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An ion-pair principle for enantioseparations of basic analytes by nonaqueous capillary electrophoresis using the di-n-butyl l -tartrate–boric acid complex as chiral selector
Journal of Chromatography A, 1284 (2013) 188–193
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
An ion-pair principle for enantioseparations of basic analytes by nonaqueous capillary electrophoresis using the di-n-butyl l-tartrate–boric acid complex as chiral selector Li-Juan Wang a,b , Xiu-Feng Liu b , Qie-Nan Lu b , Geng-Liang Yang b , Xing-Guo Chen a,c,∗ a
National Key Laboratory of Applied Organic Chemistry, Department of Chemistry, Lanzhou University, Lanzhou 730000, China Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmacy, Hebei University, Baoding 071002, China c Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, Lanzhou 730000, China b
a r t i c l e
i n f o
Article history: Received 8 November 2012 Received in revised form 16 January 2013 Accepted 4 February 2013 Available online 10 February 2013 Keywords: Chiral recognition mechanism Chiral ion-pair selector Di-n-butyl l-tartrate–boric acid complex Basic electrolyte Nonaqueous capillary electrophoresis
a b s t r a c t A chiral recognition mechanism of ion-pair principle has been proposed in this study. It rationalized the enantioseparations of some basic analytes using the complex of di-n-butyl l-tartrate and boric acid as the chiral selector in methanolic background electrolytes (BGEs) by nonaqueous capillary electrophoresis (NACE). An approach of mass spectrometer (MS) directly confirmed that triethylamine promoted the formation of negatively charged di-n-butyl l-tartrate–boric acid complex chiral counter ion with a complex ratio of 2:1. And the negatively charged counter ion was the real chiral selector in the ion-pair principle enantioseparations. It was assumed that triethylamine should play its role by adjusting the apparent acidity (pH*) of the running buffer to a higher value. Consequently, the effects of various basic electrolytes including inorganic and organic ones on the enantioseparations in NACE were investigated. The results showed that most of the basic electrolytes tested were favorable for the enantioseparations of basic analytes using di-n-butyl l-tartrate–boric acid complex as the chiral ion-pair selector. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Capillary electrophoresis (CE) represents one of the major techniques for analytical scale enantioseparations [1–10]. Nonaqueous capillary electrophoresis (NACE) is an interesting alternative to improve chiral recognition performance [11]. Solvent parameters of nonaquous solutions such as the relative permittivity, viscosity, polarity and autoprotolysis, have offered new possibilities for changes in chiral selectivity in NACE [12–17]. It has also been extended to the applications of chiral analytes or selectors with non or poor solubility in aqueous CE [18,19]. In addition, due to the special physicochemical properties of the nonaqueous solution, in terms of volatility and surface tension, an effective sample introduction to the mass spectrometer (MS) detector can be expected to further extend the use of NACE [5,15,20,21]. The ion-pair principle is one of the important chiral recognition mechanisms for the separation of charged, chiral analytes in NACE. As nonaqueous solvents with low relative permittivities provide a better environment for achieving ion-pair interactions than water,
∗ Corresponding author at: Department of Chemistry, Lanzhou University, Lanzhou 730000, Gansu, China. Tel.: +86 931 8912763; fax: +86 931 8912582. E-mail address: [email protected] (X.-G. Chen). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.02.006
the ion-pair formation as a principle for chiral separation in nonaqueous media should be optimal [22]. As reported in our previous works, various dialkyltartrate–boric acid complexes showed good chiral recognition abilities for some structurally similar basic analytes in methanolic background electrolytes (BGEs) [23,24]. The chiral recognition mechanism was assumed to be ion-pair principle. Triethylamine, a basic electrolyte, was quite useful for the formation of negatively charged dialkyltartrate–boric acid complex chiral counter ion, which was the real chiral selector in the ion-pair principle enantioseparations. However, no direct experimental data has verified this. Additionally, there are several studies that have reported the effect of some other basic electrolytes except for triethylamine in the BGEs in NACE [11,18,25–27]. Whether they could be also useful for the enantioseparations in this study has not been studied. Therefore, one aim of this work was to confirm the formation of negatively charged di-n-butyl l-tartrate–boric acid complex chiral counter ion in the presence of different concentrations of triethylamine. And the complex ratio between di-n-butyl l-tartrate and boric acid should be also determined. Those were carried out by an approach of mass spectrometer (MS). Another aim was to exactly study the effects of different inorganic or organic basic electrolytes on the enantioseparations of basic analytes by NACE using di-nbutyl l-tartrate–boric acid complex as the chiral selector.
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Fig. 1. MS spectra of NACE buffers with different concentrations of triethylamine. Buffer composition: 80 mM di-n-butyl l-tartrate, 100 mM boric acid and 0 mM, pH* 3.82 (A), 7.2 mM, pH* 7.09 (B), 36 mM, pH* 7.93 (C) triethylamine in methanol. MS conditions are shown in Section 2.1.
2. Experimental 2.1. Instrumentation NACE experiments were conducted on a TH-3100 high performance capillary electrophoresis system (Tianhui Institute of Separation Science, Baoding, China), equipped with a thermostatic system and a UV detector. Data were collected with a CXTH-3000 chromatography workstation. Uncoated fused silica capillary of 50 m I.D. (Yongnian Reafine Chromatography Co., Ltd., Hebei, China) with a total length (Ltot ) of 55.0 cm and an effective length
(Leff ) of 45.0 cm was used. The new capillary was conditioned by flushing with methanol for 10 min, 1.0 M NaOH for 20 min, distilled water for 5 min, 1.0 M hydrochloric acid for 20 min and distilled water for 5 min in sequence. Before each run the capillary was rinsed with running buffer for 3 min. Samples were introduced into the capillary tube by positive pressure at 2.9 psi for 2 s. The experiments were performed at 25 ± 0.2 ◦ C. The detection wavelength was set at 214 nm. MS experiments were performed with an Agilent 1100 series LC/MSD Trap XCT (Agilent Technologies, USA) equipped with an electrospray ionization source (ESI). The MS was controlled by
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Table 1 Effect of triethylamine concentration on migration time, effective mobility, enantioselectivity, resolution and efficiency.a Samples
Propranolol Sotalol Atenolol Bisoprolol Bambuterol
7.2 mM triethylamine (pH* 7.09)
36 mM triethylamine (pH* 7.93)
t1
t2
eff1 b
eff2 b
˛eff
Rs
5.607 5.660 6.019 6.139 5.900
5.820 5.808 6.202 6.340 6.253
18.78 18.43 16.26 15.59 16.95
17.43 17.50 15.25 14.52 14.98
1.08 1.08 1.07 1.06 1.05
1.55 0.90 0.99 1.19 1.78
N1 35,205 36,756 36,611 33,215 30,967
N2 23,634 23,529 22,417 17,892 13,894
t1
t2
eff1 c
eff2 c
˛eff
Rs
10.012 9.849 10.421 10.488 9.245
10.544 10.234 10.852 10.990 9.853
9.826 10.16 9.009 8.883 11.53
8.785 9.372 8.223 7.985 10.15
1.12 1.08 1.10 1.11 1.14
2.39 1.98 2.07 2.21 2.78
N1 49,231 48,775 44,424 48,304 51,083
N2 26,573 32,874 27,824 28,981 21,946
a Buffer component in addition to triethylamine is 80 mM di-n-butyl l-tartrate and 100 mM boric acid in methanol. CE conditions: capillary dimensions, Ltot 55.0 cm, Leff 45.0 cm, I.D. 50 m; positive pressure injection for 2 s at 2.9 psi; applied voltage, 20 kV; detection wavelength, 214 nm. b ×10−5 cm2 V−1 s−1 , EOF = 18.01 × 10−5 cm2 V−1 s−1 . c ×10−5 cm2 V−1 s−1 , EOF = 10.78 × 10−5 cm2 V−1 s−1 .
