Jizhong Yan

Enantiomeric separation of (R, S)-naproxen by recycling high speed counter-current chromatography with hydroxypropyl-β-cyclodextrin as chiral selector.

Recycling high speed counter-current chromatography (HSCCC) was successfully applied to resolution of (R, S)-naproxen (NAP) using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The two-phase solvent system composed of n-hexane-ethyl acetate-0.1 mol L(-1) phosphate buffer solution with pH=2.67 (8:2:10, v/v/v) was selected. Influence factors for the chiral separation process were investigated, including concentration of HP-β-CD, equilibrium temperature and pH of aqueous phase. Suitable elution mode was selected for HSCCC enantioseparation of (R, S)-NAP. Under optimum separation conditions, 29 mg of (R, S)-NAP was separated using preparative recycling HSCCC with the molar ratio HP-β-CD/NAP racemate 83:1. Technical details for recycling elution mode were discussed as for chiral HSCCC separation. The purities of both (S)-NAP and (R)-NAP were over 99.5% as determined by HPLC. Enantiomeric excess of (S)-NAP and (R)-NAP reached 99.4%. Recovery for NAP enantiomers from HSCCC fractions was 82-89%, yielding 13 mg of (S)-NAP and 12 mg of (R)-NAP.

Enantioseparation of phenylsuccinic acid by high speed counter-current chromatography using hydroxypropyl-β-cyclodextrin as chiral selector.

High speed counter-current chromatography (HSCCC) was successfully applied to resolution of phenylsuccinic acid (PSA) with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector (CS). The two-phase solvent system composed of n-hexane-methyl tert-butyl ether-0.1 mol L⁻¹ phosphate buffer solution with pH=2.51 (0.5:1.5:2, v/v/v) was selected. Influence factors involved in the chiral separation were investigated, including the concentration of chiral selector, pH value of the aqueous phase, the separation temperature, and the thermodynamic parameters of inclusion complex were calculated. The complex formation constants were determined using analytical instrument. Two HSCCC elution modes were studied and peak resolution equation was discussed. Under optimum separation conditions, 712 mg of PSA racemate was separated using preparative apparatus. The purities of both of the fractions including (+)-PSA and (-)-PSA from the preparative CCC separation were over 98.5% determined by HPLC and enantiomeric excess of (+)-PSA and (-)-PSA reached 97.6% and 98.6%, respectively. Recovery for the target compounds from the CCC fractions reached 80-82% yielding 285 mg of (+)-PSA and 292 mg of (-)-PSA.

Separation of α-cyclohexylmandelic acid enantiomers using biphasic chiral recognition high-speed counter-current chromatography

This work concentrates on a chiral separation technology named biphasic recognition applied to resolution of alpha-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography (HSCCC). The biphasic chiral recognition HSCCC was performed by adding lipophilic (-)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic hydroxypropyl-beta-cyclodextrin 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-water (9:1:10, v/v/v) with the above chiral selectors was selected according to the partition coefficient and separation factor of the target enantiomers. Important parameters involved in the chiral separation were investigated, namely the types of the chiral selectors (CS); the concentration of each chiral selector; pH of the mobile phase and the separation temperature. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Langmuirian isotherm was employed to estimate the loading limits for a given value of chiral selectors. Under optimum separation conditions, 3.5-22.0 mg of alpha-cyclohexylmandelic acid racemate were separated using the analytical apparatus and 440 mg of racemate was separated using the preparative one. The purities of both of the fractions including (+)-enantiomer and (-)-enantiomer from the preparative CCC separation were over 99.5% determined by HPLC and enantiomeric excess reached 100% for the (+/-)-enantiomers. Recovery for the target compounds from the CCC fractions reached 85-88% yielding 186 mg of (+)-enantiomer and 190 mg of (-)-enantiomer. The overall experimental results show that the HSCCC separation of enantiomer based on biphasic recognition, in which only if the CSs involved will show affinity for opposite enantiomers of the analyte, is much more efficient than the traditional monophasic recognition chiral separation, since it utilizes the cooperation of both of lipophilic and hydrophilic chiral selectors.

