INDIRECT DETECTION METHODS FOR CAPIL-Y SEPWRRTIONS Edward S. Yeung Ames Laboratory-U.S. Department of Energy and Department of Chemistry, Iowa State University Ames, IA 50011
Werner G. Kuhr Department of Chemistry University of California-Riverside Riverside, CA 92521
There is no need to re-enumerate the many reasons why detection plays an important role in analytical separation schemes. How else can we derive quantitative information? How else can we monitor the separation process so we can control it? Because of the nature of the mobile phase, it is generally agreed that detection is most problematic in liquid chromatography (LC) and least problematic in gas chromatography (GC), with supercritical fluid chromatography (SFC) falling somewhere in between. Surely many advances in detector technology have been reported in the last decade ( I ) , but problems still exist as new separation techniques are developed. A particularly challenging area is detection in capillary LC and capillary electrophoresis (CE). These methods have provided us with some of the highest separation efficiencies available (up to millions of theoretical plates) (2,3).The fact that small internal diameter capillaries are required is no coincidence. Small dimensions favor the plate height in LC and allow lower currents (less heating) to be used in CE. In general, we are faced with detection in 2-10-pm tubes with detector volumes on the order of 10-100 pL. Clearly not many conventional LC detection methods can be adapted for these situations. An obvious solution is to interface a UV-vis spectrophotometer to capillary columns, which was done in the early days of capillary LC ( 4 ) and permeated into the recent generation of commercial CE instruments. With ball lenses and associated optics, it is in fact possible to send a fair amount of light across capillaries with 50-pm internal diameters. The light path is usually defined 0003-2700/9 1/0363-275A/$02.50/0 @ 1991 American Chemical Society
by a slit, the width of which approaches the column i.d. The height of the slit can be on the order of 1 mm for most eluted peaks. In addition to the short optical pathlength (which equals the column i.d.), one has to fight shot noise (photon statistics at low intensity) and nonlinearity (ill-defined light paths). Realistically, even with the most highly absorbing analytes, one cannot expect to achieve concentration limits of detection (LOD) better than M of injected material. The mass LOD, mol for 50-pm capillaries, sounds impressive. The performance deteriorates rapidly when dealing with capillaries 10 pm or less in diameter. Of the common LC detectors, the two schemes that can be readily miniaturized are fluorescence and electrochemistry. They benefit from the small dimensions of focused laser beams (5) and microelectrodes (6),respectively. In fact, a concentration LOD of M and a mass LOD of mol have been reported (7,B). Spectacular as these results are, they rely on the very specific molecular properties of fluorescence and electrochemical activity. Only a small fraction of interesting analytes naturally lend themselves to these detection modes. The alternatives of preor postcolumn derivatization (9) are
not very appealing at these small dimensions. What if we can use the same instrumentation for universal detection (i.e., to detect nonfluorescent analytes by laser-excited fluorescence and to detect electrochemically inactive analytes by amperometry)? What if we can do so with reasonable detection limits? Indirect detection methods have been around for a long time. The key is that the analyte displaces a mobilephase additive in the eluted band. The signal is derived from this mobilephase additive rather than from the analyte itself. The displacement causes a decrease in the signal because the concentration of the mobile-phase additive is lower in the eluted bands when compared with its steady-state concentration (Figure 1). An early related scheme is vacancy chromatography (IO).However, here no displacement is involved and one is simply tracing a difference chromatogram. The first reported example of indirect detection involves ion chromatography (II),where the eluting ion is the chromophore being monitored by photometry. Therefore, a constant large absorbance (around unity) constitutes the baseline in the chromatogram. When analyte ions elute, the ion-exchange mechanism forces a 1:l displacement of the eluting ion in the sample zone, creating a negative peak. The nonabsorbing analyte ions are thus detected indirectly by photometry. The displaced eluting ions are not lost but are eluted in a system peak having the identical retention characteristics as if the eluting ion were injected onto the column. A corollary is that this system peak is absent in vacancy chromatography. Detector optimization Let us examine the requirements for good performance in indirect detection. We are faced with having a large background signal produced by the mobile-phase additive. The indirect signal (displacement peak) is a minor perturbation on the background signal. Normally, instruments are optimized for peak performance when the background signal is absent or very low. The main reason for the success of laser-
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REPORT excited fluorometry in ultratrace detection is the efficient rejection of stray light made possible by the highly collimated beam. In photometry, detector performance is typically shot-noiselimited. The absorbance LOD will deteriorate as the background absorbance increases because less light will reach the photodiode. The figure of merit here is the dynamic reserve (DR), which is the ability to measure a small change on top of a large signal, and is equal to the signal-to-noise ratio (S/N) of the background signal. The instruments will therefore have to be reoptimized with this in mind. Another consideration is the concentration CM of the mobile-phase additive that is being monitored. The DR limits the fractional change in the background signal that can be recognized. If the background signal is derived from a low concentration of the additive, then the fractional change will be large for a given amount of the mobile-phase additive being displaced. The third consideration is that the displacement process must be efficient. Usually a transfer ratio (TR), defined as the number of molecules of the mobile-phase additive displaced by each analyte molecule, approaching unity is desirable. For example, in ion chromatography (II), elution guarantees a 1:l (per charge) displacement. The net effect is that one can predict the concentration limit of detection (CLOD)of a given indirect detection scheme by CLOD = CM/TR X DR The three parameters are not necessarily independent. As one reduces CM, DR can also decrease, as in the case of indirect photometry (12).Equilibrium and surface effects can further reduce TR as CMis lowered (13).Thus, in addition to optimizing the detector to provide a large DR, one must also optimize the separation process to allow the use of a low CM and to achieve a large TR. This may mean altering the
