Quantitative Assessment of Retention Mechanisms of Nucleosides on a Bare Silica Stationary Phase in Hydrophilic Interaction Liquid Chromatography (HILIC)

Nucleosides are of significant interest to biomedical and pharmaceutical research and have been successfully separated in hydrophilic interaction liquid chromatography (HILIC). However, there have been few studies focusing on the retention mechanisms, and detailed retention mechanisms are not clearly understood. The quantitative assessment methodology based on the linear relationship between the observed retention factors and the phase ratio has been shown to be a new tool to investigate the retention mechanisms of polar compounds in HILIC. This study evaluated the retention mechanisms of 16 nucleosides on a bare silica column. The retention contributions by partitioning, adsorption, and electrostatic attractions are quantitatively determined, and the main retention mechanism can be unambiguously identified for each nucleoside. The study results indicate that the main retention mechanism can shift with the salt concentration in the mobile phase, but partitioning seems to dominate at higher salt concentrations. In addition, the partitioning coefficients are measured using the quantitative assessment methodology and have a relatively strong correlation with the log P values of the nucleosides. Considering large errors in the log P values for these very polar compounds, the partitioning coefficients measured experimentally in the HILIC system may provide a more accurate measure for polarity assessment.