In silico screening of off-line comprehensive two-dimensional counter-current chromatography with liquid chromatography for four saponins isolation.

Being restrained by the limited peak capacity, one-dimensional chromatography usually leads to an unsatisfactory separation with low purity of compounds in a complex mixture. To obtain more highly pure targets for standard reference and to discover new substances for structural elucidation, two-dimensional chromatography is more and more prevalent in many fields. As few metrics on assessment of the preparative capability of two-dimensional chromatographic separations are reported, a methodology of in silico screening of various two-dimensional chromatographic separations with a minimal number of experiments was demonstrated in this work, which was based on three descriptors including the occupation rate of peaks and system homogeneity of a two-dimensional separation space, and the minimal distance of all nearest-neighbor distances of peaks. Combining the advantages of counter-current chromatography and liquid chromatography, we elaborated the methodology by employing off-line comprehensive two-dimensional counter-current chromatography with liquid chromatography to be in silico screened for separation of four saponins from Panax notoginseng at an analytical scale to simulate the case of preparative scale transfer. The predictive results were presented by two-dimensional contour plots and verified by experiments. The result showed that the experimental results were in general accord with the predictive results.

Preparative separation of gypenoside XVII, ginsenoside Rd2, and notoginsenosides Fe and Fd from Panax notoginseng leaves by countercurrent chromatography and orthogonality evaluation for their separation.

The minor ginsenosides with less polarity may have more potent biological activities. Four minor saponins, i.e., gypenoside XVII, ginsenoside Rd2, notoginsenoside Fe, and notoginsenoside Fd, were successfully separated from Panax notoginseng leaves (PNL) after biotransformation by one-step countercurrent chromatography using the biphasic solvent system consisting of n-butanol-ethyl acetate-water (1:4:5, v/v/v). 30 mg of the refined extract of PNL produced 1 mg of gypenoside XVII, 4 mg of notoginsenoside Fe, 2.5 mg of ginsenoside Rd2, and 8.4 mg of notoginsenoside Fd, with purity of 74.9, 95.2, 87.3, and 97.6%, respectively. Besides, orthogonality evaluation for the separation of the four saponins using countercurrent chromatography and liquid chromatography was discussed. Four minor saponins were successfully separated from each other on a preparative scale by countercurrent chromatography from PNL, which will facilitate to provide ample of these minor saponins for further pharmacological studies.

Enantioseparation of 2-(4-chlorophenyl)succinic acid by countercurrent chromatography and investigation of injection volume on resolution.

2-(4-Chlorophenyl)succinic acid was successfully enantioseparated by countercurrent chromatography using hydroxypropyl-ß-cyclodextrin as chiral selector. A two-phase solvent system composed of n-hexane-ethyl acetate-0.1 mol/L phosphate buffer with pH 2.65 (5:5:10, v/v) was selected. Enantioselective liquid-liquid extraction was used to optimize the enantioseparation conditions. Meanwhile, the influence of injection volume on resolution in countercurrent chromatography was investigated and a linear relationship between the inflection point of injection volume and sample loading was tentatively obtained. The peak resolution will decrease significantly when the injection volume over the inflection point was used. In addition, it could be found that the smaller amount of sample loading, the larger impact of injection volume on resolution could be observed, which might serve as a good reference for the selection of sample volume in enantioseparations by countercurrent chromatography. Under optimized conditions, 20 mg of 2-(4-chlorophenyl)succinic acid racemate dissolved in 10 mL of aqueous phase was successfully enantioseparated by countercurrent chromatography. The recovery for both of the enantiomer of (±)-2-(4-chlorophenyl)succinic acid reached within 70-75% with a purity of 99.0%.

Liquid chromatographic study of two structural isomeric pentacyclic triterpenes on reversed-phase stationary phase with hydroxypropyl-ß-cyclodextrin as mobile phase additive.

