Characterization of a Thermally Induced Irreversible Conformational

Sep 20, 2003 - Department of Analytical Research, Merck Research Laboratories, Merck & Co., Inc., P.O. Box 2000, RY818 B-208, Rahway, New Jersey 07065...
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Anal. Chem. 2003, 75, 5877-5885

Characterization of a Thermally Induced Irreversible Conformational Transition of Amylose Tris(3,5-dimethylphenylcarbamate) Chiral Stationary Phase in Enantioseparation of Dihydropyrimidinone Acid by Quasi-Equilibrated Liquid Chromatography and Solid-State NMR Fang Wang,* Robert M. Wenslow, Jr.,* Thomas M. Dowling, Karl T. Mueller,† Ivan Santos, and Jean M. Wyvratt

Department of Analytical Research, Merck Research Laboratories, Merck & Co., Inc., P.O. Box 2000, RY818 B-208, Rahway, New Jersey 07065

A thermally induced irreversible conformational transition of amylose tris(3,5-dimethylphenylcarbamate) (i.e., Chiralpak AD) chiral stationary phase (CSP) in the enantioseparation of dihydropyrimidinone (DHP) acid racemate was studied for the first time by quasi-equilibrated liquid chromatography with cyclic van’t Hoff and step temperature programs and solid-state (13C CPMAS and 19F MAS) NMR using ethanol and trifluoroacetic acid (TFA)-modified n-hexane as the mobile phase. The conformational transition was controlled by a single kinetically driven process, as evidenced by the chromatographic studies. Solid-state NMR was used to study the effect of the temperature on the conformational change of the solvated phase (with or without the DHP acid enantiomers and TFA) and provided some viable structural information about the CSP and the enantiomers. The derivatized cellulose and amylose chiral stationary phases (CSPs) are widely used phases for LC enantioseparation.1-4 Column temperature is one of the critical parameters used to control enantioseparation and is a useful tool to probe the separation mechanism. Application of van’t Hoff plots (logarithm of retention or selectivity factors versus the reciprocal of absolute temperature) provides important information about the separation mechanism.5,6 Linear van’t Hoff plots indicate the same separation mechanism throughout the temperature range. Nonlinear van’t Hoff plots indicate that there are some changes in either the conformations of compounds or stationary phases, their interac* Corresponding authors. E-mail: [email protected]; robert_wenslow@ merck.com. † Current address: 152 Davey Laboratory, The Pennsylvania State University, University Park, PA 16802. (1) Okamoto, Y.; Kaida, Y. J. Chromatogr., A 1994, 666, 403-419. (2) Yashima, E.; Yamamoto, C.; Okamoto, Y. Synlett 1998, 344-360. (3) Okamoto, Y.; Yashima, E. Angew. Chem., Int. Ed. 1998, 37, 1020-1043. (4) Yashima, E. J. Chromatogr., A 2001, 906, 105-125. (5) Pirkle, W. H. J. Chromatogr. 1991, 558, 1-11. (6) Pirkle, W. H.; Murray, P. G. J. High Resolut. Chromatogr. 1993, 16, 285288. 10.1021/ac034714e CCC: $25.00 Published on Web 09/20/2003

© 2003 American Chemical Society

tions, or combinations therein. In both linear and nonlinear van’t Hoff plot cases, the general assumption for van’t Hoff plots is that the separation process is under equilibrium conditions. This assumption is based on the fact that such conformational changes are typically much faster than the time scale of the separation process so that the interactions between the compounds and the CSPs are considered under thermodynamic equilibrium. Therefore, column temperature programing (either heating or cooling) to reach a given temperature will not affect retention or selectivity factors. Recently, we reported the nonlinear van’t Hoff plots of dihydropyrimidinone (DHP) acid racemate on amylose tris(3,5dimethylphenylcarbamate) CSP (Chiralpak AD) with 15% ethanol and 0.1% trifluoroacetic acid (TFA)-modified n-hexane mobile phase.7 The van’t Hoff plots of retention factor and selectivity factor were not superimposable when the column underwent a cyclic heating (5 f 50 °C first)/cooling (50 f 5 °C second) program. On the basis of these preliminary results, we concluded that Chiralpak AD underwent a thermally induced irreversible conformational transition during the heating process. It was not clear whether there was any other transition(s) during the cooling process because the cooling curve was also nonlinear. To the best of our knowledge, the application of the cyclic van’t Hoff and step temperature programs to study such thermally induced transitions of the CSPs has not been previously reported. To date, understanding of the interactions between derivatized cellulose and amylose CSPs and chiral compounds at the molecular level is still very limited. Solid-state NMR is a noninvasive analytical tool for elucidation of structural information on molecular conformation, crystal modification, molecular dynamics, and molecular adjacency.8 It has been used as a viable technology to study the achiral stationary phases in reversed-phase LC.9-14 Solid(7) Wang, F.; O’Brien, T.; Dowling, T.; Bicker, G.; Wyvratt, J. J. Chromatogr., A 2002, 958, 69-77. (8) Voelkel, R. Angew. Chem., Int. Ed. Engl. 1988, 27, 1468-1483. (9) Sentell, K. B. J. Chromatogr., A 1993, 656, 231-263. (10) Pursch, M.; Sander, L. C.; Albert, K. Anal. Chem. 1999, 71, 733A-741A. (11) Pursch, M.; Strohschein, S.; Handel, H.; Albert, K. Anal. Chem. 1996, 68, 386-393.

