intensities also induce hyperfine pumping effects in the odd isotopes (see, e.g., ref 14), which result in a change of the measured intensities in the hyperfine components. Saturation as well as hyperfine pumping effects can be controlled by the calibration diode A, where these effects are induced in the same strength. EXPERIMENTAL R E S U L T S A N D D I S C U S S I O N M e a s u r e m e n t s of Isotope Ratios u n d e r C o n s t a n t

Flgure 3. (a) Thermionic diode signals of different barium isotopes measured simultaneously by the calibration diode A (i)and the analytical diode B (ii)under steady-state evaporation conditions. The diode laser is driven by the computer. (b) Thermionic diode signal (diode B) of the 13*Ba component when driving the semiconductor diode laser between the Ba component and the background by a square-well voltage generator. Additionally the readings of a digital multimeter are given. The signal was processed by a lock-in amplifier.

Conditions. Figure 2 shows a resonant Doppler-free twophoton spectrum of the 6s2 'S0-6s6p 'Pl-6s6d ID2 transition in Ba. The frequency of the first-step laser (dye laser) was kept fixed within the Doppler profile of the resonance line, while the second-step laser (diode laser) was tuned. The laser beams had counterpropagating directions. The line widths are about 57 MHz. About half of the widths are due to the natural widths of the short-lived P and D levels. At this level of amplification we recognize the components of the most abundant isotope '%Ba and of the odd isotopes 135,137Ba.The other hyperfine splitting components of the odd isotopes are a t the long wavelength side of the 138Bacomponent (faint structures in Figure 2) and two are within the I3*Bacompoisotopes are also blended by nent. The lines of the 134,136Ba the 138 isotope line. By use of copropagating laser beams the spectrum is stretched in the frequency scale and most of the blended components can be resolved. To improve the resolution, it would be necessary to choose an excitation scheme via longer-lived states. At least the final state should be a Rydberg state with a very long lifetime. This would also improve the detection limit by orders of magnitude. The 6s6d ID, level is about 11800 cm-' below the ionization limit of the Ba atoms. The final level in the second excitation scheme used (6s2'S04s6p 3P1-6s6d3D2)lies slightly higher (about 500 cm-') but is still far from the limit. Therefore the collisional ionization probability is expected to be very small (23,15)in both cases. The Ba number density had to be increased to get a good signal-to-noise ratio for the Ba lines in diodes A and B. Care had to be taken that under those conditions the vapors were still optically thin for the laser light inducing the resonance transition. Figure 3a shows recorder traces of three cycles of isotope ratio measurements under constant Ba vapor conditions. Trace i is the signal of the calibration diode A, trace ii is the signal of the analytical diode B when switching the diode laser between the background and the Doppler-free components. Diode A as well as diode B are loaded with Ba of natural isotopic composition (138Ba,71.7%; 13'Ba, 11.3%; '"Ba, 7.8%; '%Ba, 6.6%; '%Ba, 2.4%; 13,Ba, 0.1%; '%Ba, 0.1%). A cycle starts with the background far from the lines. Then the diode laser is tuned to the 136 isotope followed by the 138 and the 135 and 137 signals. The 134,132, and 130 isotopes were not measured, because their lines were blended by other components (mainly hyperfine components of the odd isotopes). The analog signals of both diodes were digitized and stored by the computer (see Figure 1). The line investigated is the 1SO-1P1-1D2 transition and the laser beams had copro-

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Table I. Comparison of the Ratios of Ionization Signals with Standard Deviation (SD) of Various Ba Isotopes as Measured Simultaneously in the Transient Signal of the Analytical Diode A and in the Constant Signal of the Calibration Diode B Displayed in Figure 4 analytical diode (B)

* B Q / ' ~ * B ~ ratio

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Figure 4. Thermionic diode signals of different barium isotopes measured simultaneously by the calibration diode A (i) and the analytical diode 6 (ii) when a very small Ba sample (about 50 pg) is evaporated from the Re band of the probe.

