Anal. Chem. 2008, 80, 1824-1828
Prediction for the Separation Efficiency of a Pair of Enantiomers during Chiral High-Performance Liquid Chromatography Using a Quartz Crystal Microbalance Shinsuke Inagaki, Jun Zhe Min, and Toshimasa Toyo’oka*
Division of Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
A simple and rapid screening method of the chiral stationary phase during high-performance liquid chromatography (HPLC) utilizing a quartz crystal microbalance (QCM) has been developed for the chiral separation of a pair of enantiomers. The outline of the method is as follows: a self-assembled monolayer (SAM) is constructed on the gold electrodes of the QCM sensor chips by utilizing the interaction between thiols and gold. The chiral selectors used as chiral stationary phases in the HPLC are then immobilized, and a pseudostationary phase is constructed on the electrodes. Subsequently, the sensors are equilibrated in the solutions, the targeted chiral samples are injected, and the frequency changes are observed. Four kinds of chiral molecules and three kinds of chiral stationary phases were examined in this study. When chiral separation is possible using the chiral stationary phase immobilized on the sensors, significant differences in the frequency changes are observed because the intensities based on interactions differ among the isomers. The developed method can predict not only the possibility for chiral separation but also the elution order from the chiral stationary phase column. Furthermore, the degree of the mutual separation of a pair of enantiomers seems to be roughly predictable from the difference in the frequency change (∆F) and first-order association rate constant (kobs). The method does not require several different kinds of chiral columns that are more expensive than achiral ones such as the octadecylsilica (ODS) column. The required amounts of the chiral stationary phases are extremely small, and the sensors with immobilized chiral selectors are reusable. In addition, the method requires only a few minutes to complete the analysis. Thus, considerable reductions in both cost and time are realized. By applying the developed method to many chiral molecules and chiral stationary phases, its superiority may be corroborated; thus, it is expected that the method can be effectively used for the selection of chiral stationary phases.
* To whom correspondence should be addressed. Phone: +81-54-264-5656. Fax: +81-54-264-5593. E-mail:
[email protected].
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In general, the physical and chemical characteristics of a pair of enantiomers in chiral molecule are identical; however, their behaviors in vivo and their physiological functions sometimes differ. During the development of drugs that have chiral center(s) in their chemical structures, the evaluation of the pharmacological and toxicological properties is essential. Therefore, the development of a separation method for each enantiomer is necessary to facilitate the generation of pharmacokinetic (PK) and toxicokinetic (TK) data on new chiral drug candidates. Thus, “chiral separation” is a very important task as same as “chiral synthesis”. To separate the chiral isomers, high-performance liquid chromatography (HPLC) is one of the most useful and standardized methods.1,2 A number of chiral stationary phases for the separation of enantiomers in drugs and biological compounds have already been developed, and their use is presently widespread. However, to perform chiral separations of the target enantiomers by HPLC, the chiral stationary phase must be selected through a trial-and-error process based solely on information gleaned from prior experience; thus, multiple iterations are inevitable. More specifically, a number of chiral columns that are more expensive than achiral ones (e.g., octadecylsilica (ODS) columns) must be purchased and prepared. Moreover, to obtain a good resolution, the analytical conditions (e.g., the composition of the mobile phase and the flow rate) must be optimized. These operations are laborious, time-consuming, and expensive. Consequently, the methods by which these operations can be efficiently and economically performed would be very useful, and their development is crucial. A quartz crystal microbalance (QCM), incidentally, is an extremely sensitive mass sensor, capable