Enantioseparation of mandelic acid derivatives by high performance liquid chromatography with substituted β-cyclodextrin as chiral mobile phase additive and evaluation of inclusion complex formation

The enantioseparation of ten mandelic acid derivatives was performed by reverse phase high performance liquid chromatography with hydroxypropyl-β-cyclodextrin (HP-β-CD) or sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as chiral mobile phase additives, in which inclusion complex formations between cyclodextrins and enantiomers were evaluated. The effects of various factors such as the composition of mobile phase, concentration of cyclodextrins and column temperature on retention and enantioselectivity were studied. The peak resolutions and retention time of the enantiomers were strongly affected by the pH, the organic modifier and the type of β-cyclodextrin in the mobile phase, while the concentration of buffer solution and temperature had a relatively low effect on resolutions. Enantioseparations were successfully achieved on a Shimpack CLC-ODS column (150×4.6mm i.d., 5μm). The mobile phase was a mixture of acetonitrile and 0.10molL(-1) of phosphate buffer at pH 2.68 containing 20mmolL(-1) of HP-β-CD or SBE-β-CD. Semi-preparative enantioseparation of about 10mg of α-cyclohexylmandelic acid and α-cyclopentylmandelic acid were established individually. Cyclodextrin-enantiomer complex stoichiometries as well as binding constants were investigated. Results showed that stoichiometries for all the inclusion complex of cyclodextrin-enantiomers were 1:1.

Application and comparison of high performance liquid chromatography and high speed counter-current chromatography in enantioseparation of (±)-2-phenylpropionic acid.

High performance liquid chromatography (HPLC) and high speed counter-current chromatography (HSCCC) were applied and compared in enantioseparation of 2-phenylpropionic acid (2-PPA) when hydroxypropyl-β-cyclodextrin (HP-β-CD) was used as chiral mobile phase additive. For HPLC, the enantioseparation was achieved on ODS C(18) reverse phase column and the mobile phase was 25 mmol L(-1) HP-β-CD aqueous buffer solution (pH 4.0, adjusted with triethylamine): methanol: glacial acetic acid (85:15:0.5 (v/v/v)). For HSCCC, the two-phase solvent system was composed of n-hexane-ethyl acetate-0.1 mol L(-1) phosphate buffer solution pH2.67 (5:5:10 for isocratic elution and 8:2:10 for recycling elution (v/v/v)) added with 0.1 mol L(-1) HP-β-CD. The key parameters, such as a substitution degree of HP-β-CD, the concentration of HP-β-CD, pH value of the aqueous phase and the temperature were optimized for both separation methods. Using the optimum conditions a complete HSCCC enantioseparation of 40 mg of 2-propylpropionic acid in a recycling elution mode gave 15-18 mg of (+)-2-PPA and (-)-2-PPA enantiomers with 95-98% purity and 85-93% recovery.

Profiling the Ginsenosides of Three Ginseng Products by Lc‐Q‐Tof/Ms

Ginseng is a well-known herbal medicine that has been gaining increasingly popularity as a potential chemopreventive agent. In traditional Chinese medicine practice, white ginseng (WG), red ginseng (RG), and dali ginseng (DG) are 3 different ginseng-processed products used for different purposes. Although the morphological appearance and some constituents contained in these ginseng products are similar, their pharmacological activities are significantly different due to the varied types and quantity of ginsenosides in each product. In the present study, a practical method based on rapid liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-Q-TOF/MS) was developed to identify the chemical profiles of ginsenosides in these 3 ginseng products. The results demonstrated that a total of 55, 53, and 43 compounds were unambiguously assigned or tentatively identified in DG, WG, and RG samples, respectively. The featured compounds are mainly malonyl ginsenosides in WG, and decarboxyl products of mal-ginsenosides and the dehydrated compounds from polar ginsenosides were characteristic in RG, while DG contain some characteristic components present both in WG and RG. We presume that heating processing is the major factor affecting the chemical profile of ginseng products. The difference of chemical information revealed by LC-Q-TOF/MS could be used to discriminate the WG, RG, and DG samples.