pagation directions to resolve also the 136Bacomponent. Unfortunately we found that the diode laser driven by the PC had a small frequency drift after jumping. So we decided to scan the diode laser over the peak of each line after jumping. This can be clearly seen in Figure 3a. Therefore only one value, the peak value of the line, for each jump in a measuring cycle was used for isotope ratio determination. Taking the data of many cycles, we found agreement between the isotope ratios measured in diode A and B within 1% . The frequency drift observed is most probably due to an incorrect operation of the DA converter used. When we replaced the driving PC by a square-wave voltage generator driving the diode between the background and the peak of the 138Bacomponent with a frequency of about 1 Hz,we got a very constant signal over many minutes (see Figure 3b). The readings of a precision digital multimeter are also given in the figure. In the first few minutes the signal change was less than 1%. We hope to obtain similar results with a better DA converter. Measurements of Isotope Ratios with Transient Signals. In most cases the analytical chemist has the task of measuring the isotope ratio in very small samples. If small samples are evaporated electrothermally, we get transient signals as demonstrated in our earlier paper in this journal (1)where we detected isotope-selectively Yb in solid samples. Therefore the Re band on our probe was loaded in the present case with tiny samples of barium metal. We applied the procedure where the diode laser is switched by the PC between the isotopic components and the background and where the data of both thermionic diodes are stored by the computer. Just for illustration we give the recorder traces of such a measurement in Figure 4. The trace i gives the signals of the calibration diode A while trace ii presents the isotopic signals of the evaporated Ba sample from the analytical thermionic diode B. The highest peaks are due to 138Baand the second highest to '%Ba. The importance of measuring the background simultaneously can be seen in part ii of Figure 4 where the third highest signals represent the background very near to the 138J36Bacomponents. The background is mainly due to velocity changing collisions. It is less pronounced in the

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calibrating diode A where the Ba density was much lower during that time. The same but much weaker background effect was observed near the 135J37Ba components. This is the reason why the base line in trace ii is increasing during the evaporation process in diode B. Further it can be seen that in the 13th measuring cycle there was a strong increase of Ba vapor in the interaction region with the laser beams. If we neglect this cycle, we derive the isotope ratios presented in Table I. Note that the ratios of 13*Ba to other isotopes detected in diode A and B are based only on 14 maximum values each, because the diode laser was scanned across the peaks of the lines. As discussed above, the standard deviations (SD) will be much smaller if a proper switching between the components without a drift is possible. The ratios 135J37Ba/138Ba (of specific hyperfine components of the isotopes 135 and 137) measured with diode A and B agree within 2-4%. The uncertainty in the '%Ba/l%Ba ratios is much larger because the 136Bacomponent is situated on the outer wing of the 138Baline. CONCLUSION There are two points in this correspondence that we want to stress. First, the possibility of already using the promising semiconductor diode lasers in analytical laser spectroscopy. These handy, low-cost and long-lived lasers are predestined for instruments in spectrochemistry applied for routine analysis. If, in the near future, diode lasers with shorter wavelengths are available (including second harmonic generation of diode laser radiation in nonlinear crystals), dye lasers can be replaced completely by diode lasers. Then, one can think about using more than two lasers for laser spectroscopic multielement analysis. The second point of this letter is the introduction of a laser spectroscopic isotope dilution technique which may become an alternative to mass spectrometry. Although the reliability of the presented isotope ratio data from transient signals is still not good, we have shown that with a proper frequency switching of the diode laser we can do much better. This is a technical problem that can be solved. On the other hand, the optical isotope shift in the investigated spectra is small. One should look for transitions with larger shifts. This requires laser equipment with a wider variety of wavelengths. If the isotope shift is large, the isotopic selectivity can be excellent. This has been shown recently by our group (13) where we reported on the investigation of the dynamic range of the thermionic diode detectors. In naturally abundant Ca we detected all isotopes from the rarest (46Ca, 33 ppm) to the most prominent one (40Ca,97%) within one scan. LITERATURE CITED (1) Niernax, K.; Lawrenz, J.; Obrebski, A.; Weber, K.-H. Anal. Cbem. 1988, 5 8 , 1566-1571. (2) Omenetto, N.; Nikdel, S.; Bradshaw, J. D.; Epstein, M. S.; Reeves, R. D.; Winefordner, J. D. Anal. Chem. 1979, 5 1 , 1521-1525. (3) Epstein, M. S.;Nikdel, S.; Omenetto, N.; Reeves, R. D.; Bradshaw. J. D.; Winefordner, J. D. Anal. Chem. 1979, 51. 2071-2077 (4) Travis, J. C.; Turk, G. C.; DeVoe, J. R.; Schenck, P. K.; van Dijk, C. A. Prog. Anal. A t . Specfrosc. 1984, 7 , 199-241.