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state 13C CPMAS NMR and Raman spectroscopy were used to study the polymorphs of cellulose triacetate (CTA) from different preparation methods.15 A 13C CPMAS NMR study by the column vendor showed that the coating solvents for the CTA packing determined the structure of the CSPs.16 The structure of the coated CTA polymers did not change even though the coating solvent was replaced by methanol. Recent 13C CPMAS NMR studies showed that the alcohol modifiers formed complexes with Chiralpak AD.17 The conformations of Chiralpak AD depended on the mobile-phase modifiers. At low modifier concentration, the phase crystallinity increased with an increase in the modifier’s concentration. Then, the phase was saturated by the modifiers at higher concentration. The incorporation of 2-propanol into the phase provided a more distinct/ordered conformation than that of ethanol. The authors concluded that the reversal of the elution orders of the racemates on the same CSP by changing the alcohol modifiers was caused by the change in the solvated conformations of Chiralpak AD. Mobile-phase additives also play an important role in LC enantioseparations because they can minimize peak broadening (caused by some undesired interactions between stationary phases and analytes) and change either retention factors or enantioselectivity factors. It is well recognized that the incorporation of mobile-phase additives into CSPs occurs in LC enantioseparations.18-27 The incorporation of additives has a memory effect on CSPs under certain conditions. However, this level of understanding has been indirect based on the chromatographic data. To date, research by solid-state NMR on CSPs is still at a very early stage with limited published reports. To the best of our knowledge, the effect of the temperature on the solvation of CSPs has not been studied. The interactions between CSPs and chiral compounds and the influence of the temperature on such interactions have not been reported. There has been no direct evidence at the molecular level to show the interactions between CSPs and mobilephase additives. Nevertheless, there has also been no evidence of the additive’s roles in the incorporation of analytes into CSPs to change enantioselectivity. (12) Albert, K.; Lacker, T.; Raitza, M.; Pursch, M.; Egelhaaf, H. J.; Oelkrug, D. Angew. Chem., Int. Engl. 1998, 37, 777-780. (13) Zeigler, R. C.; Maciel, G. E. J. Am. Chem. Soc. 1991, 113, 6349-6358. (14) Pursch, M.; Vanderhart, D. L.; Sander, L. C.; Gu, X.; Nguyen, T.; Wise, S. A.; Gajewski, D. A. J. Am. Chem. Soc. 2000, 122, 6997-7011. (15) VanderHart, D. L.; Hyatt, J. A.; Atalla, R. H.; Tirumalai, V. C. Macromolecules 1996, 29, 730-739. (16) Sei, T.; Matsui, H.; Shibata, T.; Abe, S. In Viscoelasticity of Biomaterials; Glasser, W. G., Hatakeyama, H., Eds.; ACS Symposium Series 489; American Chemical Society: Washington, DC, 1992; pp 53-64. (17) Wenslow, R. M., Jr.; Wang, T. Anal. Chem. 2001, 73, 4190-4195. (18) Okamoto, Y.; Kawashima, M.; Aburatani, R.; Hatada, K.; Nishiyama, T.; Masuda, M. Chem. Lett. 1986, 1237-1240. (19) Okamoto, Y.; Aburatani, R.; Hatada, K. J. Chromatogr. 1988, 448, 454455. (20) Okamato, Y.; Aburatani, R.; Kaida, Y.; Hatada, K. Chem. Lett. 1988, 11251128. (21) Okamato, Y.; Aburatani, R.; Kaida, Y.; Hatada, K.; Inotsume, N.; Nakano, M. Chirality 1989, 1, 239-242. (22) Ushio, T.; Yamamoto, Y. J. Chromatogr. 1994, 684, 235-242. (23) Tang, Y. Chirality 1996, 8, 136-142. (24) Tang, Y.; Zielinski, W.; Bigott, Y. Chirality 1998, 10, 364-369. (25) Ye, Y. K.; Lord, B.; Stringham, R. W. J. Chromatogr., A 2002, 945, 139146. (26) Ye, Y. K.; Stringham, R. W. J. Chromatogr., A 2001, 927, 47-52. (27) Ye, Y. K.; Stringham, R. W. J. Chromatogr., A 2001, 927, 53-60.

