Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 microm sorbent.
Separation by hydrophilic interaction chromatography (HILIC) with fluorescence detection utilizing a sub-2 microm glycan column for the separation of 2-aminobenzamide (2-AB) labeled N-linked glycans is described. The HILIC column packed with a 1.7 microm amide sorbent improves the peak capacity compared to a 3.0 microm HILIC column by a similar degree as observed in reversed-phase ultra-performance liquid chromatography (RP-UPLC). The results indicated that the optimal peak capacity was achieved at flow rate 0.2-0.5 mL/min. HILIC method transfer guidelines were shown to further enhance the resolution of glycans by changing initial gradient conditions, flow rate, column temperature, and different column lengths. Additionally, excellent resolution can be achieved in the separation of 2-AB labeled glycans released from fetuin, RNase B, and human IgG with a rapid analysis time.
Ahn
Joomi
J
Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
Bones
Jonathan
J
Yu
Ying Qing
YQ
Rudd
Pauline M
PM
Gilar
Martin
M
D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D003911:Dextrans / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D013058:Mass Spectrometry; D010316:Particle Size; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification*; D012015:Reference Standards; D012212:Rheology; D013194:Staining and Labeling; D013997:Time Factors; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D003911:Dextrans; D007074:Immunoglobulin G; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; C000219:anthranilamide