Anal. Chem. 1998, 70, 4563-4569
Simultaneous Separation of Acidic, Basic, and Neutral Organic Compounds, Including Strong and Moderate Acids and Bases, by Capillary Electrochromatography Ira S. Lurie,* Timothy S. Conver, and Valerie L. Ford
Special Testing and Research Laboratory, U.S. Drug Enforcement Administration, 7704 Old Springhouse Road, McLean, Virginia 22102-3494
The separation of strongly basic, moderately basic, weakly basic, strongly acidic, moderately acidic, weakly acidic, and neutral compounds in a single run using capillary electrochromatography (CEC) is presented. This is accomplished using a 3-µm CEC Hypersil C8 capillary with high organic content acetonitrile/phosphate (pH 2.5) mobile phases containing hexylamine. Fifteen basic, acidic, and neutral drugs of forensic interest are resolved using a step gradient. Strong and moderately basic drugs separate before to, apparently by a combination of free zone electrophoresis (CZE) and chromatographic phenomena. Weak bases separate after to, also by a combination of CZE and chromatographic processes. Due to large selectivity differences between CEC and CZE for bases, there is evidence that the stationary phase is playing a significant role in the separation of these solutes. The CEC approach presented offers unique selectivity, expanded peak capacity, and the ability to solubilize both hydrophilic and hydrophobic solutes in an injection solvent that is compatible with the chromatographic system. Capillary electrochromatography (CEC), which combines the best features of CE (i.e., separation efficiency) with the best features of HPLC (i.e., well-characterized retention and selectivity mechanisms, ability to handle thermally labile solutes and highly polar compounds, and increased sample capacity) has recently generated much interest.1-3 However, a recent survey of leading practitioners in CEC indicated that one of the major drawbacks of the technique is its limited ability to separate strong bases and its inability to separate strongly basic, strongly acidic, and neutral compounds in a single run.4 In order for CEC to fully realize its potential for the separation of small molecules, it must be applicable to a wide range of solutes, including weak, moderate, and strong bases, neutrals, and weak, moderate, and strong acids. This requirement is particularly significant in the pharmaceutical, (1) Dittman, M. M.; Rozing, G. P. J. Chromatogr., A 1996, 744, 63-74. (2) Dittman, M. M.; Wienand, K.; Bek, F.; Rozing, G. P. LC-GC 1995, 13, 800814. (3) Colo´n, L. A.; Guo, Y.; Fermier, A. Anal. Chem. 1997, 15, 4461A-467A. (4) Majors, R. E. LC-GC 1998, 16, 96-110. 10.1021/ac9804543 Not subject to U.S. Copyright. Publ. 1998 Am. Chem. Soc.
Published on Web 09/30/1998
clinical, and forensic fields, where all these different types of components can be present in a single sample. Some preliminary work with strong and moderate bases has been reported. Smith separated tricyclic antidepressants via CEC using a strong cation-exchange stationary phase.5 However, although extremely narrow peaks were obtained for these compounds in certain runs (presumably due to some kind of focusing effect), broad as well as multiple peaks were also observed for these solutes.6 Very recently, the use of bare silica with buffered acetonitrile/water mobile phases has been reported for the CEC separation of basic drugs.7 The separation mechanism in this latter study is presumably a mixture of cation exchange and normal phase. This article describes the simultaneous CEC separation of strongly basic, moderately basic, weakly basic, neutral, weakly acidic, moderately acidic, and strongly acidic compounds in a single run. To our knowledge, this is the first report of the successful CEC separation of this range of solutes. In addition, the CEC separation of strong and moderately basic drugs using a C8 bonded phase column at low pH is described. EXPERIMENTAL SECTION Instrumentation. A Hewlett-Packard Model HP3DCE capillary electrophoresis system (Waldbronn, Germany) was used for all CEC and CZE experiments. A Beckman Pace 5500 capillary electrophoresis system (Fullerton, CA) was used for all micellar electrokinetic capillary chromatography (MECC) experiments. Finally, a Hewlett-Packard model 1100 was employed for all HPLC experiments. For CEC, a 100 µm i.d./350 µm o.d. column with a packed bed length of 25 cm (CEC Hypersil C8, 3 µm) was obtained from Hewlett-Packard. For all separations, the total column length was the packed bed length plus 8.5 cm of polyimide-coated fused-silica tubing. The column was conditioned with mobile phase by first pressurizing the inlet at 10 bar and ramping the voltage to 25 kV (5) Smith, N. W.; Evans, M. B. Chromatographia 1995, 41, 197-203. (6) Smith, N. W. Eleventh International Symposium on High Performance Capillary Electrophoresis and Related Microscale Techniques, Orlando, FL, February 1-5, 1998; oral presentation. (7) Wei, W.; Luo, G.; Yan, C. Eleventh International Symposium on High Performance Capillary Electrophoresis and Related Microscale Techniques, Orlando, FL, February 1-5, 1998; poster presentation.