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EXPERIMENTAL SECTION Materials. Ethanol, 2-propanol, and n-hexane were HPLC grade and purchased from EM Science (Gibbstown, NJ). 1,3,5Tri-tert-butylbenzene (t0 marker) was purchased from Aldrich (Milwaukee, WI). Ethyl trifluoroacetate was also purchased from Aldrich (Milwaukee, WI). TFA was purchased from Fisher Scientific (Springfield, NJ). DHP acid and methyl ester racemates and pure enantiomers were prepared by the Process Research Department, Merck Research Laboratories (Rahway, NJ).28 Chiralpak AD (amylose tris(3,5-dimethylphenylcarbamate)) column (25 cm × 4.6 mm) was purchased from Chiral Technologies (Exton, PA). Chromatographic Conditions. All chromatographic experiments were performed on an Agilent 1100 system with a column oven and a photodiode array detector (Santa Carita, CA). The detector wavelength was set up at 220 nm with a 4-nm bandwidth. The mobile phases were prepared at ambient temperature by diluting the alcohol modifiers and TFA in n-hexane. The water content of the mobile phases was determined by a 756 Brinkmann coulometric KF instrument (Westbury, NY). Samples and the t0 marker were prepared together in the mobile phases to achieve the desired concentration. A 10-µL volume of each sample was injected. The flow rate was 1.0 mL/min. Temperature Programs. Cyclic van’t Hoff temperature program: the column temperature changed from 5 to 50 °C by a 5 °C interval during the (re-)heating/(re-)cooling processes. After each temperature change, the column was equilibrated with the mobile phase for 1 h before injections. Step temperature programs: in a single-step temperature program, the column temperature was kept at the initial temperature for 7 h with injections of the samples. Then, the temperature was immediately stepped up to 50 °C. The column was equilibrated with the mobile phase for 1.5 h before injections (because the temperature change interval was larger than that of the cyclic van’t Hoff programs). In a two-step temperature program, the first step was the same as the single-step program. After several injections of DHP acid at 50 °C, the column temperature stepped back to the initial temperature immediately. The column was equilibrated with the mobile phase for another 1.5 h before injections. Solid-State NMR. All solid-state experiments were performed on a Bruker DSX-400 NMR spectrometer. The 13C, 1H, and 19F resonance frequencies are 100.6, 400.1, and 376.5 MHz, respectively, at this magnetic field strength. Chiralpak AD packing material was flushed by the mobile phases and then removed from the stainless steel hardware for NMR measurement as previously reported.17 All 13C CPMAS experiments were performed using a Bruker double-resonance, variable-temperature CPMAS probe and a standard variable-amplitude CP pulse sequence. A contact time of 2.0 ms was utilized, 4K of data points were acquired in 60 ms for each of 3500 acquisitions and then zero-filled to 8K before transformation using 10.0 Hz of line broadening. Recycle delays for the CPMAS experiments were 7.0 s. Rotor frequency was 7.0 kHz. All 13C spectra were referenced to TMS using the carbonyl carbon of glycine (176.03 ppm) as a secondary reference. Temperature cycling included 3500 acquisitions at 20, 35, 50, and 35 °C and then a return to 20 °C, resulting in ∼7 h being spent at each temperature point. (28) Hu, E. H.; Sidler, D. R.; Dolling, U. H. J. Org. Chem. 1998, 63, 3454-3457.

Chart 1. Structure of Amylose Tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) and DHP Acid and Methyl Ester

All 19F MAS experiments were performed using a Bruker CRAMPS probe, 4-mm zirconia rotors with vespel end caps, and a standard block decay sequence. A total of 32 signal acquisitions with a 3-s recycle delay was obtained for each 19F spectrum. Rotor frequencies were 15.0 kHz, and all 19F spectra are referenced to poly(tetrafluoroethylene) (Teflon), which was assigned a chemical shift of -122 pm. T1F data were acquired using a standard pulse sequence. Dummy scans were utilized in temperature-cycling experiments to match CPMAS spectra for the time spent at each temperature (∼7 h at each temperature point). The physical mixtures of DHP acid enantiomers and Chiralpak AD were made by adding the enantiomers (∼20 mg) into 7-mL vials containing the packing material equilibrated by the mobile phase (slurry, ∼60 mg). One milliliter of the mobile phase was added into the vials after mixing the enantiomer and the phase. The vials were capped and rotated on a Glas-Col 099A Lab Rotator (Terre Haute, IN) overnight. The mixtures were dried by nitrogen into wet pastes for NMR measurement. RESULTS AND DISCUSSION Chromatographic Investigations. Although the thermally induced reversible conformational changes of xanthan and derivatized celluloses in the solution state were reported before,29-31 an irreversible conformation transition induced by the column temperature on a coated CSP (shown in Chart 1) in enantioseparation was first reported recently.7 From our preliminary chromatographic data, it was not clear whether the conformational transition was a thermodynamically or kinetically controlled process. The objective of our current studies is to investigate the effect of the temperature on the chromatographic behavior and to determine whether the thermally induced conformational change is a kinetically or thermodynamically controlled process. Cyclic van’t Hoff Temperature Programs. Figure 1 shows the effect of the cyclic van’t Hoff temperature programs on the (29) Gupta, A. K.; Burchard, W. Macromolecules 1975, 8, 843-849. (30) Maissa, P.; Seurin, M.; Sixou, P. Polym. Bull. 1986, 15, 257-263. (31) Liu, W.; Sato, T.; Norisuye, T.; Fujita, H. Carbohydr. Res. 1987, 160, 267281.

Figure 1. Cyclic van’t Hoff plot of the ratio of apparent retention factors of DHP acid on Chiralpak AD column. Programs: (A) heating cycles, solid lines, heating (open circle)/cooling (open triangle)/ reheating (open square)/recooling (open diamond); (B) cooling cycles, dashed lines, cooling (solid circle)/heating (solid triangle)/recooling (square)/reheating (empty diamond). Mobile phase: 15% ethanol + 0.1% TFA in n-hexane.

profile of the ratio of apparent retention factors (Rapp ) (k′S/k′R)app, where k′S and k′R are apparent retention factors for (S)-(+)-DHP acid and (R)-(-)DHP acid, respectively) in the enantioseparation of DHP acid on Chiralpak AD. In our previous study, only one heating (5 f 50 °C)/cooling (50 f 5 °C) cycle was performed on Chiralpak AD. An additional heating/cooling cycle was applied in this study. During the first heating process (open circle) of the two heating/cooling cyclic programs (solid lines in Figure 1A), the ratio of apparent retention factors increased slightly when the temperature was below the conformational transition temperature (∼30 °C). However, the ratio increased rapidly with increasing temperature when the column temperature was above the transition temperature. The unusual nonlinear curve indicated that the conformation of the CSP changed during the heating process. In the first cooling process (open triangle), the ratio of apparent retention factors increased with decreasing temperature when it was above the transition temperature. Then, it decreased slightly with the further decrease in the temperature. Both the heating and cooling curves were nonlinear. It is difficult to conclude if there was any additional conformational transition(s) in this cooling process. In the reheating process (open square), the overall ratio did not change much with the temperature, and the curve overlaid well with the first cooling process when the temperature was below the transition temperature. This indicated that the chromatographic processes were under thermodynamic equilibrium when the column temperature was below the transition temperature regardless of how a given temperature was Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

