Practical examination of flow rate effects and influence of the stationary phase water layer on peak shape and retention in hydrophilic interaction liquid chromatography

The effect of flow velocity on retention and peak shape of neutral, acidic and basic probe compounds was studied using seven different UHPLC hydrophilic interaction chromatography (HILIC) columns. Surprisingly on some columns, the retention factor k was found to vary somewhat with flow velocity, due to the combined effects of pressure and of frictional heating on retention. The selectivity of different HILIC columns was much greater than typically found with RP columns. The volume of the water layer on the HILIC columns was measured using the toluene exclusion procedure. For the neutral solute uridine, a good correlation was found between the volume of the water layer and retention, indicating the likely domination of a partition mechanism. For the ionogenic solutes, the correlation was generally poor, due to the presence of strong additional mechanisms such as ionic retention and repulsion. Reduced Van Deemter plots for uridine showed a negative correlation between the reduced b coefficient and the volume of the water layer, which can be attributed to reduced surface diffusion in this viscous layer. Once again, the behaviour of ionic solutes was complex on some columns making detailed interpretation difficult.