Effect of salts on retention in hydrophilic interaction chromatography.

There is a widespread belief that salts promote retention of solutes in hydrophilic interaction chromatography (HILIC) by expanding the volume of the immobilized layer of water on the surface of the stationary phase. To date, all studies of this premise have had flaws or limitations that left the question open. This study explored the effects of salt type and concentration. The effect of the anion was studied with four triethylammonium salts, ranging from the kosmotropic sulfate to the chaotropic perchlorate, at pH values of both 3 and 6. Concentrations ranged from 5-120 mM. All analytes were neutral except for cytosine and cytidine, which had (+) charge at pH 3. Sulfate markedly promoted retention of cytosine, cytidine and phloroglucinol. At high sulfate levels retention of cytosine and cytidine decreased again, presumably due to a "salting-out" effect. With perchlorate anion, retention of cytosine decreased steadily as salt concentration increased, while retention of other standards increased or was unchanged. The effect of the cation was examined by comparing the retention of a tryptic peptide containing either phosphoserine or aspartic acid at the same position. Salts of methylphosphonic acid were used at pH 2.5. The higher the hydration number of the cation, the better the selectivity between the two peptides. The best separation was obtained with the magnesium salt and the worst with the tetramethylammonium salt. The retention contributed by a highly hydrated cation exceeded retention due to electrostatic attraction. These results demonstrate that counterions that are well hydrated serve to promote partitioning of charged solutes into the immobilized aqueous layer in HILIC, while poorly hydrated counterions have the opposite effect. Effects on neutral solutes were more modest; retention times remained unchanged or increased modestly with an increase in concentration of any salt.