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reached. However, the high-temperature portion of the reheating curve did not overlay with the first cooling process when the column temperature was above the transition temperature. This is another indication of the existence of the kinetically controlled process(es). The recooling curve (open diamond) was basically a repeat of the first cooling curve but with a different intercept. In the two cooling/heating cyclic van’t Hoff programs (Figure 1B), the starting column temperature (50 °C) was over the transition temperature. The conformation of Chiralpak AD was changed to the higher temperature conformation before DHP acid racemate was injected onto the column. Therefore, the curves of the ratio of apparent retention factors were similar to those of cooling/reheating/recooling curves in the two-heating/cooling temperature program (Figure 1A). However, the magnitude of the ratio of apparent retention factors in the two-cooling/heating program was smaller than that in the two-heating/cooling program. All the curves in the two-cooling/heating program were nonlinear and nonsuperimposable with those in the two-heating/ cooling program, which indicates that the conformational transition is also irreversible. Step Temperature Programs. The nonlinear and nonsuperimposable curve profiles in the cyclic van’t Hoff temperature programs suggest that the conformational transition of the CSP is irreversible. However, it is still not clear whether the irreversible transition was controlled by thermodynamic or kinetic processes. To the best of our knowledge, no chromatographic study has shown the kinetically controlled conformational transitions on both coated cellulose and amylase CSPs so far. A literature survey shows that a chiral polymer underwent an irreversible conformational transformation in the solution state (evidenced by specific rotation measurement) after it was annealed.32 After the polymer reached its new conformation, its optical rotation changed reversibly with the change in temperature. For optically inactive polymers, helical conformation can be generated by the addition of chiral compounds. Complexed with optically active compounds, polyguanidine,32 polyphosphazene,33 and polyisocyanides34 resulted in kinetically controlled conformational transitions in solution during annealing. However, optical rotation and circular dichroism are suitable only for solution-phase studies32-34 and cannot be used to determine the conformational change of the helical structure of amylose tris(3,5-dimethylphenylcarbamate) on the surface of the treated silica gel particles. In addition, studying the interactions of the helical macromolecule and analytes in solution does not represent the real chromatographic conditions since the chemistry of column preparations has a pronounced effect on the overall chromatographic behaviors.35 Therefore, a different type of chromatographic experiment is required to provide the essential information about the transition(s). Figure 2 shows the kinetics of apparent retention factors and their ratio after a single temperature jump from 20 to 50 °C. The apparent retention factors for (S)-(+)-DHP acid increased by 45.6% in 24 h while those of (R)-(-)-DHP acid decreased by 4.2% (Figure 2A). The overall ratio of apparent retention factors increased by 52% (Figure 2B). The (32) Schlitzer, D. S.; Novak, B. M. J. Am. Chem. Soc. 1998, 120, 2196-2197. (33) Yashima, E.; Maeda, K.; Yamanaka, T. J. Am. Chem. Soc. 2000, 122, 78137814. (34) Ishikawa, M.; Maeda, K.; Yashima, E. J. Am. Chem. Soc. 2002, 124, 74487458. (35) Francotte, E.; Zhang, T. J. Chromatogr., A 1995, 718, 257-266.

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Figure 2. Apparent retention factors and their ratio of DHP acid as function of time in a single-step temperature program. (A) Apparent retention factors vs time for (S)-(+)-DHP acid (open triangle) and (R)(-)-DHP acid (open circle). (B) Ratio of apparent retention factors vs time for DHP acid. Temperature program, stepped from 20 to 50 °C. Mobile phase, as in Figure 1.

transition had not reached the plateau during this period of time. The ratio of apparent retention factors increased by 4.6% from the first to the second injection. The retention times for the two enantiomers at the first injection were around 9 and 15 min. Compared with the time required for the conformational transition to occur (>24 h), the chromatographic run time is relatively short, and the chromatographic process can be considered as a quasiequilibrated process. Previously, 13C CPMAS NMR was used to investigate the solvation effect of the alcohol modifiers on Chiralpak AD.17 To the best of our knowledge, study on the effect of the column temperature and mobile-phase additives on the derivatized cellulose and amylose CSPs by solid-state NMR has not been reported. Because CPMAS experiments must be averaged by time for adequate sensitivity, and since 7 h of data accumulation will be needed, step-and-hold experiments were performed and monitored by chromatographic separation. To mimic the timing in 13C CPMAS NMR experiments, a two-step temperature program (see the Experimental Section for details) was designed to study this transition. Figure 3 shows the change of the ratio of apparent retention factors with time in the two-step temperature program. At the initial temperature (10 °C), the increase of the ratio of apparent retention factors was only 0.5% over 7 h. After the temperature was stepped to 50 °C, the increase in the ratio was ∼38%. This was caused by the change of Chiralpak AD from the lower temperature conformation to the higher temperature conformation. The further increase of the ratio with time during

