SummaryTwo different methods have been used to investigate the retention mechanism of a series of imidazole derivatives in reversed-phase liquid chromatography (RPLC) over a range of column temperatures and with different concentrations of hydroxypropyl-fl-cyclodextrin (HPfl-CD) in the mobile phase. The first approach was the separate study of each factor affecting the retention mechanism; the second method was the simultaneous variation of all these factors. Changes in Van't Hoff plots as a function of HP-fl-CD concentration were examined. Enthalpy and entropy were determined for two physicochemical processes: (i) solute transfer from the mobile phase to the stationary phase, and (ii) solute complexation by HP-fl-CD. These thermodynamic data showed that the mechanism of retention of the solute was dependent on the concentration of HP-fl-CD in the mobile phase. For a HP-fl-CD concentration, C, greater than to 4 mM, from 28 ~ to a critical temperature, T*, solute retention was entropy-dominated because of inclusion of the solute in the HP-fl-CD cavity. Above T* retention was enthalpy-dominated, because of interaction of the solute with the RP18 stationary phase. At first 7-* increased as C was increased up to a critical value, C**; it then remained relatively constant because of auto-association of the HP-fl-CD molecules in the mobile phase. Enthalpy-entropy compensation revealed that HP-fl-CD-solute complexation had a greater effect on retention than RP18 stationary phasesolute interaction. This confirms that the main parameter determining retention in RPLC is the distribution of the solute in the mobile phase, and that interactions with the stationary phase play a minor role.