Anal. Chem. 1998, 70, 4030-4035
Capillary Electrophoresis with Electrochemical Detection for Chiral Separation of Optical Isomers Xiaoming Fang, Feiyan Gong, and Yuzhi Fang*
Department of Chemistry, East China Normal University, Shanghai, 200062, China
The enantiomers of two amine derivatives were directly separated by capillary electrophoresis (CE), employing β-cyclodxtrin (β-CD) as a chiral additive in strongly alkaline solutions. The analytes were detected by electrochemistry, using a copper disk electrode at +675 mV vs Ag/AgCl reference electrode. Both the free enantiomers and the enantiomer-cyclodxtrin inclusion complexes could be detected using this approach, although the complexed forms gave lower oxidation currents than the free forms. Factors affecting the chiral CE separation of the analytes, such as working potential, concentration of running buffer and β-CD, and applied voltage, were extensively investigated. Under the optimum conditions, baseline separation of the enantiomers could be accomplished in less than 18 min. In addition, a successful application of the method to the enantiomeric purity determination confirmed its validity and practicability. There is currently a great deal of interest in chiral separation of the enantiomers of biologically important compounds in chemistry and pharmacology, especially regarding the different bioactivities of the enantiomers in the living world.1 Chiral separation is of great importance not only when monitoring the enantiomeric purity of the drugs but also when studying the relationship between chirality and a drug’s therapeutic efficacy.2 However, chiral separation is one of the most difficult analytical techniques, since a pair of enantiomers shows no difference in chemical and physical characteristics in nonchiral media except exactly opposite optical rotations. Traditional chromatographic methods have been extensively studied for chiral separation of enantiomers, including thin-layer chromatography (TLC),3,4 high-performance liquid chromatography (HPLC),5,6 and gas chromatography (GC).7,8 Recently, capillary electrophoresis (CE) has emerged as a powerful separation technique. Because of its high efficiency, small volume requirement, facile operation, and rapid analysis, CE has been an alternative to HPLC for chiral separations. In free solution capillary electrophoresis, chiral separation is mainly based on the addition of a chiral additive into the running buffer to provide a (1) Crossley, R. Tetrahedron 1992, 48, 8155. (2) Coutts, R. T.; Baker, G. B. Chirality 1989, 1, 99. (3) Armstrong, D. W.; DeMond, W. J. Chromatogr. Sci. 1984, 22, 11. (4) Armstrong, D. W.; Zhou, Y. J. Liq. Chromatogr. 1994, 17, 1695. (5) Noggle, F. T.; DeRulter. J.; Clark, C. R. Anal. Chem. 1986, 58, 1643. (6) Fisher, C.; Modler, A.; Moine, J.-E. J. Chromatogr. A 1996, 728, 433. (7) Liu, J. H.; Ku, W. W. Anal. Chem. 1981, 53, 180. (8) Betshinger, F.; Libman, L.; Shanzer, A. J. Chromatogr. A 1996, 746, 53.
