Countercurrent chromatography: New methods for separation of natural products

High-speed countercurrent chromatography (HSCCC) is a support free liquid-liquid partitioning method which utilizes a strong centrifugal force field to contain the stationary phase within the column and to facilitate vigorous mixing and settling regions. We have developed improved techniques using this method for the extraction and separation of complex mixtures from marine natural products and biological fluids. HSCCC was used for the separation of a mixture of steroids: 4,22-stigmastadien-3-one, estrone, ;Modification of the stationary phase by the addition of small amounts of 18-crown-6 showed higher efficiency of extraction of theophylline from the urine of an asthmatic patient. The effect of increasing the ionic strength with disodium hydrogen phosphate and its influence on the extraction efficiency was also studied. To better understand the increased efficiency of extraction in the presence of a crown ether, molecular modeling calculations were performed. These calculations suggested (1) the presence of a hydrogen bonded theophylline-crown ether complex and (2) greater selectivity of extraction of theophylline could be expected when compared to other xanthine derivatives, such as caffeine and theobromine.;The usefulness of HSCCC was increased by interfacing it with high performance thin-layer chromatography (HPTLC) and CIMS to form a multidimensional continuous on-line chromatographic technique for the gradient extraction, fractionation, separation and preliminary structure determination of the methanolic extract of Terminalia belerica (Combretaceae). The technique provided valuable information about the components of the sample with minimal expenditure of the limited supply of the crude extract.;The basic fraction of Acanthophora spicifera (Rodophyta) was extracted with HSCCC to show a higher extraction efficiency when compared to classical extraction techniques. The increased yield provided sufficient quantities of isolated compounds for further analysis.