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

1. Hydrophilic interaction chromatography and analysis of glycans

1. Conditioning of UPLC instruments and preparation of mobile phases
   1. Prepare mobile phases including solvent A: 100 mM ammonium formate (pH = 4.4), solvent B: 100% acetonitrile, solvent C: 90% ultra-pure water (10% methanol), and solvent D: 50% methanol (ultra-pure water).
   2. Open the software to control the mobile phases.
   3. Wash UPLC instruments at flow rate of 0.2 mL/min (50% solvent B and 50% solvent C) balancing for 30 min, then at a flow rate of 0.2 mL/min (25% solvent A and 75% solvent B) balancing for 20 min, then a flow rate of 0.4 mL/min balancing.
2. Dissolve the N-glycans prelabeled with 2-aminobenzamide (2-AB) in 25 µL of a mixture of 100% acetonitrile and ultra-pure water at a 2:1 ratio (v/v). Then, centrifuge at 134 × g for 5 min (4 °C) and load 10 µL of the labeled N-glycans into the UPLC instruments.
3. Separate the labeled N-glycans at flow rate of 0.4 mL/min with a linear gradient of 75% to 62% acetonitrile for 25 min. Then, perform an analytical run by dextran calibration ladder/glycopeptide column on a UPLC at 60 °C (here, samples were kept at 4 °C prior to injection).
4. Detect N-glycan fluorescence at excitation and emission wave lengths of 330 nm and 420 nm, respectively.
5. Integrate the glycans based on peak position and retention time.
6. Calculate the relative value of each Glycan Peak (GP)/ all Glycan Peaks (GPs) (percentage, %) as follows: GP1: GP1/GPs*100, GP2: GP2/GPs*100, GP3: GP3/GPs*100, etc.