Retention mechanism on phosphodiester stationary phases in HILIC and purely aqueous mobile phase, Part I: The problem of hold-up volume determination

Non-destructive chromatographic methods were used to determine the hold-up volumes of four self-packed columns containing embedded phosphate groups. The stationary phases are named Diol-P-C10, Diol-P-C18, Diol-P-Benzyl and Diol-P-Chol. The hydrophobicity of organic ligands bound to the phosphate group increases in the benzyl< decyl < octadecyl <cholesterol direction. Using a stochastic model of inverse size exclusion chromatography with a wide pore size distribution, the hold-up volume, interstitial volume and pore volume of the columns were determined. Using the method of minor disturbances, the thermodynamic hold-up volume of the columns was calculated and the excess isotherm was determined for acetonitrile-water mobile phases. The results show that these stationary phases can be used effectively in RPLC and HILIC. When using hold-up volume markers, it was found that the composition of the mobile phase has to be taken into account. In the case of protic solvents, inorganic salts are excluded from the pores and their elution volume is not the total hold-up volume. Rather, in the case of aprotic mobile phase, retention occurs due to the adsorbed water layer.