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Counter-current chromatography (CCC) is a unique liquid-liquid partition chromatography and largely relies on the partition interactions of solutes and solvents in two-phase solvents. Usually, the two-phase solvents used in CCC include a lipophilic organic phase and a hydrophilic aqueous phase. Although a large number of partition interactions have been found and used in the CCC separations, there are few studies that address the role of water on solvents and solutes in the two-phase partition. In this study, we presented a new insight that H 2 O (water) might be an efficient and sensible hydrophobic agent in the n -hexane-methanol-based two-phase partition and CCC separation of lipophilic compounds, i.e., anti-cancer component mollugin from Rubia cordifolia . Although the n -hexane-methanol-based four components solvent systems of n -hexane-ethyl acetate-methanol-water (HEMWat) is one of the most popular CCC solvent systems and widely used for natural products isolation, this is an interesting trial to investigate the water roles in the two-phase solutions. In addition, as an example, the bioactive component mollugin was targeted, separated, and purified by MS-guided CCC with hexane-methanol and minor water as a hydrophobic agent. It might be useful for isolation and purification of lipophilic mollugin and other bioactive compounds complex natural products and traditional Chinese medicines.

H2O-Induced Hydrophobic Interactions in MS-Guided Counter-Current Chromatography Separation of Anti-Cancer Mollugin from Rubia cordifolia