GUESSmix-guided optimization of elution–extrusion counter-current separations

Rational strategies for the optimization of separations are vital to any chromatographic technique. In counter-current separations (CS), once a suitable solvent system is selected for a given separation, the operator is faced with the task of optimizing the separation through the manipulation of those adjustable operation parameters allowed for by the current CS technology. This study employed a mixture of 21 natural products of varying polarity, molecular mass, and functionality, termed the GUESSmix, as a tool to assess the effectiveness of optimization strategies. The behavior of the GUESSmix was observed in the hexane/ethyl acetate/methanol/water 4:6:4:6 (HEMWat +3) solvent system. The effect of operation parameters on both the elution and extrusion stages of a recently introduced CS methodology, termed elution–extrusion counter-current chromatography (EECCC), was investigated. The resulting chromatograms were plotted with K-based reciprocal symmetry plots (ReS and ReSS), which allow comparison of the K values of significant peaks and assessment of resolution of eluting compounds in the interval 0 ≤ K ≤ ∞. The operation parameters studied were: (1) the effect of temperature controlled water circulation around the centrifuge; (2) the combination of flow rate and revolution speed; (3) sample loading capacity; (4) the direction of rotation either agreeing with or opposing the direction of coil winding; (5) injection before equilibration, a practice that saves operator time and reduces solvent consumption. The GUESSmix was found to be a highly useful reference mixture to compare and contrast stationary phase retention volume ratios, resolution, K-values, peak shapes, and extrusion characteristics between CS experiments. EECCC is shown to be a robust technique that may be enhanced with appropriate temperature, rpm, flow rate, sample loading, direction of rotation, and injection timing. Plotting ReS[S] chromatograms enables systematic study of CS parameters, which are not reproducibly represented in retention time or volume plots.