Shiva Mishra
High-resolution separations of glycoforms of glycoproteins that differ in the number of glycans are possible using hydrophilic-interaction liquid chromatography of intact proteins. To maximise the contribution of the hydroxyl groups of the sugars in the glycoprotein, it is necessary to shield the positively charged sites of the proteins with acidic ion-pair reagents in order to get effective separations. Here, we looked at how different IPRs with diverse physico-chemical characteristics, like hydrophobicity and acidity, affected the capillary-scale HILIC separation of intact. The use of fluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, and heptafluorobutyric acid as diluents for sample preparation, solvents for loading samples onto a reversed-phase trap prior to the HILIC separation, and mobile-phase components for HILIC and HILICMS were all examined. To we used an acrylamide-based monolithic column to lessen the impact of ion-exchange interaction with the stationary phase (silica-based). We looked at how the various IPRs affected each phase in the separation of a mixture of proteins with varying sizes and hydrophilicity, as well as the separation of ribonuclease B's five glycoforms. It was established that the sample's IPR content had no impact on the separation or MS detection.
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