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Figure 1. Effect of hexylamine on CEC of basic compounds. Conditions: acetonitrile/25 mM phosphate buffer pH 2.5 (75:25) with voltage 25 kV and temperature 20 °C. A CEC Hypersil C8, 3 µm (100 µm × 34 cm) (25 cm length to detector) column is used. Other conditions described in Experimental Section. The hexylamine concentrations are (A) 0 (only heroin injected), (B) 1, and (C) 2 µL/mL, respectively. Compound key: (a) amphetamine; (b) methamphetamine; (c) procaine; (d) cocaine; (e) heroin; (f) quinine; (g) noscapine; (h) thiourea.
over a 30-min period. Both the inlet and outlet were pressurized at 10 bar and the voltage was maintained at 25 kV for another 30 min. Changing mobile phases was also accomplished electroosmotically with pressurization of the inlet and outlet to 10 bar. For CZE, a 33.5 cm (25 cm to detection window) × 50 µm i.d./350 µm o.d. uncoated fused-silica capillary was obtained from Polymicro Technologies (Phoenix, AZ). The capillary was conditioned with 1 N sodium hydroxide for 10 min, followed by water for 10 min, and finally run buffer for 30 min. For MECC, a 57 cm (50 cm to detection window) × 50 µm i.d./350 µm o.d. uncoated fused-silica capillary (Polymicro Technologies) was used. The capillary was conditioned as above for CZE. For HPLC, a 12.5 cm × 4.0 mm i.d. HPLC column (ODS Hypersil, 5 µm) was obtained from Hewlett-Packard. The column was conditioned with starting mobile phase for 30 minutes. Reagents. Sodium dodecyl sulfate (SDS) obtained from Mallinckrodt (Paris, KY) was used as received. Sodium phosphate (monobasic), sodium phosphate (dibasic), phosphoric acid, hexylamine, and sodium hydroxide were reagent grade. Deionized water was obtained from a Millipore Milli-Q water system (Bedford, MA). LC grade acetonitrile was used. All drug standards except for cannabinol (CBN) and ∆9-tetrahydrocannabinolic acid (∆9-THCA-A) were obtained from the reference collection of the Special Testing and Research Laboratory (McLean, VA). CBN was acquired from RTI (Research Triangle, NC), while ∆9-THCA-A was obtained from the Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi (University, MS). The solutions used for both CEC mobile phases and CZE run buffers were prepared by combining 25 mM monobasic phosphate and hexylamine so that the final amine concentration after the addition of acetonitrile was either 1 or 2 µL/mL. The phosphate/ 4564 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998
hexylamine buffer was then adjusted to pH 2.5-2.6 using phosphoric acid. The MECC run buffer was prepared by combining 85 parts of 50 mM SDS/20 mM dibasic phosphate buffer pH 8.5 with 15 parts acetonitrile. The HPLC mobile phases were mixed internally from solvent reservoirs containing acetonitrile and phosphate buffer with 3.4 mL/L hexylamine at pH 2.0. The phosphate buffer consisted of a mixture of 3480 mL of water, 120 mL of 2 M sodium hydroxide, and 40 mL of phosphoric acid. Procedures. For CEC and CZE, standard compounds were dissolved in either mobile phase or run buffer at a concentration between 0.3 and 0.5 mg/mL prior to 3-s electrokinetic injections at 5.0 kV. For MECC, standard compounds were dissolved in methanol at a concentration between 0.3 and 1.0 mg/mL prior to 1-s pressure injections at 0.5 psi. For HPLC, standard solutes were dissolved in 1 part mobile phase buffer and 1 part acetonitrile at a concentration between 0.3 and 0.5 mg/mL prior to 5-µL injections. RESULTS AND DISCUSSION Effect of Hexylamine on CEC of Strong and Moderate Organic Bases. Strongly and weakly acidic organic compounds (e.g., cannabinoids) have been previously separated using a C8 column with a mobile phase containing acetonitrile and phosphate buffer at pH 2.5.8 This system is also viable for weakly basic and neutral organic solutes, which are un-ionized at this pH. These CEC conditions were therefore investigated for strongly and moderately basic organic solutes. However, heroin (a moderately basic solute (pKa 7.6)9) exhibited very poor chromatographic (8) Lurie, I. S.; Meyers, R. E.; Conver, T. S. Anal. Chem. 1998, 70, 32553260.
