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.
Highlights
Hydrophilic interaction chromatography (HILIC) offers practical advantages to the separation of polar compounds over reversed-phase liquid chromatography (RPLC), and has been widely applied to challenging analysis in biomedical and pharmaceutical fields [1,2,3,4,5,6]
Toluene is considered unable to penetrate into the water layer adsorbed on the surface of polar stationary phases, and has been used to estimate the pore volume occupied by the water-rich liquid layer [19]
The volume ratio is proposed to be a quantitative measure of the water-rich liquid layer on the surface of the polar stationary phases in HILIC and can be determined using the toluene elution surface of the polar stationary phases in HILIC and can be determined using the toluene elution volume
Summary
Hydrophilic interaction chromatography (HILIC) offers practical advantages to the separation of polar compounds over reversed-phase liquid chromatography (RPLC), and has been widely applied to challenging analysis in biomedical and pharmaceutical fields [1,2,3,4,5,6]. Other polar interactions (e.g., adsorption and hydrogen bonding) may be involved depending on the stationary phase chemistry and solute structures [9,10,11]. If both the stationary phase and the solutes are charged in the mobile phase, electrostatic interactions—either attractive or repulsive—can have significant effects on retention [7,11,12,13]. The attractive interactions between oppositely charged stationary phases and solute molecules enhance the retention; whereas the repulsive interactions reduce the retention, but may provide a unique separation mechanism as in electrostatic repulsion - hydrophilic interaction chromatography (ERLIC) [14]
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