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Abstract
The choice of an appropriate solvent system for Countercurrent Chromatography (CCC) is a critical step in the purification of natural products. Targeted towards their high sample diversity, G.U.E.S.S. is a practical approach for the prediction of CCC distribution constants, K values, by standard thin layer chromatography (TLC). G.U.E.S.S. allows a major reduction in workload by direct use of routine TLC information. The separation capability of CCC focuses on an optimal “window of opportunity” that can be described as the “sweet spot” of CCC separation. The sweet spot of optimal CCC performance may be described as an area where compound K values are between 0.4 and 2.5. Two useful CCC solvent systems: hexane/ethyl acetate/methanol/water and chloroform/methanol/water are organized and recommended as the HEMWat and ChMWat methods of solvent system selection. The relationship of (i) P values, measured by the ratio of UV‐vis absorption, (ii) TLC R f values and (iii) CCC retention volumes for over 20 diverse commercially available natural products are described. The HEMWat method characterizes a versatile solvent selection technique. TLC R f values will often give practical predictions, even with simple single‐phase mixtures. Additional information can be acquired from equivalent solvent systems and by calibration with the G.U.E.S.S. standard compounds. The latter will also aid in the important selection of which phase will function as the mobile phase. The choice of normal vs. reverse phase will depend on the polarity of compounds that are desired to be gathered into the sweet spot. In addition, G.U.E.S.S. has been shown to be readily applicable to natural product purification necessary for drug discovery, bioassay guided fractionation, and metabolome analysis.