20.500.12556/RUL-170835 Impact of mobile phase composition on separation selectivity of labeled dextran ladder in hydrophilic interaction liquid chromatography The glycosylation process plays a crucial role in the structural integrity and biological activity of glycoproteins, where glycans are attached to a protein backbone. There are many kinds of glycans, the most common being N-glycans, which can be arranged into three classes, that is, complex, hybrid, and high mannoses, forming a structurally very diverse set of polar compounds that are difficult to detect and separate. Most commonly, N-glycans are labeled before separation by charged or fluorescence tags for better MS or fluorescence detection, respectively. This study examines the influence of ionic strength and organic modifier selection on the separation of fluorescently labeled dextran ladders in Hydrophilic Interaction Liquid Chromatography (HILIC). Using a Glycan BEH Amide column and varying the ammonium formate buffer concentration along with acetonitrile and methanol ratios, we investigated analyte retention, separation efficiency, and post-column conductivity changes. Our findings reveal that changes in the ionic strength of the mobile phase do not contribute to changes in selectivity, neither when acetonitrile nor methanol were used as organic modifiers to the mobile phase. However, the addition of methanol significantly changes the separation mechanism where two different prevailing separations mechanisms can be identified. It was assumed that the addition of methanol influences the folding pattern of dextrans around the permanent positive charge on the added tag, which influences the changes of separation selectivity. This work presents a systematic approach to altering mobile phase composition (buffer concentration, organic modifier type) to control retention and selectivity in complex glycan analysis. The discovery that methanol significantly alters separation behavior provides a potential new method for refining HILIC separations of polar compounds. HILIC dextran ladder FLD ionic strength organic modifier HILIC dekstranska lestvica FLD ionska moč organski modifikator true false true Angleški jezik Slovenski jezik Članek v reviji 2025-07-17 13:22:00 2025-07-17 13:22:02 2025-07-20 03:42:30 0000-00-00 00:00:00 2025 0 0 13 str. iss. 6, art.1327 Vol. 30 Mar. 2025 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 543.544.5 1420-3049 10.3390/molecules30061327 229482499 RAZ_Grcman_Matjaz_2025.pdf RAZ_Grcman_Matjaz_2025.pdf 1 74C1E7C0DEE86A9EE093CA5336432832 5029cf823fa6f38b96d2ada8dd2b467d38d6e4e2555d957f00dcbc179cdb9a19 8dacf2fd-62fe-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=213093 https://www.mdpi.com/1420-3049/30/6/1327 1 4ff0039e-62fe-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=213092 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0