of measuring subnanogram-level mass changes.3-6 The frequency of the QCM vibration depends on the parameters associated with the phases adjacent (1) Ward, T. J. Anal. Chem. 2006, 78, 3947-3956. (2) Sumika Chemical Analysis Service. Chiral Stationary Phases for Enantiomer Separation by HPLC, 4th ed.; 2004. (3) Dongbo, L.; Bingjun, H.; Songyan, H.; Shenqi, W.; Qingping, L.; Anzai, J.; Osa, T.; Qiang, C. Mater. Sci. Eng., C 2007, 27, 665-669. (4) Marx, K. A. Biomacromolecules 2003, 4, 1099-1120. (5) Nakanishi, K.; Muguruma, H.; Karube, I. Anal. Chem. 1996, 68, 16951700. (6) Uttenthaler, E.; Koblinger, C.; Drost, S. Anal. Chim. Acta 1998, 362, 91100. 10.1021/ac702031b CCC: $40.75
© 2008 American Chemical Society Published on Web 01/24/2008
to the QCM and also on the physical properties of the QCM itself.7 The resonance frequency decreases with an increase in the amount of material on the QCM surface in accordance with Sauerbrey’s equation.8 The QCM has been used for various purposes in the interaction analysis of many biological samples such as DNA, peptides, proteins, and oligosaccharides.9-13 However, using a QCM is a new analytical method for the evaluation of adsorption behaviors. By applying these QCM techniques, a rapid, simple, and lowcost screening system for selecting the appropriate chiral stationary phase for HPLC would be possible.14 Chiral discrimination using QCM with β-cyclodextrin, which is provided as a representative chiral stationary phase,15,16 and comparison with gas chromatographic retention data have already been reported.17-21 However, few reports have used other chiral stationary phases for HPLC. In this paper, we present a novel evaluation method for chiral stationary phases using a QCM. In detail, chiral selectors were immobilized on sensor electrodes using amido-coupling reactions and a pseudostationary phase was constructed. The targeted chiral molecules were then injected into the analytical solutions, and the frequency changes were observed. When chiral separation is possible using the chiral stationary phase immobilized on the sensors, because the intensity of the interaction differs among enantiomers, significant differences in the frequency changes are observed, which make the evaluation possible. We considered that the application for the rapid screening of chiral stationary phases would be possible, which is otherwise one of the most time-consuming steps in chiral separation using HPLC. EXPERIMENTAL SECTION Materials and Reagents. Three types of chiral selectors, cyclomaltoheptosyl-(6f1)-O-R-D-glucopyranosyl-(4f1)-O-R-D-glucopyranosiduronic acid (CGGA), N-(3,5-dinitrophenylaminocarbonyl)-D-phenylglycine (DNPAP), and N-[(R)-1-(R-naphthyl)ethylaminocarbonyl]-L-tert-leucine (NEACL), were kindly donated from Sumika Chemical Analytical Service, Ltd. (Osaka, Japan). Their chemical structures are shown in Figure 1. Sulfuric acid (H2SO4), aminoethanethiol, N-hydroxysuccinimide, and dimethyl sulfoxide (DMSO) were purchased from Wako Pure Chemicals (Osaka, Japan). The hydrogen peroxide (H2O2) and ethanol were obtained from Kanto Chemicals (Tokyo, Japan). 1-(3-Dimethy(7) Bunde, R. L.; Jarvi, E. J.; Rosentreter, J. J. Talanta 2000, 51, 159-171. (8) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (9) Caruso, F.; Furlong, D. N.; Niikura, K.; Okahata, Y. Colloids Surf., B 1998, 10, 199-204. (10) Rickert, J.; Brecht, A.; Gopel, W. Biosens. Bioelectron. 1997, 12, 567-575. (11) Ito, K.; Hashimoto, K.; Ishimori, Y. Anal. Chim. Acta 1996, 327, 29-35. (12) Morgan, C. L.; Newman, D. J.; Price, P. C. Clin. Chem. 1996, 42, 193209. (13) Rogers, K. R. Biosens. Bioelectron. 1995, 10, 533-541. (14) Toyo’oka, T.; Inagaki, S.; Suzuki, Y. Jpn. Kokai Tokkyo Koho JP 2007193462, 2007. (15) Armstrong, D. W.; Ward, T. J.; Armstrong, R. D.; Beesley, T. E. Science 1986, 232, 1132-1135. (16) Armstrong, D. W.; DeMond, W. J. Chromatogr. Sci. 1984, 22, 411-415. (17) Ng, S.-C.; Sun, T.; Chan, H. S. O. Macromol. Symp. 2003, 192, 171-181. (18) Ng, S.-C.; Sun, T.; Chan, H. S. O. Tetrahedron Lett. 2002, 43, 2863-2866. (19) Fietzk, C.; Hermle, T.; Rosenstiel, W.; Schurig, V. Fresenius’ J. Anal. Chem. 2001, 371, 58-63. (20) Weif, T.; Leipert, D.; Kaspar, M.; Jung, G.; Gopel, W. Adv. Mater. 1999, 11, 331-335. (21) May, I. P.; Byfield, M. P.; Lindstrom, M.; Wunsche, L. F. Chirality 1997, 9, 225-232.