Figure 3. Change in ratio of apparent retention factors as function of time in a two-step temperature program. Ratio of apparent retention factors vs time (open circle). Temperature program, solid line. Mobile phase, as in Figure 1.

the hold at 50 °C showed the kinetics of the transition (Figure 2). The interesting observation was that the ratio of apparent retention factors did not change after the temperature was stepped back and held at 10 °C. The magnitude of the ratio of apparent retention factors is still higher than that of the first temperature holding step at 10 °C. This shows that the higher temperature conformation was frozen (without changing back to the lower temperature conformation) below the transition temperature. The ratio increased when the temperature was stepped to 50 °C again. The effect of the starting temperatures on the kinetics showed that as long as the temperature was below the transition temperature, the curves were similar (data not shown). This provides the necessary information for the accuracy of solid-state NMR experiments in the step temperature programs. More importantly, these two-step temperature program data are consistent with the curve profile in the two cyclic van’t Hoff temperature programs in Figure 1. During the heating cycles, the conformation transition occurred over 30 °C. During the cooling cycles, the conformation was frozen as the higher temperature conformation even when the temperature was kept below 30 °C. As long as the mobile phase is pumped through the column, this conformation can be maintained at least for 48 h (data not shown). At the temperatures below the transition temperature, the enantioseparation of DHP acid with this higher temperature conformation was still a thermodynamically controlled process because these parts of the heating/cooling curves were superimposable. The conformational transition continued as long as the temperature stepped above the transition. Combined with the single-step temperature program experiment, it is clear that only one thermally induced conformational transition occurs during the two cyclic van’t Hoff temperature programs even though two nonlinear, nonsuperimposable curves were discovered. Solid-State NMR Investigations. Okamoto and co-workers studied the interactions between some model chiral compounds with the derivatized cellulose and amylose polymers (i.e., the chiral selectors before coated to silica particles) by liquid-phase NMR.36,37 (36) Yashima, E.; Yamada, M.; Yamamoto, C.; Nakashima, M.; Okamoto, Y. Enantiomer 1997, 2, 225-240. (37) Yashima, E.; Yamamoto, C.; Okamoto, Y. J. Am. Chem. Soc. 1996, 118, 4036-4048.

This type of study can provide some information that is suitable for enantioseparation in homogeneous media by a technique such as capillary zone electrophoresis (CZE).38,39 However, the nature of these chiral polymers (such as their charges and solubilities in aqueous media) determines that they are not suitable candidates for CZE enantioseparations. Moreover, even if liquid-phase NMR studies provide some favorable information for the enantioseparation, the immobilization of these chiral polymers can change the CSP’s chromatographic behavior dramatically. A subtle change in the coating chemistry (such as change in the composition of the coating solvents) can cause a large change in the CSP’s performance.35 13C CPMAS NMR. We studied the effect of temperature on Chiralpak AD under different conditions by using a two-step temperature program (from room temperature to 50 °C and back to room temperature); however, we will not discuss these spectra acquired at 50 °C in all 13C CPMAS NMR experiments because the chromatographic data clearly show the kinetic process during a 7-h data acquisition of the carbon spectra. The 13C CPMAS NMR at 50 °C can only provide the averaged results of the conformational transition. First, the dry Chiralpak AD was tested under the two-step temperature program (data not shown). The 13C CPMAS spectra displayed no changes upon cycling through 50 °C and returning to room temperature. Next, Chiralpak AD was flushed with 15% ethanol in n-hexane and tested under the two-step program while still in contact with the liquid phase. The carbon spectra for the phase before and after 50 °C are displayed in Figure 4. The majority of peaks representing the sugar backbone are unaffected by the temperature cycling. However, the C-6 peak appears to be significantly broadened by the temperature cycling (Figure 4B). After the temperature cycle, the peaks representing n-hexane within the stationary phase (14, 23, and 35 ppm) are considerably reduced (relative to their original 20 °C intensity). Additionally, the incorporated ethanol peak (Figure 4B) has been shifted downfield as a result of the temperature cycle. From the backbone modification and included solvent changes, it is clear that Chiralpak AD underwent an irreversible transition after the temperature cycle. This study clearly shows the advantage of solidstate NMR over the conventional chromatographic methods in characterizing stationary-phase materials. The same temperature cycle experiment was performed on Chiralpak AD flushed with 15% 2-propanol in n-hexane. The carbon spectra before and after the temperature cycle are identical as previously published.17 This confirms that no conformational change on Chiralpak AD or change in the solvent inclusion occurred after the phase was flushed by 15% 2-propanol in n-hexane. As shown in Figure 2, once the conformation is changed to the higher temperature conformation, it will maintain this conformation even when the temperature is changed to below the transition temperature. However, the original lower temperature conformation can be regenerated successfully by flushing Chiralpak AD with pure 2-propanol for 3 h.7 Since the conformation of the phase plays a crucial role in the enantioselectivity and resolution, understanding of the phase transition will provide vital (38) Chankvetadze, B. Capillary Electrophoresis in Chiral Analysis; John Wiley & Sons: Chichester, U.K., 1997. (39) Wang, F.; Khaledi, M. G. In High Performance Capillary Electrophoresis; Khaledi, M. G., Ed.; John Wiley & Sons: New York, 1998; Chapter 23.

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Figure 5. Full 13C CPMAS spectra of the physical mixture of Chiralpak AD and (a) (S)-(+)-DHP acid at 20 °C, (b) (S)-(+)-DHP acid after temperature cycled through 50 °C, and (c) (R)-(-)-DHP acid after temperature cycled through 50 °C equilibrated with 15% ethanol in n-hexane with 0.1% TFA (* represents DHP acid peaks).

