Unusual chromatographic enantioseparation behavior of naproxen on an immobilized polysaccharide-based chiral stationary phase

Journal of Chromatography A, 1218 (2011) 8718–8721

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Unusual chromatographic enantioseparation behavior of naproxen on an immobilized polysaccharide-based chiral stationary phase Cui Xiang a,b , Guoqing Liu a,b , Shanshan Kang a,b , Xueping Guo a,b , Bixia Yao a,b , Wen Weng a,b,∗ , Qingle Zeng c a

Department of Chemistry and Environmental Science, Zhangzhou Normal University, Zhangzhou 363000, China Key Laboratory for Analytical Science of Fujian Province University, Zhangzhou 363000, China c College of Materials and Chemistry & Chemical Engineering, Chengdu University of Technology, Chengdu 610059, China b

a r t i c l e

i n f o

Article history: Received 16 July 2011 Received in revised form 4 October 2011 Accepted 7 October 2011 Available online 12 October 2011 Keywords: Enantiomer separation Chiralpak IA column Naproxen Reversal of elution order Peak deformation

a b s t r a c t Enantioseparation of naproxen was performed on an immobilized polysaccharide-based chiral stationary phase (CSP), Chiralpak IA, in the normal-phase mode. The effects of polar alcohol modifier in mobile phase and column temperature on retention, enantioseparation, and elution order were investigated. Two unusual phenomena were observed. One was solvent-induced reversal of elution order for the two enantiomers. Not only the type but also the content of polar alcohol modifier could induce the reversal. Another uncommon phenomenon was peak deformation under some chromatographic conditions. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Elution order between a pair of enantiomers is a key theme in the field of chiral high-performance liquid chromatography (HPLC) [1]. Elucidation of chiral recognition mechanism is closely related to this issue. It is also desirable to elute minor enantiomer before major isomer when enantiomer purity needs to be evaluated accurately. But till now prediction of elution order remains very difficult. Elution order reversal makes this prediction more complex [2]. Several types of elution order reversal between enantiomers in a given selector/selectant system have been found. The first type is temperature-induced reversal. Koppenhöfer and Bayer predicted the reversal based on the Van’t Hoff equation in 1984 [3]. Sparse evidence has been reported in HPLC because of the narrow operational temperature range [4–8]. The second type is solvent-induced reversal that can be induced by type and content of mobile phase modifier. This type of reversal has been reported primarily on polysaccharide-based CSPs [8–16]. Wang and coworkers observed the concentration of alcohol modifier caused reversal for some pharmaceutical compounds on Chiralpak AD or Chiralpak AS col-

umn [17,18]. They further utilized solid-state nuclear magnetic resonance (NMR) to identify modifier’s structural factor causing the inversion on the Chiralpak AD [19]. Vibrational circular dichroism (VCD) was also used to investigate the interactions between analytes and polysaccharide-based CSPs, and elucidated the reversal was due to conformational change of the CSPs [20,21]. The third type is sample load-induced reversal. Only one example has been reported [22]. Naproxen is a representative of non-steroidal antiinflammatory drug. The pharmacological activity of S-isomer for naproxen is about 35 times stronger than R-enantiomer. It is preferable to use pure S-enantiomer in drugs in order to decrease dosage and undesirable side effects. Naproxen enantioseparations have been achieved on a variety of CSPs [23–27]. In this paper, the enantioseparation of naproxen was performed on an immobilized polysaccharide-based chiral stationary phase, Chiralpak IA, in the normal-phase mode. Two unusual chromatographic behaviors, solvent-induced elution order reversal and peak deformation, were observed. 2. Experimental

∗ Corresponding author at: Department of Chemistry and Environmental Science, Zhangzhou Normal University, Zhangzhou 363000, China. Tel.: +86 596 2521389; fax: +86 596 2520035. E-mail address: weng [email protected] (W. Weng). 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.10.014

2.1. Chemicals Solvents (n-hexane, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 1-pentanol, 1-hexanol, and tetrahydrofunan (THF))

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Table 1 Effects of 2-propanol amount in n-hexane and column temperature on the retention and enantioseparation of naproxen. T (◦ C)

20 25 30 35 40 45 50 T (◦ C)