Agilent software A.10.02. The experiments were performed in negative ESI-mode in a mass range of m/z 100–800 with the following ionizer parameters: spray voltage 3000 V, nebulizer gas (N2 ) flow set at 15 psi, dry gas (N2 ) flow 5 L/min with a temperature of 325 ◦ C. A spectral scan range of 100–800 m/z with a maximum accumulation time of 200 ms and an ion charge control (ICC) target setting of 200,000 were applied. The sample was infused with a syringe pump at a flow rate of 3.0 L/min. A pH meter (pHS-3C, Shanghai Precision & Scientific Instrument Co., Ltd., China) equipped with a glass electrode was used to measure the apparent pH (pH*) values of the CE running buffers in NACE. The electrode was calibrated using standard aqueous buffers, pH 4.00, 6.86 and 9.18 at 25 ± 0.2 ◦ C. 2.2. Chemicals and materials Racemic sotalol hydrochloride, esmolol hydrochloride, clenbuterol hydrochloride, cycloclenbuterol hydrochloride, bambuterol hydrochloride, and tulobuterol hydrochloride were purchased from National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). (S)-Propranolol hydrochloride was purchased from Sigma–Aldrich (St. Louis, MO, USA). The following racemic compounds were extracted by methanol from medicine tablets: propranolol hydrochloride (LI® , Tianjin Lisheng Pharmaceuticals Co., Ltd., China), atenolol (YJ® , Beijing Yanjing Pharmaceuticals Co., Ltd., China), bisoprolol fumarate (BOSU® , Wellso Pharmaceuticals Co., Ltd., China), metoprolol tartrate (BETALOC® , AstraZeneca Pharmaceuticals Co., Ltd., China), and terbutaline sulphate (BRICANYL® , AstraZeneca Pharmaceuticals Co., Ltd., China). Di-n-butyl l-tartrate (purity ≥ 98 wt%, water content ≤ 0.5 wt%) was purchased from Acros (New Jersey, USA). Boric acid was the product of Baoding Chemical Reagent Factory (Baoding, China). Triethylamine (water content ≤ 0.2 wt%) and sodium hydroxide (≥98%, pellets, anhydrous) were supplied by Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). Methanol (water content ≤ 0.02 wt%), chromatographic reagent grade, was purchased from Tianjin Concord Technology Co., Ltd. (Tianjin, China). Ammonium hydroxide (≥28 wt% NH3 in water), methylamine (30–33 wt% in absolute ethanol), ethylamine (68.0–72.0 wt% in water) and trimethylamine (30 wt% in water) were all of analytical reagent grade, made in China and used as received. The other reagents and chemicals were all of analytical reagent grade, made in China and used as received. Their water contents were all lower than 0.5 wt%. 2.3. Buffer and sample preparation NACE running buffer solutions were prepared by weighing the desired quantities of boric acid, di-n-butyl l-tartrate, and dissolving them in methanol to the desired volume in a flask. The pH* values were measured and adjusted by adding appropriate concentration of some kind of inorganic or organic bases to the running
buffers. The NACE sample solutions were prepared by dissolving an appropriate quantity of each racemic sample in methanol to a concentration of 0.1 mg/mL. The composition of MS sample solutions was 80 mM di-n-butyl l-tartrate, 100 mM boric acid and the desired quantities of triethylamine in methanol. All of the solutions were filtered through a 0.45 m syringe type filter prior to use. 3. Results and discussion In this study, enantioseparations were evaluated in terms of resolution (Rs ) and selectivity (˛eff ). All of the calculations of performance parameters referred to Ref. [23]. 3.1. The MS verification for the effect of triethylamine concentration in BGEs on the formation of chiral ion-pair selector MS experiments were performed to examine the quantity of din-butyl l-tartrate–boric acid complex and its complex ratio. NACE running buffers with different concentrations of triethylamine in BGEs were directly used as the MS samples. As shown in Fig. 1(A), MS sample without triethylamine obtained a spectrum with four MS peaks at m/z 205.0, 363.1, 419.2, and 475.2, which were consistent with the pseudo-molecular formulas [C8 H14 O6 −H+ ]− , [C12 H17 BO12 −H+ ]− , [C16 H25 BO12 −H+ ]− , and [C20 H33 BO12 −H+ ]− , respectively. And no enantioseparations of basic analytes could be achieved with the same BGE in NACE. Maybe this could be explained from two aspects. On the one hand, the chiral selectors detected here might have no chiral recognition abilities for the analytes tested; on the other hand, maybe there were not sufficient number of chiral selectors in the BGE to achieve good enantioseparations. Obviously different from Fig. 1(A), the MS peaks at m/z 531 were the most intense signals in the spectra of samples containing different concentrations of triethylamine [Fig. 1(B) and (C)]. The MS peak at m/z 531 agreed with the pseudo-molecular formula [C24 H41 BO12 −H+ ]− , with a complex ratio of 2:1 between di-n-butyl l-tartrate and boric acid, and its structural formula was shown in Fig. 2. As shown in Fig. 1(B), when 7.2 mM triethylamine was added to the MS sample, a relatively intense signal of MS peak at m/z 531 appeared in the spectrum. The other new MS peaks at m/z 333.2, 375.0, 433.2, and 563.2 were found to corroborate pseudomolecular formulas of [C14 H27 BO8 −H+ ]− , [C17 H33 BO8 −H+ ]− , [C17 H27 BO12 −H+ ]− , and [C24 H41 BO12 −H+ +CH3 OH]− . Meanwhile, certain levels of enantioseparations for most of the chiral analytes were achieved using the same BGE in NACE, as shown in Table 1. The absolute signal of MS peak at m/z 531 increased with the concentration of triethylamine from 7.2 mM to 36 mM. And the chiral resolutions of analytes also increased with the increase of triethylamine concentration in NACE. As shown in Fig. 1(C), the most intense signal of MS peak at m/z 531 appeared in the spectrum when 36 mM triethylamine was added. The best enantioseparations were also achieved for all of the analytes at that concentration of triethylamine in NACE. When triethylamine
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Fig. 2. Structural formula of pseudo-molecular ion [C24 H41 BO12 −H+ ]− with m/z 531 at MS spectra.
