Basic thermodynamic functions responsible for retention of new 1,2,4-triazole derivatives exhibiting varied antiepileptic activity on cholesterol-based stationary phase were determined. Evaluation of the Gibbs energy change, the change in enthalpy and the change in entropy was based on the van't Hoff relationship representing lnk versus 1/T. A detailed discussion of the van't Hoff equation, exploring the influence of the phase ratio on deviations from linearity in a van't Hoff plot is presented. We show chromatographic evidence to the question of how a varied mobile phase composition may cause different thermodynamic phase ratios. The analysis of data from a differential scanning calorimetry excluded any phase transitions of either the individual solutes or cholesterol stationary phase suspended in the mobile phase components within the studied temperature range.