Anal. Chem. lQQ1,63, 1733-1737
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Indirect Fluorescence Detection in Micellar Electrokinetic Chromatography Lawrence N. Amankwa and Werner G. Kuhr*
Department of Chemistry, University of California, Riverside, California 92521
Detection of neutral analytw separated by mlceUar electroklnetlc caplllary chromatography (YECC) has been accomplbhed by Wed fh”daectkn udng quWne sullate srthevbualdngagent. Theseparatknbufferwascompowd of 100.0 mM sodium dodecyl sulfate (SDS) and 0.50 mM qulnlne sulfate at a pH of 6.8. The detectlon mechanlsm Involves a comblnatlon of dlsplacement of the fluorophore from the mlcelle by the analytes and net reductlon of the quanhmefkbncyofthefluorophorolnthesampkzone. The selectlvlty of this detectlon technique Is demonstrated wlth data drowlng the separation of several aHphatk alcohols and some phenolic compounds. The effect of quenchlng by the analyte on the sensltlvlty of detectlon Is also dlscussed.
INTRODUCT10N Micellar electrokinetic capillary chromatography (MECC) (1-5) is a new separation technique that has a separation efficiency comparable to that of free solution capillary electrophoresis, CZE (6). MECC, however, distinguishes itself from CZE in that it can separate neutrals as well as ionic analytes. The different classes of compounds that have been successfully separated by this method include, among others, chlorinated phenols (1-7), PTH amino acids (3), ionic and nonionic catechols (8,9), dansylated methylamines (IO),vitamins (11), and /3-lactam antibiotics (12). The fundamental principle of MECC has previously been described (2, 3), and it involves micellar solubilization and electrokinetic migration. Solutes are partitioned between the micelles that are under electrokinetic migration and the bulk aqueous phase that is moving because of electroosmotic flow in a direction opposite to that of the micelles. Solute resolution is therefore dependent upon the differences in the residence times of the solutes in the micelles, yielding a separation similar to those obtained with reversed-phase HPLC. More hydrophilic solutes spend most of their time in the more polar aqueous phase and elute with a time closer to that of the electroosmotic flow, while more hydrophobic solutes stay longer inside the micelles and hence elute with a time closer to that of the micelle (2). Like other efficient separation techniques utilizing micron-sized capillaries, the efficiency of MECC can only be fully realized when very selective and sensitive detection methods are employed. In the past, detection techniques have primarily involved W absorption ( I ) , with some use of electrochemical (8, 9) and fluorescence measurements (10). Although electrochemical and fluorescence detectors are very sensitive and also highly selective, they are not universal in that not all compounds have either an electrochemical activity or possess a high enough fluorescence quantum yield. Recently, Kuhr and Yeung (13,14) have described a sensitive technique involving indirect fluorescence detection in CZE. A fluorescing ion (14,15) was used as the CZE buffer component, and therefore, a large fluorescence signal was present at the detector at all times. When an analyte ion of the same charge as that of the buffer ion is injected, there is 0003-2700/91/0363-1733$02.50/0
a one-to-one displacement of the fluorescing ion in the sample zone to maintain local charge neutrality. The displacement of the fluorophore ions resulta in a decrease of the fluorescence background signal even though the analyte neither absorbs nor fluoresces. Reported limits of detection obtained by using laser-inducedindirect fluorescence detection of inorganic ions, proteins, and nucleotides were in the 50-100 amol range (0.1 pM sample) (14). The displacement mode of indirect fluorescence detection cannot be used for detecting neutral analytes that are easily separable by MECC, since charge displacement is required. Takeuchi and Yeung have demonstrated the principle of indirect fluorescence detection in reversed-phase liquid chromatography (16),where the analyte perturbs the partitioning of the visualizing agent between the mobile and stationary phases. Thus, neutral analytes can alter the concentration of the visualizing agent and allow indirect detection. The same mechanism should be operant in an MECC separation, where the analyte disturbs the partitioning between the aqueous and the micellar phases. Additionally, micelles have several other unique