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chiral environment in which not only the analyte-additive association constants of enantiomers are different but also the mobilities of the free forms and complexed forms of enantiomers are unequal. Several compounds have been used as chiral additives, such as crown ethers,9,10 bile salts,11,12 surfactants,13,14 glycopeptides,15 and, most frequently, cyclodextrins (CDs).16-18 CE with UV detection employing a chiral additive is currently the dominant technique for chiral separations of enantiomers possessing appreciable UV-visible absorbance;9-18 other methods of detection, such as fluorescence,19 MS,20,21 and NMR,22 are also used. Electrochemical detection (ED) can provide higer sensitivity and selectivity than classical UV detection, which has been developed as an alternative to unsatisfasctory photometric methods. Although much work has been done for the determination of a variety of compounds,23-30 together with the improvement of the electrode-capillary alignment,31-34 only one paper reporting CE-ED for chiral separations was published.35 A fairly significant extension of CE-ED would be needed to explore its applicability to chiral separations. In this work, we applied CE-ED employing native β-CD to perform chiral separa(9) Wagner, J.; Walbroehl, Y. J. Chromatogr. A 1994, 680, 253. (10) Martin, G. S.; Gerald, G. J. Chromatogr. A 1995, 709, 81. (11) Terabe, S.; Shibata, M.; Miyashita, Y. J. J. Chromatogr. 1989, 480, 403. (12) Nishi, H.; Fukuyama, T.; Matsuo, M.; Terabe, S. Anal. Chim. Acta 1990, 236, 281. (13) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1989, 61, 491. (14) Ozaki, H.; Ichihara, A.; Terabe, S. J. Chromatogr. A 1995, 709, 3. (15) Carpenter, J. L.; Camilleri, P.; Dhanak, D.; Goodall, D. M. J. Chem. Soc., Chem. Commun. 1992, 804. (16) Tait, R. J.; Thompson, D. O.; Stella, V. J.; Stobaugh, J. F. Anal. Chem. 1994, 66, 4013. (17) Stalcup, A. M.; Gahm, K. H. Anal. Chem. 1996, 68, 1360. (18) Fanali, S.; Camera. E. J. Chromatogr. A 1996, 475, 17. (19) Stefansson , M.; Novotny, M. J. Am. Chem. Soc. 1993, 115, 11573. (20) Sheppard, R. L.; Tong, X.; Cai, J.; Henion, J. D. Anal. Chem. 1995, 67, 2054. (21) Lamoree, M. H.; Sprang, A. F. H.; Tjaden, U. R.; Van der Greef, J. J. Chromatogr. A 1996, 742, 235. (22) Chankvetadze, B.; Endresz, G.; Schalte, G.; et al. J. Chromatogr. A 1996, 732, 143. (23) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1986, 60, 1872. (24) Olefirowicz, T. M.; Zare, R. N. Anal. Chem. 1990, 62, 1872. (25) Olefirowicz, T. M.; Ewing, A. G. J. Chromatogr. A 1990, 499, 713. (26) Colon, L. A.; Dadoo, R.; Zare, R. N. Anal. Chem. 1993, 65, 476. (27) Zhou, J.; O’Shea, T. J.; Lunte, S. M. J. Chromatogr. A 1994, 680, 271. (28) Ye, J.; Baldwin, R. P. J. Chromatogr. A 1994, 687, 141. (29) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1994, 66, 307. (30) Fang, X.; Ye, J.; Fang, Y. Anal. Chim. Acta 1996, 329, 49. (31) Huang, X.; Zare, R. N. Anal. Chem. 1991, 63, 2193. (32) Ye, J.; Baldwin, R. P. Anal. Chem. 1993, 65, 3525. (33) Zhou, J.; Lunte, S. M. Anal. Chem. 1995, 67, 13. (34) Matysik, F.-M. J. Chromatogr. A 1996, 742, 229. (35) Hadwiger, M. E.; Torchia, S. R.; Park, S.; Biggin, M. E.; Lunte, C. E. J. Chromatogr. B 1996, 681, 241. S0003-2700(97)00796-8 CCC: $15.00
© 1998 American Chemical Society Published on Web 08/25/1998
Figure 1. Structures of the and DANP (R ) CH3).