8%

7%

5%

kS

˛R,S

kR

kS

˛R,S

kR

kS

˛R,S

kR

kS

˛R,S

2.738 2.614 2.501 2.396

2.996 2.798 2.623 2.477

1.093 1.070 1.048 1.034 1.000 1.000 1.000

3.117 3.008 2.888

3.330 3.167 2.985

1.068 1.052 1.033 1.000 1.000 1.000 1.000

3.594 3.462 3.312

3.822 3.620 3.412

1.063 1.045 1.030 1.000 1.000 1.000 1.000

4.325 4.173

4.545 4.312

1.050 1.033 1.000 1.000 1.000 1.000 1.000

2.341 2.264 2.150

2.829 2.687 2.573 2.477

4%

3.232 3.105 2.990 2.899

3%

kS 20 25 30 35 40 45 50

6%

kR

4.647 4.442 4.331

2%

kR

˛S,R

kS

kR

4.785 4.591 4.500

1.000 1.000 1.000 1.000 1.029 1.033 1.038

6.990 6.583 6.296 6.066 5.832 5.617 5.389

7.647 7.324 7.019 6.771 6.546 6.323a 6.044a

5.533 5.275 5.049 4.816

4.067 3.890 3.749 3.622 3.540

˛S,R 1.093 1.112 1.114 1.116 1.122 a a

kR

˛S,R

10.27 9.637 9.156 8.677 8.386

1.161 1.133 1.108 1.084 1.065

kS 8.849 8.502 8.257 8.000 7.872 7.561 7.282

a

a

a

a

–1

Conditions: flow rate, 1.0 mL min . a Peak deformation.

were HPLC grade. Isoamyl alcohol, 2-butanol, cyclohexanol, cyclopentanol, and glacial acetic acid were analytical reagents. They were used as received without further purification. Racemic and S-naproxen were purchased from Alfa Aesar China (Tianjin). A methanol solution enriched with S-naproxen was used to facilitate peak assignment in this study. 2.2. Chromatography An Agilent 1200 chromatographic system was used to perform the experiments. A Chiralpak IA column (25 cm × 4.6 mm I.D., 5 ␮m particle size), an immobilized amylose tris(3,5dimethylphenylcarbamate) CSP, was purchased from Daicel (Tokyo, Japan). A corresponding guard column (1.0 cm × 4.6 mm I.D.) was connected. Chromatographic experiments were carried out under normal-phase conditions. The mobile phase consisted of n-hexane, a polar alcohol modifier, and glacial acetic acid (0.5% by volume). All solvents were degassed in an ultrasonic bath prior to use. The flow rate was 1.0 mL min−1 , and the ultraviolet (UV) detection wavelengths were 220, 254, and 280 nm. The injection volume was 10 ␮L. The hold-up time was determined from the first perturbation of the baseline. The retention factor, k, was calculated as (tR − t0 )/t0 , where tR is the retention time and t0 is the holdup time. The separation factor ˛ was calculated as k2 /k1 , where k1 and k2 are the retention factors for the first- and the second-eluted enantiomer, respectively. To eliminate some unexpected memory effects, a column regeneration procedure according to the vendor’s instruction was performed when a new alcohol modifier was utilized. The column was flushed with pure ethanol (0.5 mL min−1 for 2 h), followed by 100% THF (0.5 mL min−1 for 2 h). Once a new chromatographic condition was adopted, the column was equilibrated for at least 1 h before injection. 3. Results and discussion Temperature-induced elution order reversal was observed for binaphthol enantioseparated on Chiralpak IA column [28]. To gather more information about the reversal, naproxen enantioseparation was conducted on the same CSP. The effects of type and concentration on enantioseparation and elution order from polar alcohol modifier were specifically investigated. Temperature effect was also studied simultaneously for each mobile phase system. The