concentration increased from 36 mM to 144 mM, the absolute signal of MS peak at m/z 531 no longer increased. And the higher pH* also decreased the degree of the ionization of basic analytes and weakened their interactions with negatively charged chiral selector, so that the chiral resolutions decreased. Contrast MS and NACE results, it was concluded that the negatively charged di-n-butyl l-tartrate–boric acid complex counter ion with a complex ratio of 2:1 was the real chiral selector in the NACE chiral separation system. The presence of triethylamine in methanolic BGEs promoted the formation and deprotonation of din-butyl l-boric acid complex by adjusting the pH* of the BGEs to a higher value. A certain number of negatively charged chiral counter ions were necessary for the ion-pair principle enantioseparations of basic analytes in NACE [15,28].
Fig. 3. Effect of NH4 OH concentration on NACE chiral resolution. Buffer composition in addition to NH4 OH is 80 mM di-n-butyl l-tartrate and 100 mM boric acid in methanol. Other conditions are the same as in Table 1.
3.2. Effects of various basic electrolytes on the chiral separation From the experiments above, it was assumed that triethylamine should promote the formation and deprotonation of di-n-butyl ltartrate–boric acid complex by increasing the pH* value of BGEs. Therefore, the other basic electrolytes might also be used instead of triethylamine in the chiral separations. Consequently, effects of
Fig. 4. Electropherograms of analytes under optimized conditions. Buffer composition is 80 mM di-n-butyl l-tartrate, 100 mM boric acid and 45 mM NH4 OH in methanol, pH* 8.65. Other conditions are the same as in Table 1.
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inorganic and organic basic electrolytes on the enantioseparations of some basic analytes were exactly investigated in the following studies. 3.2.1. Effects of inorganic basic electrolytes on the chiral separation Sodium hydroxide (NaOH) is an inorganic base commonly used to adjust the buffer pH* in NACE [16,22,29]. In this study, NaOH at three different concentration levels (5, 10 and 20 mM) were used to evaluate its effect on the enantioseparations of the basic analytes. The presence of 5 mM NaOH could only obtain small peak splittings for most of the tested analytes. Among the three concentration levels, when 10 mM NaOH was used, the best chiral resolutions were achieved. At all concentrations evaluated, different peak splittings were achieved, but no baseline enantioseparation appeared. Another inorganic base, ammonium hydroxide (NH4 OH), was also used to adjust the buffer pH*. The effect of NH4 OH concentration was investigated from 0 to 60 mM. As shown in Fig. 3, NH4 OH concentration had great impact on the chiral separation. The chiral resolutions increased with the concentration of NH4 OH from 0 to 45 mM, but decreased with the concentration increased from 45 mM to 60 mM. As shown in Fig. 4, all of the analytes obtained baseline enantioseparations when NH4 OH was at a concentration of 45 mM in the running buffer. The chiral separation performance was much better than that when NaOH was used. In this study, both NaOH and NH4 OH could increase the buffer pH* and promote the deprotonation of the acidic chiral ion-pair selector. This was favorable for the ion-paring between the chiral selector and the analytes. The two bases also had great impact on the EOF. As shown in Fig. 5, the EOF decreased with the increase of base concentration, especially for NH4 OH. This may be advantageous because a decrease of EOF should increase the difference of eff of the enantiomers. All of these above mentioned were advantageous for the improvement of the chiral separation performance. However, with higher concentration, the coexist counter ions, Na+ and NH4 + , formed competing nonstereoselective ion-pairs to a different extent with the chiral selector, which changed the amount of selector available for complexation with the enantiomers [15,30]. When NH4 OH was used, its influence on EOF and ionization of chiral selectors were dominant, so better results could be achieved; contrary to the case above, when NaOH was used, the competing nonstereoselective ion-pairing was dominant, and no good enantioseparations appeared.
Fig. 5. Effects of NaOH and NH4 OH concentrations on the EOF in NACE. Buffer composition in addition to the basic electrolyte is 80 mM di-n-butyl l-tartrate and 100 mM boric acid in methanol. Other conditions are the same as in Table 1.
3.2.2. Effects of organic basic electrolytes on the chiral separation Triethylamine was an aliphatic tertiary amine and it was used to adjust the buffer pH* in the NACE experiment. It was predicted that other ultraviolet transparent aliphatic amines or alcohol amines should also be useful for the chiral separations of basic analytes using di-n-butyl l-tartrate–boric acid complex as the chiral selector in this study. Twelve aliphatic amines and four aliphatic alcohol amines were evaluated. The concentrations of different amines or alcohol amines were investigated and optimized concentrations for each of them were obtained. In order to estimate their effects on the enantioseparations exactly, the separation parameters for the eleven pairs of analytes were calculated. It was found that the effects of the concentration of different amines or alcohol amines on the chiral separations were similar to that of triethylamine. Before the optimized concentration, the resolutions increased with the concentration increased, but decreased after the optimized concentration. The reason might be the same as that when triethylamine was used. Certainly the trends for every analyte had some differences, so the mean values of Rs for the eleven analytes were used to evaluate
Fig. 6. Effects of various organic bases on the mean chiral resolutions in NACE at the optimized conditions. Buffer composition in addition to the basic electrolyte is 80 mM di-n-butyl l-tartrate and 100 mM boric acid in methanol. Other conditions are the same as in Table 1.
