Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis

Hydrophilic-interaction liquid chromatography (HILIC), reversed-phase chromatography (RPC) and porous graphitic carbon (PGC) chromatography are typically applied for liquid chromatographic separations of protein N-glycans. Hence the performances of these chromatography modes for the separation of fluorescently labeled standard glycan samples (monoclonal antibody, fetuin, ribonuclease-B) covering high-mannose and a broad range of complex type glycans were investigated. In RPC the retention of sialylated glycans was enhanced by adding an ion-pairing agent to the mobile phase, resulting in improved peak shapes for sialylated glycans compared to methods recently reported in literature. For ion pairing RPC (IP-RPC) and HILIC ultra-high performance stationary phases were utilized to maximize the peak capacity and thus the resolution. But due to the shallow gradient in RPC the peak capacity was lower than on PGC. Retention times in HILIC and IP-RPC could be correlated to the monosaccharide compositions of the glycans by multiple linear regression, whereas no adequate model was obtained for PGC chromatography, indicating the significance of the three-dimensional structure of the analytes for retention in this method. Generally low correlations were observed between the chromatography methods, indicating their orthogonality. The high selectivities, as well as the commercial availability of ultra-high performance stationary phases render HILIC the chromatography method of choice for the analysis of glycans. Even though for complete characterization of complex glycan samples a combination of chromatography methods may be necessary.