Enantiomeric Separation Of Chiral Compounds By Pressurized Capillary Electro-Chromatography System

Molecular chirality is a fundamental phenomenon that plays an important role in biological processes. A wide range of biological and physical functions are generated through precise molecular recognition because enzymes, vectors, receptors, proteins and polysaccharides interact with different enantiomersin decisively different ways. As a result of such chiral recognition, drug enantiomers may differ in their pharmacodynamic handling, pharmacokinetic handling and toxicological profiles. Accordingly, the development of a rapid and precise method to separate forms of different chirality has been a research focus for many separation scientists.There are numerous analytical techniques have been developed for chiral separation, such as high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), gas chromatography (GC), capillary electrophoresis (CE), capillary electrochromatography (CEC), supercritical fluid chromatography (SFC). Among them, CEC is a relative new separation technique that combines the separation efficiency of CE with the selectivity of HPLC. Analytes can be separated on the basis of the chromatographic and electromotive principle between the stationary and the mobile phase. As its superexcellent feature, CEC has attracted much attention during the last years. Now more people focus on chiral separation by using CEC.The pressurized capillary electrochromatography (pCEC) system was developed by Unimicro Technologies, in which the mode of pCEC, CLC and CE can be carried out. In this study, we developed CLC, pCEC and CE methods using the system for enantiomeric separation of some compounds. Different experimental factors were investigated to optimize the chiral separation of the studied compounds. The structural analysis of some compounds was carried out by QSERR study. The paper was divided into three parts.Part 1 Enantiomeric separation of compounds by CLCSection one Enantiomeric separation of compounds by CLC using Chiralpak AD aschiral stationary phaseObjective: To evaluate enantiomeric separation methods for some acidic and basic compounds by CLC using amylose tris (3,5-dimethylphenylcarbamate) (Chiralpak AD) as chiral stationary phase (CSP). And the structural analysis of some basic compounds was carried out by QSERR study. Methods: 1) The conditions for chiral separation of acid compounds: mobile phase was isopropanol- trifluoroacetic acid (TFA) (100:0.1, v/v);capillary column was packed with chiralpak AD (effective length 200 mmxlOOum ID, Unimicro Technologies Shanghai Co., Ltd);the flow rate was set at 0.02ml/min;theexperiment was performed at the temperature of 12°C. 2) The conditions for chiral separation of basic compounds: mobile phase was isopropanol-n-hexane adding different amount of triethylamne (TEA);the flow rate was set at 0.02ml/min;the experiment was performed at the room temperature. Results: 1) Chiral separation of acid compounds: baseline separation was obtained for ketoprofen and mandelic acid when mobile phase was isopropanol-TFA (100:0.1, v/v), and the resolution was 1.8 and 1.5 respectively. While ibuprofen, flurbiprofen and 2-chloromandelic acid were partly separated. However naproxen and etodolac couldn't be separated. Several factors affecting on chiral separation were investigated, including the ratio of isopropanol to n-hexane, the content of TFA, temperature and flow rate, in which the ratio of isopropanol to n-hexane and the temperature were the predominant influencing factors. 2) Chiral separation of basic compounds and QSERR study: Most of the studied compounds were separated successfully or partly separated at the definite conditions. Among them, the baseline separation was achieved for chiral separation of esmolol, propafenone, cetirizine, cinildipine, five of SPE compounds and twelve of BZYT compounds. The influencing factors that affected on chiral separation were the ratio of isopropanol to n-hexane and the content of TEA. The structural parameters of the studied compounds were calculated by Hyperchem7.52 software. Stepwise multiple regression analysis was utilized to establish the correlation between experimental parameter (a or Rs) and structural parameters of the 19 compounds in training set. Then the predicted process was performed for the extrapolating set. The results were shown that the correlation coefficient was 0.984 and 0.912 when a and Rs as the dependent variable respectively. It showed that the established model could be used to predict the potentiality of enantiomeric separation of SPE group compounds and their analogues. Conclusion: the established methods could be used for the chiral separation of some acid and