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.

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.

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A large number of polar stationary phases with diverse chemistry have been developed for various applications in hydrophilic interaction chromatography (HILIC). However, column manufacturers employ different testing procedures to evaluate retention of the polar stationary phases. This renders the retention data impossible for comparison and makes it difficult for the users to select the right stationary phase based on retention. We have evaluated 25 polar stationary phases using cytosine and uracil as the model compounds in various mobile phase conditions. These stationary phases show a wide range of retention characteristics for the model compounds. The ranking of the stationary phases does not change drastically with the acetonitrile level in the mobile phase.