L.-J. Wang et al. / J. Chromatogr. A 1284 (2013) 188–193
the chiral separation performance, as shown in Fig. 6. For the seven primary amines, under the optimized conditions, except for ethylamine and n-propylamine, the other five ones were effective for the enantioseparations in this study. Desirable enantioseparations were also achieved with all of the aliphatic secondary and tertiary amines. The chiral separation performance in terms of both enantioselectivity (˛) and resolution (Rs ) were similar with them. For the four alcohol amines, the chiral separation performance decreased with the increase of the number of hydroxyethyl group. On the whole, except for ethylamine and triethanolamine, relatively good resolutions were achieved when most of the amines or alcohol amines were used. Owing to the lack of more physicochemical properties of these amines and alcohol amines in methanol, the reason for their different effects on the enantioseparations could not be explained exactly. 3.2.3. Migration orders of two enantiomers Migration order is one of the important parameters for the study of chiral recognition mechanism in CE [31–33]. In this work, the migration orders of propranolol enantiomers were the same when various basic electrolytes were used. It was that the (S)-enantiomer of propranolol migrated later. 4. Conclusions In this study, an ion-pair principle which can rationalize the enantioseparations of some basic analytes using the complex of din-butyl l-tartrate and boric acid as the chiral selector in methanolic background electrolytes (BGEs) by nonaqueous capillary electrophoresis (NACE) has been proposed. The MS approach directly confirmed that triethylamine promoted the formation of negatively charged di-n-butyl l-tartrate–boric acid complex chiral counter ion with a complex ratio of 2:1, which was the real chiral selector in the ion-pair principle enantioseparations. The effects of various basic electrolytes including inorganic and organic ones on the enantioseparations in NACE were investigated. The results showed that most of the basic electrolytes tested were favorable for the enantioseparations of basic analytes using di-n-butyl l-tartrate–boric acid complex as the chiral ion-pair selector. The achiral coexist counter ions could form competing nonstereoselective ion-pair with the chiral selector, which changed the amount of selector available for complexation with the enantiomers and caused an undesirable resolution. This work is an advance in the research on the chiral discrimination mechanism using tartrate–boric acid complexes as chiral selectors in NACE.
Acknowledgement The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (NSFC) (Grant Nos. 21075056 and 21175031). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2013.02.006. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
[16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]
Conflict of interest statement The authors declared no conflict of interest.
193
[32] [33]
G. Gübitz, M.G. Schmid, J. Chromatogr. A 1204 (2008) 140. Z.-Z. Wang, J. Ouyang, W.R.G. Baeyens, J. Chromatogr. B 862 (2008) 1. B. Preinerstorfer, M. Lämmerhofer, W. Lindner, Electrophoresis 30 (2009) 100. N.W. Frost, M. Jing, M.T. Bowser, Anal. Chem. 82 (2010) 4682. ´ ˜ C. Simó, V. Garcla-Ca nas, A. Cifuentes, Electrophoresis 31 (2010) 1442. Z.-B. Zhang, R.-A. Wu, M.-H. Wu, H.-F. Zou, Electrophoresis 31 (2010) 1457. T.J. Ward, K.D. Ward, Anal. Chem. 82 (2010) 4712. S. Fanali, Electrophoresis 30 (2009) S203. B. Chankvetadze, Electrophoresis 30 (2009) S211. G.K.E. Scriba, J. Pharm. Biomed. Anal. 55 (2011) 688. L. Geiser, J.-L. Veuthey, Electrophoresis 28 (2007) 45. M.-L. Riekkola, M. Jussila, S.P. Porras, I.E. Valkól, J. Chromatogr. A 892 (2000) 155. L. Geiser, J.-L. Veuthey, Electrophoresis 30 (2009) 36. J. Tjørnelund, S.H. Hansen, J. Biochem. Biophys. Methods 38 (1999) 139. Y. Hedeland, C. Pettersson, in: A. Van Eeckhaut, Y. Michotte (Eds.), Chiral Separation by Capillary Electrophoresis, CRC Press, Taylor & Francis Group, Boca Raton, 2009, p. 271. Y. Carlsson, M. Hedeland, U. Bondesson, C. Pettersson, J. Chromatogr. A 922 (2001) 303. M. Fillet, A.-C. Servais, J. Crommen, Electrophoresis 24 (2003) 1499. A. Karbaum, T. Jira, J. Biochem. Biophys. Methods 48 (2001) 155. ˇ ˇ cík, E. Tesaˇrová, D.W. Armstrong, V. Maier, V. Ranc, M. Svidrnoch, J. Petr, J. Sevˇ J. Chromatogr. A 1237 (2012) 128. K. Lomsadze, A. Salgado, E. Calvo, J.A. López, B. Chankvetadze, Electrophoresis 32 (2011) 1156. A.-C. Servais, M. Fillet, R. Mol, A. Rousseau, J. Crommen, G.W. Somsen, G.J. de Jong, Electrophoresis 31 (2010) 1157. I. Bjørnsdottir, S.H. Hansen, S. Terabe, J. Chromatogr. A 745 (1996) 37. L.-J. Wang, S.-Q. Hu, Q.-L. Guo, G.-L. Yang, X.-G. Chen, J. Chromatogr. A 1218 (2011) 1300. L.-J. Wang, J. Yang, G.-L. Yang, X.-G. Chen, J. Chromatogr. A 1248 (2012) 182. M. Lämmerhofer, J. Chromatogr. A 1068 (2005) 3. F. Wang, M.G. Khaledi, Anal. Chem. 68 (1996) 3460. E. Zarbl, M. Lämmerhofer, P. Franco, M. Petracs, W. Lindner, Electrophoresis 22 (2001) 3297. F. Wang, M.G. Khaledi, J. Chromatogr. A 875 (2000) 277. G.-H. Du, S.-Z. Zhang, J.-W. Xie, B.-H. Zhong, K.-L. Liu, J. Chromatogr. A 1074 (2005) 195. Y. Hedeland, J. Haglöf, P. Beronius, C. Pettersson, Electrophoresis 27 (2006) 4469. A.-C. Servais, A. Rousseau, M. Fillet, K. Lomsadze, A. Salgado, J. Crommen, B. Chankvetadze, Electrophoresis 31 (2010) 1467. B. Chankvetadze, Electrophoresis 23 (2002) 4022. A.-C. Servais, A. Rousseau, G. Dive, M. Frederich, J. Crommen, M. Fillet, J. Chromatogr. A 1232 (2012) 59.
