Abstract: The present study focuses on the preparation and application of zwitterionic stationary phases with controllable ratios of positively charged amino groups and negatively charged phosphonic acid groups. Successful grafting was confirmed by elemental analysis and evaluation of pore structure characteristics. Additionally, the proportion of amino and phosphonic acid groups could be easily determined by the mass fractions of N and P. In this study, three types of amino phosphate-based zwitterionic surface-bonded stationary phases (APS) were prepared with different ratios of amino and phosphonic acid groups. The resulting APS silica materials were slurry-packed into stainless-steel columns (150 mm×4.6 mm). Various parameters such as column temperature, water content, pH, and ionic strength of the mobile phase were investigated to evaluate the chromatographic characteristics of the APS columns. A mixed-mode retention mechanism involving surface adsorption, partitioning, and electrostatic interactions was revealed. Finally, a set of test mixtures (nucleosides, water-soluble vitamins, benzoic acids, and basic compounds) were employed to evaluate the separation selectivity and retention characteristics of the three prepared APS stationary phases. The APS phases demonstrated entirely different selectivity and retention behavior for various polar analytes under the same conditions, thus demonstrating excellent application potential for the analysis of polar compounds in hydrophilic interaction chromatography (HILIC).

Key words: chromatographic separation, hydrophilic interaction chromatography (HILIC), polar compounds, retention mechanism, zwitterionic stationary phase