Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn

Abstract

Pure compounds isolated from complex natural plants are important for drug discovery. This study describes a novel two-dimensional hyphenation of counter-current chromatography and high-performance liquid chromatography (2D CCC×HPLC) with heart-cutting and stop-and-go techniques for preparative isolation of multiple targets components from Peucedanum praeruptorum Dunn (Umbelliferae) crude extracts in a single step. The CCC and HPLC were hyphenated via a 4-port valve equipped at the post-end of the CCC column, to heart cut the impure fractions to the 2nd dimensional HPLC for further separation. Furthermore, the stop-and-go flow scheme was applied in the 1st dimensional CCC to fit with the time constraints of the 2nd dimensional preparative HPLC. Last but not least, an optimal biphasic solvent system composed of n-heptane/acetone/water (31:50:19, v/v/v) with suitable Kd values and a higher retention of the stationary phase was chosen to separate target compounds, resulting in the improvement of the CCC column efficiency. By taking the advantages of this rationally designed system, sixteen coumarins were isolated from 1.0g of P. praeruptorum crude extract, with HPLC purity from 90.1% to 99.5%, in a single 2D separation run. More interestingly, two minor linear coumarins and one angular coumarin were isolated from P. praeruptorum Dunn for the first time. As far as we known, this is the first report on the combination of heart-cutting technique and stop-and-go protocol in 2D CCC×HPLC system, by which good separations on comprehensive matrix were achieved. We expect that this approach may have broad applications for simultaneous isolation and purification of multiple components from other complex plant-derived natural products.