Evaluating the Adsorbed Water Layer on Polar Stationary Phases for Hydrophilic Interaction Chromatography (HILIC)

Abstract

The water-rich liquid layer immobilized on the surface of the polar stationary phases is critical to the retention of polar compounds in hydrophilic interaction chromatography (HILIC). Although the presence of the adsorbed water layer has been investigated and confirmed by multiple techniques, there is a lack of quantitative measures that can be easily determined and linked to chromatographic parameters. This study proposes a simple measure termed volume ratio (the ratio of the adsorbed water layer volume and the mobile phase volume) that can be easily determined using toluene elution volume. The volume ratio values measured using the proposed method indicate that the volume of the adsorbed water layer varies in a wide range in the stationary phases commonly used in HILIC separation. It was observed that the volume ratio increases with the acetonitrile content and ammonium acetate concentration in the mobile phase. In addition, increasing the column temperature had the effect of reducing the volume ratio and diminishing the adsorbed water layer.

A survey of polar stationary phases for hydrophilic interaction chromatography and recent progress in understanding retention and selectivity

“…The iHILIC-Fusion(+) column carries net positive charges due to additional quaternary amine groups on the ligand. Similar to the sulfobetaine and phosphocoline type of zwitterionic phases, the newer zwitterionic phases also have a thick adsorbed water layer and strong retention for polar compounds (Guo, Bhalodia, Fattal, & Serris, 2019).…”

“…The phase ratio values determined by various methods are not drastically different for the same column (e.g., ZIC-HILIC column). Figure 5 shows the phase ratio of 25 polar stationary phases in different mobile phase conditions measured using toluene marker (Guo, Bhalodia, Fattal, & Serris, 2019). Zwitterionic phases tend to have higher phase ratio; in comparison, bare silica phases have a relatively lower phase ratio.…”

“…and superficially porous silica (Poroshell 1200 HILIC, Ascentis Express OH5, Speedcore HILIC, etc.). Sub-2 micro and superficially porous silica supports have been shown to provide higher separation efficiencies (Heaton et al, 2012;Heaton et al, 2014); however, the superficially porous silica particles have relatively thinner adsorbed water layer and less retentivity probably due to the reduced surface area (Dolzan et al, 2014;Guo, Bhalodia, Fattal, & Serris, 2019).…”

“…Jandera argues that the phase ratio of the polar stationary phases should be calculated using the volume of the adsorbed water layer as the volume of the stationary phase, V S (Jandera & Hajek, 2021). In fact, this idea has been employed to determine the phase ratio of 25 polar stationary phases using toluene marker (Guo, Bhalodia, Fattal, & Serris, 2019).…”

A New Definition of the Stationary Phase Volume in Mixed-Mode Chromatographic Columns in Hydrophilic Liquid Chromatography

“…Dinh et al [ 14 ] presume that desorption and partitioning actually coexist as retention promoters for neutral solutes. Guo et al [ 15 ] proposed determination of the sorbed water layer as the difference between the elution volumes of toluene in the aqueous/organic mobile phase and in pure acetonitrile. The distribution constant in LC depends on the retention mode and—deliberately or unintentionally—leads to the phase definition by accepting a convention.…”