Transferring RapiFluor-MS Labeled N-Glycan HILIC Separations Between UPLC And HPLC

By Stephan M. Koza, Matthew A. Lauber, Kenneth J. Fountain, Waters Corporation

In 2009, Waters introduced a revolutionary UPLC HILIC Column designed specifically for achieving high resolution glycan separations. This column technology was based on stationary phase constructed from 1.7 μm diameter, 130Å pore size, ethylene bridged hybrid (BEH) particles with an optimized amide ligand  bonding that has exhibited exceptional resolution for a broad range of N-glycans ranging from small neutral structures to highly sialylated extended structures.¹ In addition to this UPLC-based column, Waters has also introduced HPLCbased XBridge Glycan BEH Amide Columns based on 2.5 μm particles and has demonstrated that these columns provide selectivity for 2-AB labeled N-glycans comparable to that observed in UPLC separations.² Most recently, Waters has introduced a novel labeling reagent, RapiFluor-MS, that provides both a fast and efficient sample preparation workflow and unsurpassed fluorescent and MS sensitivity.³

In the following work, we demonstrate that Glycan BEH Amide Columns packed with 1.7 μm and 2.5 μm particle sizes afford scalability between RapiFluor-MS labeled glycan separations performed under UPLC and HPLCcompatible conditions. Using standard LC method transfer principles to account for differences in particle diameter (dp), column length, and column internal diameter, we show that comparable chromatographic profiles and relative quantitation can be achieved with the larger particle size column at HPLC-compatible pressures, albeit with an increase in sample load, mobile phase use, and most importantly, analysis time.