Effects of alcoholic solvents as organic modifiers on separation of hydrophilic compounds in hydrophilic interaction liquid chromatography

Although alcoholic solvents are suggested as low-level organic modifiers in the mobile phase, the effects of alcoholic solvents on HILIC separation have not been evaluated. This study systematically investigated the effects of alcoholic solvents on the adsorbed water layer, mobile phase polarity, retention mechanisms, and selectivity using the quantitative assessment methodology. The study results clearly show that low-level methanol or IPA in the mobile phase reduces the adsorbed water layer, thus the phase ratio, and it decreases partitioning coefficients when the aqueous content is kept constant. These two factors lead to a decrease in the retention contributed by hydrophilic partitioning. The retention contributed by adsorption, on the other hand, increases with the level of methanol in the mobile phase for most nucleoside model compounds. IPA seems to have less effect on adsorption. Hydrophilic partitioning is found to be the main retention mechanism for all the model nucleosides in the mobile phase containing only acetonitrile; however, adding alcohol modifiers reduces the significance of the partitioning mechanism and enhances adsorption. The presence of alcohol modifiers in the mobile phase seems to have a small, but significant effect on the selectivity for some closely eluting pairs of nucleosides. The change in selectivity can be explained by the effect of alcohol on the retention mechanisms. In addition, separation of selected uric acid and methylated uric acids demonstrates that the use of low-level alcohol modifiers offers an alternative to changing acetonitrile levels to modulate the retention time in HILIC.