Figure 2. Effect of percent acetonitrile on CEC of basic compounds. Conditions identical to Figure 1C, except for percent acetonitrile (ACN) and voltage (V). Compound key: as per Figure 1.
performance, with multiple peaks being obtained (see Figure 1A). Adsorption of heroin onto the unbonded silanol groups is presumably contributing to this phenomenon. Hexylamine has been previously used in HPLC as a modifier to minimize tailing of basic solutes.10,11 This additive minimizes silanophilic interactions by competing with the solutes for the unbonded silanol groups. A major concern for its use in CEC would be the minimization and/or reversal of osmotic flow. However, as shown in Figure 1B and C, appreciable osmotic flow still exists after the addition of hexylamine, as evidenced by the time of the neutral marker (to); the retention time of thiourea only increased from 5.2 to 7.7 min after the addition of 2 µL/mL hexylamine. In addition, as shown in Figure 1C, improved chromatographic performance was also obtained for the individual moderate and strong bases such as amphetamine (pKa 9.9),9 methamphetamine (pKa 10.1),9 procaine (pKa 9.0),9 cocaine (pKa 8.6),9 heroin (pKa 7.6),9 quinine (pKa 4.1, pKa 8.5),9 and noscapine (pKa 6.2).9 These solutes all migrated before to, indicating that electrophoresis is playing a major role in the separation process. It has been postulated that the CEC separation process for charged solutes would include CZE;3 these results confirm that postulation. As shown in Figure 1B, lowering the hexylamine concentration from 2 to 1 µL/mL resulted in peak splitting for certain solutes (cf. amphetamine, procaine, and cocaine). However, higher concentrations of hexylamine would result in lower osmotic flow, higher current and increased risk of bubble formation, and possible band spreading due to Joule heating. Therefore, 2 µL/ mL appears to be the optimal concentration. Effect of Acetonitrile Concentration on CEC of Strong and Moderate Organic Bases. The effect of acetonitrile concentra(9) Moffat, A. C., Ed. Clarke’s Isolation and Identification of Drugs, 2nd ed.; The Pharmaceutical Press: London, 1986. (10) Gill, R.; Alexander, S. P.; Moffat, A. C. J. Chromatogr. 1982, 247, 15-37. (11) Lurie, I. S.; Carr, S. M. J. Liq. Chromatogr. 1983, 6, 1617-1630.
