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.

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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.

2‐(4‐Chlorophenyl)succinic acid was successfully enantioseparated by countercurrent chromatography using hydroxypropyl‐β‐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%.

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