Unusual effect of column temperature on chromatographic enantioseparation of dihydropyrimidinone acid and methyl ester on amylose chiral stationary phase

This paper reports an unusual effect of column temperature on the separation of the enantiomers of dihydropyrimidinone (DHP) acid and its methyl ester on a derivatized amylose stationary phase by normal-phase liquid chromatography. The separation of the DHP acid enantiomers was investigated using both carbamate-derivatized amylose and cellulose stationary phases (Chiralpak AD and Chiralcel OD) with an ethanol–n-hexane (EtOH–n-Hex) mobile phase. On the amylose phase, the van ’t Hoff plot of the retention factor of the S-(+)-DHP acid was observed to be non-linear while that of R-(−)-DHP acid was linear. Likewise, the van ’t Hoff plot for DHP acid enantioselectivity was non-linear with a transition occurring at approximately 30 °C. Furthermore, the van ’t Hoff plot for the DHP acid enantioselectivity factor for data taken when heating the column from 5 to 50 °C was not superimposable with the same plot prepared with data from the cooling process from 50 to 5 °C. This observation suggested that the stationary phase was undergoing a thermally induced irreversible conformational change that altered the separation mechanism between the heating and cooling cycles. Similar phenomena were observed for the separation of the enantiomers of the DHP ester probe compound. The conformational change of the AD phase was shown to depend on the polar component of the mobile phase. When 2-propanol (2-PrOH) was used as the modifier instead of EtOH, the van ’t Hoff plots for DHP acid were linear and thermally reversible, suggesting that no such irreversible conformational change occurs with this modifier. Conversely, when the AD phase was pre-conditioned with a more polar methanol (MeOH) or water containing mobile phase, thermal irreversibility of DHP acid enantioselectivity was once again observed. Interestingly, when the stationary phase was changed to its cellulose analogue, the Chiralcel OD, all van ’t Hoff plots for the retention and selectivity of DHP acid were thermally reversible for both EtOH–n-Hex and 2-PrOH–n-Hex mobile phases.