Analysis of glycan has many challenges since even the purified protein contains non-uniform glycans and the analysis requires identifying branched structures and isomers specific to glycans. HPLC analysis is an effective method to solve those problems. Various glycans had been identified and quantified using two-dimensional liquid chromatography combining affinity and HILIC modes as their separation. The application below shows the separation of 2-Aminobenzoic acid (2AA) labeled glycans. Asahipak NH2P-40 2D, a polymer-based amino HILIC column, was used with an ESI-TOF-MS to identify the glycans.
Sample : Sample : 2μL
Human IgG (1μg protein)
Column : Shodex Asahipak NH2P-40 2D (2.0mmI.D. x 150mm) Eluent : (A); 95% MeCN/0.1 % Formic acid (B); 5% MeCN/0.1 % Formic acid Linear gradient: B%, 30 % (0-2.5min), 30-95 % (2.5-20min) Flow rate : 0.2mL/min Detector : ESI-TOF MS (Polarity : Negative, Full MS range : 2000, CDL temp.: 190°C, Detector voltage: 1.7kV, Neuburising gas: 1.5L/min) Column temp. : 45°C
Data provided by Dr. Mitsuhiro Kinoshita, School of Pharmacy, Kinki University