High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)
HPAEC-PAD - High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection separates carbohydrates via specific interactions between the hydroxyl groups of the glycan and the stationary phase of the column at high pH. The glycans chromatograph as anionic species and interact with the column based on glycan size, composition and linkage. The data obtained provides a profile of the overall glycosylation or oligosaccharide population present on a product, or at a specific glycosylation site, which can be used for batch comparison studies.
Identification of each component can be performed either by analysis of well characterised standards (these standards are not always available and co-elution of components cannot be ruled out), or by collecting and characterising the peaks of interest using mass spectrometry. The chromatogram itself can be viewed as a “fingerprint” for the glycosylation pattern of the glycoprotein. Characterization of a reference standard can then allow this pattern to be used as a point of comparison for test batches.
This method is used in carbohydrate analysis in preference to methods which require labelling of glycans using fluorescent tags, such as 2-aminobenzoic acid or 2-amino acridone as:
- Labelling efficiency is different for different glycan structures. This could potentially skew the results in favour of the more efficiently labelled structures, and
- The HPAEC-PAD provides better glycan resolution.