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 measure 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 provides a relative, but quantitative information on the adsorbed water layer and may be linked to the phase ratio. The volume ratio can be easily determined using toluene elution volume. The volume ratio values are measured in 25 polar stationary phases in various mobile phase conditions. In addition to the acetonitrile content in the mobile phase, ammonium acetate concentration in the mobile phase and column temperature also have significant influences on the volume ratio and the adsorbed water layer.

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

“…The salt concentration in the mobile phase is another factor that affects the volume of the adsorbed water layer significantly. Water uptake is found to increase with ammonium acetate concentration in the mobile phase, as shown in Figure 6, and consequently the phase ratio of the amide phase increases with ammonium acetate concentration at various acetonitrile levels (Dinh et al, 2013; Guo, Bhalodia, Fattal, & Serris, 2019). In addition, the volume of the adsorbed water layer is observed to be influenced by column temperature.…”

“…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).…”

Evaluation of multiple hydrophilic interaction chromatography columns and surrogate matrix for arginine quantification in saliva by high‐resolution mass spectrometry

“…The third way to decrease tailing factor issues is by increasing the water layer thickness. Most recently, Prof. Guo's team has quantitatively compared the water layer on different HILIC column stationary phase surfaces by using the volume ratio concept [46]. The thick water layer on a HILIC stationary surface could provide an environment for hydrophilic partitioning, hydrogen bonding, and electrostatic interactions and diminish the adsorptive interactions between analytes and silanol groups of the silica core surface.…”