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.
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Sample : 2 μL
Human IgG (1 μg protein)
2AA-labeled N-Glycan

Column       : Shodex Asahipak NH2P-40 2D (2.0 mm I.D. x 150 mm)
Eluent       : (A); 95 % MeCN/0.1 % Formic acid
               (B); 5 % MeCN/0.1 % Formic acid
               Linear gradient;
               (B %) 30 % (0 - 2.5 min), 30 - 95 % (2.5 - 20 min)
Flow rate    : 0.2 mL/min
Detector     : ESI-TOF MS 
               (Polarity : Negative, Full MS range : 2000, CDL temp.: 190 °C, 
               Detector voltage: 1.7 kV, Neuburising gas: 1.5 L/min)
Column temp. : 45 °C

Data provided by Dr. Mitsuhiro Kinoshita, School of Pharmacy, Kinki University