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(5) Hurst. G. S.;Payne, M. G.; Kramer, S. D; Young, J. P. Rev. Mod. Phys. 1979, 51, 767-819. (6) . . Bekov. G. I.: Letokhov, V. S. TrAC, Trends Anal. Chem. (Pers. Ed.) 1983, 2, 252-256. (7) Fassett, J. D.; Moore, L. J.; Travis, J. C.; De Voe, J. R. Science (Washington, D.C.) 1986, 230, 262-267. (8) Hino, 1.: Kawata, s.; Gomyo, A.: Kobayashi, K.; Suzuki, T. A@. Phys. Lett. 1986, 4 8 , 557-558. (9) Harnagel, G.;Welch, D.: Cross, P.; Scifres, D. R. Laser A p p l . 1986, 5(6\. 135-138. (IO) s t Z f e r , - ~ . ;Burnham, R. D.; Paoli, T. L.; Scifres, D. R. Laser FOCUS Elecfro-ODt. Mau. 1984. 20(6). 100-109. 111) Baumert.’J.-C.: kunter. P.: Melchior, H. Oot. Commun. 1983. 4 8 , 215-220. (12) Technology Report Laser focus f/ectro-Opt. Mag. 1986, 22(10), 42-46. (13) Niemax, K. Appl. Phys. 6 1985, 838, 147-157. (14) Demtrijder, W. Laser Spectroscopy; Springer-Verlag: New York, 1981. .

(15) Niemax, K. Appl. Phys. 6 1983, 832, 59-62.

J o r g Lawrenz Andrea; Obrebski Kay Niemax* Institut fiir Spektrochemie und Angewandte Spektroskopie (ISAS) Bunsen-Kirchhoff-Strasse 11 D-4600 Dortmund 1, Federal Republic of G~~~~~~

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RECEIVED for review October 20, 1986. Accepted January 2, 1987. This project is supported by the Deutsche Forschungsgemeinschaft. The financial support is gratefully acknowledged.

