Contribution of ionic interactions to stationary phase selectivity in hydrophilic interaction chromatography

Abstract

We compared the separation selectivities of 19 different hydrophilic interaction chromatography columns. The stationary phases included underivatized silica and hybrid particles, cyano-bonded silica, materials with neutral ligands such as amide, diol, pentahydroxy, and urea, zwitterionic sorbents, and mixed-mode materials with amine functionalities. A set of 77 small molecules was used to evaluate the columns. We visualized the retention behavior of the different columns using retention time correlation plots. The analytes were classified as cations, anions, or neutral based on their estimated charge under the separation conditions. This involved adjusting the dissociation constants of the analytes for the acetonitrile content of the mobile phase and experimentally determining the pH of the mobile phase containing 70% acetonitrile. The retention correlation plots show that the selectivity differences strongly depended on ionic interactions. Comparisons of the neutral stationary phases (e.g., diol vs. amide) showed more similar selectivity than did comparisons of neutral columns versus columns with cation or anion exchange activity (bare silica or amine columns, respectively). The zwitterionic columns did not behave as perfectly neutral. The correlation plots indicated that they exhibited either cation or anion exchange activity, although to a lesser degree than the silica and amine-containing stationary phases.

Retention mechanisms of amitriptyline and its impurities on amide, amino, diol, and silica columns in hydrophilic interaction liquid chromatography

“…The amino column did not exhibit a dual retention mechanism, being attributed to the strong repulsive forces between the positively charged column surface and cationic analytes (Figure S3) [10]. Additionally, the dual retention mechanism was not present with impurities A and G on any column, because they were not ionized (molecular form), showing almost negligible retention [38].…”

Quantitative analysis of the glutathione pathway cellular metabolites by targeted liquid chromatography‐tandem mass spectrometry

“…While NEM‐derivatized thiol compounds were sufficiently retained, RP did not provide adequate retention for free amino acids especially Glu, Ser, and CysCys, as expected (Figure S2). Therefore, HILIC was further considered and 4 distinct HILIC columns were evaluated, two of them with sulfobetaine chemistry (Z‐HILIC, and HILIC‐Z), one amide bonded (BEH Amide), and one mixed‐mode positive and negative charge‐modulated hydroxyethyl amide stationary phase (iHILIC Fusion) [36]. In this screening, mobile phase composition and column compartment temperature were kept constant, whilst for this column screening also the ratio t g /t 0 was kept constant.…”

A robust ultra‐performance liquid chromatography‐tandem mass spectrometry method for simultaneous determination of 10 components in glutathione cycle

“…The generation and transformation of GSH mainly depend on the GSH cycle, which involves many highly polar endogenous substances. Previous methods were typically performed on a C18 column to analyze similar compounds, which had a low separation performance and required multimodal resin treatment to meet the separation requirements [16,[41][42][43][44]. Thus, an amide column was selected in this study to analyze these analytes.…”