In the current work, a 14 different types of stationary phases with specific structural properties (eight commercially available stationary phases and six home-made) have been studied. We used the minor disturbance method to measure the excess adsorption isotherms of water onto surface of chemically bonded stationary phases from water-acetonitrile mixtures. The presence of polar and hydrophobic groups in the structures of adsorbents as well as changes in the mobile phase composition causes the excess adsorption of given solvent when its concentration in the mobile phase is low. The excess adsorption of water is observed in acetonitrile-rich mobile phase and the excess adsorption of acetonitrile is observed in water-rich mobile phase. The maximum excess of adsorbed water is connected with a negative excess of adsorbed acetonitrile. However, the scale of these excess adsorption depend on the type of the stationary bonded phases. The retention factors of three test solutes (tryptophan, glycine and proline) are correlated with the maximum amount of excessively adsorbed water on the stationary bonded phase surface. Linear dependence of retention factor with excess amount of water suggest, that the amount of adsorbed water ("hydrophilic pillow") play an important role in the retention mechanism in HILIC. All tested stationary phases were divided into several groups according to the retention factors of 16 different biologically active compounds (selected amino acids, pesticides, vitamin B6). Principal component analysis (PCA) and cluster analysis (CA) analysis were used in column comparison and grouping. CA results indicated that all stationary phases may be generally grouped into several clusters, due to structure and properties of stationary phases. Interesting results were obtained also with the use of PCA. Presented methodologycan provide useful information on the hydrophilic properties of various polar columns and their suitability for HILIC applications.
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