Hydrophilic-interaction chromatography of complex carbohydrates

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.