standard mobile and stationary phases, such as lowering the column capacity in ion chromatography (14). Why is there a need for indirect detection methods? They offer universal detection for analytes sharing the same displacement mode. In fact, if the displacement mechanism is clearly defined, there may exist a single calibration curve for that group of analytes. The analytical procedure is simplified because there is no need for pre- or postcolumn derivatization to convert the analyte of interest into a species that gives a response at the detector. Furthermore, we would not need to redevelop a separation scheme for the derivatized analytes, which are likely to behave quite differently compared with the underivatized analytes. Because the analytes are not chemically altered, collection and further studies are facilitated. This is particularly important for rare and expensive biotechnological products. Also, there is the possibility for improved detection power. Whereas indirect methods are unlikely to provide the high level of performance typical of the corresponding direct methods (because of the degraded S/N in the presence of a large background), often the compromised LOD is still quite impressive. Finally, because selection of the mobile-phase additive is based mainly on the displacement mechanism, we can impose other requirements that favor the instrumentation used (such as absorption wavelength or electrode potential) rather than be bound by the properties of individual analytes. It is apparent that to become the method of choice, indirect detection schemes must provide an improvement in LOD when compared with standard detection schemes and allow us to maintain good performance in the separation process even though the chromatographic conditions may need to be altered to achieve good detection. For indirect photometry in ion chromatography, the LOD is comparable to but not better than that obtained with conductivity detectors. Absorbing eluting ions such a6 phthalate and benzoate are well behaved, but so are many eluting ions optimized for conductivity detection. One gains the convenience of using the more common absorbance detector and perhaps better immunity against temperature changes, but indirect photometry is not likely to replace conductivity detection in analyticalscale ion chromatography.
Flgure 1. General scheme for indirect fluorescence detection.
Indirect fluorometry
The mobile-phase additive provides a large background signal at the phototube. In the analyte zone, displacement occurs and a lower fluorescence intensity is observed.
In the last few years, indirect fluorometry has been shown to be a viable detection method in chromatography. The
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first demonstration was in ion chromatography (15), where the absorbing eluting ion in indirect photometry is essentially replaced with a fluorescing eluting ion. The experiment was much more difficult, however, because fluorescence gives rise to a more noisy baseline than does absorption, especially when laser excitation is employed. The intensity stability of common continuous lasers is roughly 1% (DR = loo), and this stability is improved by using a fancy double-beam configuration to a level comparable to current-controlled light bulbs (DR = lo4). The end result is that the LOD was not any better than for indirect photometry or conductivity detection. The advantages of indirect fluorometry become apparent as one tries to decrease CMto improve concentration LOD or to miniaturize the chromatography to improve mass LOD. The 1-cm optical pathlength in analytical-scale separations allows a reasonably small CM M) to be used for typical absorption strengths of the eluting ions ( e = lo3L molv1cm-l). DR for photometry will degrade as optical pathlength, CM, or absorption strengths decrease from the above values. Indirect photometric detection for CE therefore has limited detection power (16). On the other hand, even though fluorescence intensity also decreases with decreasing pathlength, concentration, and molar absorptivity, there is such a large signal (especially for laser excitation) that it is easy to maintain a large DR over a wide range of conditions. The first step toward miniaturization involved the use of microbore (1-mm i.d.) columns. The silica gel stationary phase was dynamically modified to become a low-capacity anion exchanger. This allowed CMto be lowered to 5 X M to provide a mass LOD of 6-15 pmol (subnanogram levels) for common inorganic anions (13). A 50-pm open tubular capillary column similarly modified lowered the mass LOD to 1 pg of NO, injected (17). There the eluting ion concentration was 2.5 X M. Recently, even 10-pm capillaries have been successfully employed, resulting in a mass LOD of 10 fg for NO, when CM = 2 X M (14). For the smaller capillaries, double-beam correction of the background fluctuations was not possible because of mechanical (alignment) instabilities and difficulties in matching two micrometer-scale flow systems. The laser beam was first stabilized to DR = lo3 by external servo control, and a single-beam arrangement was used. A particularly good candidate for indirect fluorescence detection is capil-
ges for improved cap
horesis separations.
