Two-dimensional countercurrent chromatography × high performance liquid chromatography for preparative isolation of toad venom

In this work, a new on-line two-dimensional chromatography coupling of flow programming counter-current chromatography and high-performance liquid chromatography (2D CCC × HPLC) was developed for preparative separation of complicated natural products. The CCC column was used as the first dimensional isolation and a preparative ODS column operated in reversed-phase (RP) mode as the second dimension. The CCC was operated at a controlled flow rate to ensure that each fraction eluted within one hour, corresponding to the isolation time of the 2nd dimensional preparative HPLC. The eluent from the 1st dimensional CCC was diluted using a makeup pump and trapped onto holding column, before been eluted and transferred to the 2nd dimensional HPLC. The performance of the holding column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure, for the solution to the issue of online trapping of low pressure eluent from a CCC column. Satisfactory trapping efficiency and tolerable CCC pressure can be achieved using a commercially available 15 mm × 30 mm i.d. ODS pre-column. The present integrated system was successfully applied in a one-step preparative separation of 12 compounds, from the crude methanol extract of venom of Bufo bufo gargarizans. Compounds 1-12 were isolated in overall yield of 1.0%, 0.8%, 2.0%, 1.3%, 2.0%, 1.5%, 1.9%, 3.6%, 6.1%, 4.8%, 3.5% and 4.1%, with HPLC purity of 99.9%, 99.7%, 90.6%, 99.9%, 77.0%, 99.9%, 90.4%, 99.9%, 52.0%, 99.9%, 99.3%, and 85.0%, respectively. All the results demonstrate that the flow programming CCC × HPLC method is an efficient and convenient way for the separation of compounds from toad venom and it can also be applied to isolate other complex multi-component natural products.