uR2 2.1x50mm UHPLC Column

The LudgerSep uR2 2.1x50mm UHPLC column was developed for the analysis of monosaccharides labeled with 2-AA using the LudgerTag Monosaccharide Labeling Kit.

The uR2 column gives very good separation of the seven main monosaccharides found in most N-link and O-link glycans. Samples are diluted into the butylamine/ortho-phosphoric acid/tetrahydrofuran (BPT) HPLC running solvent.

The LudgerSep uR2 column is designed for use with the latest generation of UHPLC instruments capable of withstanding high flow pressures and fast sample analyses. In order to take advantage of the high resolving power of sub 3 μm particle size containing columns, we recommend keeping sample injection volumes at or below 5 μL and minimising system void volumes. Ideally use full loop injection. Tubing should be narrow bore (about 0.13 mm diameter or less) and detector flow cell volumes should be 10 μl or less. Although an example chromatogram is shown in the product guide, retention times will vary dependent on the UHPLC system used.

Overview of the Procedue

The method for monosaccharide analysis is as follows:
1. Release of monosaccharides from the glycoprotein by mild acid hydrolysis.
2. Fluorescent labelling of released monosaccharides with 2-aminobenzoic acid (2-AA).
3. Relative quantitative analysis of 2AA-labelled monosaccharides by HPLC or UHPLC

The kit also contains the LudgerTag MonoMix, a monosaccharide standard containing glucosamine, galactosamine, galactose, mannose and fucose. Xylose is included for use as an internal standard.

There are two hydrolysis acids provided. 2 molar trifluoroacetic acid (2M TFA) and 6 molar hydrochloric acid (6M HCl). 2M TFA is good for releasing neutral monosaccharides but is less effective with the N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) monosaccharides for which we recommend using 6M HCl. We recommend using these acids on separate replicates of your samples.

8 minute 2-AA fluorescence chromatogram of monosaccharides glucosamine (GlcN), galactosamine (GalN), galactose (Gal), mannose (Man), glucose (Glc), xylose (Xyl) and fucose (Fuc).
Peaks: 1 = Neu5Gc; 2 = Neu5Ac; 3 = Neu5,7Ac2; 4 = Neu5Gc,9Ac; 5 = Neu5,8Ac2; 6 = Neu5,9Ac2; 7 = Neu5,x,xAc3 (where x is an unknown acetyl position); * = Reagent