Selectivity Tests of Stationary Phases for Reversed-Phase HPLC

Abstract

The retention and selectivity properties of 17 columns for reversed-phase HPLC were characterized using classical Galushko, Engelhardt, and Walters chromatographic tests; homologous alkylbenzene and LFER tests in aqueous acetonitrile and aqueous methanol mobile phases; and, finally, isomeric naphthalene disulfonic acids (NDSA) tests. The column properties were compared using the chemometric approach—cluster analysis. The results of simple hydrophobicity and silanol activity tests using relative retention of phenol, aniline, isomeric toluidines, and other compounds strongly depend on the mobile phase. The results of the alkylbenzene methylene and phenyl selectivity tests take into account the effects of the composition of the two-component aqueous-organic mobile phase. The LFER tests distinguish the selective polar interactions of the stationary phase. The alkylbenzene and LFER tests allow column characterization with respect to the column hydrophobicity and polar selectivity in a medium composition range of aqueous-organic mobile phases, but the results strongly depend on the type of the organic solvent (acetonitrile or methanol) and cannot be extrapolated to highly aqueous mobile phases. The NDSA tests performed in highly aqueous mobile phases take into account the effects of column hydrophobicity and ionic exclusion. The results are not affected by the solvation of stationary phase by organic solvents and can be used to characterize the column properties in highly aqueous mobile phases.