Figure 1. Chemical structures of the chiral selectors used.
laminopropyl)-3-ethylcarbodiimide‚hydrochloride (EDC) was purchased from Aldrich (Milwaukee, WI). Naproxen (NAP; R- and S-forms), N-acetylphenylalanine (APA; D- and L-forms), and BOCphenylalanine (BOCPA; D- and L-forms) were obtained from Wako Pure Chemicals. 1,1′-Bi-2-naphthol (BNP; R- and S-forms) was purchased from Kanto Chemicals. Distilled water was produced by an AQUARIUS GSR-200 system (Advantec, Tokyo, Japan) and purified by an AQUARIUS PWU-200 system (Advantec). Apparatus. The QCM analysis was carried out using an Initium AffinixQ system (Initium, Inc., Tokyo, Japan). The system consisted of a QCM sensor, a reaction bath (11 mL), an oscillation detector, and a notebook computer. A 27 MHz AT-cut quartz crystal with gold electrodes (diameter, 2.5 mm; area, 4.9 mm2) was driven by an oscillator circuit. The diameter of the quartz plate was 8 mm, with gold electrodes deposited on each side. One side of the quartz crystal was exposed to the aqueous buffer solution. However, the other side was sealed in a rubber casing in order to prevent both sides of the sensor from coming into contact with the ionic aqueous solution (Figure 2). Immobilization of Chiral Selectors on the QCM Sensor. The gold surface of the QCM sensor was cleaned by exposure to a piranha solution (97% H2SO4/30% H2O2 ) 3:1) for 5 min, followed by rinsing with distilled water. After drying the gold surface in air, these procedures were repeated twice. The cleaned gold QCM sensor electrode was soaked in an aqueous solution of 5 mM aminoethanethiol for 30 min at room temperature, then rinsed with distilled water, and finally dried in air. At the same time, 300 µL of the chiral selector in a DMSO solution (3 mg/mL) was reacted with 30 µL of N-hydroxysuccinimide (100 mg/mL) and 30 µL of EDC (100 mg/mL) for 30 min to activate the carboxyl groups of the chiral selector. This solution was deposited on the sensor electrode for 1 h at room temperature. The sensor with Analytical Chemistry, Vol. 80, No. 5, March 1, 2008
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Figure 2. Reaction bath in the QCM instrument.
the immobilized chiral selector was then rinsed with distilled water and dried in air again. Determination of Frequency. The sensor was stabilized in 8 mL of distilled water at 25 °C for approximately 20 min. Exactly 8 µL of the sample solution (5 mg/mL) was then injected, and the frequency change was observed (Figure 2). The first-order association rate constant (kobs) was estimated by the equation: kobs ) 0.693/t1/2, where t1/2 is the time when the frequency was half that compared to the maximum one. The separation factors (R) were estimated by the following equation: R ) (tR1 - t0)/(tR2 t0), where tR1 and tR2 are the retention times of a pair of enantiomers (tR1 > tR2) and t0 is the elution time of the mobile phase. RESULTS AND DISCUSSION Figure 1 shows the chemical structures of the chiral stationary phases, and Figure 2 shows the reaction bath in the QCM instrument. A self-assembled monolayer (SAM) was constructed on the gold electrode of the QCM sensor chip using the interaction between the thiol and gold. Thiols are known to form stable SAMs due to the strong S-Au covalent bond.22 Thus, the chiral selector was immobilized using the amido-coupling reaction, and the pseudostationary phases were constructed on the electrode. Figure 3 shows the method used to immobilize them on the QCM sensor electrode. Subsequently, the sensor was equilibrated in the water, the targeted chiral molecule was injected, and the frequency change was observed. Differences among the values of these changes for different enantiomers indicated that chiral separation by HPLC could be achieved using the stationary phase immobilized on the QCM sensor chips. Figure 4 shows the frequency changes of the sensor immobilized with the chiral selector upon injection of the chiral molecules. With the use of CGGA as the chiral selector and NAP as the chiral molecule, the frequency immediately decreased after the injection of the samples and exhibited almost constant values for 4 min after the injection. The average frequency decreases 4 min after the injection were 50.5 Hz for the (S)-NAP and 70.1 Hz for the (R)-NAP. No frequency changes occurred when blank (22) Akram, M.; Stuart, M. C.; Wong, D. K. Y. Anal. Chim. Acta 2004, 504, 243-251.
