On-line comprehensive two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for preparative isolation of toad venom

• Vacuum evaporation assisted adsorption (VEAA) interface was designed for a 2D preparative NPLC × RPLC system.
• The NPLC and RPLC mobile-phase immiscibility was solved by the new interface.
• The VEAA interface based 2D NPLC × RPLC system was evaluated with a crude extract of toad venom.
• Nineteen compounds were isolated using the 2D-LC system.

An on-line comprehensive preparative two–dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography (2D NPLC × RPLC) system was constructed with a newly developed vacuum evaporation assisted adsorption (VEAA) interface, allowing fast removal of NPLC solvent in the vacuum condition and successfully solving the solvent incompatibility problem between NPLC and RPLC. The system achieved on-line solvent exchange within the two dimensions and its performance was illustrated by gram-scale isolation of crude extract from the venom of Bufo bufo gargarizans. Within separation time of ∼20 h, 19 compounds were obtained with high purity in a single run. With the VEAA interface, the 2D system exhibited apparent advantages in separation efficiency and automation compared with conventional methods, indicating its promising application in the routine separation process for complicated natural products.