High-speed countercurrent chromatography (HSCCC) has been widely applied to separation and purification of small molecules, but it has not popularly been used to separate polysaccharides [1]. An aqueous biphasic system (ABS) plays a vital role in the HSCCC separation process, but the investigation about ABS selection in previous works was not comprehensive. In this work, a comparison of binodal curves was performed, which allowed the analysis of the tunable kind of the inorganic salt (i.e., anionic, cationic) and the influence of the degree of the PEG polymer on the ability of these solutes to induce an ABS [2]. According to the phase diagram, ratio of phase, demixing time and the ratio of sugar contents in the uppper and lower layers, an ABS of PEG400-Na2SO4-H2O (20%-16%-64%, w/w/w) was determined for the separation of crude polysaccharides from pericarpium granati (PGP). The lower phase was used as the mobile phase at a flow rate of 0.8 mL/min. The effluent was collected with a fraction collector by every 8 min and examined with phenol-sulfuric acid method at 492 nm. An elution profile was obtained as shown in Fig.1. This study demonstrated that phase diagrams could provide an excellent means to search solvent system for CCC and that an aqueous biphasic system based on lower degree of PEG could be used for effective separation of polysaccharides by HSCCC.

Acknowledgments The project was supported by National Natural Science Foundation of China (No. 21275098) and Doctor Base Foundation of Chinese Ministry of Education (No. 20110202110005). References [1] J. Y. Yin, Z. H. Jiang, H. Yu, M. Y. Xie, W. L. Hsiao, A. P. Lu, Q. B. Han. J. Chromatogr. A. 2012, 1262: 92-97. [2] M. G. Freire, J. F. B. Pereira, M. Francisco, H. Rodriguez, L. P. N. Rebelo, R. D. Rogers, J. A. P. Chem. Eur. J. 2012, 18:1831-1839.