Retention behavior of two structural isomeric pentacyclic triterpenic acids, maslinic acid and corosolic acid, was investigated by reverse phase high performance liquid chromatography (HPLC) with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as mobile phase additive. Inclusion complexation of maslinic acid, corosolic acid with hydroxypropyl-ß-cyclodextrin was evaluated under different concentration of hydroxypropyl-ß-cyclodextrin. Apparent formation constant (Km) between methanol and hydroxypropyl-ß-cyclodextrin was determined to be 13.82 L mol-1 under 25 °C using UV-spectrophotometry. Two retention models were employed individually for evaluation of inclusion complexation between the two pentacyclic triterpenic acids and hydroxypropyl-ß-cyclodextrin. It was found that a higher apparent formation constant (Kf) for corosolic acid and hydroxypropyl-ß-cyclodextrin was obtained, 19115 L mol-1, indicating that a greater affinity of hydroxypropyl-ß-cyclodextrin with corosolic acid was produced compared with that of maslinic acid, 11775 L mol-1, in the selected mobile phase, and stoichiometric ratio for both of inclusion complex was found to be 1:1. Thermodynamic analysis showed that a negative standard enthalpy change (ΔH) and an entropy change (ΔS*) for analyte transfer were obtained, where ΔH of maslinic acid and corosolic acid was found to be -10.188 kJ mol-1 and -10.650 kJ mol-1, ΔS* of two compounds was -2.092 and -2.180, respectively, indicating that transfer of structural isomers from mobile phase to stationary phase was enthalpically driven. Meanwhile, positive values were obtained for standard enthalpy change and standard entropy change, 136 kJ mol-1 and 274 kJ mol-1 and 536 J mol-1 K-1and 1004 J mol-1 K-1, for inclusion complexation between maslinic acid, corosolic acid and hydroxypropyl-ß-cyclodextrin, while negative values were obtained for Gibbs free energy during formation of inclusion complex, -160 kJ mol-1 and -299 kJ mol-1, indicating a spontaneous inclusion reaction happened.

Preparative separation of structural isomeric pentacyclic triterpenes from Eriobotrya japonica (Thunb.) leaves by high speed countercurrent chromatography with hydroxypropyl-ß-cyclodextrin as additive.

Maslinic acid and corosolic acid with high purity were successfully separated from Eriobotrya japonica (Thunb.) leaves by two-step countercurrent chromatographic separation. Two biphasic solvent systems composed of petroleum ether-ethyl acetate-ethanol-water (6:4:5:5, v/v) and petroleum ether-ethyl acetate-ethanol-0.10 mol/L of hydroxypropyl-ß-cyclodextrin with pH 7.0 (8:2:3.5:6.5, v/v) were selected according to the partition performance of the main structural isomeric pentacyclic triterpenes. The influences of pH value and concentration of hydroxypropyl-ß-cyclodextrin in separation of two isomers were investigated. In first step countercurrent chromatographic separation, a mixture of two target structural isomers (14.12 mg of sample I) was separated from 40.00 mg of a partially purified sample. In second step countercurrent chromatographic separation, maslinic acid and corosolic acid were completely isolated from 12.00 mg of sample I with hydroxypropyl-ß-cyclodextrin as aqueous phase additive. The recoveries of the two isomers were over 90%, yielding 5.18 mg of maslinic acid and 5.47 mg of corosolic acid, respectively.

Orthogonality in the selection of biphasic solvent systems for off-line two-dimensional countercurrent chromatography from Polygonum cuspidatum Sieb. et Zucc.

Off-line two-dimensional countercurrent chromatography has been widely applied to the isolation of complex samples, but little research on the investigation of orthogonality in the selection of biphasic solvent systems is available. In the present work, the orthogonality in the selection of a biphasic solvent system for liquid-liquid chromatographic separation of aqueous extract and ether extract from the traditional Chinese medicinal plant Polygonum cuspidatum Sieb. et Zucc was evaluated by the correlation coefficient and space occupancy rate. In total, 25 different biphasic solvent systems were tested, and 313 system combinations were analysed. A convex hull methodology was used to determine the separation space and to optimize separation conditions. The correlation coefficient matrix was transformed into dendrograms and a colour map to visualize the dissimilarity between, and orthogonality for, all solvent systems. The aqueous extracts from Polygonum cuspidatum were separated using selected biphasic solvent systems with high orthogonality: ethyl acetate-ethanol-water (70:1:70, v/v) and petroleum ether-ethyl acetate-water (1:5:5, v/v). The ether extracts from Polygonum cuspidatum were also separated using selected biphasic solvent systems with high orthogonality: petroleum-ethyl acetate-methanol-aqueous 0.25 M NH3•H2O (5:5:5:5, v/v) and petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v). Thirteen compounds were successfully obtained. The experimental results demonstrated that the evaluation of orthogonality provided an alternative strategy to select an applicable solvent system for the separation of complex samples using off-line two-dimensional countercurrent chromatography.