D
and
L
enantiomers of ANP (R ) H)
tion of the enantiomers of two amine derivatives which, to our best knowledge, had not been reported. The effects of working potential, concentrations of running buffer and β-CD, and applied voltage had been exensively investigated. Furthermore, we applied the method to determine the enantiomeric purity of laboratory synthetic batches, and the results were quite satisfactory. EXPERIMENTAL SECTION Chemicals. The enantiomers of threo-2-amino-1-(4-nitrophenyl)-1,3-propanediol (ANP) were kindly supplied by Shanghai Sixth Pharmaceutical Factory (Shanghai, China), and the enantiomers of threo-2-(dimethylamino)-1-(4-nitrophenyl)-1,3-propanediol (DANP) were gifts from Associate Professor B. Liu (Department of Chemistry, East China Normal University, Shanghai, China). Their structures are illustrated in Figure 1. β-CD was purchased from Sigma (St. Louis, MO). Other chemicals were of analytical grade and used as received. Running buffers were prepared by mixing β-CD with NaOH solution. Enantiomers were dissolved in 0.1 M HCl solution to give the stock solutions (0.050 M) of D-ANP, L-ANP, D-DANP, and L-DANP, and standard solutions were diluted to the desired concentration with the running buffer. The water used was doubly distilled. All buffers and samples were filtered through a 0.45-µm membrane syringe filter immediately prior to use. Apparatus. The assembly of the laboratory-made CE with wall-jet electrochemical detection was described in detail elsewhere.32,36 In brief, a high-voltage power supply (Shanghai Institute of Nuclear Research, China) provided a voltage of up to 30 kV. The inlet end of the capillary was held at a positive potential, while the outlet end was grounded. It was housed in an interlock box to prevent the operator from accidental shock. A fused-silica capillary (Yongnian Optical Fiber Factory, Hebei, China) of 25 µm i.d. and 80 cm length was used for separation. Amperometric measurements were performed on a potentiostat (ZF-3, Shanghai Second Component Factory, China) coupled to a picoammeter (WD-1, Shanghai Yanzhong Instrument Factory, China), and the electropherograms were recorded using a chart (36) Fang, Y.; Fang, X.; Ye, J. Chem. J. Chin. Univ. 1995, 16, 1514.
Figure 2. Hydrodynamic voltammograms of (a) 0.10 mM D-ANP and (b) 0.30 mM D-DANP in 0.10 M NaOH solution containing 12 mM β-CD. Capillary, 25 µm i.d. × 80 cm; Cu disk electrode, 220 µm; applied voltage, 20 kV; injection, 20 kV for 10 s; temperature, 30 °C.
recorder (XWTD-164, Shanghai Dahua Instrument Factory, China). The three-electrode system was composed of a copper disk working electrode (220 µm), a platinum auxiliary electrode, and a Ag/AgCl reference electrode (3 M KCl). Before the experiment, the surface of the working electrode was polished with emery sandpaper, sonicated in doubly distilled water, and then carefully positioned opposite to the capillary outlet with the aid of a micromanipulator to minimize the gap between the electrode tip and the capillary outlet. The potential applied to the working electrode was set at +675 mV vs Ag/AgCl reference electrode. Just prior to each series of measurements, the capillary was sequentially washed with the NaOH solution (0.4 M), doubly distilled water, and running buffer, each for 4 min, and then was given 10 min to equilibrate under the applied voltage of 20 kV. Samples were injected electrokinetically at 20 kV for 10 s. All separations were carried out at ambient temperature, ∼30 °C. RESULTS AND DISCUSSION Electrochemistry. The cyclic voltammetry (CV) of ANP and DANP in 0.10 M NaOH solution containing 12 mM β-CD was tested at variable disk working electrodes, such as copper, platinum, gold, titanium dioxide, nickel, and graphite electrodes. CV demonstrated that the copper disk electrode was well suited to the detection of the two compounds, with higher response and better reproducibility. The hydrodynamic peak current as a function of the working potential in CE was investigated. Figure 2 shows the hydrodynamic voltammograms (HDVs) of ANP and DANP. From Figure 2, it can be seen that ANP showed plateau-shaped HDVs, while DANP exhibited an increasing current HDV up to +850 mV. At more positive potential, both the background current and the noise became higher due to the oxidation of the electrolyte buffer. Therefore, +675 mV was chosen as the optimum potential, giving better signal-to-noise ratio and lower background current. The effect of the β-CD concentration on the oxidation currents is shown in Figure 3. Both the enantiomers of ANP and DANP Analytical Chemistry, Vol. 70, No. 19, October 1, 1998
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Figure 3. Effect of β-CD concentration on the peak current: (a) 0.10 mM D-ANP, (b) 0.10 mM L-ANP, (c) 0.10 mM D-DANP, (d) 0.10 mM L-DANP. Buffer, 0.10 M NaOH solution containing β-CD; working potential, 675 mV; other conditions are as in Figure 2.