Figure 4. 13C CPMAS spectra of Chiralpak AD equilibrated with 15% ethanol in n-hexane at (a) 20 °C and (b) after temperature cycled through 50 °C. (A) Full spectra. (B) Expanded spectra (dashed line at ∼66 ppm represents C-6 and dashed line at ∼57 ppm represents included ethanol).

information for enantioseparation method development and largescale preparative chromatography. These carbon spectra clearly show why 2-propanol can be used to regenerate the original conformation. To monitor the effect of TFA on the interactions of the DHP acid enantiomers with Chiralpak AD, separate physical mixtures of DHP acid enantiomers with Chiralpak AD flushed by 15% ethanol in n-hexane with TFA were prepared. The 20 °C carbon spectra (not shown) are similar compared to the physical mixtures without TFA. When the mixtures were cycled through 50 °C (Figure 5), the carbon spectra displayed some major differences. Primarily, the DHP acid carbon peaks in the physical mixture of (R)-(-)-DHP acid with Chiralpak AD have vanished. The disappearance of the DHP acid carbon peaks suggests that the acid is no longer in the solid state. Signal received from CPMAS is mediated through dipole coupling. In the solution state, dipole coupling is averaged away, resulting in a null signal from the CPMAS experiment. Since the acid cannot escape the sealed rotor, the lack of signal for the acid in the CPMAS experiment must be due to the acid being dissolved in the flushed solvent. This phenomenon only occurs with (R)-(-)-DHP enantiomer when TFA is present in the flush and after the material was cycled through 50 °C. This indicates that (R)-(-)-DHP acid was excluded from 5882 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

the solid phase and became a “liquidus” state. The chromatographic data show that the higher temperature conformation retained less of this enantiomer than the lower temperature conformation (Figure 2). The ease of dissolution for (R)-(-)-DHP enantiomer suggests a more “loose” binding of this enantiomer to Chiralpak AD in the presence of TFA. From the chromatographic results, after the temperature program, the complexation between (S)-(+)-DHP acid and the flushed phase in the higher temperature conformation should be stronger than that of the lower temperature. Overall, the changes of the carbon environment of the sugar backbone of Chiralpak AD complexed with the enantiomers can be used to characterize the interactions between the phase and the compounds. 19F MAS NMR. Previously, the effect of the additives in LC enantioseparation on the derivatized cellulose and amylose CSPs was studied by chromatography.18-27 However, there is no direct evidence of the change of the additive’s chemical environment in the presence of the CSPs. Because TFA has been commonly used as an additive in enantioseparation of both acidic and basic racemates and has three fluorines, it becomes an ideal probe to examine such effect by 19F solid-state NMR. Figure 6 displays the 19F MAS NMR spectra of Chiralpak AD after flushing by ethanol in n-hexane phase with 0.1% TFA at 20 °C compared to liquid TFA, liquid ethyl trifluoroacetate in hexane, and liquid ethanol in n-hexane with 0.1% TFA at 20 °C and after 50 °C. The 19F spectra for these samples uniquely display the TFA environments due to the lack of fluorine atoms in the solvent, the stationary phase, and the silica support. The fluorine spectra for liquid TFA consists of a single peak at -77.2 ppm, while the fluorine spectrum for liquid ethyl trifluoroacetate in n-hexane consists of a single peak at -75.5 ppm. The spectra for liquid ethanol in n-hexane with 0.1% TFA and Chiralpak AD flushed with ethanol in n-hexane and 0.1% TFA display two resolved 19F peaks representing two distinct CF3 environments at 20 °C. It is clear that the peak at -75.5 ppm is due to ethyl trifluoroacetate, indicating significant conversion of TFA to ethyl trifluoroacetate in n-hexane/ethanol mixtures. Without additional relaxation data, however, the minimal information can be abstracted concerning the physical state of the CF3 environment at -76.3 ppm in Figure 6d. Spin-lattice relaxation

Table 1. T1G Values for

19F

TFA Peaks (Mobile Phase, 15% Ethanol in Hexane with 0.1% TFA)

sample TFA (liquid) ethyl trifluoroacetate (in n-hexanes) EtOH/Hex/0.1% TFA (liquid) Chiralpak AD flushed with mobile phase

temp (°C)

19F

20 20 20 20 20 20

peak (ppm)

T1F (ms)

temp (°C)

-77.2 -75.5 -76.3 -75.5 -76.3 -75.5

240 250 231 162 27 260

after 50 after 50

19F

peak (ppm)

T1F (ms)

-76.2 -75.6

15 300

Figure 6. 19F MAS spectra of (a) liquid TFA, (b) 0.1% liquid ethyl trifluoroacetate in n-hexane, (c) 15% ethanol in n-hexane with 0.1% TFA at 20 °C, (d) Chiralpak AD equilibrated with 15% ethanol in n-hexane with 0.1% TFA at 20 °C, and (e) after temperature cycled through 50 °C.

