Effect of temperature on selectivity in reversed-phase liquid chromatography, a thermodynamic analysis Original Research Article

This paper describes the results of a theoretical analysis of the effect of temperature on selectivity for several complicated retention processes in reversed-phase liquid chromatography (RPLC). Situations included in this study are: (1) chromatography of ionizable compounds such as carboxylic, amine, amino acid, and peptide compounds; (2) chromatography of solutes that experience two independent retention modes such as amine compounds; and (3) ion-pair chromatography of carboxylic acids. The focus of the study is to evaluate the effect of temperature-dependent enthalpy change of the more retained solute on selectivity. We find that for chromatography of these ionizable compounds an improvement in selectivity at elevated temperature may be expected at high mobile phase pH, and the degree of the improvement for carboxylic compounds is roughly related to the mobile phase pH, with basic mobile phase more beneficial. Furthermore, it is found that buffers can play a very important role in affecting the retention, selectivity and change in selectivity with temperature, particularly for co-eluted compounds. For solutes that undergo two independent retention processes, a similar but more significant change in selectivity can be expected if the secondary interaction is not fully eliminated. For ion-pair chromatography of carboxylic acids, high temperature can either significantly improve or further decrease selectivity, depending on the ion-pair complex formation and its retention enthalpy. If the ion-pair complex formation and its retention enthalpy is dominant, a significant decrease in selectivity is expected with increasing temperature, otherwise an improvement in selectivity may be seen.