exhibited relatively large currents in the absence of β-CD in the buffer, because the molecules of the analytes possess electroactive groups, adjacent hydroxyl and amino groups, which are readily oxidized at the copper electrode.37 The equality of currents for each pair of enantiomers reveals that the enantiomers of the same compound possess the same electrochemical properties in nonchiral media. In addition, ANP showed higher currents than DANP. The reason might be the substitution of two methyl groups (DANP) for two protons on the amino group (ANP), to become a tertiary amino group, which is unfavorably oxidized. Additionally, two methyl groups hinder the nearby hydroxyl groups on C-1 and C-3 from being oxidized, which is probably another important factor in the decrease in current for DANP. A significant decrease in current occurred, however, when β-CD was added into the running buffer. As was shown in Figure 3, as an example, the currents dropped ∼41% for D-ANP and 47% for D-DANP when the β-CD concentration was 4 mM, compared to those when there was no β-CD in the buffer. The reason for this is that, once the inclusion complexation between the analytes and β-CD occurs, the hydroxyl and amino groups on the molecules of the analytes are partially hidden by the β-CD cavity, which prevents the electroactive groups from effectively making contact with the electrode surface; therefore, the response for the complexed form is much smaller than that for the uncomplexed form. Figure 3 also clearly showed that the peak currents decreased with the increase in β-CD concentration, which agreed with the fact that more complexation occurs at higher β-CD concentration. It was interesting to note that, although the D and L enantiomers showed the same currents in the NaOH buffer, the D enantiomers of both ANP and DANP gave higher currents than the corresponding L enantiomers in the buffer containing β-CD. This indicates that the L enantiomers of the two compounds associate with β-CD more strongly than the corresponding D (37) Luo, M. Z.; Baldwin, R. P. J. Electroanal. Chem. 1995, 387, 87.
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Figure 4. Electropherograms of (A) ANP and (B) DANP enantiomers mixed with en in 0.05 M NaOH solution: (a) 0.050 mM D-ANP, (b) 0.050 mM L-ANP, (c) 0.15 mM D-DANP, (d) 0.15 mM L-DANP, (e) 0.50 mM en. Conditions are as in Figure 3.
Figure 5. Electropherograms of the enantiomeric separation of (A) ANP and (B) DANP in 0.10 M NaOH solution containing 12 mM β-CD: (a) 0.10 mM D-ANP, (b) 0.10 mM L-ANP, (c) 0.25 mM D-DANP, (d) 0.25 mM L-DANP. Conditions are as in Figure 3.
enantiomers; consequently, the fraction of the free form of the D enantiomers in the buffer was slightly greater than that of the corresponding L enantiomers when the concentrations were identical. Therefore, the observed currents of the two D enantiomers were somewhat higher than those of the corresponding L enantiomers. Separation Mode. The enantioseparations of ANP and DANP were studied by free solution capillary electrophoresis in NaOH solutions with concentration ranging from 0.01 to 0.20 M, containing native β-CD as a chiral additive. Within this NaOH concentration range, the molecules of ANP and DANP were almost neutral, since they nearly migrated together with ethylenediamine (en), as shown in Figure 4. The en served as a neutral marker because it demonstrates no ionization in buffers of pH > 12, owing to its pKb of 7.56 and 10.71.38 Figure 5 shows the (38) CRC Handbook of Chemistry and Physics; Robert, C. W., Ed.; CRC Press: Boca Raton, FL, 1980; p D-161.