properties that make them very useful systems for applications in spectroscopic (I7-19) and chromatographic (18,20,21) studies. Among these are their ability to alter the fluorescence quantum yields of a variety of fluorophores. Fluorophore-micelle interactions, either by ion pairing or micellar solubilization of a fluorophore into the amphiphatic compartments of the micelle, greatly affect excitation and emission spectra, fluorescence lifetimes, depolarization, and quenching effects. As a consequence, the fluorescence intensities of most fluorophores are significantly enhanced in micellar solutions (I 7-19). In this work, we have taken advantage of these properties of micelles in devising an indirect fluorescence detection technique for detecting neutral analytes separated by MECC. The separation buffer consists of sodium dodecyl sulfate (SDS), the micelle forming surfactant, and quinine sulfate, which is present as the fluorophore. The enhanced fluorescence of the quinine ion because of its interaction with the micelles leads to a large fluorescence background signal continuously present at the detector. When an analyte that interacts with the micelle is injected, the fluorophoremicelle complex is perturbed, resulting in a decrease in the fluoreecence signal to produce a negative peak. The signal obtained in this manner is probably due to a combination of displacement of the fluorophore and a net reduction of the quantum efficiency of the remaining fluorophore molecules. Application of this detection technique to the detection of some aliphatic alcohols and some phenols separated by MECC is presented. The effect of quenching on detection sensitivity is also discussed. EXPERIMENTAL SECTION Apparatus. The work was done on a modified version of the Applied Biosystems capillary electrophoresis system, Model 270A, which has already been described (22,23). The detection system on the instrument was modified to enable both absorbance and fluorescence detection. In addition, the instrument was equipped with a 75-Wxenon arc lamp (UVP Inc.), which was powered by an auxiliary Hamamatsu Model C2177-01 power supply. Data 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
were recorded on a stripchart recorder (Model EV41-885835,Kipp & Zonen, Holland). Reagents. Water was distilled and deionized (Millipore, Bedford, MA). Electrophoresis-grade sodium dodecyl sulfate was purchased from National Diagnostics (Highland Park, NJ). Quinone sulfate dihydrate (Matheson Coleman & Bell, Norwood, OH) was of analytical grade and used as received. The separation buffer was composed of 0.05 mM quinine sulfate in 100.0 mM aqueous SDS solution. The two pK, values of quinine are 8.52 and 4.13. Therefore, at pH 6.0-7.0, the quinine acts as its own buffer component. In some of the separations, the buffer was modified to also contain 10% v/v methanol. When necessary, the pH of the solution was adjusted to the desired pH with 0.1 M H&O4 Prior to use, the buffer was filtered through an 0.45pm Gelman No. 60173 filter (Fisher Scientific, Fair Lawn, NJ). Compounds employed as solutes were of analytical grade and were used without further purification. They include phenol, 1chlorophenol, o-cresol, m-cresol, p-cresol, 1-butanol,1-propanol, 2-butanol, 2-propanol, 2-methyl-1-propanol,and 1-hexanol. Stock solutions of the solutes were made by diluting 5.0 p L of each solute to 5.0 mL with buffer. Working sample solutions were made by diluting appropriate volumes of the stock solutions with buffer to the desired concentration. The concentrations of samples ranged from 0.2 to 8.0 mM. For calibration plots, analyte concentrations up to 60 mM were used. Procedure. Fused silica capillary tubing (Polymicro Tech., Phoenix Arizona) was used as the separation column. It was 80 cm long (60cm to the detector),50pm i.d., and 360-mm 0.d. Prior to use, the column was treated by washing with 1.0 M NaOH for 10 min followed by a 10-min water rinse. Washing was accomplished by applying a low-pressure (10 in. Hg) vacuum at the cathodic end of the column while the anodic end was immersed in the appropriate wash solution. Vacuum injections were performed with 5.0 in. Hg vacuum applied to the cathodic end for a specified length of time (- 1or 2 s) while the anodic end of the column was immersed in the appropriate sample solution. The excitation wavelength of the xenon arc lamp was isolated at 325 nm, while the emitted fluorescence was collected on a PMT after passing through a 345-nm-long wave-pass glass filter.
