Mobile-phase selection for isomer selectivity in reversed-phase liquid chromatography: Evaluation of conventional and green, binary and ternary mobile phases for the separation of carvacrol and thymol

To map out the mobile-phase parameter space for the separation of the positional isomers carvacrol and thymol, naturally occurring monoterpenoid phenols of astonishingly wide biological activity, by reversed-phase liquid chromatography, we tested conventional and green candidates for the organic solvent (OS) in the mobile phase. Binary and ternary compositions were evaluated at different solvent ratios regarding resolution, selectivity, and analysis time of the isomer separation on a C₁₈ column. Baseline separation with binary compositions was obtained with ethanol (EtOH), acetonitrile (ACN), or tetrahydrofuran (THF) as OS, but not with water‒methanol (W‒MeOH) mobile phases. The sensitivity of the isomer selectivity to the OS volume fraction in the mobile phase strongly depended on the OS candidate, yielding a narrower or wider range of OS volume fractions over which the isomer separation succeeded. Among the binary compositions, W‒ACN mobile phases performed best, but W‒EtOH mobile phases were an acceptable green alternative. The addition of THF or MeOH as third solvent to W‒ACN mobiles phase offered little or no selectivity enhancement, whereas the addition of THF or ethyl acetate (EtOAc) to W‒MeOH or W‒EtOH mobile phases, respectively, delivered large selectivity improvements compared to all other binary and ternary compositions. The quantification of carvacrol and thymol in oregano leaf extract using a W‒EtOH‒EtOAc mobile phase proved that green methods can easily replace conventional ones in this application. Regarding the physicochemical properties of the OS candidates, the study showed that the absence of hydrogen-bond donor properties favored, but did not create, the isomer selectivity with binary mobile phases, and that ternary mobile phases combining two OS candidates with large differences in molecular volume, solute polarity, and hydrogen-bond capability achieved the best results overall.