Table 1. Effect of β-CD Concentration on Migration Times and Resolution for Enantiomersa 4 mM β-CD
8 mM β-CD
12 mM β-CD
16 mM β-CD
molecule
tb,d
Rsc,d
t
Rs
t
Rs
t
Rs
ANP DANP
15.8 ( 1.0 15.9 ( 0.9
0.0 ( 0.0 0.0 ( 0.0
16.7 ( 1.2 17.1 ( 1.2
1.0 ( 0.1 1.2 ( 0.2
17.3 ( 1.3 18.2 ( 1.5
2.0 ( 0.3 2.2 ( 0.4
18.5 ( 1.6 20.2 ( 1.9
3.5 ( 0.6 3.8 ( 0.5
a Conditions: buffer, 0.10 M NaOH solution with β-CD concentration ranging from 4 to 16 mM; other conditions as in Figure 3. b Migration times of first eluting enantiomers, in minutes. c The resolution, Rs, was calculated using the formula, Rs ) 1.17(t2 - t1)/[W0.5(2) + W0.5(1)], where the migration times t1 and t2 refer to the first- and secend-eluting enantiomers, respectively, as do the widths at half height W0.5(1) and W0.5(2). d Values are mean symbol ( standard deviation for three injections.
Table 2. Effect of NaOH Concentration on Migration Times and Resolution for Enantiomersa 0.04 M NaOH
0.10 M NaOH
0.16 M NaOH
molecule
t
Rs
t
Rs
t
Rs
ANP DANP
14.7 ( 0.7 15.2 ( 0.8
1.1 ( 0.1 1.4 ( 0.2
17.3 ( 1.3 18.2 ( 1.5
2.0 ( 0.3 2.2 ( 0.4
18.5 ( 1.8 19.1 ( 1.7
1.6 ( 0.2 19.1 ( 0.3
a Conditions: buffer, NaOH concentration ranging from 0.04 to 0.20 M with 12 mM β-CD; other conditions as in Figure 3. See Table 1 for definitions of t and Rs.
separation of the 1:1 standard mixture of the D and L enantiomers of (A) ANP and (B) DANP in the 0.10 M NaOH buffer containing 12 mM β-CD. Complete baseline separations of the two pairs of enantiomers were obtained, and the D enantiomers migrated faster than the corresponding L enantiomers. The separations were based on the fact that the native β-CD takes negative charge in strongly alkaline solutions39 and moves in the direction opposite to the electroosmotic flow. The difference in the dynamic equilibrium between the neutral enantiomers and charged β-CD resulted in chiral discrimination. Although charged CD derivatives have been investigated for chiral separation applications,40-42 nothing about using native β-CD as the anionic chiral additive has been reported. Owing to the use of native β-CD, this technique is quite inexpensive and practical for use in the chiral separation of neutral enantiomers, compared with the use of other charged CDs, which required chemical modifications. Optimization of the CE Conditions. To obtain the optimal separations of ANP and DANP enantiomers, several parameters, such as β-CD concentration, NaOH concentration, and applied separation voltage, were investigated. Table 1 showed the effect of the β-CD concentration on the migration times and resolutions for ANP and DANP enanatiomers. At the extralow concentration of β-CD (i.e., below 4 mM), no chiral separations occurred because the chiral additive concentration was too low to form enough diastereomeric associations to achieve enantioseparations. Addition of β-CD to the running buffer was found to increase the migraton times and resolutions due to two simultaneously occurring effects: (1) the concentration of diastereomeric associates increased with elevating the β-CD concentration, so a significant difference in the resolutions of the enantiomers was obtained; (2) the buffer viscosity was increased upon increasing the β-CD concentration, which reduced the electrophoretic mobolities of (39) Li, S.; Purdy, W. C. Chem. Rev. 1992, 92, 1457. (40) Sioni, H.; Riekkola, M. L.; Novotny, M. V. J. Chromatogr. 1992, 608, 265. (41) Rawjee, Y. Y.; Vigh, G. Anal. Chem. 1994, 66, 619. (42) Gahm, K.; Stalcup, A. M. Anal. Chem. 1995, 67, 19.