REPORT lary zone electrophoresis (CZE), which is a special case of CE. We have discussed the limitations of standard detectors above. Basically, we need a universal detector for CZE that will work for capillaries < 25 pm in diameter and for analyte concentrationsbelow M. In CZE, an ionic buffer is present in the capillary tube. This provides electrical conductivity so that a constant electric field gradient can be maintained along the length of the column. The chemical composition of this buffer solution has only a secondary effect on the separation process. It is therefore straightforward to use a fluorescing ion as part of the buffer. As in ion chromatography, one has a large fluorescence background a t all times. When the analyte ion elutes, charge displacement creates a negative peak because the local concentration of the fluorescing ion is reduced. In actual operation, there are a few subtle points. The displacement TR is close to, but not exactly, 1:l. The more exact value can be derived from the Kolralsch function (18) and from the fractional charge. However, because all analytes separated by CZE are charged, displacement is guaranteed and universal detection is possible. The electrophoretic mobility of the analyte ion versus that of the buffer ion affects the separation.efficiency (18). Ideally, one wants to match these as closely as possible to achieve the narrowest zones. This is generally possible in indirect fluorescence detection. In contrast, conductivity detection (19), which also functions in a displacement mode, requires the equivalent conductance of the ions to be very different for sensitive detection. We have already shown that to lower concentration LOD, CMmust be reduced. As one decreases CM,eventually the buffer capacity of the solution (to maintain a constant pH and therefore a constant fractional charge) will degrade. More importantly, the surface of the column walls will begin to affect detection because retention (e.g., via ion exchange or adsorption onto the column walls) can significantly alter the fluorophore concentration. Band broadening caused by a mismatch in conductivity in the analyte zone will also be more pronounced. Also, a low CM precludes the use of low or high pH conditions, as H+ and OH- ions, respectively, will dominate the displacement process. Indirect fluorescence detection in CZE works quite well. In general M buffers can be used with untreated fused-silica capillaries. With a DR of lo3 for externally stabilized continuous-wave lasers, concentration LOD is in the €O-7 M range, or 5 X mol 278 A
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injected onto 10-pm columns (20).The low ionic strength of the buffer can actually be an advantage because the amount of heating induced by the low operating currents is minimized. One concern is distortion of the analyte zones (18)when buffers with low ionic strengths are used. This depends critically on the difference in electrophoretic mobilities of the buffer and analyte ions. When they have identical mobilities, sharp zones can be obtained even if the analyte concentration approaches that of the buffer ion. Experimentally, a linear response is obtained for the peak areas over a 500-fold range of concentrations (21),even though the peaks do become broader when the analyte ion concentration approaches that of the buffer ion. The universal nature of the response was verified in studies involving native amino acids (22),nucleotides (20),and common inorganic anions (21). The displacement mechanism is further confirmed using a variety of fluorophores for detecting the same group of analytes (22). Naturally, the same scheme is applicable to the detection of cations as well. The signal can be derived from a cationic fluorophore in the buffer, still producing negative peaks when nonfluorescing cationic analytes elute (23). An interesting application is the separation and detection of peptides derived from trypsin degradation of proteins. These tryptic digest maps can be used for characterizing proteins-for example, in the quality control of biotechnological products. The corresponding standard procedure is gradient HPLC, which can take up to 2 h per run and requires picomole levels of protein. Figure 2 shows two electropherograms of the same tryptic digest sample, &casein. In both cases, the number of observed peaks corresponds to the expected number of peptide fragments in the digest sample. Figure 2a is obtained using an anionic fluorophore (salicylate) in the buffer (24). At pH 10.9, the peptides are anionic and displace the salicylate ions to produce peaks. Figure 2b is obtained using a cationic fluorophore (quinine) in the buffer (23).At pH 3.7, the peptides are cationic and displace the quinine ions to produce peaks. Because one depends on the pKa of the acid groups and the other depends on the p& of the amine groups, the separations are complementary. This illustrates that just by changing the pH one can achieve a range of selectivity in these,separations.The separation times are quite different, because mobilities and the direction of elec63, NO. 5, MARCH 1, 1991
