Dynamic pH junction high-speed counter-current chromatography coupled with microwave-assisted extraction for online separation and purification of alkaloids from Stephania cepharantha.

A simple and efficient dynamic pH junction high-speed counter-current chromatography method was developed and further applied to the online extraction, separation and purification of alkaloids from Stephania cepharantha by coupling with microwave-assisted extraction. Mineral acid and organic base were added into the mobile phase and the sample solution, respectively, leading to the formation of a dynamic pH junction in the column and causing focus of alkaloids. Selective focus of analytes can be achieved on the basis of velocity changes of the pH junction through appropriate selection of solvent systems and optimization of additive concentrations. The extract can be directly introduced into the HSCCC for the online extraction, separation and purification of alkaloids from S. cepharantha. Continuous separation can be easily achieved with the same solvent system. Under the optimum conditions, 6.0 g original sample was extracted with 60 mL of the upper phase of hexane-ethyl acetate-methanol-water (1:1:1:1, v/v/v/v) containing 10% triethylamine under 50 °C and 400 W irradiation power for 10 min, the extracts were directly separated and purified by high-speed counter-current chromatography. A total of 5.7 mg sinomenine, 8.3mg 6,7-di-O-acetylsinococuline, 17.9 mg berbamine, 12.7 mg isotetrandrine and 14.6 mg cepharanthine were obtained with purities of 96.7%, 93.7%, 98.7%, 97.3% and 99.3%, respectively. The online method provides good selectivity to ionizable compounds and improves the separation and purification efficiency of the high-speed counter-current chromatography technique. It has good potential for separation and purification of effective compounds from natural products.