Hydrophillic Interaction Chromatography (HILIC) has been widely used to separate small polar compounds, yet its application to large biomolecules, other than released glycans, has been surprisingly limited. With the widepore Waters ACQUITY UPLC Glycoprotein BEH Amide 300A 1.7μm column and some new ideas on separation methods, it is now possible to use HILIC to glean previously unattainable information from intact proteins (with or without glycosylation), protein fragments, and complex, released glycans.
View our series of "Tips and Tricks" videos for some pratical considerations to take when using HILIC columns for large biomolecule analyses.
Glycoprotein Column Chemistry
The ACQUITY UPLC Glycoprotein BEH Amide 300Å 1.7 µm Column allows scientists to obtain novel yet complementary glycan related information of biotherapeutic proteins at the intact glycoprotein, glycoprotein subunit, or glycopeptide level.
The figure to the left shows the separation of the Glycoprotein Performance Test Standard(RNase A + RNase B glycoforms) using an ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm, 2.1 x 150 mm Column. Fluorescence detection at Ex 280 nm and Em 320 nm and a column temperature of 45 ˚C.
Intact Glycoprotein Analyses
The ACQUITY UPLC Glycoprotein BEH Amide 300Å, 1.7 µm Columns separate individual intact protein glycoforms as well as deliver information about glycan occupancy. Using a high organic solvent concentration with TFA ion pairing and elevated temperature, one is able to successfully enhance the solubility of intact glycoproteins for this HILIC-based, gradient separation.
Best Practices for HILIC: Featuring Novel HILIC Techniques for Glycan Analysis
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