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,...
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2025-03-01
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| author | Matjaž Grčman Niko R. Pompe Drago Kočar Matevž Pompe |
| author_facet | Matjaž Grčman Niko R. Pompe Drago Kočar Matevž Pompe |
| author_sort | Matjaž Grčman |
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| description | 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. |
| format | Article |
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| institution | Kabale University |
| issn | 1420-3049 |
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| publishDate | 2025-03-01 |
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| spelling | doaj-art-612cb1b1f3a243beaea545e12f94f20d2025-08-20T03:43:40ZengMDPI AGMolecules1420-30492025-03-01306132710.3390/molecules30061327Impact of Mobile Phase Composition on Separation Selectivity of Labeled Dextran Ladder in Hydrophilic Interaction Liquid ChromatographyMatjaž Grčman0Niko R. Pompe1Drago Kočar2Matevž Pompe3Faculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, SloveniaFaculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, SloveniaFaculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, SloveniaFaculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, SloveniaThe 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.https://www.mdpi.com/1420-3049/30/6/1327HILICdextran ladderFLDionic strengthorganic modifier |
| spellingShingle | Matjaž Grčman Niko R. Pompe Drago Kočar Matevž Pompe Impact of Mobile Phase Composition on Separation Selectivity of Labeled Dextran Ladder in Hydrophilic Interaction Liquid Chromatography Molecules HILIC dextran ladder FLD ionic strength organic modifier |
| title | Impact of Mobile Phase Composition on Separation Selectivity of Labeled Dextran Ladder in Hydrophilic Interaction Liquid Chromatography |
| title_full | Impact of Mobile Phase Composition on Separation Selectivity of Labeled Dextran Ladder in Hydrophilic Interaction Liquid Chromatography |
| title_fullStr | Impact of Mobile Phase Composition on Separation Selectivity of Labeled Dextran Ladder in Hydrophilic Interaction Liquid Chromatography |
| title_full_unstemmed | Impact of Mobile Phase Composition on Separation Selectivity of Labeled Dextran Ladder in Hydrophilic Interaction Liquid Chromatography |
| title_short | Impact of Mobile Phase Composition on Separation Selectivity of Labeled Dextran Ladder in Hydrophilic Interaction Liquid Chromatography |
| title_sort | impact of mobile phase composition on separation selectivity of labeled dextran ladder in hydrophilic interaction liquid chromatography |
| topic | HILIC dextran ladder FLD ionic strength organic modifier |
| url | https://www.mdpi.com/1420-3049/30/6/1327 |
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