Estimation of the extent of the water-rich layer associated with the silica surface in hydrophilic interaction chromatography.

The possible presence of a mobile phase layer rich in water on the surface of silica columns used under conditions typical in hydrophilic interaction chromatography was investigated by the injection of a small hydrophobic solute (benzene) using acetonitrile-water mobile phases of high organic content. Benzene does not partition into this layer and is thus partially excluded from the pores of the phase up to a water content of about 30%, after which hydrophobic retention of the solute on siloxane bonds is observed. In 100% acetonitrile, the retention volume of benzene was smaller than that estimated either by pycnometry or by calculation from the basic physical parameters of the column. This result might be attributable to the larger size of the benzene molecule: the elution volume of a molecule is the pore volume minus a surface layer half the diameter of the analyte molecule. However, some influence of strongly adsorbed water that remains on the surface of the phase even after extensive purging with dry acetonitrile cannot be entirely discounted. The results suggest that about 4-13% of the pore volume of a silica phase is occupied by a water-rich layer when using acetonitrile-water containing 95-70% (v/v) acetonitrile.