Effect of salts on retention in hydrophilic interaction chromatography

Abstract

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.

Analysis of Nucleotide Pools in Bacteria Using HPLC-MS in HILIC Mode

“…In the case of temperature this phenomenon was constant for a wide temperature range [44] whereas for buffer concentration it was apparent only above certain concentration value. At lower concentration levels the retention factor decreased with increasing buffer concentration, whereas at higher concentration levels the retention factor increased with increasing buffer concentration [45] (compare Fig. 3).…”

Evaluating Relative Retention of Polar Stationary Phases in Hydrophilic Interaction Chromatography

“…Cytosine has been used to evaluate the performance of various polar stationary phases in HILIC [25]. Recently, Alpert demonstrated by comparing the retention of cytosine and cytidine that cytosine might have specific interactions with the Hydroxyethyl A phase [26]; however, the unusual elution pattern was not observed on Altantis HILIC silica, YMC Pack amino, TSKgel Amide-80, and ZIC-HILIC phases [25]. Uracil has two pKa values: pKa 1~9 .36 and pKa 2~1 3.49.…”

“…In addition to the acetonitrile level, previous studies have demonstrated that the salt concentration can have significant effect on the retention of the neutral compounds in HILIC [25][26][27]. We selected three stationary phases, namely, ZIC-HILIC 3, XBridge Amide, and LUNA HILIC 2 to represent different levels of retentivity based on the previous results (Figures 1 and 2).…”

Salt and solvent effects in the microscale chromatographic separation of heparan sulfate disaccharides

“…Discussing the possible interactions governing retention is rather difficult due to the unknown stationary phase chemistry and the mixed-mode operation. The solvation of the analytes and their dissociation states are strongly influenced by the acetonitrile ratio in the mobile phase [37]; and the fact that besides the effective pH [38,39], the ionic strength may also have an influence [29], further complicating the picture. Instead of the pure HILIC and WAX operation conditions, their resultant determines the retention.…”

“…Although frequently used in ion (exchange) chromatography, salt gradients are almost entirely neglected in HILIC-based chromatography, mainly because of the lack of theoretical understanding of the retention mechanisms which still need theoretical and experimental elucidation [29][30][31]. Using salt gradients in reversed-phase chromatography is also uncommon, even for pH-gradient there are only a few reported examples [32,33].…”