A strategy to process hundred-gram level complex sample using liquid-liquid-refining extraction and consecutive counter-current chromatography: Toona sinensis case study.

Large-scale preparation of target compounds from complex samples is facing great challenges. In the present study, an efficient strategy for large-scale preparation of target compound was proposed and successfully applied in the separation of active components from Toona sinensis. The pretreatment technology of liquid-liquid refining extraction (LLRE) combined with consecutive high-speed counter-current chromatography (HSCCC) was used to process hundred grams of extractions. Firstly, two phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (5:5:5:5, v/v) and (2:5:2:5, v/v) were used to remove low polar and high polar impurities from 100 g crude extracts of T. sinensis, respectively, and 9.25 g of crude sample was obtained. And then, n-hexane-ethyl acetate-methanol-water (2.5:5:2.5:5, v/v) was used as the solvent system for HSCCC separation. The isocratic elution mode with max loading and consecutive injections mode were investigated to obtain more target compound. As a result, ethyl gallate with purity of 97% was successfully separated by 5 times consecutive counter-current chromatography. The separation was repeated once. Finally, ethyl gallate (3.73 g) was isolated from 9.25 g of crude sample (100 g crude extracts). The results demonstrated that the yield increased from 0.26 g/h/L of untreated crude extract to 0.93 g/h/L of LLRE pre-treated sample for single injection, and further increased to 1.62 g/h/L for 5 consecutive injections mode with the present method.