RESULTS AND DISCUSSION Quinine sulfate is a widely used standard in fluorescence spectrometry. It has unique characteristics, such as a broad emission spectrum and minimal susceptibility to oxygen quenching (24). The fluorescence quantum yield of quinine sulfate depends upon the pH of the medium. A 10% increase in the fluorescence quantum yield was obtained by lowering the pH from 6.2 to 3.7 with 0.1 M H2S04 (24, 25). In an aqueous solution of SDS, the positively charged quinine ions (Q&) interact by electrostatic attraction with the SDS micelles (M) to form quinine-micelle aggregates as follows: aQ*+ Mn- (;j [Q,M](n-a*)(1)
+
where a is the number of quinine ions that interact with one micelle moiety, b is the charge of the quinine ion, and n is the micelle aggregation number. The stability of the quininemicelle aggregate depends on the magnitude of the interaction or binding constant as well as the presence or absence of impurities or other additives (20). Although the exact structure of the quinine-micelle aggregate is not known, the quinine ions are most likely located inside the hydrophilic layer of the micelles. Therefore, the microenvironment of the micelle-complexed quinine ion is entirely different from that in the bulk aqueous phase. The micelle acts as a pseudophase and shields the quinine ions from possible vibrational quenching by the hydrogen-bond structure of water. Simply, the micellar environment protects the excited state of the quinine ions from quenchingldeactivation processes such that the radiative proceases are favored (28).The fluorescence intensity of the SDS micellar solution of quinine sulfate is thus significantly enhanced over that of a free aqueous solution. There is also a significant shift of the quinine emission maximum (from 384 to 458 nm) in the
presence of SDS. The enhancement factor of 0.50 mM quinine sulfate in 100.0 mM SDS was measured as the ratio of the fluorescence intensity of this solution to that of 0.50 mM quinine sulfate in water. A t pH 6.2, the measured enhancement factor was 1.3, corresponding to an increase in the total fluorescence (emission at all wavelengths longer than 345 nm) of 30%. The magnitude of the enhancement factor obtained in this work is similar to the signal enhancement observed in oncolumn fluorescence detection of anthracene in open-tubular capillary liquid chromatography by Takeuchi and Yeung (26). In their case, anthracene in the stationary phase coated onto the inside of the capillary had a higher quantum efficiency than that in the mobile phase. In the present work, the ability of the micelle to organize the quinine ions into its amphiphatic compartments leads to a means of detection as well as separation. The presence of a solute that can either decrease the stability of the quinine-micelle aggregate or decrease the distribution ratio of quinine between the aqueous and micellar phases will result in the lowering of the fluorescence intensity in the sample zone via a chromatographic displacement mechanism (i.e., ref 27) and via direct alteration of the net fluorescence quantum yield of quinine remaining in the sample zone. A pH of 6.8 was used in this work, since quinine is partially ionized at this pH, and also the pH is moderate enough to provide adequate electroosmotic flow to allow separation. Excitation of the buffer at 325 nm results in a high stable fluorescence background signal at the detector. Owing to the fact that detection is performed on column, the fluorescence intensity recorded at the detector is the s u m of two intensities, that of quinine in the aqueous phase and that of quinine in the micellar phase. When an analyte is injected, it interacts with the micelle (e.g., solubilization) and thus perturbs the distribution equilibrium of the quinine between the two phases, displacing quinine from the micellar phase into the aqueous phase. The number of fluorescencing molecules in the aqueous phase is thus increased, while that in the micellar phase is decreased. Since the fluorescence quantum efficiency of quinine is higher in the micelle than in the aqueous phase, there is a net decrease in the fluorescence intensity in the analyte zone. Under electrophoretic conditions, the aqueous portion of the analyte zone migrates at the rate of the electroosmotic flow toward the detector (placed at the cathodic end of the capillary), while those analytes partitioned into micelles migrate at a slower rate. Eventually, there is spatial resolution between the analytes and the aqueous fluorophore displaced from the original