R2 4.6x150mm HPLC Column

The LudgerSep R2 4.6x150mm HPLC column was developed for the analysis of monosaccharides labeled with 2-AA using the LudgerTag Monosaccharide Labeling Kit.

The R2 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 R2 column can be used with any HPLC pumping system capable of delivering accurate gradients at a flow rate of 0.3 to 2.0 ml/min. In general, systems that mix eluants at high pressure (after the pump head) have lower dead volumes and supply more accurate gradients that are appropriate at the flow rate needed for LudgerSep columns. Low dead volume injectors should be used (Rheodyne 7125 / 9125 or similar are recommended). The loop size to be used depends on the separation mode and amount of sample. For analytical runs it is desirable to minimise the sample volume and, typically, a 10 μl loop is used with sample injection volumes of 1 to 5 μl (partial fill) or > 10 μl (complete fill). For charge mode separations, generally, anionic glycans that are retained by the column (and are therefore effectively concentrated on the column) are reasonably tolerant of larger injection volumes whereas non-anionic glycans are not retained by the column matrix and will elute in a volume proportional to the injection volume.

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 labeling of released monosaccharides with 2-aminobenzoic acid (2-AA).
3. Relative quantitative analysis of 2-AA labeled 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.

2-AA fluorescence chromatogram of monosaccharides glucosamine (GlcN), galactosamine (GalN), galactose (Gal), mannose (Man), glucose (Glc), xylose (Xyl) and fucose (Fuc).