Preparative Isolation and Purification of Amygdalin from Prunus armeniaca L. with High Recovery by High‐Speed Countercurrent Chromatography

Abstract The bioactive component, amygdalin, was successfully separated from the crude extract of Prunus armeniaca L. using high‐speed countercurrent chromatography (HSCCC) with high recovery. A polar two‐phase solvent system, containing n‐butanol‐ethyl acetate‐water (4∶1∶6, v/v/v) was run on a preparative scale. The lower phase was used as the mobile phase in the head to tail elution mode. A 208 mg quantity of the crude extract, containing 36.1% amygdalin, was loaded onto a 270 mL HSCCC column, which yielded 69 mg amygdalin of 98.7% purity with 90.7% recovery. The chemical structure of amygdalin was determined by electrospray ionization MS‐MS and 1H NMR.

Preparative enantioseparation of β-blocker drugs by counter-current chromatography using dialkyl L-tartrate as chiral selector based on borate coordination complex.

Counter-current chromatography (CCC) was applied for preparative enantioseparation of three β-blocker drugs, including propranolol, pindolol and alprenolol. The two-phase solvent system was composed of chloroform-0.05 mol L(-1) acetate buffer containing 0.10 mol L(-1) boric acid (1:1, v/v), in which 0.10 mol L(-1) di-n-hexyl L-tartrate was added in the organic phase as chiral selector. Influence factors in the enantioseparation of propranolol were investigated. The chromatographic retention mechanism based on borate coordination complex was proposed. 116 mg of racemic propranolol was completely enantioseparated using conventional high speed CCC in a single run, yielding 48 mg of (+)-propranolol with HPLC purity of 98.9% and 47 mg of (-)-propranolol with HPLC purity of 96.3%. Recovery for propranolol enantiomers from CCC fractions was in the range of 75-82%. pH-zone-refining CCC was also successfully applied in enantioseparation of propanolol and it was found that 356 mg of racemic propranolol could be completely enantioseparated. 145 mg of (+)-enantiomer with HPLC purity of 95.6% and 148 mg of (-)-enantiomer with HPLC purity of 98.2% were recovered from pH-zone-refining mode. Separation mechanism about chiral separation by pH-zone-refining CCC was discussed.

Preparative Isolation and Purification of Two Coumarins from Edgeworthia chrysantha Lindl by High Speed Countercurrent Chromatography

Abstract Two bioactive coumarins including 7‐hydroxycoumarin (umbelliferone) and 7‐hydroxyl‐6‐methoxy‐3, 7′‐dicoumarylether (daphnoretin) were separated from the ethyl acetate extract of the stems of Edgeworthia chrysantha Lindl (E. papyrifera) by high speed countercurrent chromatography (HSCCC). The two‐phase solvent system used was composed of n‐hexane‐ethyl acetate–methanol–water at an optimized volume ratio of 4∶6∶4∶6 (v/v/v/v). Preparative HSCCC yielded, from 317 mg of the crude extract, 21 mg umbelliferone and 41 mg of daphnoretin each, at over 95% purity by high performance liquid chromatography (HPLC) analysis. Their structures were identified by EI‐MS and 1H NMR.

Chiral ligand exchange high-speed countercurrent chromatography: mechanism and application in enantioseparation of aromatic α-hydroxyl acids.

This work concentrates on the separation mechanism and application of chiral ligand exchange high-speed countercurrent chromatography in enantioseparation of ten racemic aromatic α-hydroxyl acids, including mandelic acid, 2-chloromandelic acid, 4-methoxymandelic acid, 4-hydroxymandelic acid, α-methylmandelic acid, 4-hydroxy-3-methoxy-mandelic acid, 3-chloromandelic acid, 4-bromomandelic acid, α-cyclopentylmandelic acid and α-cyclohexylmandelic acid, in which five of the racemates were successfully enantioseparated by analytical apparatus with an optimized solvent system. The two-phase solvent system was composed of butanol-water (1:1, v/v) or hexane-n-butanol-water (0.5:0.5:1, v/v), to which N-n-dodecyl-l-proline was added in the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transition metal ion. Various influence factors in high-speed countercurrent chromatography were optimized by enantioselective liquid-liquid extraction. The separation mechanism for chiral ligand exchange high-speed countercurrent chromatography was proposed based on the results of present studies. Successful enantioseparations of 72mg of mandelic acid, 76mg of 2-chloromandelic acid and 74mg of 4-methoxymandelic acid were achieved individually with high resolution by preparative high-speed countercurrent chromatography. The HPLC purity of all enantiomers was over 96% with the recovery in the range of 82-90% from the collected fractions.