sample zone. As these zones elute pass the detector, these variations in the background fluorescence intensity are recorded. Thus, the system peak (28) results from the net increase in the fluorescence intensity as a result of the local increase in the concentration of displaced quinine in the aqueous phase. The analyte peak, on the other hand, results from the net decrease in the fluorescence intensity due the local decrease in the concentration of quinine (caused by the presence of the analyte) in the micellar phase. The capacity factor (k') for quinine is very large, due to its interaction with the SDS micelles. This was indicated by the long elution time (>60min) of quinine, when quinine was injected into a buffer containing only SDS (i.e., no quinine in the eluent phase). When the original buffer contains quinine in addition to SDS but the concentration of the quinine is not enough to completely complex all of the micelles in the buffer, any additional quinine added to the aqueous phase (either injected directly or created due to the injection of a solute that can displace quinine already in the micelles) is repartitioned back into free (uncomplexed) micelles. This
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
0
4
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8
TIME(min)
Electropherogram showing the separation of a mixture of alcohols by MECC and indirect fluorescence detection. Buffer was composed of 0.50 mM quinlne sulfate in 100.0 mM SDS aqueous solution, pH 6.8. Instrumental parameters were as follows; Capillary was 80-cm fused silica (60 cm to detector), 50-pm i.d. and 360-pm 0.d. Excitation wavelength was at 325 nm. Voltage was 25 kV (current = 26 MA). Temperature was 30 O C . Sample was composed of 0.06% vlv (-7.0 mM) of each component and was Injected for 1.0 s by applying 5 In. Hg vacuum to the cathodic compartment. Peak Identity Is as follows: (a) 2-propanol, (b) 1-propanol,(c) 2-butanol, (d) 1-butanol: (e) 2-methyl-1-propanol. Flgure 1.
repartitioning process will continue to take place as the aqueous portion of the sample zone migrates toward the detector. If the repartitioning process is not completed before the zone containing the excess fluorophore reaches the detector, a positive signal (system peak) will be recorded at a time corresponding to the elution time of uncomplexed quinine. Therefore, the intensity of the system peak will be strongly dependent on the initial concentration of quinine relative to the concentration of micelle. An electropherogram of the separation of five aliphatic alcohols is shown in Figure 1. The large pmitive peak at time 3.4 min is the system peak,which corresponds to the migration rate of quinine ions that were not complexed by the micelle. Quinine ions that were displaced from the micelle by the analyte accumulate in the system peak, which is migrating at the rate of electroosmotic flow, resulting in a local increase in the background signal. The identity of the system peak was confirmed by injecting a sample that also contains methanol, which is not solubilized by SDS micelles and hence will migrate only at the rate of the electroosmotic flow. Methanol eluted just prior to the elution of the system peak, and because of the overlap, the positive system peak was distorted to a derivative peak as depicted in Figure 2. The negative methanol peak resulted from the change in the refractive index of the buffer due to the presence of methanol. The derivative shape is characteristic of a phase-distribution-based displacement process, where both the fluorophore and the analyte have the same migration velocity &e., that of electroosmotic flow) (28). Since SDS micelle has a negative electrophoretic mobility under the present conditions, it elutes at a much later time than the system peak. Consequently, an analyte that either partitions into or forms a complex with the micelle will also elute later than the system peaks. Comparison of the separations obtained in Figures l and 2 indicates that modification of the buffer to also contain 10% v/v methanol has only a slight improvement in analyte resolution. The retention times are longer in the methanol-modified phase (Figure 21, probably due to the decrease in the electroosmotic flow induced by
0
4
8
M(fi)
Flgure 2. Electropherogram showing the separation of a mixture of alcohols by MECC and indirect fluorescence detection. Buffer was composed of 0.50 mM qulnine sulfate in 100.0 mM SDS aqueous solution which has been modified with 10% v/v methanol, pH 6.8. Other parameters were as given In Figure 1.
