Hydrophilic Interaction Liquid Chromatography: A Technique to Separate Hydrophilic Polar Analytes Using Hydrophilic Beads
Published: April 30, 2023
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 Conditioning of UPLC instruments and preparation of mobile phases 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). Open the software to control the mobile phases. Wash UPLC instruments at flow rate of 0.2 mL/min (50% solvent B and 50% solvent C) balanci…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Tags
Cite This Article
Hydrophilic Interaction Liquid Chromatography: A Technique to Separate Hydrophilic Polar Analytes Using Hydrophilic Beads. J. Vis. Exp. (Pending Publication), e21097, doi: (2023).