| Polyphenols, especially hydrophilic polyphenols are major active components in traditional Chinese medicine. Irreversible adsorption behavior of polyphenols on the current separation and purification technologies, duo to the structures of hydroxyl and glycosly groups, get many difficulties in separation and purification, and low purity and yield of finished product was a popular problem. In view of the fact that a new and practical technology platform was established for the separation and purification of hydrophilic polyphenols, high-speed countercurrent chromatography and hydrogen bond adsorption chromatography based on agarose media were introduced in this research for the separation and purification of hydrophilic polyphenols. Three-phase HSCCC was introduced in this research for separation hydrophilic polyphenols.1. In this study, the method of selection and adjustment the solvent system of HSCCC was improved; a new dual-rotation elution mode was forwarded established and two new concepts of normal phase and reversed phase chromatography in HSCCC separation were introduced. The universal process was drawn from the examples of separation and purification of isomers of dihydroxybenzoic acid, hydrophilic polyphenols of Salvia miltiorrhiza Bunge and hydrophilic polyphenols of Geranium Wilfordii Maxim with HSCCC, in which many fractions were obtained, respectively.Five isomers of dihydroxybenzoic acids were separated by HSCCC with dual-rotation elution method. Salvianolic acid B was isolated and purified effectively in one-step separation from crude extract of Salvia miltiorrhiza Bunge at the purity and yield of 99.4% and 85.5%. Five batches of sample were loaded consequently in one run leading to low consuming of organic solvents, high advantage of solvent system, stable running and high preparation of targets. Gallic acid, corilagin and geraniin were separated in one-step separation and purification from crude extact of Geranium Wilfordii Maxim with purities of 85.9%,92.1% and 94.2%, and yields of 88.1%,70.3% and 92.2%, respectively。A stable liquid/liquid/solid HSCCC system was established creatively for the separation of low molecular weight substances with similar structure from natural resources, particularly hydrophilic polyphenols which was difficult to be separated by other chromatographies. In this study, a higher stationary was obtained by the optimizing the operating parameters. Flavonols (Flavon, quercetin and Myricetin) and rutin and quercitrin belong to polyphenols used as model compounds were separated by liquid/liquid/solid HSCCC. 2. The method and mechanism of high concentration of agarose, highly cross-linked agarose gels, include of Superose Prototype gel (hydrogen bond acceptor) and Tris-Novarose SE 40/100 gel (hydrogen bond donor), were studied in the separation of hydrophilic polyphenols from natural resources. It is helpful for laid the foundation of subsequent modification of agarose gel. Superose Prototype gel was used to separation and purification of hydrophilic polyphenols with the phase of ethanol-acetic acid-water and grandient elution method.3,4-dihydroxyphenyllactic acid, protocatechualdehyde and salvianolic acid B were isolated and purified effectively in one step separation from crude extact of Salvia miltiorrhiza Bunge with Superose Prototype gel at the purity of 98.1%,99.2% and 94.3%, and yield of 94.3%,98.2% and 94.1%. Furthermore, this agarose gel has high sample loading capacity at a sample load of 15 mL(1/4 column volume) crude extract of Salvia miltiorrhiza Bunge, corresponding to 600 mg. Salvianolic acid B got a good separation with purity and yield of 84.9% and 78.3%. Gallic acid, corilagin and geraniin were first separated in one-step separation and purification from crude extract of Geranium Wilfordii Maxim at purities of 90.4%,90.2% and 84.3% and yields of 82%,87% and 77.1%。Superose Prototype on the average diameter of 30μm was of good resolution and rigidity and was suitable for industrial separation.
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