0
4
8
12
16
TIME(&)
Flgure 3. Electropherogram showing the separation of a mixture of phenols by MECC and Indirect fluorescence detection. Other parameters were as given in Flgure 2. Peak idenwy is as fdbws: (a)phenol, (b) o-cresol, (c) m-cresol, (d) pcresol, (e) 1-chlorophenol.
methanol (29). The separation of a number of phenols is also shown in Figure 3, demonstrating the selectivity of the separation. The sensitivity of this detection technique is based on the stability of the fluorescence background signal (dynamic reserve; DR), the effectiveness of displacement of the fluorophore from the micelle (transfer number; TR), and the concentration of the fluorophore (Cmu). These quantities are related in the equation
where CmDis the concentration limit of detection (15). The dynamic reserve under the present conditions was approximately 350 when using a xenon arc lamp excitation source. This compares favorably to an unstabilized laser excitation source (DR 100) (13) but is somewhat less than that obtained once power stabilization is implemented (DR
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Table I. Relative Fluorescence Intensities of Various Solutions of 0.50 m M Quinine Sulfate (Q)
solution"
70re1 fluorescence int a7 51 100 25
Q
Q + phenol Q + SDS
Q + SDS + phenol "I~henoll= 11.2 mM. lSDSl = 100.0 mM. 1W
en
(-)
I . . . 0
4
8
12
TIME(min)
Flgurr 4. Electropherogramshowing the separation of a mixture of alcohols and phenols by MECC and Indirect fluorescence detection. Other conditions were as given In Flgure 2. Peak !dewIs as folbws: (a) 1-butanol; (b) 2-methyl-1-propanol, (c) phenol, (d) lchlorophenol, (e) l-hexanol.
80G1000) (14). Considering the simplicity and low cost of the arc lamp source, this is quite a satisfying result. It is difficult to evaluate the transfer number for indirect detection involving partition phenomena. Ishii and Takeuchi have derived an expression relating the capacity factors of the analyte and visualizing agent to sensitivity in reverse-phase HPLC (27) and have found that the best sensitivity is obtained when the two capacity factors are identical. Indeed, sensitivity was found to fall off rapidly when the analyte had either higher or lower k' than the visualizing agent when a nonsaturating concentration of visualizing agent is used. Typical transfer numbers ranged from 1:200 to 1:lOOO in this study (27). In contrast, the data presented here (Figures 1 and 2) demonstrate that sensitivity is independent of k'of the analyte in this separation. Virtually identical peak heights were observed for equal concentrations of the various test samples. This is due to the fact that in MECC the pseudostationary phase is also mobile. Consequently, all analytes encounter fresh fluorophore-saturated micelles; therefore; displacement of the fluorophore by analytes having different k' values yields similar deficiency peaks. Considerably higher sensitivity was obtained for the phenols (Figure 4). Peak heights were roughly 5 times larger for the phenols than those obtained for equal concentrations of aliphatic alcohols, even though their capacity factors (indicated by their retention times) were comparable (Figure 4). This is a strong indication that other phenomena (e.g., quenching) are also occurring. This anomaly is explained by the fact that phenol has a strong quenching effect on the quinine fluorescence. The fluorescence intensity of an 0.50 mM solution of quinine sulfate in water (pH 6.2) is 70% higher than that observed in the presence of a 11.2 mM aqueous solution of phenyl (Table I). Similar results were obtained for solutions that also contain 100 mM SDS. The observed phenol peaks are thus the sum of the effects of quenching and the perturbation of the quinine-micelle complex by the the andyte. Although fluorescence quenching by the analyte allows increased sensitivity,it leads to a nonlinear response (16). The concept of quenching by phenol was tested by running an equivalent concentration (5.0 mM) of benzaldehyde, which is a strong spin-orbit coupler and therefore a strong quencher. Exactly similar peak heights were obtained as those of the
E 60
I-
I
P Lu
I X
5n
40
20
0 I
0 5
1.o
1 5
2.0
2.5
[ QUININE SULFATE ] (mM)
Flgure 5. Plot of peak height of 1-butanol (27.3 mM) versus the concentration of quinine sulfate In 100 mM SDS. Electrophoretic condfflons were as given In Flgure 1.
