Orthogonal separation on one beta-cyclodextrin column by switching reversed-phase liquid chromatography and hydrophilic interaction chromatography

A dual retention combined with reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) has been observed on beta-cyclodextrin (β-CD) bonded stationary phase. A typical U-shaped retention curve was achieved owing to dual retention mechanism. Based on this observation, a β-CD column can be operated under reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) modes. Two-dimensional liquid chromatography (2D-LC) analysis can be realized on just a β-CD column by switching these two different separation modes. In this study, off-line 2D-LC analysis for a natural product was carried out to prove the orthogonal separation between RP-LC and HILIC modes on a Click β-CD column. Herba Hedyotis Diffusae, the whole grass of Hedyotis Diffusae wild was extracted with water, pretreated with macroporous resin and then first separated at RP-LC mode on the Click β-CD column to obtain successive fractions, which were then reanalyzed at HILIC mode on the same Click β-CD column. The result proved that both separation modes on the Click β-CD column have good retention and peak shape, and these two separation modes have good orthogonality. 2D-LC analysis revealed abundant information in the natural product. Especially numerous minor components were enriched and separated. The mobile phase used in RP-LC and HILIC modes can be same and the switch between these two separation modes is easily realized by changing the ratio of the acetonitrile and water. Hence the mobile phase in this 2D-LC system is completely compatible. This advantage makes this combination is an appropriate 2D-LC method for the solutes having retention at both separation modes.