Use of O-carboxymethyl chitosan in high-speed counter-current chromatography: a novel additive for a biphasic solvent system

Abstract

A novel additive, O-carboxymethyl chitosan (O-CMC), for high-speed counter-current chromatography (HSCCC) was found and investigated. It was shown that O-CMC can improve peak resolution (R_s). The mechanism of O-CMC as a solvent additive to improve resolution was studied using quercetin, luteolin and kaempferol as model analytes. It was indicated that O-CMC improved resolution not by increasing the retention of the stationary phase but by introducing intermolecular forces: hydrogen bonding interaction and electrostatic interaction. Steviol glycosides (SGs) were separated by analytical HSCCC using a biphasic solvent system composed of n-hexane/n-butanol/water at a volume ratio of 1.5 : 3.5 : 5 with O-CMC (1.0 mg mL⁻¹) in the lower phase. HPLC analysis showed that acceptable separation of SGs was achieved by using O-CMC as a solvent additive and the purities of ST, RC and RA were 61.5%, 97.5% and 74.1%, respectively.