3-Methylcyclohexanone thiosemicarbazone: Determination of E/Z isomerization barrier by dynamic high-performance liquid chromatography, configuration assignment and theoretical study of the mechanisms involved by the spontaneous, acid and base catalyzed pr

Here, we report on the simultaneous direct HPLC diastereo- and enantioseparation of 3-methylcyclohexanone thiosemicarbazone (3-MCET) on a polysaccharide-based chiral stationary phase under normal-phase conditions. The optimized chromatographic system was employed in dynamic HPLC experiments (DHPLC), as well as detection technique in a batch wise approach to determine the rate constants and the corresponding free energy activation barriers of the spontaneous, base- and acid-promoted E/Z diastereomerization of 3-MCET. The stereochemical characterization of four stereoisomers of 3-MCET was fully accomplished by integrating the results obtained by chemical correlation method with those derived by theoretical calculations and experimental investigations of circular dichroism (CD). As a final goal, a deepened analysis of the perturbing effect exercised by the stationary phase on rate constant values measured through DHPLC determinations as a function of the chromatographic separation factor α of the interconverting species was successfully accomplished. This revealed quite small deviations from the equivalent kinetic values obtained by off-column batch wise procedure, and suggested a possible effective correction of rate constants measured by DHPLC approach.

► HPLC separation of four stereoisomers of 3-methylcyclohexanone thiosemicarbazone (3-MCET).
► Discussion on the mechanism of the E/Z diastereomerization process.
► Correction of the rate constants of the diastereomerization reaction measured by DHPLC approach.