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Figure 3. Immobilization scheme for the chiral selector.
solutions were injected, and the frequency changes were found to depend on the sample concentrations and their injection volumes. In addition, when using the aminoethanethiol-modified electrode that did not contain the immobilized CGGA, the frequency slightly decreased after the sample injection, but no difference in the frequency changes were observed between the R- and S-forms (data not shown). As shown in Figure 4B-D, the frequency changes were also observed using BNP, APA, and BOCPA as the chiral molecules (Figure 4B-D, respectively). Figure 5 compares the frequency changes of the sensors immobilized with the chiral selector after injection of the chiral molecules. With the use of NAP, BNP, and APA as chiral molecules, significant differences in frequency changes were clearly observed between both enantiomers. These results corresponded with the elution order obtained from a chiral selectormodified column. On the other hand, no significant difference was observed for this method using BOCPA as the chiral molecule for which the separation factor was low using the NEACL-modified column (R ) 1.11). Table 1 indicates the estimated first-order association rate constant (kobs) between the chiral molecules and the chiral selectors on the QCM sensor chips. The kobs values between a pair of enantiomers tend to be high when the frequency changes were large, that is, the interactions with the chiral selectors were strong. Furthermore, the greater their difference is, the larger are the separation factors in the HPLC analysis. On the other hand, the differences in the kobs values are negligible when the separation factors by the HPLC analysis using chiral columns were small. These observations indicate that the results obtained by the QCM
Figure 4. Online monitoring of frequency on the sensor immobilized with the chiral selector. Conditions: chiral selectors are (A) CGGA, (B) DNPAP, and (C and D) NEACL; chiral molecules are (A) (R)-NAP (solid lines) and (S)-NAP (dashed lines), (B) (R)-BNP (solid lines) and (S)BNP (dashed lines), (C) D-APA (solid lines) and L-APA (dashed lines), and (D) D-BOCPA (solid lines) and L-BOCPA (dashed lines). Table 1. Mean Frequency Changes, First-Order Association Rate Constants (kobs), and the Separation Factors QCM chiral molecule
chiral selector
frequency decrease F/Hz
(R)-BNP (S)-BNP
DNPAP
(R)-NAP (S)-NAP
HPLC
∆F/Hz
kobs/s-1
∆kobs/s-1
separation factor (R)
elution time
27.9 59.5
21.6
1.96 × 10-1 2.34 × 10-1
3.8 × 10-2
1.84 (1)a
RS
D-APA L-APA
NEACL
59.6 73.7
14.1
1.13 × 10-2 1.20 × 10-2
7.1 × 10-4
1.38 (3)a
D
L
a The mobile phases used for HPLC analysis were as follows: (1) hexane/2-propanol/methanol ) 70:20:10, (2) 40% acetonitrile in 20 mmol L-1 potassium phosphate buffer pH 3.0, (3) hexane/2-propanol/methanol/TFA ) 90:5:5:0.2, (4) hexane/2-propanol/methanol/TFA ) 98:1:1:0.2. The flow rate of the mobile phase was 1.0 mL/min. The size of the column was 4.6 mm i.d. × 250 mm. The UV absorbance was monitored at 254 nm (2).
method have a close relation to that of the HPLC analysis. Furthermore, the rough speculation of the elution orders and the degree of separation efficiency seem to be possible with the proposed method. CONCLUSION This study showed significant possibilities for using the QCM method as a rapid and convenient screening technique for the appropriate stationary phase in the separation of enantiomers. The developed method can predict not only the possibility for chiral separation but also the elution order from the chiral column. The larger difference between the frequency changes indicated higher separation factors in the HPLC analysis using a chiral column. On the contrary, we have confirmed that differences in the frequency change cannot be observed when the chiral separation is impossible or the separation factor is extremely low. The present method does not require several kinds of chiral columns, and moreover, the required amount of the chiral stationary phase is extremely small. Furthermore, the sensors can be reused after
Figure 5. Comparison of the frequency (mean ( SD) at 240 s after the sample injections (/ p < 0.01, // p < 0.05). The conditions are defined as in Figure 4.
cleaning with a piranha solution and reconstructing pseudostationary phases. Even though the immobilization of the sensors and their stabilization are time-consuming steps, the time required for the analysis is on the order of only a few minutes. In Analytical Chemistry, Vol. 80, No. 5, March 1, 2008
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comparison with the optimization of the HPLC conditions using a chiral column, significant reductions in both time and cost are possible. By applying the method to many more chiral molecules and chiral stationary phases, the efficiency of the method may be determined; thus, it is expected to be used for the screening and selecting of chiral stationary phases in actual practice. ACKNOWLEDGMENT The authors are grateful to Initium, Inc., for the loan of the AffinixQ system. Thanks are also due to Sumika Chemical
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Analytical Service, Ltd. for the generous gift of several chiral molecules. This work was supported in part by the Global COE Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Received for review September 29, 2007. Accepted December 13, 2007. AC702031B