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
An ion-pair principle for enantioseparations of basic analytes by nonaqueous capillary electrophoresis using the di-n-butyl l-tartrate–boric acid complex as chiral selector Li-Juan Wang a,b , Xiu-Feng Liu b , Qie-Nan Lu b , Geng-Liang Yang b , Xing-Guo Chen a,c,∗ a
National Key Laboratory of Applied Organic Chemistry, Department of Chemistry, Lanzhou University, Lanzhou 730000, China Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmacy, Hebei University, Baoding 071002, China c Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, Lanzhou 730000, China b
a r t i c l e
i n f o
Article history: Received 8 November 2012 Received in revised form 16 January 2013 Accepted 4 February 2013 Available online 10 February 2013 Keywords: Chiral recognition mechanism Chiral ion-pair selector Di-n-butyl l-tartrate–boric acid complex Basic electrolyte Nonaqueous capillary electrophoresis
a b s t r a c t A chiral recognition mechanism of ion-pair principle has been proposed in this study. It rationalized the enantioseparations of some basic analytes using the complex of di-n-butyl l-tartrate and boric acid as the chiral selector in methanolic background electrolytes (BGEs) by nonaqueous capillary electrophoresis (NACE). An approach of mass spectrometer (MS) directly confirmed that triethylamine promoted the formation of negatively charged di-n-butyl l-tartrate–boric acid complex chiral counter ion with a complex ratio of 2:1. And the negatively charged counter ion was the real chiral selector in the ion-pair principle enantioseparations. It was assumed that triethylamine should play its role by adjusting the apparent acidity (pH*) of the running buffer to a higher value. Consequently, the effects of various basic electrolytes including inorganic and organic ones on the enantioseparations in NACE were investigated. The results showed that most of the basic electrolytes tested were favorable for the enantioseparations of basic analytes using di-n-butyl l-tartrate–boric acid complex as the chiral ion-pair selector. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Capillary electrophoresis (CE) represents one of the major techniques for analytical scale enantioseparations [1–10]. Nonaqueous capillary electrophoresis (NACE) is an interesting alternative to improve chiral recognition performance [11]. Solvent parameters of nonaquous solutions such as the relative permittivity, viscosity, polarity and autoprotolysis, have offered new possibilities for changes in chiral selectivity in NACE [12–17]. It has also been extended to the applications of chiral analytes or selectors with non or poor solubility in aqueous CE [18,19]. In addition, due to the special physicochemical properties of the nonaqueous solution, in terms of volatility and surface tension, an effective sample introduction to the mass spectrometer (MS) detector can be expected to further extend the use of NACE [5,15,20,21]. The ion-pair principle is one of the important chiral recognition mechanisms for the separation of charged, chiral analytes in NACE. As nonaqueous solvents with low relative permittivities provide a better environment for achieving ion-pair interactions than water,
∗ Corresponding author at: Department of Chemistry, Lanzhou University, Lanzhou 730000, Gansu, China. Tel.: +86 931 8912763; fax: +86 931 8912582. E-mail address: [email protected] (X.-G. Chen). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.02.006
the ion-pair formation as a principle for chiral separation in nonaqueous media should be optimal [22]. As reported in our previous works, various dialkyltartrate–boric acid complexes showed good chiral recognition abilities for some structurally similar basic analytes in methanolic background electrolytes (BGEs) [23,24]. The chiral recognition mechanism was assumed to be ion-pair principle. Triethylamine, a basic electrolyte, was quite useful for the formation of negatively charged dialkyltartrate–boric acid complex chiral counter ion, which was the real chiral selector in the ion-pair principle enantioseparations. However, no direct experimental data has verified this. Additionally, there are several studies that have reported the effect of some other basic electrolytes except for triethylamine in the BGEs in NACE [11,18,25–27]. Whether they could be also useful for the enantioseparations in this study has not been studied. Therefore, one aim of this work was to confirm the formation of negatively charged di-n-butyl l-tartrate–boric acid complex chiral counter ion in the presence of different concentrations of triethylamine. And the complex ratio between di-n-butyl l-tartrate and boric acid should be also determined. Those were carried out by an approach of mass spectrometer (MS). Another aim was to exactly study the effects of different inorganic or organic basic electrolytes on the enantioseparations of basic analytes by NACE using di-nbutyl l-tartrate–boric acid complex as the chiral selector.
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Fig. 1. MS spectra of NACE buffers with different concentrations of triethylamine. Buffer composition: 80 mM di-n-butyl l-tartrate, 100 mM boric acid and 0 mM, pH* 3.82 (A), 7.2 mM, pH* 7.09 (B), 36 mM, pH* 7.93 (C) triethylamine in methanol. MS conditions are shown in Section 2.1.
2. Experimental 2.1. Instrumentation NACE experiments were conducted on a TH-3100 high performance capillary electrophoresis system (Tianhui Institute of Separation Science, Baoding, China), equipped with a thermostatic system and a UV detector. Data were collected with a CXTH-3000 chromatography workstation. Uncoated fused silica capillary of 50 m I.D. (Yongnian Reafine Chromatography Co., Ltd., Hebei, China) with a total length (Ltot ) of 55.0 cm and an effective length
(Leff ) of 45.0 cm was used. The new capillary was conditioned by flushing with methanol for 10 min, 1.0 M NaOH for 20 min, distilled water for 5 min, 1.0 M hydrochloric acid for 20 min and distilled water for 5 min in sequence. Before each run the capillary was rinsed with running buffer for 3 min. Samples were introduced into the capillary tube by positive pressure at 2.9 psi for 2 s. The experiments were performed at 25 ± 0.2 ◦ C. The detection wavelength was set at 214 nm. MS experiments were performed with an Agilent 1100 series LC/MSD Trap XCT (Agilent Technologies, USA) equipped with an electrospray ionization source (ESI). The MS was controlled by
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Table 1 Effect of triethylamine concentration on migration time, effective mobility, enantioselectivity, resolution and efficiency.a Samples
Propranolol Sotalol Atenolol Bisoprolol Bambuterol
7.2 mM triethylamine (pH* 7.09)
36 mM triethylamine (pH* 7.93)
t1
t2
eff1 b
eff2 b
˛eff
Rs
5.607 5.660 6.019 6.139 5.900
5.820 5.808 6.202 6.340 6.253
18.78 18.43 16.26 15.59 16.95
17.43 17.50 15.25 14.52 14.98
1.08 1.08 1.07 1.06 1.05
1.55 0.90 0.99 1.19 1.78
N1 35,205 36,756 36,611 33,215 30,967
N2 23,634 23,529 22,417 17,892 13,894
t1
t2
eff1 c
eff2 c
˛eff
Rs
10.012 9.849 10.421 10.488 9.245
10.544 10.234 10.852 10.990 9.853
9.826 10.16 9.009 8.883 11.53
8.785 9.372 8.223 7.985 10.15
1.12 1.08 1.10 1.11 1.14
2.39 1.98 2.07 2.21 2.78
N1 49,231 48,775 44,424 48,304 51,083
N2 26,573 32,874 27,824 28,981 21,946
a Buffer component in addition to triethylamine is 80 mM di-n-butyl l-tartrate and 100 mM boric acid in methanol. CE conditions: capillary dimensions, Ltot 55.0 cm, Leff 45.0 cm, I.D. 50 m; positive pressure injection for 2 s at 2.9 psi; applied voltage, 20 kV; detection wavelength, 214 nm. b ×10−5 cm2 V−1 s−1 , EOF = 18.01 × 10−5 cm2 V−1 s−1 . c ×10−5 cm2 V−1 s−1 , EOF = 10.78 × 10−5 cm2 V−1 s−1 .
