Sweet spot matching: A thin-layer chromatography-based countercurrent solvent system selection strategy

- TLC method of solvent system selection for countercurrent separation is revisited.
- A broader approach to the GUESS method is implemented.
- The theory supporting TLC and CCS sweet spot matching is presented.
- Application of the GUESS method to natural product separation is demonstrated.

TLC-based strategies were proposed in 1979 (Hostettmann et al.) and 2005 (Friesen & Pauli; GUESS method) to minimize the number of partitioning experiments required for countercurrent separation (CCS) solvent system selection. As semi-empirical approaches, both proposed that the K values defining the sweet spot of optimal CCS corresponded to a matching Rf value range from the silica gel TLC plate developed in the organic phase of a biphasic or a corresponding monophasic solvent system. Despite their simplicity, there has been an absence of theoretical support and a deficiency of reported experimental evidence. The present study explores the theory required to develop correlations between Rf and K. All theoretical models surmise that the optimal Rf value range should be centered at 0.5. In order to validate the feasibility of the concept of matching Rf and K values, 43 natural products and six solvent system families were investigated. Out of 62 correlations, 45 resulted in matched Rf and K values. Based on this study, practical guidelines for the TLC-based prediction strategy are provided. These approaches will equip CCS users with an updated understanding of how to apply the TLC-based solvent system selection strategy to accelerate a targeted selection of CCS conditions.