the all analytes, hence, smaller mobility gave longer times for enantiomer-chiral additive discrimination. According to a simple model based on the host-guest complexation for optimization of chiral additive concentration proposed by Wren and Rowe,43 the optimum concentration of the chiral additive relies on the magnitude of the association constants. Generally, the greater the association constants are, the lower the optimum concentration will be. On the other hand, the resolution will be reduced when the concentration is extremely low or high. Nevertheless, in this study, the observed resolutions monotonically increased with higher β-CD concentration, indicating that the β-CD concentration did not reach the optimal value. Yet a further increase in the β-CD concentration could not be realized due to β-CD’s low solubillity in water (∼16.5 mM). Thus, we chose 12 mM β-CD to perform our further research. The effect of NaOH concentration on migration time and resolution was more complicated because native β-CD in strongly alkaline solutions is partially deprotonated. Therefore, the NaOH concentration influenced not only the electroosmotic flow but also the percentage of the negatively charged β-CD. In this study, only the anionic diastereomeric complexes could be resolved, since they migrated by their own electrophoretic mobilities; therefore, the enantioseparations were dependent on the amount of the negatively charged β-CD. Elevating the NaOH concentration increased the amount of the charged β-CD, which enhanced the enantioseparations. However, an opposite effect occurred simultaneously: both the electroosmotic flow and the electrophoretic current increased. The former reduced the migration times of the analytes, and the latter generated Joule heating that gave rise to the peak broadening. Therefore, the proper NaOH concentraiton was compromised between the two opposite effects. Table 2 illustrates that the optimal enantioseparations for ANP and DANP were obtained when the NaOH concentration was around 0.10 M. The applied voltage also significantly affected the migration times and resolutions. According to the description of the (43) Wren, S. A. C.; Rowe, R. C. J. Chromatogr. 1992, 603, 235.
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Table 3. Effect of Applied Voltage on Migration Times and Resolution for Enantiomersa 14 kV
a
20 kV
26 kV
molecule
t
Rs
t
Rs
t
Rs
ANP DANP
30.3 ( 2.7 31.7 ( 2.5
3.1 ( 0.4 3.9 ( 0.5
17.3 ( 1.3 18.2 ( 1.5
2.0 ( 0.3 2.2 ( 0.4
11.9 ( 0.6 12.2 ( 0.6
1.2 ( 0.2 1.4 ( 0.3
Conditions: buffer, 0.10 M NaOH solution with 12 mM β-CD; other conditions as in Figure 3. See Table 1 for definitions of t and Rs.
Table 4. Data of Linear Regression and Accuracy Testa
range of L enantiomersb (%)
regression eqn,c y ) a + bx
ANP
1.0-10.0
DANP
1.0-10.0
a ) -0.08 ( 0.01 b ) 29.94 ( 0.86 a ) -0.11 ( 0.02 b ) 32.62 ( 1.03
molecule
correl coeff 0.9987 0.9990
amount of L enantiomersb (%) added determined 2.0 8.0 2.0 8.0
recovery (%)
2.1 8.3 1.9 8.2
105.0 103.8 95.0 102.5
a Conditions are as described in Figure 4. b The percentages of the L enantiomers in their optical isomers. c The regression equations (n ) 6) are expressed in the form of y ) a + bx, where x and y are the peak current (in, nanoamperes) and the percentage of L-enantiomers, respectively. The regression test was repeated three times.
