HILIC for Large Biomolecules : Waters

Waters offers a comprehensive range of analytical system solutions, software, and services for scientists. Liquid Chromatography. Mass Spectrometry. Waters is the leading provider of lab equipment, supplies and software for scientists across the world. Easily research and order everything your lab needs! Waters offers a comprehensive range of analytical system solutions, software, and services for scientists. Liquid Chromatography. Mass Spectrometry. Waters is the leading provider of lab equipment, supplies and software for scientists across the world. Easily research and order everything your lab needs!

An alternative, high resolution separation technology

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.

Optimized wide-pore, HILIC stationary phase for resolving glycoforms from intact or digested glycoproteins

Generation of domain specific glycan linkages with or without MS

Elucidation of site specific glycan occupancy

High resolution glycopeptide mapping without limitations due to peptide/glycan size or composition

Improved resolution in separations of large, released N-glycans (EPO, Factor IX)

QC tested with Waters Glycoprotein Performance Test Standard

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.