troosmotic flow are different. Note that Figure 2a shows a complete separation in only 3 min. The concentration LOD in each case is also in the 5 X mol range. Recently, immobilization of trypsin in capillary tubes was demonstrated (25).Thus it should be possible to start with subfemtomole amounts of protein and eventually obtain a tryptic digest map with CZE and indirect fluorescence detection. The performance of indirect fluorescence detection under extreme conditions can be seen in Figure 3. Sugars are normally neutral molecules that are not separated by CZE. At pH 11.5, they are in fact slightly ionized. That is sufficient for separation based on differences in fractional charge, and at the same time for indirect detection (22). Because of competition for displacement with OH- ions and because of the small extent of ionization, detection is only in the femtomole range. This is still a competitive LOD for separations in capillaries that are 10 pm or smaller. Indirect fluorescence detection is also possible with micellar electrokinetic capillary chromatography
Figure 2. Electropherograms showing the separation of peptides obtained from a tryptic digest of @-casein. (a) The buffer is 0.5 mM saiicylate-3-cyciohexoamino-l-propane sulfonic acid, pH 10.9. The column was 10-pm i.d., 70 cm overall length, and 60 cm from injector to detector. Displacement of salicylate results in negative fluorescence peaks. (Adaptedwith permissionfrom Reference 24.) (b) The buffer is 0.38 mM quinine sulfate and 0.58 mM H2S04,pH 3.7. The column is 18-pm i.d., 82.3 cm overall length, and 70.7 cm from injector to detector. Displacement of quinine resuits in negattve fluorescence peaks. (Adapted from Reference 23.)
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REPOR7 (MECC) (26). A micellar phase (e.g., sodium dodecyl sulfate [SDS])is added to the electrophoresis buffer, such that nonpolar analytes can partition between aqueous and micellar phases as they are swept along by electroosmosis. Indirect detection is possible when the fluorophore (quinine) also partitions between the aqueous and micellar phases, where it has different quantum efficiencies (27). Addition of a nonpolar analyte alters the partitioning of
Figure 3. Electropherogramshowing the separation and indirect fluorescence detection of sugars. Buffer contains 1 mM coumarinat pH 11.5. Column used is 90 cm long with 18-pm i.d. Peaks: S, sucrose; G, glucose; F, fructose. (Adapted with permissionfrom Reference 22.)
the quinine between these phases and thereby changes the concentration of the fluorophore in each phase, altering the total fluorescence observed. Because this induces only a partial change in the fluorescence signal observed at the detector, the concentration sensitivity is not as good as that obtained by direct displacement. The separation of a mixture of alcohols is shown in Figure 4, indicating a concentration LOD of approximately 10-5 M. These data were obtained using a commercial CE instrument (Model 270A, Applied Biosystems, Inc., San Jose, CA) equipped with a prototype fluorescence detector using a 75-W Xe arc lamp as the excitation source. The dynamic reserve for this system ranged between 350 and 850 with 0.5 mM quinine, which is adequate for most analyses.
Indirect electrochemical detection Electrochemical detection has been shown to have virtually the same level of sensitivity as fluorescence when used with CZE (28-30). The principle of indirect electrochemical detection has been demonstrated with LC (31, 32) and with ion chromatography (33,34).Although the former employed
Time (min
Figure 4. Electropherogram showing the separation of a mixture of alcohols by MECC with indirect fluorescence detection. Buffer composition: 0.5 mM quinine sulfate dihydrate, 100 mM SDS in 10% (v/v) methanol solution, pH 6.8. CZE parameters: capillary: 75-cm fused silica (60 cm to detector), 50-pm i.d. and 360-pm 0.d.; sample: composed of 0.06% (vh) of each component;injection: 1.O s at 5 psi (vacuum);voltage: 25 kV; current: 26 pA; temperature: 30 OC; excitation wavelength: 325 nm. Peaks: A, 2-propanol; B, 1-propanol; C, 2-butanol; D, 1-butanol; E, 2-methyl-1-propanol.
