Retention mechanism of pH-peak-focusing in countercurrent chromatographic separation of baicalin and wogonoside from Scutellaria baicalensis Georgi

Abstract

An efficient and target-oriented pH-peak-focusing countercurrent chromatographic method was established for large-scale separation of baicalin and wogonoside from the crude exact of traditional Chinese medicinal herb Scutellaria baicalensis Georgi. An optimized two-phase solvent system composed of n-butanol–ethyl acetate–methanol–water (1:4:0.5:5, v/v) was selected. Trifluoroacetic acid (10 mmol/L) was added to the upper organic phase, used as the stationary phase. One liter of the aqueous lower phase was used as the mobile phase for 0–350 min, and then 10 mmol/L ammonia was added to remaining 1 L of the aqueous lower phase and used as the mobile phase for 350–600 min. In total, 493.2 mg of baicalin with 98.6% purity and 88.6 mg of wogonoside with 98.9% purity were obtained from 1.0 g of crude exact of S. baicalensis by countercurrent chromatography in a single run. The acid dissociation constant (pKa) and oil–water partition coefficient values of two components were measured to better understand the mechanism of separation. Results showed that pH-peak-focusing countercurrent chromatography with a polar solvent system added with trifluoroacetic acid could be an efficient method for large-scale isolation of organic acids, which are difficult to separate with conventional countercurrent chromatography due to their poor solubility in non-polar solvents.