concentration of polar alcohol was in the range of 1–14% by volume. Column temperature was in the range of 20–50 ◦ C. When ethanol was used, baseline enantioseparation was obtained with R-naproxen as the first-eluted enantiomer. The effect of ethanol content on the enantioseparation was unconspicuous. The separation factor decreased with column temperature increase, while temperature-induced elution order reversal was not observed. When 2-propanol was used, solvent-induced elution order reversal took place (Table 1). At 20 ◦ C, R-naproxen eluted first when 2-propanol content was in the range of 5–8%, but the two enantiomers coeluted with 4% of 2-propanol. Then, S-naproxen eluted first with lower 2-propanol content (2–3%). The overlapped chromatograms were shown in Fig. 1. Peak deformation, the other unusual phenomenon, was observed at elevated temperature with low 2-propanol content. With 3% of 2-propanol at 45 and 50 ◦ C, the peak fronting of the second-eluted enantiomer became serious (Fig. 2A). The peak tailing of the second-eluted enantiomer became serious instead with 2% of 2-propanol at these two temperatures (Fig. 2B). S-Naproxen always eluted first with 1-propanol in the range of 2–8%. The effect of 1-propanol content on the enantioseparation was also unconspicuous. Similarly, peak deformation was observed at elevated temperature when the 1-propanol content was lower than 5%. With 3% of 1-propanol, peak deformation for the second-eluted enantiomer was visible under all experimental temperatures (Fig. 2C). The peak profile was similar to that with 3% of 2-propanol. With 2% of 1-propanol, peak tailing of the second-eluted enantiomer reemerged. At 50 ◦ C, a small peak appeared unexpectedly after the first peak (Fig. 2D). This unusual phenomenon was barely discernible at 45 ◦ C with 2% of 2-propanol (Fig. 2B). It is worthy of being mentioned that this peak deformation was not observed with other eight alcohols (ethanol, 1-butanol, 2-butanol, 1-pentanol, isoamyl alcohol, 1-hexanol, cyclopentanol, and cyclohexanol). A HPLC peak deviating from classical Gaussian Langmuirian and anti-Langmuirian profiles was defined as deformed peak, including compressed, broadened, distorted or split peak from a normal isocratic peak [29–31]. It is generally considered that peak compression, broadening, and deformation are created by local change in mobile phase concentration in comparison with the equilibrium composition, resulted from either system peak or gradient elution. Previous studies have

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Fig. 1. Elution order reversal of naproxen enantiomers induced by the 2-propanol content on Chiralpak IA column. Conditions: mobile phase, n-hexane/2-propanol/acetic acid (0.5%); column temperature, 20 ◦ C.

Fig. 2. Peak deformations in the naproxen enantioseparation on Chiralpak IA column. Conditions: (A) n-hexane/2-propanol/acetic acid, 96.5/3/0.5 (v/v/v); (B) n-hexane/2propanol/acetic acid, 97.5/2/0.5 (v/v/v); (C) n-hexane/1-propanol/acetic acid, 96.5/3/0.5 (v/v/v); (D) n-hexane/1-propanol/acetic acid, 97.5/2/0.5 (v/v/v); column temperature from the top down, 20, 25, 30, 35, 40, 45, 50 ◦ C.

Fig. 3. Elution order reversal of naproxen enantiomers induced by the different types of alcohol modifier on Chiralpak IA column. Conditions: n-hexane/alcohol/acetic acid, 91.5/8/0.5 (v/v/v); column temperature, 20 ◦ C.

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demonstrated that adsorbing additives, in particular strongly bound, can have an extreme impact on the peak sharp [32,33]; the utilization of the effects for increased productivity in process chromatography was also recently demonstrated [34,35]. In this work, acetic acid was added to the mobile phases to accelerate the eluting process. Moreover, the negative system peak of acetic acid was observed at 220 nm. This system peak may bring about the peak deformation in this chromatographic system. It seems there is no regular trend for the effect of polar alcohol modifier on the elution order (Fig. 3). When 1-butanol, 1-pentanol or isoamyl alcohol was used as a polar modifier, Rnaproxen was eluted first. Only slight enantioseparation with S-naproxen coming out first was obtained with low content of 2-butanol. The 1-hexanol modification presented the similar performance but R-naproxen eluted first. Inconsistently, no enantioseparation was found with low content of cyclohexanol (2%), but with high content of cyclopentanol (6–10%). R-Naproxen eluted first with high content of cyclohexanol (4–12%), but S-naproxen eluted first with low content of cyclopentanol (2–4%). The solvent-induced reversal increased the complexity of elucidation of chiral recognition mechanism. No enantioseparation was found when 2-propanol content was 4% at 20 ◦ C. The loss of enantioseparation with around the modifier at this content can be described as solvent-induced blind zone of chiral recognition, the same way as for the temperature-induced blind zone of chiral recognition [28]. The farther from the blind zone, the better enantioseparation can be obtained. Elution order reversal would happen when the polar alcohol concentration changes from below to above the [D]iso , and vice versa. More experiments are needed to elucidate the origin of elution order reversal. 4. Conclusion In summary, two unusual chromatographic phenomena were observed for the enantioseparation of naproxen on an immobilized polysaccharide-based CSP. One was solvent-induced elution order reversal. Not only the type but also the content of polar alcohol modifier in the mobile phase could induce the reversal. Another unusual phenomenon was peak deformation under some chromatographic conditions. The system peak of acetic acid likely caused the peak deformation in the chromatographic system.

