Separation and purification of four compounds from Desmodium styracifolium using off-line two-dimensional high-speed counter-current chromatography.

An off-line 2D high-speed counter-current chromatography technique in preparative scale has been successfully applied to separate and purify the main compounds from the ethyl acetate extract of Desmodium styracifolium. A two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water at an optimized volume ratio of 1:2:1:2 v/v/v/v was used. Conventional high-speed counter-current chromatography was used as the first dimension, and the upper phase of the solvent system was used as the stationary phase in the head-to-tail elution mode at a flow rate of 2.0 mL/min and a rotation speed of 900 rpm. Recycling high-speed counter-current chromatography served as the second dimension to separate an impure fraction of the first dimension. A total of four well-separated substances including vanillic acid (1), β-sitosterol (2), formononetin (3), and aromadendrin (4) were obtained, and their purities and structures were identified by HPLC-MS and (1) H NMR spectroscopy. The results illustrated that off-line 2D high-speed counter-current chromatography is an effective way to isolate compounds in complex samples.