tion on the CEC of strong and moderate bases is shown in Figure 2. Lower separation voltages were used at lower acetonitrile concentrations in order to operate at smaller currents. In general, lowering the amount of acetonitrile in the mobile phase increases resolution and alters selectivity, especially at 30% acetonitrile. Except for quinine, the elution order remains the same with changes in acetonitrile concentration. As the acetonitrile concentration is increased, the relative hydronium ion concentration is lowered, leading to an increase in apparent pH. In addition, the pKa of bases is lowered with increasing acetonitrile concentration. The phenomena for quinine can be explained by considering that the apparent pH of the mobile phase is near its first pKa. It is less likely that selectivity effects for the other basic drugs can be explained by pH and pKa considerations. These solutes all have pKa values greater than 6.2, and in addition, acetonitrile (a relatively nonbasic solvent) would not be expected to have such a large effect on lowering the pKa. At all acetonitrile concentrations, the solutes eluted before to. The time of the neutral marker (thiourea) significantly increased with a decrease in acetonitrile concentration (this effect will be discussed in more detail later in the paper). As shown in Figure 2, the best separation of the bases in terms of resolution and speed of analysis was obtained at 60% acetonitrile. Good plate counts of 82 000, 85 000, 101 000, 110 100, and 130 000 plates/m were obtained for heroin, cocaine, procaine, noscapine, and amphetamine, respectively, under these separation conditions. Methamphetamine and quinine (which both exhibit significant tailing) have plate counts of 34 000 and 42 000, respectively. These results compare quite favorably with the 14 000 plates/m obtained for heroin via HPLC using a 3-µm C18 column with a similar mobile phase.11 It is noted that this stationary phase might be expected to have less unbonded silanol groups than the one used for CEC, since it is end capped12 (unlike (12) Lurie, I. S.; Allen, A. C. J. Chromatogr. 1984, 317, 427-442.
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Figure 3. Effect of percent acetonitrile on CZE of basic compounds. Conditions identical to Figure 2 except a 50 µm × 33.5 cm (25 cm length to detector) uncoated fused-silica capillary was used. Compound key: as per Figure 1.
the CEC phase).1 For CEC, no general trends were observed for the effect of acetonitrile concentration on plate height, except that the highest efficiencies were obtained at 60% acetonitrile. The effects shown in Figure 2 could represent a combination of electrophoretic and chromatographic phenomena. Electrophoresis would involve a CZE mechanism while chromatography considerations include hydrophobic and silanophilic interactions. Hydrophobic interactions involve the mobile phase and the C8 ligands, while the silanophilic interactions include the mobile phase and unbonded silanol groups. The peak tailing observed for the basic solutes indicates the latter interaction is occurring. Comparison of CZE and CEC. To gain some insight into the mechanism of the CEC separation of moderate and strong bases, CZE was performed using conditions identical to those used for CEC, except that a 50-µm-i.d. open-tubular fused-silica capillary was used. As shown in Figure 3, major changes in selectivity were obtained for the various solutes at all acetonitrile concentrations vs CEC (cf. Figures 2 and 3). These selectivity differences include changes in separation order, especially at 75% acetonitrile. For CZE, the largest change in migration relative to other solutes occurs for quinine. Again this phenomenon can be explained by considering that the apparent pH of the mobile phase is near the first pKa of quinine. However, other significant selectivity effects, such as the reversal in order of amphetamine and methamphetamine, and heroin and noscapine, cannot be explained by pKa considerations. Acetonitrile not having a large effect on lowering the pKa is confirmed by the fact that the weak bases diazepam (pKa 3.3)9 and methaqualone (pKa 2.5)9 are clearly charged at all concentrations of organic modifier, since they migrate before to in all cases. Therefore, it appears that processes other than free zone electrophoresis are contributing to the CZE separations. These other selectivity effects could be explained by silanophilic and/or hydrophobic interactions with the silanol groups on the capillary surface. Although the non-Gaussian peaks are for the 4566 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998
most part triangular in shape (which indicates that electrodispersion is occurring), there is also some evidence of tailing. This latter phenomenon could indicate that adsorption onto the unbonded silanol groups is occurring. Hydrophobic interactions could also be occurring between the epoxide moiety of fused silica and the hydrophobic portion of a solute. These effects are minimized by the relatively large amount of acetonitrile present in the run buffer. The large selectivity differences between CEC and CZE indicates that the packed bed is playing a significant role in the separation process in CEC. The major differences in selectivity that occur between CZE and CEC could be explained by larger silanophilic and/or hydrophobic interactions in CEC due to the much larger surface area of the packed capillary. In agreement with a previous study,13 the CZE electroosmotic flow (EOF) at a given acetonitrile concentration is 2-3 times higher than the CEC EOF. This effect is not explained by the relative amount of silanol groups present in both CE columns. It had been previously shown that the EOF is higher for CZE than CEC when the packing material was underivatized silica.14 The magnitude of the EOF decreases because of nonalignment of the flow channels in the packing material with the capillary axis and the lack of electrodrive inside the particle pores.15 At all acetonitrile concentrations, significantly higher plate counts were obtained using CZE vs CEC. In general, the difference in efficiencies between the two techniques increased with an increase in acetonitrile concentration. The increase in efficiency of CZE over CEC ranged from ∼20 times at 75% acetonitrile to ∼7 times at 30% acetonitrile. Despite the lower plate counts for the latter technique, however, the overall resolution for CEC at a given acetonitrile concentration was equal or (13) Choudhary, G.; Horvath, C. J. Chromatogr., A 1997, 781, 161-183. (14) Govindaraju, K.; Ahmed, A.; Lloyd, D. K. J. Chromatogr., A 1997, 768, 3-8. (15) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317-328.