basic compounds. The prediction of enantiomeric separation can be carried out by QSERR study.Section two Enantiomeric separation of compounds by CLC using vancomycin aschiral stationary phaseObjective: Enantiomeric separation of chiral compounds was carried out in CLC using vancomycin as CSP. Methods: A lOOum I.D. fused-silica capillary column packed with 5um diameter silica particles modified with vancomycin was used (effective length 200mmx lOOum I.D. x375^im O.D., Unimicro Technologies Shanghai Co., Ltd). Enantiomeric separation of fifteen P-receptor blockers and analogues was studied by CLC mode using mobile phases containing methanol-isopropanol- acetic acid- TEA. The experiment was carried out at the room temperature. Results: Most of the studied compounds were successfully or partly separated at the definite conditions except for phenylpropanolamine and methylephedrine by polar organic CLC mode. Several factors affecting on chiral separation were investigated, including the organic modifier type, the ratio of methanol to isopropanol, temperature, the content of acetic acid and TEA in mobile phase. Among them, the organic modifier type and the ratio of methanol to isopropanol were the predominant influencing factors. Conclusion: Most of studied chiral compounds could be completely or partly separated employing polar organic CLC mode.Part 2 Enantiomeric separation of compounds by pCECObjective: To establish methods for enantiomeric separation of p-receptor blockers and analogues by polar organic pCEC and reversed phase pCEC using vancomycin as CSP. Methods: A lOOum I.D. fused-silica capillary column packed with 5um diameter silica particles modified with vancomycin was used (effective length 200mmx lOOum I.D. x375um O.D., Unimicro Technologies Shanghai Co., Ltd). Enantiomeric separation of some P-receptor blockers and analogues was studied by polar organic pCEC mode and reversed phase pCEC mode using mobile phase containing methanol-isopropanol-TEA-acetic acid and TEAA buffer-methanol respectively. Results: 1) The baseline separation of enantiomers of atenolol,bisoprolol, clenbuterol and terbutaline were obtained by polar organic pCEC mode. The influencing factors, such as the different type of organic modifier, ratio of methanol to isopropanol, the content of acetic acid and TEA in mobile phase, voltage, flow rate and sample concentration, were investigated during the chiral separation of studied compounds, in which the former three were the predominant influencing factors. 2) The baseline separation of 7 P-receptor blockers was achieved. Compared to polar organic CLC and pCEC mode, the resolution of aryloxypropanolamines compounds increased, while that of phenylethanolamines compouds decreased by reversed phase pCEC mode. It was surprised that the enantiomers of terbutaline couldn't be separated under any condions in reversed phase pCEC mode. The influencing factors were studied, including the ratio of TEAA buffer to methanol, pH value of TEAA buffer, voltage and the concentration of TEAA. Among them, the ratio of TEAA buffer to methanol and pH value of TEAA buffer were the major factors that affected on enantiomeric separation of the studied compounds. Conclusion: Most of studied chiral compounds could be completely or partly separated employing pCEC mode. And there was a complementarity between polar oranic pCEC and reversed phase pCEC mode.Part 3 Enantiomeric separation of carvedilol by CEObjective: The enantiomeric separation of carvedilol by CE using P-cyclodextrin as the chiral selector was studied. Methods: The separation was performed at room temperature on a fused-silica capillary of 61cm (effective length 50cm). The buffer solution used in this method was phosphoric acid- trihydroxyethylamine buffer. The detection wavelength was 240nm. The effects of the concentration of P-cyclodextrin, different kinds of buffer, the concentration of phosphoric acid, different capillaries, pH of background electrolyte, sample injection time and the voltage were investigated. Results: The baseline separation of carvedilol enantiomers was achieved with background electrolyte of lOOmmol-L"1 phosphoric acid -trihydroxyethylamine containing Smmoll/'p-cyclodextrin (pH 3.0) and applied high voltage of-18kv. The resolution of two enantiomers was 2.1, the migration time of R-carvedilol and S-carvedilol was 32.42min and 33.88min respectively. The results demonstrated that the concentration of P-cyclodextrin, kinds of buffer and pH of background electrolyte were the predominant influencing factors for the enantiomeric separation of carvedilol. Conclusion: The enantiomers of carvedilol were successfully separated by the established method and the method was simple and cost-effective.