Figure 7. Expanded 19F MAS spectra of the DHP acid region for pure (S)-(+)-DHP acid (a) solid and (b) dissolved in 15% ethanol in n-hexane with 0.1% TFA compared to the physical mixture of Chiralpak AD with (c) (S)-(+)-DHP acid and (d) (R)-(-)-DHP acid at 20 °C equilibrated with 15% ethanol in n-hexane with 0.1% TFA.

in the rotating frame (T1F) offers a quick method to probe the structural significance of the CF3 peaks. The T1F values for all the peaks in the 19F spectra in Figure 6 are displayed in Table 1. For liquid TFA and ethyl trifluoroacetate, the 19F peaks display the relatively long T1F values expected for liquid samples. The 19F peaks for the spectra of ethanol in n-hexane with 0.1% TFA yield similarly long T1F values. In the Chiralpak AD sample, the peak at -75.5 ppm displays a typical liquid like T1F value; however, the peak at -76.3 ppm yields a significantly shortened T1F value. The shortened 19F T1F value suggests that this peak represents a CF3 environment with significantly decreased molecular motion. One possibility for this decrease in motion is that the peak represents the included TFA associated with ethanol. From the carbon data on this sample (see Supporting Information, S-3), direct evidence of the ethanol inclusion is seen from the presence of the ethanol peaks in the CPMAS spectrum. The TFA associated with this included ethanol would possess significantly decreased molecular motion as well as incur an isotropic CF3 peak shift compared to liquid TFA. After Chiralpak AD sample has been cycled through 50 °C (Figure 6e), a reversal in the relative peak intensity for the CF3 peaks is witnessed. This suggests a conversion of the included TFA to ethyl ester. This is supported by the high-temperature 13C data indicating a decrease in the ethanol inclusion at the elevated temperatures. TFA associated with the included ethanol would also be excluded and eventually convert to ethyl trifluoroacetate. Additionally, the included CF3 peak is shifted slightly to -76.2 ppm, representing a nonreversible change in the TFA inclusion. This is consistent with the carbon data, which suggested

that Chiralpak AD, as well as the included ethanol, undergoes an irreversible structural change after being heated through 50 °C. Subsequently, the physical mixtures of DHP acid with Chiralpak AD flushed by the mobile phase with TFA were analyzed by 19F MAS and T solid-state NMR. This provides in-depth char1F acterization of the binding strength of (S)-(+)- and (R)-(-)-DHP acids, as well as the significance of TFA. Both 19F peaks from TFA (∼ -76 ppm) and the acid (∼ -140 ppm) can be monitored simultaneously. Figure 7 displays the 20 °C 19F MAS spectra for solid (S)-(+)-DHP acid, (S)-(+)-DHP acid, dissolved in ethanol in n-hexane with 0.1% TFA, and the physical mixtures of DHP acid with Chiralpak AD flushed with the mobile phase. The corresponding 19F T1F values for the peaks in these spectra are displayed in Table 2. Solid DHP acid displays extensively broadened 19F peaks (due to incomplete averaging of solid interactions using the current spectrometer conditions) with fast 19F T values (8 ms). When the acid is dissolved in the mobile 1F phase, the peak width decreases as expected while the 19F T1F values are extended. The spectrum from the physical mixture of (S)-(+)-DHP acid with Chiralpak AD flushed with the mobile phase clearly displays broadened components combined with sharp peaks representing the acid. Comparing the spectrum of the pure solid acid to those of the physical mixtures of the acid and Chiralpak AD, it appears the broadened components in the physical mixture of (S)-(+)-DHP acid and Chiralpak AD are probably due to the acid with significantly less mobility. This is confirmed with the 19F T1F values that were too fast to be measured using typical delay times. One hypothesis that accounts for the Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

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Table 2. T1G Values for

19F

DHP Acid Peaks (Mobile Phase, 15% Ethanol in Hexane with 0.1% TFA)

sample

temp (°C)

(S)-(+)-DHP acid solid

20

(S)-(+)-DHP acid dissolved in mobile phase

20

(S)-(+)-DHP acid + Chiralpak AD + mobile phase

20 20 20 20 20 20 20 20 20 20 20 20 20

(R)-(-)-DHP acid + Chiralpak AD + mobile phase

Figure 8. Expanded 19F MAS spectra of the TFA region for (a) Chiralpak AD at 20 °C equilibrated with 15% ethanol in n-hexane with 0.1% TFA compared to the physical mixtures of Chiralpak AD with (S)-(+)-DHP acid at (b) 20 °C and (c) after temperature cycled through 50 °C and (R)-(-)-DHP acid at (d) 20 °C and (e) after temperature cycled through 50 °C equilibrated with 15% ethanol in n-hexane with 0.1% TFA.

results of the 20 °C fluorine spectra for the physical mixtures is that (S)-(+)-DHP acid is involved in stronger binding to the stationary phase. The stronger binding would result in both the observed decrease in the 19F T1F values as well as the increased less-mobile components. Figure 8 displays the temperature effect on the TFA 19F spectra for both physical mixtures. The accompanying 19F T1F values are displayed in Table 2. The major TFA component in the physical mixture of (S)-(+)-DHP acid and Chiralpak AD is the short 19F T1F site at -76.3 ppm. From the discussion above, this site represents the included TFA associated with ethanol. Obviously, the enantioseparation involves the inclusion of (S)-(+)-DHP acid together with both TFA and ethanol into the CSP. After the material is cycled through 50 °C, the major TFA component is now the peak at -75.6 ppm, which has a significantly longer 19F T1F (400 ms) and is assigned to free ethyl trifluoroacetate. This indicates that (S)-(+)-DHP acid was included more into the CSP grooves (longer retention factors) while TFA was excluded from the phase when the temperature was above the transition 5884 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

19F

peak (ppm)

T1F (ms)

temp (°C)