Control of Dispersion and Variation of Reaction Coil Length in Flow Injection Analyzers by Flow Reversals Sir: This preliminary communication reports a means by which the reaction coil length seen by samples in flow injection analysis (FIA) may be automatically varied, while maintaining the same system geometry. A number of unusual FIA arrangements are reported here in which, by one or more reversals in the flow direction, the plug may be moved effectively any distance. This freedom of movement has not been reported on conventional FIA systems. The dispersion characteristics that result from flow reversal(s) differ significantly from those usually observed in FIA. Existing physical models for dispersion in FIA will only apply after some modification. Thus, flow reversal flow injection analysis (FRFIA) methodology is a significant departure from normal FIA practice and is formally proposed here. On manual flow injection analyzers, flow reversals are difficult to carry out reproducibly since they require precise timing sequences for switching valves and/or pumps. However, on an FIA automated methods development system ( 1 4 ,the operation of any pump or valve is under computer control and timing is strictly repeatable. The carrier stream pH, buffer strength, concentrations of reagents, and flow rate(s) are all specified by using several computer-controlled variable-speed pumps (1-3). Sample size may be varied either by hydrodynamic means ( 4 ) or by partial flushing of a large sample loop (2, 3 ) . This latter has been achieved on our apparatus by means of timing sequences for pumps and for valves. Reaction coil length plays a major role in determining the characteristics of any FIA system and significantly interacts with other factors ( 5 ) ;tubing internal diameter, system geometry, flow rate, and temperature-related variables all play their part in determining the amount of dispersion that a sample plug undergoes as it traverses a flow injection manifold. FRFIA may be used in conjunction with choice of flow rate and sample size to determine the amount of dispersion that a sample undergoes. Three simple approaches to the problem of automated variation of reaction coil length for conventional FIA are shown in Figure 1. In the first (Figure la) (which is a variant of that in ref 6) the length of tubing between injector and detector is physically changed. In the second (Figure l b ) , several detectors are spaced a t intervals down a long length of tubing (7-9). Here the geometry of the flow system remains fixed and the peak profile information obtained is similar in format to that from computer simulations ( I O , 1 1 ) . The third

(Figure IC)uses a closed-loop flow injection system (12). In each case, the effective coil lengths obtainable are limited to discrete values. Thus, FRFIA, while “nonstandard”, may sometimes have significant advantages over such systems. We have effected FRFIA on an automated flow injection methods development system by control of pumps (Figure 2a) and a valve (Figure 2b). The first design (Figure 2a) is limited to a single flow reversal. Several flow reversals may be effected on the design shown in Figure 2b, thus allowing multidetection of peaks. In principle a simple single-pump, single-valve FIA system (Figure 2c) could be used for flow reversal studies. However, on the apparatus used here the pump direction was not under computer control. On reflection, it becomes obvious that Figure 2c could be accomplished on the existing apparatus by replacing the bidirectional pump with a tee, the arms of which lead to two unidirectional variable speed pumps, set up so that one can push or the other pull. EXPERIMENTAL S E C T I O N Apparatus. The apparatus used here (known as “AFID”,for automated flow injection development) was constructed in this laboratory ( 2 ) and is shown schematically in Figure 3. For the purposes of this communication it can be considered as functionally equivalent to our previous system recently reported in this journal ( I ) , except that no custom-built electronics were necessary. The control computer was an Apple I1 Europlus microcomputer equipped with 48 kbytes of RAM, two 5.25-in. disk drives, a 3.6-MHz 6502C accelerator card (Titan Technologies Inc., MI), and a printer (Epson MX8OF/T 111). A graphics plotter (Hewlett-Packard 7470A) was interfaced to the Apple I1 via a standard IEEE interface card. A nonstandard IEEE card (CIL Electronics, Ltd., Worthing UK) allowed communication with two CIL equipment interfaces (PCI 6380, CIL). These each contained a 280 microprocessor and had four analog outputs + eight analog inputs (each with a precision of 1 part in 32767) and four relays suitable for switching mains voltages (240 V ac). The analog outputs were used to control up to six variable-speed peristaltic pumps (Watson-Marlow, Ltd., Falmouth, UK). Relay outputs controlled two injection valves (Rheodyne Type 5020; Rheodyne, Inc., Cotati, CA), which were built into a unit designed in this laboratory and were actuated pneumatically. The unit also contained a f 15-V 200-mA power supply (Product No. 591-124; RS Components, L a . ) needed by the photometric detector. The photometric detector used was of a design reported previously (13)and employed a flow cell of path length 0.8 mm; as before (13),detection was via a light emitting diode and phototransistor combination, mounted transversely across a short length of transparent plastic tubing. Its output was routed to one of the

0003-2700/87/0359-1236$01.50/0 0 1987 American Chemical Society