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

Abstract

Source: Liu, D., et al. Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography. J. Vis. Exp. (2020).

This video demonstrates the principle of separation of hydrophilic polar compounds using hydrophilic interaction liquid chromatography. This technique helps in the purification of various polar analytes, including N-glycans.

Protocol

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-aminobenzamide, 2-AB	Sigma, China
Acetonitrile	Huihai Keyi Technology Co., Ltd, China
Ammonium formate	Beijing Minruida Technology Co., Ltd.
Dextran Calibration Ladder/Glycopeptide column	Watts technology Co., Ltd, China
Empower 3.0	Waters technology Co., Ltd, America
GlycoProfile 2-AB Labeling kit	Sigma, China
Methanol	Huihai Keyi Technology Co., Ltd, China
Milli-Q pure water meter	Millipore Co., Ltd, America	202-2AB
Transfer liquid gun	Smer Fell Science and Technology Co., Ltd, China	Advantage A10
Ultra-low temperature refrigerator	Thermo Co., Ltd, America	PB-10
Ultra-performance liquid chromatography	Watts technology Co., Ltd, China
Vacuum Pump	Watts technology Co., Ltd, China	4672100, 0.5-10μl & 10-100μl & 20-200μl & 1000μl