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

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.

Evaluating MISER chromatography as a tool for characterizing HILIC column equilibration.

Hydrophilic Interaction Liquid Chromatography (HILIC) is a technique for retaining polar analytes that uses polar stationary phases and acetonitrile-rich mobile phases. While this technique has several advantages over reversed-phase liquid chromatography (RPLC), one main drawback is the reported need for longer column equilibration. The reason for this is not fully understood and is a topic of current investigation. In order to better understand and reduce the equilibration needs, accurate characterization of column equilibration under varying conditions is required. The current method of characterizing HILIC column equilibration produces limited data points per test, or low time resolution, and is highly dependent on the column and probe compounds being used. There is a need for an improved method for characterizing HILIC column equilibration, especially if trends across stationary phases are to be observed. In this work, MISER, or Multiple Injections in a Single Experimental Run, is evaluated as a possible tool for characterizing HILIC column equilibration. MISER improves time resolution by allowing for replicate injections without interruption of data collection, enabling a more thorough evaluation of column equilibration compared to traditional techniques. Experimental results gathered using MISER show that equilibration of a BEH Amide column is notably shorter when equilibrating from acetonitrile to mobile phases containing higher percentages of water. Column equilibration to a 10% aqueous mobile phase was found to be approximately 5-fold faster than equilibration to a 3% aqueous mobile phase.