Estimation of the phase ratio in reversed-phase high-performance liquid chromatography

Phase ratio Φ for an HPLC column is a common parameter being defined as the ratio between the volume of the stationary phase Vst and the void volume of the column V0. Although apparently simple, the evaluation of phase ratio presents difficulties because there is no sharp boundary between the mobile phase and the stationary phase, and different mobile phases lead to different “effective” phase ratios. A considerable number of studies have been dedicated to the evaluation of Vst and V0 with the goal of obtaining the value for Φ. However, the parameter is seldom reported for common commercially available columns and key information for its calculation (e.g. the weight of column packing) is not typically reported by the vendors. Present study describes a novel procedure for the evaluation of phase ratio for a chromatographic column when used with a given mobile phase. The procedure allows the calculation of Φ only from the measurements of retention factors k' for two hydrocarbons i and j, for which the octanol/water partition coefficients log Kow are known. A theoretical support for this procedure based on solvophobic theory of interactions in solution is presented, and several experimental results reported in the literature are used to demonstrate the validity of the procedure.