Practical investigation of the factors that affect the selectivity in hydrophilic interaction chromatography

Retention data for a series of 29 compounds comprising acids, bases and neutrals were obtained on six different hydrophilic interaction (HILIC) columns including bare silica, zwitterionic and those bonded with neutral bonded ligands. The principal aim of the work was to evaluate the effect of various experimental variables such as the nature of the stationary phase, buffer pH, buffer concentration, organic solvent and its concentration, and temperature, in order to determine which factors gave the most effect on the selectivity of the separation. The influence of solute properties on the separation, such as log D values, was also considered. In this way, it was hoped to provide a practical guide to aid in the selection of conditions to achieve a separation in HILIC. The nature of the stationary phase was found to have the greatest effect on selectivity.

► We compare the retention characteristics of 6 different HILIC columns. ► We measure the effects of experimental variables on selectivity. ► The stationary phase has the greatest effect on selectivity. ► Buffer pH also has considerable influence for ionised solutes. ► Solute log D values are only a partial predictor of retention.