Polyacrylamide brush layer for hydrophilic interaction liquid chromatography of intact glycoproteins

A chromatographic column of nonporous silica particles with a bonded phase of linear polyacrylamide chains is evaluated for hydrophilic interaction liquid chromatography (HILIC) of intact glycoproteins. The column is shown to retain glycoproteins significantly more strongly than non-glycoproteins. A particle diameter of 700 nm gives two-fold higher resolution than does a 1.4 μm particle diameter, and the column efficiency is found to be mostly limited by packing heterogeneity. LCMS is able to resolve the five glycoforms of ribonuclease B and give high quality mass spectra, but there is loss of resolution of the isomers of glycoforms due to the lower amount of TFA. Compared to two leading commercial HILIC columns operated at 60 °C, the polyacrylamide column operated at 30 °C provided at least two-fold higher resolution for intact ribonuclease B, and showed peaks for glycoforms of prostate specific antigen, although not resolved.