Figure 4. CEC step gradient of basic, neutral, and acidic compounds. Initial conditions: (for first minute) acetonitrile/25 mM phosphate buffer pH 2.5 (60:40) with 2 µL/mL hexylamine. Final conditions: acetonitrile/25 mM phosphate buffer pH 2.5 (75:25) with 2 µL/mL hexylamine. A voltage of 25 kV and temperature of 20 °C was used. Other conditions described in Experimental Section. Compound key: Same as Figure 1 except (i) phenolbarbital, (j) diazepam, (l) testosterone, (m) cannabinol, (n) testosterone propionate, (o) ∆9-tetrahydrocannabinol, and (p) ∆9tetrahydrocannabinolic acid.
better than that obtained via CZE. This is directly attributable to the increase in selectivity that is obtained by combining a free zone mechanism with enhanced chromatographic phenomena obtained by using a packed bed. As expected, in CZE the solutes eluted before to at all acetonitrile concentrations. The time of the neutral marker (thiourea) significantly increased with a decrease in acetonitrile concentration. For both CEC and CZE, the µeo increased with increasing acetonitrile concentration. These results can be attributed to decreases in viscosity16 and ionic strength at higher acetonitrile concentrations. The relative conductivity (calculated from Ohm’s law from CE measurements) and therefore ionic strength decreased with increasing acetonitrile concentration for both CEC and CZE. Again as the acetonitrile concentration is increased, the relative hydronium ion concentration is lowered, leading to an increase in apparent pH and an increase in ionization of silanol groups, which increases the µeo. There are some conflicting data in the literature on the effect of acetonitrile concentration on µeo. Some previous CEC studies performed at both decreasing ionic strength1,17 and constant ionic strength13,18 also showed that µeo increases with increased acetonitrile concentration. However, other investigations showed a decrease in µeo with increasing acetonitrile concentration and decreasing ionic strength.19,20 Similar CZE studies performed at both decreasing ionic strength13,21 and constant ionic strength13,16 (16) Schwer, C.; Kenndler, E. Anal. Chem. 1991, 63, 1801-1807. (17) Lelie′vre, F.; Yan, C.; Zare, R. N.; Gareil, P. J. Chromatogr., A 1996, 723, 145-156. (18) van den Bosch, S. E.; Heemstra, S; Kraak, J. C.; Poppe, H. J. Chromatogr., A 1996, 755, 165-177. (19) Yamamoto, H.; Baumann, J.; Erni, F. J. Chromatogr. 1992, 593, 313-319. (20) Yan, C.; Schaufelberger, D.; Erni, F. J. Chromatogr., A 1994, 670, 15-23. (21) Wright, P. B.; Lister, A. S.; Dorsey, J. G. Anal. Chem. 1997, 69, 3251-3259.