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Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 20705031), the Education Bureau of Fujian Province (No. JK2011030), the Science Research Project of Zhangzhou Normal University (No. SX1006), and funded by the Base for Postgraduate Student Educational Innovation of Zhangzhou Normal University. References [1] R. Cirilli, R. Ferretti, B. Gallinella, L. Zanitti, F. La Torre, J. Chromatogr. A 1061 (2004) 27. [2] F.P. Zhan, G.Y. Yu, B.X. Yao, X.P. Guo, T. Liang, M.G. Yu, Q.L. Zeng, W. Weng, J. Chromatogr. A 1217 (2010) 4278. [3] B. Koppenhöfer, E. Bayer, Chromatographia 19 (1984) 123. [4] W.H. Pirkle, P.G. Murray, J. High Res. Chromatogr. 16 (1993) 285. [5] K. Fulde, A.W. Frahm, J. Chromatogr. A 858 (1999) 33. [6] M. Schlauch, A.W. Frahm, Anal. Chem. 73 (2001) 262. [7] A. Aranyi, I. Ilisz, Z. Pataj, I. Szatmári, F. Fülöp, A. Péter, J. Chromatogr. A 1218 (2011) 4869. [8] L. Chankvetadze, N. Ghibradze, M. Karchkhadze, L. Peng, T. Farkas, B. Chankvetadze, J. Chromatogr. A 1218 (2011) 6554. [9] K.S.S. Dossou, P.A. Edorh, P. Chiap, B. Chankvetadze, A.-C. Servais, M. Fillet, J. Crommen, J. Sep. Sci. 34 (2011) 1772. [10] M. Okamoto, J. Pharm. Biomed. Anal. 27 (2002) 401. [11] M. Okamoto, H. Nakazawa, J. Chromatogr. 588 (1991) 177. [12] K. Balmér, B.-A. Persson, P.-O. Lagerström, J. Chromatogr. A 660 (1994) 269. [13] O. Gyllenhaal, M. Stefansson, J. Pharm. Biomed. Anal. 46 (2008) 860. [14] B.A. Olsen, D.D. Wirth, J.S. Larew, J. Pharm. Biomed. Anal. 17 (1998) 623. [15] S. Svensson, J. Vessman, A. Karlsson, J. Chromatogr. A 839 (1999) 23. [16] B.-A. Persson, S. Andersson, J. Chromatogr. A 906 (2001) 195. [17] T. Wang, Y.W. Chen, J. Chromatogr. A 855 (1999) 411. [18] T. Wang, Y.W. Chen, A. Vailaya, J. Chromatogr. A 902 (2000) 345. [19] T. Wang, R.M. Wenslow, J. Chromatogr. A 1015 (2003) 99. [20] S. Ma, S. Shen, H. Lee, M. Eriksson, X. Zeng, J. Xu, K. Fandrick, N. Yee, C. Senanayake, N. Grinberg, J. Chromatogr. A 1216 (2009) 3784. [21] S. Ma, S. Shen, H. Lee, N. Yee, C. Senanayake, L.A. Nafie, N. Grinberg, Tetrahedron: Asymmetry 19 (2008) 2111. [22] C. Roussel, J.-L. Stein, F. Beauvais, A. Chemlal, J. Chromatogr. 462 (1989) 95. [23] L. Asnin, Pharm. Chem. J. 42 (2008) 435. [24] L. Asnin, K. Horváth, G. Guiochon, J. Chromatogr. A 1217 (2010) 1320. [25] Y.-C. Guillaume, C. André, Talanta 76 (2008) 1261. [26] C.F. Zhao, S. Diemert, N.M. Cann, J. Chromatogr. A 1216 (2009) 5968. [27] Y. Liu, H. Zou, J. Chromatogr. A 1178 (2008) 118. [28] B.X. Yao, F.P. Zhan, G.Y. Yu, Z.F. Chen, W.J. Fan, X.P. Zeng, Q.L. Zeng, W. Weng, J. Chromatogr. A 1216 (2009) 5429. [29] W. Lao, J. Gan, J. Chromatogr. A 1217 (2010) 6545. [30] J. Samuelsson, R. Arnell, T. Fornstedt, Anal. Chem. 78 (2006) 2765. [31] J. Lindholm, T. Fornstedt, J. Chromatogr. A 1095 (2005) 50. [32] J. Samuelsson, R. Arnell, T. Fornstedt, J. Sep. Sci. 32 (2009) 1491. [33] T. Fornstedt, J. Chromatogr. A 1217 (2010) 792. [34] P. Forssén, R. Arnell, M. Kaspereit, A. Seidel-Morgenstern, T. Fornstedt, J. Chromatogr. A 1212 (2008) 89. [35] P. Forssén, R. Arnell, T. Fornstedt, J. Chromatogr. A 1216 (2009) 4719.