Separation and purification of six biosurfactant rhamnolipids by high-speed countercurrent chromatography utilizing novel solvent selection method

Abstract

In this study, an original solvent selection method was developed for the separation of biosurfactant rhamnolipids from fermentation broth by high-speed countercurrent chromatography. For this method, calculations were based on solubility and functional group parameters that enabled the rapid selection of suitable solvent systems at no more than 10% of average time by traditional method. Based on selection results, a three-phase solvent system composed of n-hexane-methyl acetate-acetonitrile-water (2:2:2:5, v/v) was efficiently used for one-step separation of a mixture of six rhamnolipids. Thereby, RhaC10C10 (0.73 mg), RhaC10 (0.51 mg), Rha2C10 (0.12 mg), RhaC10C12 (1.26 mg), Rha2C10C10 (1.03 mg), and Rha2C10C12 (0.87 mg) were extracted from prepared Pseudomonas aeruginosa (100.0 mg) with purities of 96.37, 95.20, 91.25, 84.41, 89.8, and 90.26%, respectively. The purified compounds were identified by high-performance liquid chromatography/mass spectroscopy, plus 1H and 13C nuclear magnetic resonance studies. This study provides a new perspective on concepts involving efficient solvent system selection.