HPLC Column System Suitability Application Note
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The normal phase column contains particles coated in a robust amide polymer. These bind to glycans under conditions of high organic solvent. Glycans are separated and eluted from the column using solvent gradients with decreasing organic solvent content.
HPLC chromatogram from two N2 2x150mm in series of 2-AB labeled GHP
LudgerSep N2 2.0mm column can be used with an HPLC system capable of delivering accurate gradients at a flow rate of 50 to 300 µl/min. In general, systems which 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 the LudgerSep N2 normal phase column.
For the 2mm column inject the sample in up to 25 μl of the starting buffer (i.e. the solvent mixture used at the very start of the HPLC gradient). Acetonitrile is recommended as solvent A and a 50 mM ammonium formate pH 4.4 buffer Cat. No. LS-N-BUFFX40 for solvent B is recommended.
Either fluorescence, mass spectrometry or UV-absorbance depending on the dye used (see the appropriate LudgerTagTM instruction guide).
For optimal detection, use wide slit widths (e.g. 10 – 20 nm). Sub-picomole levels of 2-AB or 2-AA labelled glycans can be detected with good signal-to-noise (depending on the sensitivity of the detector used).
To improve repeatability and intermediate precisions for glycan analyses use a column temperature controller. Good results can be obtained with a column temperature of 35˚C.
Normal Phase Column Use with Sialylated Glycans
Sialylated glycans can become desialylated if exposed to acidic conditions and elevated temperatures. Avoid desialylation with such samples by minimizing exposure to acid (if possible, keep the pH between 5 – 8), and minimizing exposure to temperatures greater than 25˚C.
Base Matrix 3 µm with 80 angstrom pores and polymeric amide coating
Column dimensions 2.0mm x 150mm
Typical flow rate 0.15 – 0.22 ml / min
Pressure limit 2900 psi
Column tube Stainless steel, Valco compatible end fittings
pH compatibility pH 2 to pH 7.5