Solvent systems with n-hexane and/or cyclohexane in countercurrent chromatography—Physico-chemical parameters and their impact on the separation of alkyl hydroxybenzoates

• A CCC method was developed for the separation of alkyl hydroxybenzoates.
• Solvent systems with n-hexane and/or cyclohexane and 3 other components were used.
• Physico-chemical parameters of the solvent systems were determined.
• Structurally similar compounds could be resolved by CCC.

Countercurrent chromatography (CCC) is an efficient preparative separation technique based on the liquid–liquid distribution of compounds between two phases of a biphasic liquid system. The crucial parameter for the successful application is the selection of the solvent system. Especially for nonpolar analytes the selection options are limited. On the search for a suitable solvent system for the separation of an alkyl hydroxybenzoate homologous series, we noted that the substitution of cyclohexane with n-hexane was accompanied with unexpected differences in partitioning coefficients of the individual analytes. In this study, we investigated the influence of the subsequent substitution of n-hexane with cyclohexane in the n-hexane/cyclohexane/tert-butylmethylether/methanol/water solvent system family. Exact phase compositions and polarity, viscosity and density differences were determined to characterize the different mixtures containing n-hexane and/or cyclohexane. Findings were confirmed by performing CCC separations with different mixtures, which led to baseline resolution for positional isomers when increasing the amount of cyclohexane while the resolution between two pairs of structural isomers decreased. With the new methodology described, structurally similar compounds could be resolved by choosing a certain ratio of n-hexane to cyclohexane.