Kinetic analysis and subambient temperature on-line on-column derivatization of an active aldehyde

The chromatographic analysis of aldehydes under typical reversed-phase conditions may be a challenging task due to an equilibrium process leading to the formation of a gem diol species regardless of acidic or basic conditions. Initially, a reversed-phase HPLC gradient elution was developed to determine the amount of an acetylenic aldehyde in a reaction mixture. Significant fronting was observed under acidic and basic conditions even at −5 °C. In order to circumvent this problem, a reversed-phase HPLC gradient method on a C18 column at subambient temperature was developed using diethylamine as a mobile phase additive for on-line on-column derivatization of the aldehyde moiety. The on-line on-column reaction rate for the derivatization of the aldehyde with diethylamine was determined as a function of column temperature. An Arrhenius plot was constructed and the activation energy was calculated. The chromatographic behavior of the derivatized acetylenic aldehyde and products formed in-situ in the chromatographic system were studied at various temperatures ranging from −10 to 60 °C. It was found that the reaction products could be controlled by adjusting the column temperature. Different reaction pathways were identified as a function of temperature. The products and the reaction pathways were characterized by NMR, LC–MS and UV spectra.