charge displacement of hydroquinone (31) or 2,5-dihydroxybenzoicacid (34),
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the latter required the complexation of metal ions to an electroactive ligand (dithiocarbamate). Once the electroinactive complex was formed, the consumption of the reagent was monitored electrochemically. Although these reports indicate the feasibility of indirect measurements, the sensitivity of the measurement must be enhanced through careful selection of the electrophore and further optimization of the electrochemical dynamic reserve. Ewing and co-workers (28-30) demonstrated that extremely sensitive amperomefric measurements can be made in CZE by electrically decoupling an amperometric detector from the electrophoretic power supply. Basically, a small break in the separation capillary several centimeters before the detector serves as a conductive joint, which removes the current from the separation without disturbing the flow of buffer along the capillary. A carbon fiber (radius 5-10 pm, length 100-500 pm) is inserted into the end of the column and the reference electrode is placed in buffer at the end of the capillary, thus forming an amperometric detector with a volume of only a few tens of picoliters. The apparatus is housed in a Faraday cage to minimize noise.
Because nanomolar detection limits were found for indoles and catecholamines (28), indirect electrochemical detection uses a similar experimental paradigm. Olefirowicz and Ewing (35) demonstrated the feasibility of indirect amperometric detection using 26-pmi.d. capillaries and 5-pm-diameter carbon fiber working electrodes. Dihydroxybenzylamine (DHBA) was used as the cationic electrophore, which provided a stable background at concentrations as low as 10p M in the presence of 25 mM 2-morpholinoethanesulfonic acid (MES) (Figure 5). Several amino acids and peptides were detected in this manner with 500-attomole detection limits. Direct amperometric detection of catechols could also be performed simultaneously, allowing both electroactive and electroinactive constituents of the sample to be monitored in the same run. Alternatively, an ion-selective electrode (ISE) of micrometer dimensions may serve as a sensitive detector ideally suited for indirect detection. Potentiometric measurements of many cations have been performed on column in capillary HPLC by placing the micrometer-wide tip of the ion-selective microelectrode directly into the end of
Flg"3ctrop herogram of amino acids with indirect detection. Buffer: 0.1 mM DHBA-0.025 M MES (pH 5.50)10% (v/v) ethanol; 72.1-cm-long, 26-pm i.d. separation capillary; 0.6-cm detection capillary: injection by electromigration,3 s at 15 kV; separation voltage, 15 kV: electrode potential, 0.7 V versus SSCE. Peaks: A, Lys; B, Arg; C, His; S, system peak. (Adapted with permission from Reference 35.)
the separation capillary (36).A potassium-selective microelectrode (5-pm tip
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REPORT diameter), using valinomycin as the ionophore in a liquid membrane ISE, provides a selective potentiometric measurement of potassium concentration when placed at the end of the electrophoresis capillary. Again, the electrophoresis current is shunted off via a small break in the capillary several centimeters above the detector. If a potassium salt is used as the principal buffer component (e.g., KCl), other cations will displace the potassium ions and allow indirect detection (37). An additional advantage of indirect potentiometric detection lies in the fact that it is a differential measurement. The analytical signal is simply a reflection of the difference in potassium concentration between analytical and reference solutions. Therefore, if the potassium concentrations of these solutions are matched, there is essentially no background recorded, yet high indirect sensitivity can be obtained.
cell to the beginning of a 5-10 pm capillary, burst open the cell, and proceed with capillary separation. We have already documented the suitability of indirect detection of biologically important materials such as anions (Cl-, HCO,), cations (Na+/K+/ Li+, Ca2+/Mg2+),nucleotides (ATP/ ADP), peptides, and simple sugars. The recipes are in place for single-cell studies. It should not be long before we can monitor pharmacokinetics, for instance, on a single-cell basis, or correlate biological activity with the chemical content of selected cells.
