Abstract
Counter-current chromatography is a chromatographic technique with a support-free liquid stationary phase. In the present study, a successful application of linear gradient counter-current chromatographic method for preparative isolation of bioactive components from the crude ethanol extract of Zanthoxylum planispinum was presented. The application of n-hexane/ethyl acetate/methanol/water quaternary solvents, in terms of "HEMWat" or "Arizona" solvent families, in gradient elution mode was evaluated. Results indicated that slightly proportional changes of biphasic liquid systems provided the possibility of gradient elution in counter-current chromatography, maintaining stationary phase retention in the column. With the selected quaternary solvent systems composed of n-hexane/ethyl acetate/methanol/water (2:1:2:1 and 3:2:3:2, v/v), and optimized gradient programs, in total seven fractions were separated in 4.5 h. Most of the purified compounds could be obtained at the milligram level with over 80% purity. The present study indicated that the linear gradient counter-current chromatographic approach possessed unique advantages in terms of separation efficiency, exhibiting great potential for the comprehensive separation of complex natural extracts.
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