Hydrophilic Interaction Liquid Chromatography: A Technique to Separate Hydrophilic Polar Analytes Using Hydrophilic Beads

First, open the software to control the mobile phases. Wash UPLC instruments with 50% solvent B and 50% solvent C at the flow rate of 0.2 milliliters per minute, for 30 minutes. Then wash, with 25% solvent A and 75% solvent B at a flow rate of 0.2 milliliters per minute, for 20 minutes, then, at a flow rate of 0.4 milliliters per minute.

Dissolve the labeled N-glycans with 25 microliters of a mixture of 100% acetonitrile and ultra-pure water at a 2:1 (volume/volume) ratio. Then, centrifuge at 134 x g for 5 minutes at 4 degrees Celsius, and use a drive pipe to load 10 microliters of the supernatant into the UPLC instruments.

Separate the labeled N-glycans at a flow rate of 0.4 milliliters per minute, with a linear gradient of 75% to 62% acetonitrile for 25 minutes. Then, perform an analytical run by Dextran Calibration Ladder/Glycopeptide column on a UPLC at 60 degrees Celsius.

Detect N-glycan fluorescence at excitation and emission wavelengths of 330 nanometers and 420 nanometers respectively.