Agilent software A.10.02. The experiments were performed in negative ESI-mode in a mass range of m/z 100–800 with the following ionizer parameters: spray voltage 3000 V, nebulizer gas (N2 ) flow set at 15 psi, dry gas (N2 ) flow 5 L/min with a temperature of 325 ◦ C. A spectral scan range of 100–800 m/z with a maximum accumulation time of 200 ms and an ion charge control (ICC) target setting of 200,000 were applied. The sample was infused with a syringe pump at a flow rate of 3.0 L/min. A pH meter (pHS-3C, Shanghai Precision & Scientific Instrument Co., Ltd., China) equipped with a glass electrode was used to measure the apparent pH (pH*) values of the CE running buffers in NACE. The electrode was calibrated using standard aqueous buffers, pH 4.00, 6.86 and 9.18 at 25 ± 0.2 ◦ C. 2.2. Chemicals and materials Racemic sotalol hydrochloride, esmolol hydrochloride, clenbuterol hydrochloride, cycloclenbuterol hydrochloride, bambuterol hydrochloride, and tulobuterol hydrochloride were purchased from National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). (S)-Propranolol hydrochloride was purchased from Sigma–Aldrich (St. Louis, MO, USA). The following racemic compounds were extracted by methanol from medicine tablets: propranolol hydrochloride (LI® , Tianjin Lisheng Pharmaceuticals Co., Ltd., China), atenolol (YJ® , Beijing Yanjing Pharmaceuticals Co., Ltd., China), bisoprolol fumarate (BOSU® , Wellso Pharmaceuticals Co., Ltd., China), metoprolol tartrate (BETALOC® , AstraZeneca Pharmaceuticals Co., Ltd., China), and terbutaline sulphate (BRICANYL® , AstraZeneca Pharmaceuticals Co., Ltd., China). Di-n-butyl l-tartrate (purity ≥ 98 wt%, water content ≤ 0.5 wt%) was purchased from Acros (New Jersey, USA). Boric acid was the product of Baoding Chemical Reagent Factory (Baoding, China). Triethylamine (water content ≤ 0.2 wt%) and sodium hydroxide (≥98%, pellets, anhydrous) were supplied by Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). Methanol (water content ≤ 0.02 wt%), chromatographic reagent grade, was purchased from Tianjin Concord Technology Co., Ltd. (Tianjin, China). Ammonium hydroxide (≥28 wt% NH3 in water), methylamine (30–33 wt% in absolute ethanol), ethylamine (68.0–72.0 wt% in water) and trimethylamine (30 wt% in water) were all of analytical reagent grade, made in China and used as received. The other reagents and chemicals were all of analytical reagent grade, made in China and used as received. Their water contents were all lower than 0.5 wt%. 2.3. Buffer and sample preparation NACE running buffer solutions were prepared by weighing the desired quantities of boric acid, di-n-butyl l-tartrate, and dissolving them in methanol to the desired volume in a flask. The pH* values were measured and adjusted by adding appropriate concentration of some kind of inorganic or organic bases to the running
buffers. The NACE sample solutions were prepared by dissolving an appropriate quantity of each racemic sample in methanol to a concentration of 0.1 mg/mL. The composition of MS sample solutions was 80 mM di-n-butyl l-tartrate, 100 mM boric acid and the desired quantities of triethylamine in methanol. All of the solutions were filtered through a 0.45 m syringe type filter prior to use. 3. Results and discussion In this study, enantioseparations were evaluated in terms of resolution (Rs ) and selectivity (˛eff ). All of the calculations of performance parameters referred to Ref. [23]. 3.1. The MS verification for the effect of triethylamine concentration in BGEs on the formation of chiral ion-pair selector MS experiments were performed to examine the quantity of din-butyl l-tartrate–boric acid complex and its complex ratio. NACE running buffers with different concentrations of triethylamine in BGEs were directly used as the MS samples. As shown in Fig. 1(A), MS sample without triethylamine obtained a spectrum with four MS peaks at m/z 205.0, 363.1, 419.2, and 475.2, which were consistent with the pseudo-molecular formulas [C8 H14 O6 −H+ ]− , [C12 H17 BO12 −H+ ]− , [C16 H25 BO12 −H+ ]− , and [C20 H33 BO12 −H+ ]− , respectively. And no enantioseparations of basic analytes could be achieved with the same BGE in NACE. Maybe this could be explained from two aspects. On the one hand, the chiral selectors detected here might have no chiral recognition abilities for the analytes tested; on the other hand, maybe there were not sufficient number of chiral selectors in the BGE to achieve good enantioseparations. Obviously different from Fig. 1(A), the MS peaks at m/z 531 were the most intense signals in the spectra of samples containing different concentrations of triethylamine [Fig. 1(B) and (C)]. The MS peak at m/z 531 agreed with the pseudo-molecular formula [C24 H41 BO12 −H+ ]− , with a complex ratio of 2:1 between di-n-butyl l-tartrate and boric acid, and its structural formula was shown in Fig. 2. As shown in Fig. 1(B), when 7.2 mM triethylamine was added to the MS sample, a relatively intense signal of MS peak at m/z 531 appeared in the spectrum. The other new MS peaks at m/z 333.2, 375.0, 433.2, and 563.2 were found to corroborate pseudomolecular formulas of [C14 H27 BO8 −H+ ]− , [C17 H33 BO8 −H+ ]− , [C17 H27 BO12 −H+ ]− , and [C24 H41 BO12 −H+ +CH3 OH]− . Meanwhile, certain levels of enantioseparations for most of the chiral analytes were achieved using the same BGE in NACE, as shown in Table 1. The absolute signal of MS peak at m/z 531 increased with the concentration of triethylamine from 7.2 mM to 36 mM. And the chiral resolutions of analytes also increased with the increase of triethylamine concentration in NACE. As shown in Fig. 1(C), the most intense signal of MS peak at m/z 531 appeared in the spectrum when 36 mM triethylamine was added. The best enantioseparations were also achieved for all of the analytes at that concentration of triethylamine in NACE. When triethylamine
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Fig. 2. Structural formula of pseudo-molecular ion [C24 H41 BO12 −H+ ]− with m/z 531 at MS spectra.
