C3 Anion Exchange Column

The LudgerSep C3 anion exchange column is a highly sensitive strong anion exchange column designed for charge-based analysis and purification of 2-AB and 2-AA labeled glycans. Sub-picomole quantities can be analysed.

The outline of the procedure is as follows :
• The oligosaccharides are labeled by reductive amination with 2-AB or 2-AA.
• Excess labeling reagents are then removed using LudgerClean S cartridges, or T1 cartridges.
• The labeled oligosaccharides are analysed by anion exchange HPLC on a LudgerSep C3 column with fluorescence detection.

LudgerSep C3 anion exchange columns contain particles with a macroporous polymeric anion exchange coating optimized for anion exchange chromatography of complex glycan mixtures. Developed as a replacement for weak anion exchange columns, the C3 columns deliver reliable, reproducible separations. Samples are applied in water and are typically eluted with a sodium acetate or ammonium formate aqueous salt gradient.

Picomole quantities (or less depending on the sensitivity of the detector) of oligosaccharides may be analysed by fluorescent labeling with 2-AB or 2-AA, followed by separation using the LudgerSep C3 strong anion exchange column.

For the 7.5 mm diameter column inject the sample in up to 100 µl of the starting buffer (i.e. the solvent mixture used at the very start of the HPLC gradient). The maximum amount of glycan sample that can be loaded on the column depends on the number and type of glycan components as well as the nature of any non-glycan material. The typical range for successful analytical runs is 0.1pmol – 1 nmol per sample peak and up to 200 nmol of total glycans.

For optimal detection, use wide slit widths (e.g. 10 – 20 nm). Sub-picomole levels of 2-AB labelled glycans can be detected with good signal-to-noise (depending on the sensitivity of the detector used).

We recommend using LudgerSep C buffer (500 mM ammonium formate buffer pH 9, 20 % acetonitrile v/v) rather than lower pH solvents because this higher pH ensures that all anionic glycans (sialic acids, sulphated and phosphorylated sugars) are in a negatively charged state. A lower pH solvent (e.g. pH 4.4) can be used for separation of sialylated glycans, but this is below the pKa value for phosphorylated glycans.

Separation of 2-AB labeled N-glycans from fetuin on C3 7.5 x 75mm using LS-C pH 4.4 solvent
S, 2S, 3S & 4S indicate the mono-, di-, tri- and tetra-sialylated glycan peaks. N = neutral glycans

Separation of 2AB labeled glycan standards (picomolar concentration) on LudgerSepC3 7.5 x 75mm using ammonium acetate based solvent.

LudgerSep C3 Anion Exchange Column Specifications

Base Matrix 10 µm polymer based hydrophilic particles
Functional Group –CH₂CH₂⁺(CH₂H₅)₂
Column dimensions – volume
7.5 x 75 mm- 3.3 ml
Column volume
3.3 ml
Typical flow rate 0.3 – 1.0 ml/min
Pressure limit 150 psi
Column tube Stainless steel, Valco compatible end fittings
pH compatibility pH 2 to pH 12