On-line two-dimensional countercurrent chromatography × high performance liquid chromatography system with a novel fragmentary dilution and turbulent mixing interface for preparation of coumarins from Cnidium monnieri

• A novel fragmentary dilution and turbulent mixing (FD-TM) interface was developed for online 2D CCC × HPLC system.
• Online 2D CCC × HPLC enables simultaneous separation of coumarins in complex matrices.
• Solvent system of n-heptane/acetone/water was optimized for the CCC separation.
• One step separation of 8 pure coumarins from 0.5 g of Cnidium monnieri crude extract was achieved.

This study describes a novel on-line two-dimensional countercurrent chromatography × high performance liquid chromatography (2D CCC × HPLC) system for one-step preparative isolation of coumarins from the fruits of Cnidium monnieri. An optimal biphasic solvent system composed of n-heptane/acetone/water (31:50:19, v/v) with suitable Kd values and a higher retention of the stationary phase was chosen to separate target compounds. In order to address the solvent incompatibility problem between CCC and RP-HPLC, a novel fragmentary dilution and turbulent mixing (FD-TM) interface was successfully developed. In detail, the eluent from the first dimensional CCC column was divided into fractions to form ‘sample-dilution’ stripes in the two switching sample loops, by the dilution water from the makeup pump. Following this, a long, thin tube was applied to mix the CCC eluent with water by in-tube turbulence, to reduce the solvent effect. Each CCC fraction was alternately trapped on the two holding columns for further preparative HPLC separation. This nationally designed FD-TM strategy effectively reduced post-column pressure and allowed a higher water dilution ratio at the post end of CCC, leading to improved sample recovery and a robust 2D CCC × HPLC isolation system. As a result, in a single 2D separation run (6.5 h), eight target compounds (1–8) were isolated from 0.5 g crude extract of C. monnieri, in overall yields of 1.3, 2.0, 0.5, 0.5, 0.8, 1.5, 8.2, and 15.0%, with HPLC purity of 90.1, 91.1, 94.7, 99.1, 99.2, 98.2, 97.9, and 91.9%, respectively. We anticipate that this improved 2D CCC × HPLC system, based on the novel FD-TM interface, has broad application for simultaneous isolation and purification of multiple components from other complex plant-derived natural products.