conditions found (in contrast to our findings) that the µeo decreased with increasing acetonitrile concentration. It was also found that pure acetonitrile/water mobile phases’ µeo exhibited varying behavior with increases in acetonitrile concentration.21 However, between 20 and 60% acetonitrile, µeo increased with increasing acetonitrile concentration. The reasons for these discrepancies are not clear; however, our studies were performed at a buffer pH of ∼2.5 vs >6 for these other investigations. CEC of Basic, Neutral, and Acidic compounds. Chromatographic conditions similar to those described in Figure 1C were also viable for the analysis of weak bases, weak acids, strong acids, and neutral solutes. The simultaneous separation of the strong bases (amphetamine, methamphetamine, procaine), moderate bases (cocaine, heroin, noscapine, quinine), weak bases (diazepam, methaqualone), strong acids (∆9-tetrahydrocannabinolic acid (pKa1′ ∼3.0, pKa2′ ∼13.4)9 (pKa values were derived from the structurally similar salicylic acid), moderate acids (phenobarbital (pKa 7.4)9), weak acids (∆9-tetrahydrocannabinol (pKa 10.6)9, cannabinol (pKa ∼10.6)9), and neutral solutes (testosterone, testosterone propionate) in a single run is shown in Figure 4. The excellent separation of all 15 compounds was accomplished using a step gradient. This separation combines the increased resolution of the moderate and strong bases at a lower acetonitrile concentration with the good resolution and higher speed of analysis of all the other solutes at a higher acetonitile concentration. As indicated in Figure 4, all solutes except for the strong and moderate bases elute after to, indicating that chromatography is playing a major role in the separation process for the weakly basic, acidic, and neutral solutes. This is not surprising, since unlike the moderate and strong bases (which exist as cations at pH 2.2), these other solutes are either uncharged or partially charged at this pH. Analytical Chemistry, Vol. 70, No. 21, November 1, 1998
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Figure 5. MECC of basic, neutral, and acidic compounds. Acetonitrile/50 mM SDS/20 mM phosphate buffer pH 8.5 (15:85) with voltage of 25 kV and temperature 20 °C. An uncoated fused-silica capillary (50 µm × 57 cm) (50 cm length to detector)) column is used. Other conditions described in Experimental Section. Compound key: as per Figure 4.
It is interesting that the weak bases diazepam and methaqualone migrate just before to by CZE, with an elution order opposite to that obtained by CEC. These solutes elute just after to by CEC, indicating that they are separating by a combination of CZE and chromatographic phenomena. Comparison of CEC with MECC. MECC was also performed on the same mixture of solutes investigated by CEC (see Figure 5). A comparison between CEC and MECC (cf. Figures 4 and 5) reveals major differences in selectivity between the two techniques. Unlike CEC, where moderate and strong bases elute before to, these solutes elute after to (for the most part as a group) via MECC, and (in this case) following the elution of phenobarbital and methaqualone. In addition, the elution order of the moderate and strong bases is vastly different for both techniques. Similar to CEC, MECC could involve a combination of electrophoretic and chromatographic phenomena (which, for MECC, involves partitioning into the micelle and possible ionic interactions with the micelle.) Due to differences in the pH values between the buffers used in both techniques (pH 2.5 vs pH 8.5), the degree of
ionization of the solutes could be different in CEC vs MECC. Changes in pH, partitioning into the micelle, and ion pairing between basic solutes and the micelle all contribute to the differences in selectivity between the two techniques. The weak bases methaqualone and diazepam also exhibit large selectivity differences between the two techniques. This is not surprising, considering that these solutes are un-ionized in MECC (unlike CEC.) The weak acids ∆9-tetrahydrocannabinol and cannabinol, and the neutral compounds testosterone and testosterone propionate, all exhibited similar selectivity in CEC vs MECC; this is because they are all uncharged in both techniques. This would indicate that the hydrophobic interactions occurring between the mobile phase and C8 ligands by CEC are similar to the hydrophobic interactions between the run buffer and the micelle that occur by MECC. The weak acid phenobarbital and the strong acid ∆9tetrahydrocannabinolic acid, which are un-ionized via CEC and ionized using MECC, exhibit different selectivities by both techniques. A contributing factor is the repulsion