Retention and Selectivity on Hydrophilic Interaction Liquid Chromatography Columns Modified with Polyethylene Glycol of Different Chain Length

In this work, in order to investigate retention and selectivity mechanism on hydrophilic interaction liquid chromatography (HILIC), one of the most common hydrophilic stationary phases, polyethylene glycols (PEG) of different molecular weights (200, 400, 800 and 2000 Dalton) were covalently bonded to porous silica spheres via isocyanate silane. The PEG stationary phase was then characterized in detail by infrared spectroscopy, scanning electron microscope, nitrogen adsorption–desorption isotherms, and thermogravimetric analysis, and the PEG bonding density was determined as 2.7, 2.3, 1.4, 0.26 μmol m⁻², respectively. The HILIC retention mechanism was systematically conducted by quantitative evaluation of the effect of partitioning and adsorption, hydrogen bonding and hydrophilicity, and temperature on the retention of nucleosides and nucleobases. It was found that the retention was highly relevant on the partitioning, hydrogen bonding between solutes and PEG, and strong electrostatic interactions (attractive/repulsive) between solutes and silica. To further assess the separation performance, three kinds of polar compounds (nucleosides and nucleobases, organic acids and β-blockers) were chosen as probe compounds. Among the four PEG stationary phases, it was found that the higher PEG ligand density generally resulted into more superior separation performance to these polar compounds, that is, PEG200-bonded stationary phase with highest ligand density of 2.7 μmol m⁻² generated better resolution. Furthermore, higher ligand bonding mass would increase the retention of nucleosides, nucleobases and organic acids except β-blockers. Generally, the HILIC stationary phase with continuous and dense hydrophilic layer is beneficial to the separation of polar analytes.