Other considerations
References
So far, our discussion has centered around capillary ion chromatography and CZE. Indirect detection works in reversed-phase capillary chromatography as well (14). There the analyte alters the partitioning of the mobilephase additive (fluorophore) between the stationary and the mobile phases to create positive and negative peaks. Displacement is less efficient in general, and concentration LOD suffers. For other types of CE, the 1:l charge displacement inherent in CZE is not applicable. Displacement based on partitioning can in certain cases produce indirect signals in MECC (27), cyclodextrin-inclusion electrokinetic chromatography (38), or polymeric ion-exchange chromatography (39). However, the effect is not expected to be general and the detection power is not expected to approach that achieved for CZE. It should, however, be possible to incorporate indirect detection into capillary gel electrophoresis and other variations in which CZE is combined with other retention mechanisms (e.g., size) based on nonionic additives. Looking ahead, there are unique applications that can be addressed by indirect detection. We have already mentioned sample-limited situations as particularly in need of detection schemes. Direct detection by fluorescence after chemical derivatization (40) and by electrochemistry (41) have been applied to single-cell studies. Mammalian cells (e.g., red blood cells) are on the order of several micrometers in diameter. They serve nicely as the injection plugs for capillary chromatography or CZE. One might, for example, hydrodynamically transfer a single
(1) Yeung, E. S. LCIGC 1989,7(2), 118-28. (2) Jorgenson, J. W.; Guthrie, E. J. J. Chromatogr. 1983,255,335-48. (3) Laner, H. H.; McManigill, D. Anal. Chem. 1986,58,166-70. (4) Yang, F. J. J. HRC&CC J. 1981,4, 8385. (5) Yeung, E. S.; Sepaniak, M. J. Anal. Chem. 1980,52,1465 A-1481 A. (6) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984,56,479-82. (7) Cheng, Y. F.; Dovichi, N. J. Science 1988,242,562-64. (8) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1989,61,98-100. (9) Tsuda, T.; Kobayashi, Y.; Hori, A.; Matsumoto, T.; Suzuki, 0. J. Chromatogr. 1988,456,375-81. (10) Scott, R.P.W.; Scott, C. G.; Kucera, P. Anal. Chem. 1972,44,100-04. (11) Small, H.; Miller, T. E. Anal. Chem. 1982,54,462-69. (12) Wilson, S. A.; Yeung, E. S. Anal. Chim. Acta 1984,157,53-63. (13) Takeuchi, T.; Yeung, E. S. J. Chromatogr. 1986,370,83-92. (14) Pfeffer, W. D.; Yeung, E. S. J. Chromatogr. 1990,506,401-08. (15) Mho, S.; Yeung, E. S. Anal. Chem. 1985,57,2253-56. (16) Foret, F.; Fanali, S.; Ossicini, L.; Bocek, P. J. Chromatogr. 1989,470,299-308. (17) Pfeffer, W. D.; Takeuchi, T.; Yeung, E. S. Chromatographia 1987,24,123-26. (18) Mikkers, F.E.P.; Everaerts, F. M.; Verhemen. Th. P.E.M. J. Chromatoar. 1979. 165; 11-20. (19) Huang, X.; Pang, T.; Gordon, M.; Zare, R. Anal. Chem. 1987,59,2747-49. (20) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988,60,2642-46. (21) Gross, L.; Yeung, E. S. J. Chromatogr. 1989,480,169-78. (22) Garner, T. W.; Yeung, E. S. J. Chromatogr. 1990,515,639-44. (23) Gross, L.; Yeung, E. S. Anal. Chem. 1990,62,427-31. (24) Hogan, B. L.; Yeung, E. S. J. Chromatogr. Sci. 1990,28,15-18. (25) Cobb, K. A.; Novotny, M. Anal. Chem. 1989,61,2226-31. (26) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56.111-13.
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The authors thank the many co-workers in their laboratories who contributed to this article. Work at Iowa State University is supported by the U.S. Department of Energy, Director of Energy Research, Office of Basic Energy Sciences, and Office of Health and Environmental Research. Work at the University of California-Riverside is supported by the National Science Foundation.
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Edward S. Yeung (left) received his A.B. degree from Cornell University in 1968 and his Ph.D. from the Universit y of California at Berkeley in 1972.He is Distinguished Professor in Sciences and Humanities at ISU and program director of Environmental Sciences in the Ames Laboratory. His research interests include nonlinear spectroscopy, high-resolution atomic spectroscopy, laser-based detectors for LC, CZE, trace gas monitoring methods, photochemistry, and data treatment procedures in chemical measurements. Werner G . Kuhr has been an assistant professor at the University of California-Riverside since 1988. He earned B.S. and M.S. degrees from Stevens Institute of Technology and his Ph.D. in analytical chemistry from Indiana University. His current research is centered on the development of microchemical techniques for in vivo measurement of chemical dynamics in the mammalian brain in real time.