phenols. In contrast, no measurable quenching effect on the quinine fluorescence was observed for the aliphatic alcohols. Peaks for these samples were induced only by the perturbation of the quinine-micelle complex. Consequently, the detector sensitivity for the phenols is better than that of the aliphatic alcohols. A calibration graph, obtained by using peak heights, for 1-butanol was linear (coefficient of correlation = 0.998, n = 4,slope = 0.97 f 0.04 mm/mM, intercept = 1.88 f 1.25 mm) over the concentration range 1.0-60.0 mM. The corresponding calibration graph for phenol is only linear (coefficient of correlation = 0.997, n = 5, slope = 14.63 f 0.66 mm/mM, intercept = 16.59 f 3.36 mm) over the concentration range 1.0-10.0 mM. Beyond 10.0 mM, the calibration plot exhibits a negative deviation probably due to the nonlinear quenching effects. The concentration of displacable fluorophore (Le., the fluorophore that is complexed with micelle) limits the sensitivity of indirect detection in MECC. The micelle concentration in the buffer (Cmic= 1.50 mM) was estimated from the initial concentration of SDS (100 mM), the critical micelle concentration (cmc) of 8.1 mM, and an average micelle aggregation number (nsDs) of 62 where Cmic = (CSDScmc)/nsm. Figure 5 illustrates the effect of the concentration of fluorophore on the peak height of analyte while maintaining the concentration of micelle constant. It is apparent that the peak height increases with increasing concentration of fluorophore until the concentration of fluorophore equals that of the micelle. Beyond this concentration, the peak height remains constant, but peaks become broader. The fact that the peak height increases only to the point where the concentrations of fluorophore and micelle are the same suggests that
(In,
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
the fluorophore and micelle interact to form only a one-bone complex. The interaction of the analyte with the fluorophore-micelle complex is believed to be via micellar solubilization, which is controlled by the equilibrium partition ratio of the andyte between the bulk aqueous phase and the micelle. The value of the equilibrium partition ratio relates inversely to TR. In this work, TR is difficult to evaluate; however, it is expected to be less than unity since most likely there is multiple-analyte partitioning of analyte with a single micelle. By using the values of CLOD= 10 MM,CnU = 0.5 mM, and DR = 350 and using eq 2, TR is estimated to be 0.14, which implies that one fluorophore ion is displaced by approximately seven analyte molecules. In order to elucidate exactly how the negative analyte signal (nonquencher) is generated in indirect fluorescence detection in MECC, we increased the concentration of quinine sulfate to 2.0 mM in 100 mM SDS (Cmic= 1.5 mM). Assuming a 1:l interaction between quinine and micelle, there is sufficient quinine to completely complex all of the micelles in the buffer. Thus, the quinine displaced during the separation will not be repartitioned back into other micelles and will be completely eluted in the system peak. If no other mechanisms are operant, the height of the system peak should be equal to the sum of the heights of all analytes. If other phenomena occur (i.e., a change in the fluorescence efficiency of quinine fluorescence between phases), this will be indicated by the relative magnitudes of the system and analyte peaks. A plot of the height of the analyte peak versus the concentration of 2-butanol injected yielded a slope of 0.594 f 0.055 mm/mM (intercept = 2.74 f 1.99 mm, ? = 0.984, n = 4), while a plot of the system peak height yielded a slope of 0.385 f 0.011 mm/mM (intercept = 4.42 f 0.136 mm, t2= 0.999, n = 3). These slopes, which represent the response factors for each peak, are significantly different. The ratio of the response of the analyte peak to system peak is 1.54 f 0.14, indicating the presence of another mechanism, which we attribute to a change in quantum efficiency of the displaced quinine. This is likely because of the very close agreement of this value with the enhancement factor (1.3) for quinine fluorescence found under these conditions. Thus, it is apparent that the negative analyte signal is significantly affected by the difference between the fluorescence quantum efficiencies of the fluorophore in the bulk aqueous and the micellar phases. Since the mechanism of detection is based upon the net lowering of the fluorophore intensity, the sensitivity and dynamic range will be increased by this effect. Although increasing the fluorophore concentration will result in higher peak heights and thus a lower value of the LOD, it will not significantly affect the dynamic range. Other
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parameters that control the LOD are the micelle enhancement factor of the fluorescence intensity and the quenching capabilities of the analyte on the fluorophore fluorescence. Under the present experimental conditions, the concentration LOD for our test solutes was found to be lo6 M, leading to a mass sensitivity of mol of injected sample. This compares favorably to the sensitivity obtained with indirect UV detection in capillary HPLC (10-l2 mol injected (27).