concentration increased from 36 mM to 144 mM, the absolute signal of MS peak at m/z 531 no longer increased. And the higher pH* also decreased the degree of the ionization of basic analytes and weakened their interactions with negatively charged chiral selector, so that the chiral resolutions decreased. Contrast MS and NACE results, it was concluded that the negatively charged di-n-butyl l-tartrate–boric acid complex counter ion with a complex ratio of 2:1 was the real chiral selector in the NACE chiral separation system. The presence of triethylamine in methanolic BGEs promoted the formation and deprotonation of din-butyl l-boric acid complex by adjusting the pH* of the BGEs to a higher value. A certain number of negatively charged chiral counter ions were necessary for the ion-pair principle enantioseparations of basic analytes in NACE [15,28].
Fig. 3. Effect of NH4 OH concentration on NACE chiral resolution. Buffer composition in addition to NH4 OH is 80 mM di-n-butyl l-tartrate and 100 mM boric acid in methanol. Other conditions are the same as in Table 1.
3.2. Effects of various basic electrolytes on the chiral separation From the experiments above, it was assumed that triethylamine should promote the formation and deprotonation of di-n-butyl ltartrate–boric acid complex by increasing the pH* value of BGEs. Therefore, the other basic electrolytes might also be used instead of triethylamine in the chiral separations. Consequently, effects of
Fig. 4. Electropherograms of analytes under optimized conditions. Buffer composition is 80 mM di-n-butyl l-tartrate, 100 mM boric acid and 45 mM NH4 OH in methanol, pH* 8.65. Other conditions are the same as in Table 1.
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inorganic and organic basic electrolytes on the enantioseparations of some basic analytes were exactly investigated in the following studies. 3.2.1. Effects of inorganic basic electrolytes on the chiral separation Sodium hydroxide (NaOH) is an inorganic base commonly used to adjust the buffer pH* in NACE [16,22,29]. In this study, NaOH at three different concentration levels (5, 10 and 20 mM) were used to evaluate its effect on the enantioseparations of the basic analytes. The presence of 5 mM NaOH could only obtain small peak splittings for most of the tested analytes. Among the three concentration levels, when 10 mM NaOH was used, the best chiral resolutions were achieved. At all concentrations evaluated, different peak splittings were achieved, but no baseline enantioseparation appeared. Another inorganic base, ammonium hydroxide (NH4 OH), was also used to adjust the buffer pH*. The effect of NH4 OH concentration was investigated from 0 to 60 mM. As shown in Fig. 3, NH4 OH concentration had great impact on the chiral separation. The chiral resolutions increased with the concentration of NH4 OH from 0 to 45 mM, but decreased with the concentration increased from 45 mM to 60 mM. As shown in Fig. 4, all of the analytes obtained baseline enantioseparations when NH4 OH was at a concentration of 45 mM in the running buffer. The chiral separation performance was much better than that when NaOH was used. In this study, both NaOH and NH4 OH could increase the buffer pH* and promote the deprotonation of the acidic chiral ion-pair selector. This was favorable for the ion-paring between the chiral selector and the analytes. The two bases also had great impact on the EOF. As shown in Fig. 5, the EOF decreased with the increase of base concentration, especially for NH4 OH. This may be advantageous because a decrease of EOF should increase the difference of eff of the enantiomers. All of these above mentioned were advantageous for the improvement of the chiral separation performance. However, with higher concentration, the coexist counter ions, Na+ and NH4 + , formed competing nonstereoselective ion-pairs to a different extent with the chiral selector, which changed the amount of selector available for complexation with the enantiomers [15,30]. When NH4 OH was used, its influence on EOF and ionization of chiral selectors were dominant, so better results could be achieved; contrary to the case above, when NaOH was used, the competing nonstereoselective ion-pairing was dominant, and no good enantioseparations appeared.
Fig. 5. Effects of NaOH and NH4 OH concentrations on the EOF in NACE. Buffer composition in addition to the basic electrolyte is 80 mM di-n-butyl l-tartrate and 100 mM boric acid in methanol. Other conditions are the same as in Table 1.
3.2.2. Effects of organic basic electrolytes on the chiral separation Triethylamine was an aliphatic tertiary amine and it was used to adjust the buffer pH* in the NACE experiment. It was predicted that other ultraviolet transparent aliphatic amines or alcohol amines should also be useful for the chiral separations of basic analytes using di-n-butyl l-tartrate–boric acid complex as the chiral selector in this study. Twelve aliphatic amines and four aliphatic alcohol amines were evaluated. The concentrations of different amines or alcohol amines were investigated and optimized concentrations for each of them were obtained. In order to estimate their effects on the enantioseparations exactly, the separation parameters for the eleven pairs of analytes were calculated. It was found that the effects of the concentration of different amines or alcohol amines on the chiral separations were similar to that of triethylamine. Before the optimized concentration, the resolutions increased with the concentration increased, but decreased after the optimized concentration. The reason might be the same as that when triethylamine was used. Certainly the trends for every analyte had some differences, so the mean values of Rs for the eleven analytes were used to evaluate
Fig. 6. Effects of various organic bases on the mean chiral resolutions in NACE at the optimized conditions. Buffer composition in addition to the basic electrolyte is 80 mM di-n-butyl l-tartrate and 100 mM boric acid in methanol. Other conditions are the same as in Table 1.
