1H-Tetrazole-5-amine Immobilized on Substituted Polymer Gel/Silica as a New Stationary Phase for Hydrophilic Interaction Chromatography

1H-Tetrazole-5-amine immobilized on substituted polymer gel/silica as a new stationary phase has been newly suggested for application in hydrophilic liquid chromatography (HILIC). The newly investigated column demonstrated a satisfactory retention of very polar compounds. The column showed mixed separation modes: reversed-phase (RP) in water-rich mobile phases and HILIC at high concentrations of acetonitrile content (> 60-80%) in aqueous-organic mobile phases. A continuous change in retention was observed at decreasing concentration of acetonitrile in water, the rise resulting in characteristic U-shape plots of retention factors versus the concentration of acetonitrile in the mobile phase, with minima corresponding to the transition between the mechanisms controlling the retention. The retention mechanism of the new column was investigated by the models (partitioning and surface adsorption) through adjustment of the water/acetonitrile ratio in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of tested analytes. The retention behavior of tested compounds on three different stationary phases (Atlantis HILIC-silica phase; TSKgel Amide-80-amide phase; Kromasil 60-5 DIOL-diol phase) was investigated to compare the selectivity with the new DCpak PTZ HILIC prototype column. The applicability of the new HILIC column for the chromatography of polar compounds was proven by the separation of mixtures of nucleobases (uracil, thymine, guanine, cytosine, and adenine) and organic acids (ascorbic, isoascorbic, pantothenic, and nicotinic acid). The new DCpak PTZ HILIC column could be a good alternative for the separation of the polar compounds.