ACKNOWLEDGMENT The loan of the capillary electrophoresis system (Model 270A) by Applied Biosystems, Inc., is gratefully acknowledged.
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(11) Nishi, H.; Tsumagari, N.; Kakimoto, T.; Terabe, S. J . Chrmtogr. 1989. 465. 331-343. 112) . . Nishi,' H.;Tsumaoari.. N.:. Kakimoto.. T.:. Terabe. S. J . Chfmtoa. 1989. 447, 259-270. (13) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988, 6 0 , 1832-1834. (14) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988, 6 0 , 2642-2646. (15) Yeung, E. S. Acc. Chem. Res. 1989, 22(4), 125-130. (16) Takeuchi, T.; Yeung, E. S. J . Chomatogr. 1988, 366. 145-152. (17) Hinze, W. L.; Slngh, H. N.; Babe, Y.; Harvey, N. 0. Trends Anal. Chem. 1984. 3 (8), 193-199. (18) . . Armstrono. D. W.: Hinze. W. L.: Bui. K. H.;Sinoh. - H. N. Anal. Lett. 1981, 4 Gig), 1659-1667. (19) Beayens, W.; Lin, 6.; Corblsier, V. Analyst. 1990, 715, 359-363. (20) Hinze, W. L. In &&red M i a in Chemhl Separations; Hinze. W. L., Armstrong. D. W., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987; Chapter 1. (21) Cline Love, L. J.; Habarta, J. G.; Dorsey. J. 0. Anal. Chem. 1984, 56, I 132A- 1146A. (22) Amankwa, L. N.; Schoil, J.; Kuhr, W. G. Anal. Chem. 1990, 6 2 ,
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2 189-2 193. (23) Albln, M.; Weinberger, R.; Sapp, E.; Moring, S. Anal. Chem. 1991, 6 3 , 417-422. (24) Stclndards in Fluorescence Spectromehy; Miller, J. N., Ed.; Chapman and Hall: London, 1981; Voi. 2, Chapter 7, p 57. (25) Gross, L.; Yeung, E. S. Anal. Chem. 1990, 6 2 , 427-431. (26) Takeuchi, T.; Yeung. E. S. J . Chfomatogr. 1987. 389, 3-10. (27) Ishll. D.; Takeuchi, T. J . Llq. Chromatogr. 1988. 1 1 , 1865-1874. (28) Stranahan, J. J.; Deming, S. N. Anal. Chem. 1982. 5 4 , 1540-1546. (29) Aitria, K. D.; Simpson, C. F. Anal. Roc. 1986, 23, 453-454.
RFCEIVED for review October 29,1990. Accepted May 29,1991. This work was supported, in part, by the donors of the Petroleum Research Fund, administered by the American Chemical Society, the Society of Analytical Chemists of Pittsburgh, and the National Science Foundation (Grant No. CHE-8957394).
