Further investigations of the effect of pressure on retention in ultra-high-pressure liquid chromatography

In this study, we investigated further the large increases in retention with pressure that we observed previously in RP-LC especially for ionised solutes. These findings were initially confirmed on a conventional silica C₁₈ column, which gave extremely similar results to the hybrid C₁₈ phase originally used. Large increases in retention factor of ∼50% for a pressure increase of 500 bar were also shown for high MW polar but neutral solutes. However, experiments with the same bases in ionised and non-ionised forms suggest that somewhat greater pressure-induced retention increases are found for ionised solutes. Retention increases with pressure were found to be considerably smaller for a C₁ column compared with a C₁₈ column; decreases in retention with increasing pressure were noted for ionised bases when using a bare silica column in the hydrophilic interaction chromatography (HILIC) mode. These observations are consistent with the partial loss of the solvation layer in RP-LC as the solute is forced into the hydrophobic environment of the stationary phase, and consequent reduction in the solute molar volume, while the water layer on the surface of a HILIC packing increases the hydration of a basic analyte. Finally, retention changes with pressure in RP-LC can also be observed at a mobile phase pH close to the solute pK_a, due to changes in pK_a with pressure. However, this effect has no influence on the results of most of our studies.