-137.0 -139.1 -137.5 -139.7 -76.3 -75.6 -140.4 -139.8 -139.1 -137.7 -75.7 -140.1 -139.5 -139.1 -138.0 -137.1 -136.7

8 8 115 115 38 140 5 6 5 7 38 14 15 13 16 8 15

after 50 after 50 after 50 after 50 after 50 after 50 after 50 after 50 after 50

19F

peak (ppm)

T1F (ms)

-75.6

400

-140.1 -139.3 -137.8 -137.3 -75.4 -140.1 -138.0

19 17 16 19 28 48 55

Figure 9. Expanded 19F MAS spectra of the DHP acid region for the physical mixtures of Chiralpak AD with (S)-(+)-DHP acid at (a) 20 °C and (b) after temperature cycled through 50 °C and (R)-(-)DHP acid at (c) 20 °C and (d) after temperature cycled through 50 °C equilibrated with 15% ethanol in n-hexane with 0.1% TFA.

temperature. The excluded TFA converted to its ester during the temperature cycles. Conversely, the TFA environment in the (R)(-)-DHP acid mixtures before and after temperature cycling through 50 °C is dominated by a single, broad component at -75.6 ppm with a short 19F T1F. This type of 19F environment has a chemical shift similar to ethyl trifluoroacetate, yet a T1F value indicating the decreased mobility, suggesting that this site represents the bound ethyl trifluoroacetate. Therefore, in the presence of (R)-(-)-DHP acid, TFA converts to ethyl trifluoroacetate that is tightly associated with the stationary phase and does not become “liquidus” after cycling through 50 °C. The data support the claim that, in the physical mixture of (R)-(-)-DHP acid and Chiralpak AD, TFA acts to block the strong binding of (R)-(-)-DHP acid to the CSP. By focusing on the DHP acid region of the 19F MAS NMR spectra, we can gain direct insight into the structural effects of the temperature cycle on DHP acid in these physical mixtures. Corresponding T1F values to DHP acid peaks are displayed in Table 2. The 19F spectra at 20 °C (Figure 9) display a significant broadened component for the (S)-(+)-DHP acid mixture compared to the (R)-(-)-DHP acid mixture. The broadened peaks are

at shift position similar to that of the solid (S)-(+)-DHP acid. The 19F T values for these broadened components (∼6 ms) indicate 1F the reduction in mobility. The presence of a significant amount of these components in the (S)-(+)-DHP acid mixture combined with the lack of these broadened resonances in the (R)-(-)-DHP acid mixture suggests that (S)-(+)-DHP acid is more strongly bound to Chiralpak AD compared to (R)-(-)-DHP acid. When both mixtures are cycled through 50 °C, an increase in the number of resolved 19F sites is displayed. This probably represents an increase in the mobility of both acid species caused by the temperature increase. However, the fluorine spectrum for the (S)(+)-DHP acid mixture remains relatively broad compared to the spectrum for the (R)-(-)-DHP acid mixture. Additionally, the 19F T1F values for the peaks of both mixtures (Table 2) indicate a more significant increase in the mobility for (R)-(-)-DHP acid (∼50 ms T1F) compared to (S)-(+)-DHP acid (∼ 17 ms T1F). These data combined with the structural effects of the TFA as displayed earlier indicate that, in the (S)-(+)-DHP acid mixture, the acid effectively competes with TFA for binding sites in the Chiralpak AD whereas (R)-(-)-DHP acid cannot bind as effectively. CONCLUSIONS In the cyclic van’t Hoff temperature program, the ratio of apparent retention factors showed that the curve profile of DHP acid enantiomers depended on the starting column temperature. In a single-step temperature program, when the column temperature stepped from 20 to 50 °C (transition temperature ∼30 °C), the ratio of apparent retention factors increased by 52% during a period of 24 h. In a two-step temperature program, the ratio of apparent retention factors increased with time when the temperature was stepped above 30 °C; however, the ratio remained unchanged when the temperature was stepped below 30 °C. Solid-

state 13C CPMAS NMR showed that Chiralpak AD flushed by ethanol in n-hexane underwent an irreversible conformational change when the temperature was cycled through 50 °C. For the physical mixture of (R)-(-)-DHP acid with Chiralpak AD flushed by the mobile phase with TFA, after the same temperature change, the carbon peaks of (R)-(-)-DHP acid disappeared (i.e., (R)(-)-DHP acid was excluded from the solid phase) while those of (S)-(+)-DHP acid and Chiralpak AD mixture remained the same. In the presence of (S)-(+)-DHP acid, 19F MAS NMR shows that TFA is included into the CSP at room temperature but is excluded from the CSP at the elevated temperatures. An opposite effect is witnessed in the presence of (R)-(-)-DHP acid, where ethyl trifluoroacetate (converted from TFA and ethanol) blocks the incorporation of (R)-(-)-DHP acid into the CSP at all temperatures. ACKNOWLEDGMENT We thank Dr. Daniel R. Sidler for providing DHP acid and methyl ester racemates and pure enantiomers and Dr. Peter Dormer for liquid-phase NMR analysis. We also thank Drs. Richard S. Egan, Tao Wang and Thomas O’Brien for their helpful discussions. SUPPORTING INFORMATION AVAILABLE Solid-state NMR data of Chiralpak AD flushed with the mobile phase with TFA; the physical mixtures of Chiralpak AD with (S)(+)- and (R)-(-)-DHP acids without TFA. This material is available for free of charge via the Internet at http://pubs.acs.org. Received for review June 30, 2003. Accepted August 19, 2003. AC034714E

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