Abstract
A low-cost and gradual gradient online two-dimensional preparative liquid chromatography system was developed for the preparative separation of compounds of interest from complex natural products. Medium-pressure liquid chromatography (MPLC) was applied as the first dimension and preparative high-performance liquid chromatography (HPLC) was employed as the second dimension. Two trapping columns and a makeup pump were also used. Each of the two dimension columns was packed with reversed-phase ostadecyl-silca (ODS), and eluted by an isocratic pump with a gradient mixing device, in which the solvent of the first and the second dimensions were gradiently changed to fit the demand of the sample elution. The instrument operation and the performance of this MPLC × preparative HPLC system were evaluated via gram-scale isolation of a crude methanol extract of toad venom from Bufo bufo gargarizans. As a result, in a single 2D separation run (345 min), 18 bufadienolides (1–18) were isolated from 0.5 g crude extract, and the purity of each compound was higher than 90%. We anticipate that this improved low-cost preparative 2D-LC system has broad applications for the simultaneous isolation and purification of multiple components from other complex sample matrices.
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