In the past ten years, LC on polar stationary phases with mobile phases containing organic solvent and less than 5–40% water (HILIC) has become preferred method for separation of polar compounds, namely pharmaceuticals or biopolymers. On polar columns, sample interactions, retention and separation selectivity may strongly change across the full composition range of aqueous-organic mobile phases, HILIC retention mechanism predominating in the organic-rich, and reversed-phase (RP) mechanism in more aqueous media. We determined the accumulation of water from acetonitrile-rich aqueous-organic mobile phases in the water-rich diffuse layer filling the pores of eighteen polar columns, using frontal chromatography combined with Karl Fischer titration. The sorption can be described by Langmuir isotherm and it strongly depends on the type of stationary phase, ranging from less than one monomolecular water layer up to 6 - 10 water layers equivalents. Some water present in polar solvents is accumulated even on non-polar columns. Considering the actual concentration of water in calculations largely improves the accuracy of prediction of gradient elution data in HILIC systems. The dual retention mechanism enables using a single polar column alternatively in the HILIC and RP retention modes of various polar sample compounds such as phenolic acids and flavones. This approach enhances separation selectivity and peak capacity in single- and two-dimensional HPLC.
Presenting author:
Pavel Jandera
Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, CZ-53210 Pardubice, Czech Republic
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Authors:
Pavel Jandera - University of Pardubice, Czech Republic
Jan Soukup - University of Pardubice, Czech Republic
Petr Janás - University of Pardubice, Czech Republic
Magda Staňková - University of Pardubice, Czech Republic