resolution between adjacent analytes given by Jorgonson and Lukacs,44
Rs ) 0.177∆µep[V/D(µep (1,2) + µeo)]1/2 where Rs is resolution, µep(1,2) is the average electrophoretic mobility, µeo is the electoosmotic mobility, D is the diffusion coefficient, ∆µep is the electrophoretic mobility difference, and V is the applied separation voltage. From this equation, it is clear that the resolution is directly proportional to the square root of the applied voltage; hence, increasing the applied voltage will improve the resolution. Nevertheless, a disadvantage of the increase in the applied voltage is that Joule heating will increase concomitantly, especially when high-conductivity buffers are employed. Therefore, below a certain range of the applied voltage, the resolution is intially enhanced with an increase in the applied voltage and reaches a maximum, and then it declines with further increase in the applied voltage, since Joule heating effects become severe above this voltage. Under our experimental conditions, because the electrophoresis was performed in the 0.10 M NaOH solution, the electrophoretic current was relatively large, ∼43 µA at a field strength of 0.25 kV/cm. Therefore, the capillary produced more Joule heating than it could dissipate, and Joule heating has significant effects on the resolution. The results in Table 3 show that a decrease in the applied voltage enhanced the resolution but at the expense of longer analysis time. To obtain satisfying separations and prevent long time consumption, 20 kV was selected as the optimal separation voltage. Application. We applied the method to determine the enantiomeric purity of ANP and DANP. ANP is the last prodrug of chloramphenicol, and DANP is a relatively new chiral reagent, the D enantiomers of which are both efficient matrixes.45,46 The (44) Jorgenson, J. W.; Lukacus, K. D. Science 1983, 222, 266. (45) Explanatory Notes of the Pharmacopoeia of the People’s Republic of China; Chemical Industry Press: Beijing, 1993; Part 2, p 833.
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Figure 6. Electropherograms of two samples from laboratory synthetic batches of (A) ANP and (B) DANP in 0.10 M NaOH solution containing 12 mM β-CD: (a) D-ANP, (b) L-ANP, (c) D-DANP, (d) L-DANP. The concentrations of ANP and DANP were 0.5 and 1.0 mM, respectively. Conditions are as in Figure 3.
electropherograms of ANP and DANP samples from laboratory synthetic batches are depicted in Figure 6A and B, respectively, which clearly show that the two samples were both D enantiomers containing a small amount of L enantiomers. To quantitatively determine the purities of D-ANP and D-DANP, i.e., the percentages of L-ANP and L-DANP in optical isomers, we prepared two series of standard solutions with fixed concentrations of the two compounds and variable percentages of the two L enantiomers and performed CE-ED under the optimal conditions to obtain two series of calibration points. The concentrations of the two compounds were 0.5 mM for ANP and 1.0 mM for DANP, and the percentages of the two L enantiomers were both in the range 1.0-10.0% in their corresponding optical isomers. Linear regression analysis, plotting the percentages of L enantiomers (y) versus the corresponding peak current (x), gave equations with which the quantitative determinations were acchieved. The data (46) Tang, C.; Wu, G. Chin. J. Chem. Reagents 1988, 10, 104.
from the linear regression and accuracy test are listed in Table 4, which indicate that this method was practicable and valid. The determined percentages of L-ANP and L-DANP after three injections were 4.1 ( 0.3% and 5.4 ( 0.4%, respectively. The results were quite satisfactory. CONCLUSIONS The combination of capillary electrophoresis with electrochemical detection was potentially used for the chiral separation of optical isomers. The wall-jet electrochemical detection, permitting the use of a normal-size electrode, tended to fit for chiral CE-ED applications. Native β-CD as anionic chiral additive in strong alkaline solutions is very inexpensive and practical for use
in the enantioseparations of ANP and DANP. Future work in this laboratory will focus on exploring other chiral additives and separation modes for chiral CE-ED applications. ACKNOWLEDGMENT The authors are grateful for partial financial support received from the Laboratory of the Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Academia Sinica.
Received for review July 29, 1997. Accepted March 25, 1998. AC970796T
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