Hydrophilic interaction chromatography for selective separation of isomeric saponins

• An efficient HILIC method was developed to separate isomeric saponins.
• A Click XIon column with improved hydrophilicity and polar selectivity was used.
• Methanol was the weak eluent, and symmetrical peak in high sample loading was obtained.
• Eleven saponins were isolated and identified.
• A new saponin has been isolated and identified.

A method based on a Click XIon zwitterionic stationary phase was developed to separate isomeric saponins efficiently in hydrophilic interaction chromatography (HILIC) mode using methanol as a weak eluent. The retention times of a set of isomeric saponins, ginsenoside Rc (S1), ginsenoside Rb2 (S2), and ginsenoside Rb3 (S3) on five kinds of HILIC columns were compared using acetonitrile/water or methanol/water as mobile phase. All results indicated that the Click XIon column showed the highest retention times of all these isomeric saponins. Then the retention behaviors of these isomeric saponins on the Click XIon column were investigated. The results demonstrated that the retention behaviors of saponins on Click XIon in methanol/water mobile phase were different from that in acetonitrile/water. Methanol/water binary eluent offered enhanced selectivity to these isomeric saponins (αS2/S3(MeOH) = 1.16 > αS3/S2(ACN) = 1.04), and the elution order of ginsenoside Rb2 (S2) and ginsenoside Rb3 (S3) was reversed compared with acetonitrile/water. Moreover, methanol/water solvent has better solubility for saponins than acetonitrile/water, resulting in improved preparative peak shape, which is greatly beneficial for saponin purification. Application to the preparative separation of saponins from leaves of P. notoginseng was also investigated, and eleven saponins, including three sets of isomeric saponins with one new saponin were isolated and identified. All these results indicated that this method was efficient for analytical and preparative separation of saponins, especially for isomeric saponins, containing xylopyranosyl, arabinofuranosyl or arabinopyranosyl units.