The retention mechanism of a weak polar solute, a series of 10 benzodiazepines in reversed phase liquid chromatography, was investigated over a wide range of mobile phase compositions. The values of enthalpy (ΔH°) and entropy (ΔS°) of transfer from the mobile to the stationary phases were determined. The method studied each factor (water fraction Φ in the acetonitrile (ACN)/water mixture and column temperature) controlling the retention mechanism. The changes in ΔH° and ΔS° as a function of the water fraction Φ in the ACN/water mixture were examined. These variations are explained using the organization of organic modifier (ACN) in clusters in the ACN/water mixture. A change in the retention mechanism thus indicated when the ACN/water mixture was used instead of the hydrogen-bonded mobile phase such as CH(3)OH/water. Enthalpy-entropy compensation revealed that the retention mechanism was independent of the water fraction Φ but showed that differences between the molecular structures of the benzodiazepines contributed more significantly to changes in the retention process in the CH(3)OH/water mixture than in the ACN/water mixture.