title: 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.
abstract: 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 (6.5h), eight target compounds (1-8) were isolated from 0.5g 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.
authors.affiliation: School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.affiliation: School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
authors.forename: Long-Jiang
authors.initials: LJ
authors.lastname: Chen
authors.affiliation: School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
authors.forename: Jing-Lan
authors.initials: JL
authors.lastname: Liu
authors.affiliation: School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
authors.forename: Xin-Yuan
authors.initials: XY
authors.lastname: Wang
authors.affiliation: School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
authors.forename: Yun-Long
authors.initials: YL
authors.lastname: Wu
authors.affiliation: School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
authors.forename: Mei-Juan
authors.initials: MJ
authors.lastname: Fang
authors.affiliation: School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wu
authors.affiliation: School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen, 361102, China. Electronic address: qyk@xmu.edu.cn.
authors.forename: Ying-Kun
authors.initials: YK
authors.lastname: Qiu
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D031048:Cnidium / Q000737:chemistry*; D003374:Coumarins / Q000737:chemistry / Q000302:isolation & purification*; D003377:Countercurrent Distribution; D007201:Indicator Dilution Techniques; D010936:Plant Extracts / Q000737:chemistry; D012997:Solvents / Q000737:chemistry
chemical_list: D003374:Coumarins; D010936:Plant Extracts; D012997:Solvents
keywords: Cnidium monnieri; Coumarins; Fragmentary dilution and turbulent mixing interface; On-line 2D CCC×LC