| This work concentrates on the application of two chiral selectors including hydrophilic chiral selector hydroxypropyl-β-cyclodextrin (HP-β-CD) and lipophilic chiral selector dialkyl tartrate in enantioseparation of ten racemic drugs or intermediates by high speed counter-current chromatography (HSCCC). The chromatographic retention mechanism of racemates by HSCCC with HP-β-CD as chiral selector was proposed and discussed. Three stereoisomeric caffeoylquinic acids were selected and separated by pH-zone-refining counter-current chromatography (CCC) to study on elution sequence and zone overlap phenomena, which were frequently observed during pH-zone-refining CCC separation. The ten racemic drugs or intermediates were as follows:intermediates phenylsuccinic acid and a-cyclohexylmandelic acid,2-arylpropionic acid drugs including naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen and carprofen, andβ-blocker drugs propranolol and pindolol.Firstly, HSCCC was applied to resolution of phenylsuccinic acid (PSA) enantiomers with HP-β-CD as chiral selector and chromatographic retention mechanism was proposed. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether-0.1 mol/L phosphate buffer solution with pH=2.51 containing 0.05 mol/L HP-β-CD (0.5:1.5:2, v/v). Influence factors involved in the chiral separation were investigated, including concentration of chiral selector, pH of aqueous phase and separation temperature. Thermodynamic parameters of inclusion complex were calculated. The correlation between both separation factor (a) and peak resolution (Rs) and the concentration of HP-β-CD was investigated. The chromatographic retention mechanism of PSA by HSCCC with HP-P-CD as chiral selector was discussed. A mathematical model showing the relationship of capacity factor (k') with HP-β-CD concentration, hydrogen ion concentration, complex formation constant (Kf) and dissociation constants (Ka) of PSA was proposed, in which the complex formation constants were calculated. Loading limits for HSCCC separation run with a given value of chiral selector was determined and Langmuirian isotherm for the enantiomers in the two phase solvent system was investigated. Under optimum separation conditions,712 mg of PSA racemate was separated using preparative apparatus. Meanwhile, a novel chiral separation technology named biphasic chiral recognition HSCCC was proposed and applied to resolution of racemic a-cyclohexylmandelic acid (CHMA). The biphasic chiral recognition HSCCC was performed by adding lipophilic (-)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic HP-β-CD in the aqueous mobile phase, which preferentially recognized the (-)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether-0.1 mol/L phosphate buffer solution with pH=2.51 (9:1:10, v/v/v) was selected. Totally 14 dialkyl tartrate derivatives were synthesized. Factors affecting the chiral separation were investigated. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Loading limits for a given value of chiral selectors were estimated as for HSCCC separation. Under optimum separation conditions,365 mg of sample was separated using the preparative HSCCC. Enantioseparation of racemic CHMA by HSCCC using only HP-β-CD as chiral selector in the aqueous phase was also compared. The chromatographic retention mechanism was studied and an efficient method was developed for calculation of complex formation constant between HP-β-CD and CHMA enantiomer. Experimental results showed that a 2:1 stoichiometry between the cyclodextrin host and the guest molecule was found during chiral recognition instead of 1:1 stoichiometry.In the second place, closed loop recycling mode (CLRM) HSCCC was successfully applied to resolution of 2-arylpropionic acid drug (R,S)-naproxen (NAP) using HP-β-CD as chiral selector. The two-phase solvent system composed of n-hexane-ethyl acetate-0.1 mol/L phosphate buffer solution (8:2:10, v/v/v) was selected. Influence factors for the chiral separation process were investigated and several thermodynamic parameters of NAP-HP-β-CD inclusion complex were calculated.29 mg of (R,S)-NAP was separated using preparative recycling HSCCC. Resolution factor for NAP enantiomers could be improved from 0.7 to 1.2 when CLRM HSCCC was used. The successful enantioseparation of other five 2-arylpropionic acid drugs were not be achieved by HSCCCC using HP-[3-CD as chiral selector because enantioseparation factor was too small as for HSCCC. Theoretical aspects of HSCCC were demonstrated in order to improve resolution factor of NAP enantiomers. Exact equation for resolution factor of HSCCC was discussed in detail, which might be helpful for chiral separation by HSCCC.Then, dialkyl L-tartrate was used as chiral selector for enantioseparation ofβ-blockers propranolol and pindolol by HSCCC as well as pH-zone-refining counter-current chromatography. Dialkyl L-tartrate exhibited a distinctive chiral discrimination with respect to racemic P-amino-alcohols drugs propranolol and pindolol in the liquid-liquid two phase system in the presence of boric acid because a borate complex of the 1,2-diol group of the tartrate and the amino-alcohol was formed in the system. A two-phase system chloroform:aqueous (1:l,v/v) consisting of a chloroform solution of 0.1 mol/L dihexyl L-tartrate and an aqueous solution of 0.1 mol/L boric acid was successfully used for enantioseparation of propranolol and pindolol by HSCCC. pH-zone-refining CCC was also successfully used for preparative enantioseparation of racemic propranolol and 356 mg of propranolol racemate was separated by this method.Finally, principles about the elution sequence and zone overlaps during pH-zone-refining CCC were discussed. The chances of obtaining zone overlaps increase when the amount of injection in the CCC machine increase, the difference between the compounds to be separated is low (similar pKa and similar distribution coeffincients). The typical way to solve the problem is to increase both retainer and eluter concentrations. Two isomeric dicaffeoylquinic acids, including 3, 5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid along with 3-caffeoylquinic acid were studied using pH-zone-refining CCC. The elution sequence of the isomeric dicaffeoylquinic acids was discussed in terms of their acidities, hydrophilicity, and steric configuration. The possible explanations were offered for the mechanism of separation.Although HSCCC is a powerful preparative technique with its high capacity, low cost of stationary phases and low solvent consumption, it yields low efficiency compared to classic liquid chromatography. The current studies demonstrated that biphasic chiral recognition would give much higher separation factor for enantiomers than monophasic chiral recognition during HSCCC enantioseparation, which could make up its disadvantage to some extent. Closed loop recycling elution mode and pH-zone-refining counter-current chromatography could also be applied to improve resolution factor. Under constant theoretical plates and the same chiral selector, better resolution factor could be obtained by improving distribution factor or retention of stationary phase in the separation column.
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