Does phase ratio in reversed phase high performance liquid chromatography vary with temperature?

•Evaluates the phase ratio for three C18 columns at seven temperatures between 20 °C and 50 °C.•Shows that phase ratio in RP-HPLC varies with temperature.•Describes the implications of phase ratio variation with temperature on the use of vant't Hoff plots for the evaluation of enthalpy and entropy for RP-HPLC separations. Phase ratio Φ for an high performance liquid chromatography (HPLC) column is a parameter defined as the ratio between the volume of the stationary phase Vs and the void volume of the column Vm. Together with the equilibrium constant K of the separation process, phase ratio is part of the retention factor k (k = KΦ). Although a considerable number of studies have been dedicated to the evaluation of Vs and Vm with the goal of obtaining the value for Φ, there are still debatable results regarding the true value of Φ, which for a column with a specific stationary phase may vary with the composition of the mobile phase. One route for the evaluation of the value of Φ uses the measurements of retention factors k on a specific column and mobile phase for two or more hydrocarbons for which the octanol/water partition coefficients log Kow are known. This procedure has been applied in the present study for the evaluation of Φ for three commercially available C18 columns and two mobile phase compositions water/acetonitrile, in the temperature range 20 °C to 50 °C. It was found that phase ratio does change depending on the temperature, its “effective value” decreasing as the temperature increases which is in accordance with the decrease of retention times in reversed phase HPLC when the temperature increases. Besides other factors that may affect the correct calculation of thermodynamic functions, the change of phase ratio with temperature has implications regarding the possibility to calculate the enthalpy and entropy values from van't Hoff plots of the variation of log k as a function of 1/T, even when the retention process is dominated by unique hydrophobic type interactions.