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the chiral separation performance, as shown in Fig. 6. For the seven primary amines, under the optimized conditions, except for ethylamine and n-propylamine, the other five ones were effective for the enantioseparations in this study. Desirable enantioseparations were also achieved with all of the aliphatic secondary and tertiary amines. The chiral separation performance in terms of both enantioselectivity (˛) and resolution (Rs ) were similar with them. For the four alcohol amines, the chiral separation performance decreased with the increase of the number of hydroxyethyl group. On the whole, except for ethylamine and triethanolamine, relatively good resolutions were achieved when most of the amines or alcohol amines were used. Owing to the lack of more physicochemical properties of these amines and alcohol amines in methanol, the reason for their different effects on the enantioseparations could not be explained exactly. 3.2.3. Migration orders of two enantiomers Migration order is one of the important parameters for the study of chiral recognition mechanism in CE [31–33]. In this work, the migration orders of propranolol enantiomers were the same when various basic electrolytes were used. It was that the (S)-enantiomer of propranolol migrated later. 4. Conclusions In this study, an ion-pair principle which can rationalize the enantioseparations of some basic analytes using the complex of din-butyl l-tartrate and boric acid as the chiral selector in methanolic background electrolytes (BGEs) by nonaqueous capillary electrophoresis (NACE) has been proposed. The MS approach directly confirmed that triethylamine promoted the formation of negatively charged di-n-butyl l-tartrate–boric acid complex chiral counter ion with a complex ratio of 2:1, which was the real chiral selector in the ion-pair principle enantioseparations. The effects of various basic electrolytes including inorganic and organic ones on the enantioseparations in NACE were investigated. The results showed that most of the basic electrolytes tested were favorable for the enantioseparations of basic analytes using di-n-butyl l-tartrate–boric acid complex as the chiral ion-pair selector. The achiral coexist counter ions could form competing nonstereoselective ion-pair with the chiral selector, which changed the amount of selector available for complexation with the enantiomers and caused an undesirable resolution. This work is an advance in the research on the chiral discrimination mechanism using tartrate–boric acid complexes as chiral selectors in NACE.
Acknowledgement The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (NSFC) (Grant Nos. 21075056 and 21175031). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2013.02.006. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
[16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]
Conflict of interest statement The authors declared no conflict of interest.
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[32] [33]
G. Gübitz, M.G. Schmid, J. Chromatogr. A 1204 (2008) 140. Z.-Z. Wang, J. Ouyang, W.R.G. Baeyens, J. Chromatogr. B 862 (2008) 1. B. Preinerstorfer, M. Lämmerhofer, W. Lindner, Electrophoresis 30 (2009) 100. N.W. Frost, M. Jing, M.T. Bowser, Anal. Chem. 82 (2010) 4682. ´ ˜ C. Simó, V. Garcla-Ca nas, A. Cifuentes, Electrophoresis 31 (2010) 1442. Z.-B. Zhang, R.-A. Wu, M.-H. Wu, H.-F. Zou, Electrophoresis 31 (2010) 1457. T.J. Ward, K.D. Ward, Anal. Chem. 82 (2010) 4712. S. Fanali, Electrophoresis 30 (2009) S203. B. Chankvetadze, Electrophoresis 30 (2009) S211. G.K.E. Scriba, J. Pharm. Biomed. Anal. 55 (2011) 688. L. Geiser, J.-L. Veuthey, Electrophoresis 28 (2007) 45. M.-L. Riekkola, M. Jussila, S.P. Porras, I.E. Valkól, J. Chromatogr. A 892 (2000) 155. L. Geiser, J.-L. Veuthey, Electrophoresis 30 (2009) 36. J. Tjørnelund, S.H. Hansen, J. Biochem. Biophys. Methods 38 (1999) 139. Y. Hedeland, C. Pettersson, in: A. Van Eeckhaut, Y. Michotte (Eds.), Chiral Separation by Capillary Electrophoresis, CRC Press, Taylor & Francis Group, Boca Raton, 2009, p. 271. Y. Carlsson, M. Hedeland, U. Bondesson, C. Pettersson, J. Chromatogr. A 922 (2001) 303. M. Fillet, A.-C. Servais, J. Crommen, Electrophoresis 24 (2003) 1499. A. Karbaum, T. Jira, J. Biochem. Biophys. Methods 48 (2001) 155. ˇ ˇ cík, E. Tesaˇrová, D.W. Armstrong, V. Maier, V. Ranc, M. Svidrnoch, J. Petr, J. Sevˇ J. Chromatogr. A 1237 (2012) 128. K. Lomsadze, A. Salgado, E. Calvo, J.A. López, B. Chankvetadze, Electrophoresis 32 (2011) 1156. A.-C. Servais, M. Fillet, R. Mol, A. Rousseau, J. Crommen, G.W. Somsen, G.J. de Jong, Electrophoresis 31 (2010) 1157. I. Bjørnsdottir, S.H. Hansen, S. Terabe, J. Chromatogr. A 745 (1996) 37. L.-J. Wang, S.-Q. Hu, Q.-L. Guo, G.-L. Yang, X.-G. Chen, J. Chromatogr. A 1218 (2011) 1300. L.-J. Wang, J. Yang, G.-L. Yang, X.-G. Chen, J. Chromatogr. A 1248 (2012) 182. M. Lämmerhofer, J. Chromatogr. A 1068 (2005) 3. F. Wang, M.G. Khaledi, Anal. Chem. 68 (1996) 3460. E. Zarbl, M. Lämmerhofer, P. Franco, M. Petracs, W. Lindner, Electrophoresis 22 (2001) 3297. F. Wang, M.G. Khaledi, J. Chromatogr. A 875 (2000) 277. G.-H. Du, S.-Z. Zhang, J.-W. Xie, B.-H. Zhong, K.-L. Liu, J. Chromatogr. A 1074 (2005) 195. Y. Hedeland, J. Haglöf, P. Beronius, C. Pettersson, Electrophoresis 27 (2006) 4469. A.-C. Servais, A. Rousseau, M. Fillet, K. Lomsadze, A. Salgado, J. Crommen, B. Chankvetadze, Electrophoresis 31 (2010) 1467. B. Chankvetadze, Electrophoresis 23 (2002) 4022. A.-C. Servais, A. Rousseau, G. Dive, M. Frederich, J. Crommen, M. Fillet, J. Chromatogr. A 1232 (2012) 59.