of the negatively charged species by the negatively charged micelle. Due to the large selectivity differences between the two techniques, CEC and MECC are complementary for the separation of acidic, neutral, and basic drugs. The use of multiple techniques is clearly helpful for drug screening. One advantage CEC has over MECC is an infinite elution range; for MECC, the elution range is the time of the micelle (tmc) divided by to. It is not uncommon in MECC for multiple solutes to elute at the tmc, especially nonpolar solutes. The hydrophobic compounds ∆9-tetrahydrocannabinol and cannabinol, which are well separated by CEC, coelute via MECC either because of limited elution range or by being fully incorporated into the micelle. In the presence of an organic modifier, it is difficult to measure tmc. Another advantage of CEC over MECC is the solubility of the solutes in an injection solvent that is compatible with the separation system. For CEC, the injection solvent is the mobile
Figure 6. HPLC gradient of basic, neutral, and acidic compounds. Initial conditions: acetonitrile/phosphate buffer with 3.4 µL/mL hexylamine pH 2.0 (2:98) with 20 min linear ramp. Final conditions: acetonitrile/phosphate buffer with 3.4 µL/mL hexylamine pH 2.0 (65:35) with 10-min hold. Flow rate of 1.5 mL/min with temperature of 20 oC. An HPLC Hypersil ODS, 5 µm (12.5 cm × 4.0 mm i.d.) column is used. Other conditions described in Experimental Section. Compound key: as per Figure 4. 4568 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998
phase (or starting mobile phase for step gradient), which contains a relatively high amount of acetonitrile. To solubilize the more hydrophobic compounds for injection by MECC, it is necessary to dissolve the solutes in methanol. As a result, even at the lowest pressure injection allowed by the instrument, several solutes such as amphetamine, methamphetamine, and cocaine exhibit peak splitting (see Figure 5.) Comparison of CEC with Gradient HPLC. Gradient HPLC was performed on the same solute mixture using chromatographic conditions similar to those employed for CEC (∆9-tetrahydrocannabinolic acid elutes only if the hold at the end of the gradient is extended) (see Figure 6). As shown in Figures 4 and 6, major differences in selectivities were obtained between the two techniques for the strong and moderate bases. In comparison to CEC, where these solutes eluted before to, these compounds eluted after to in HPLC. Electrophoresis plays a major role in the former technique, while chromatographic processes dominate in HPLC. Silanophilic interactions may be playing a larger role in CEC than HPLC, since the former phase has a larger amount of unbonded silanol groups.1 Weak bases, weak acids, a moderate acid, a strong acid, and neutral solutes all exhibited similar selectivity via CEC and HPLC (cf. Figures 4 and 6). Except for diazepam and methaqualone, which have pKa’s near the pH of the mobilephase buffer, the other solutes are uncharged at pH 2.0. This would indicate that hydrophobic interactions between the mobile phase and the C8 ligands are dominant in both techniques for these uncharged solutes. It is also of interest that these additional solutes elute later relative to to using HPLC vs CEC. Due to the significant selectivity differences between the two techniques, CEC and HPLC are complementary for the separation of acidic, neutral, and basic drugs. Again, the use of multiple techniques are useful for drug screening. (22) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; John Wiley & Sons, Inc.: New York, 1979; Chapter 7.
Unlike HPLC, where a full gradient is employed, only a step gradient is required for the separation of acidic, neutral, and basic solutes in CEC. This is a direct consequence of using a mobile phase with a high organic content coupled with free zone electrophoresis for basic solutes. The extra peaks present in the HPLC chromatogram are primarily due to impurities present in the hexylamine. It is of interest to note that the CEC run is devoid of these extraneous peaks. These impurities are soluble at the higher acetonitrile concentrations used in CEC and thus remain constant during the run. Due to solubility considerations, it was necessary to dissolve the mixture of solutes in a mobile phase than is stronger than the starting mobile phase for the gradient HPLC run. This is often required in gradient HPLC, especially when chromatographing a mixture of hydrophilic and hydrophobic compounds. To avoid adverse effects such as peak distortion and loss in resolution (especially of the early eluting bands), the amount of sample that can be injected is limited to small volumes (i.e.,
