Carbohydrate analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD)

Importance of the Topic

Carbohydrate analysis is critical in biotechnology, food quality, pharmaceutical, and environmental monitoring.
High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) enables sensitive, direct quantification of native sugars without derivatization.
Its high resolution and low picomole detection limits have driven its widespread adoption and ongoing method refinements.

Aims and Study Overview

This Technical Note updates Dionex Technical Note 20 by outlining HPAE-PAD principles, column options, best practices, limitations, and key applications.
The document provides guidance on method development, sample preparation, eluent selection, and data interpretation.

Methodology and Used Instrumentation

HPAE-PAD relies on high-pH mobile phases to deprotonate neutral sugars, enabling anion-exchange separation on polymeric CarboPac columns stable at pH>14.
CarboPac column family includes PA1, PA10, PA20, PA100, PA200, PA300, MA1, and SA10, each tailored for specific analyte classes from monosaccharides to large glycans.
Detection uses pulsed amperometry with a four-potential waveform to oxidize carbohydrates at a gold electrode and clean the surface by alternating potentials.
Eluents are prepared from sodium hydroxide and sodium acetate or generated electrolytically (EGC mode) to produce carbonate-free KOH and potassium methanesulfonate mixtures.
Sample cleanup may involve filtration, protein precipitation, and OnGuard cartridges for removal of proteins, phenolics, halides, or anionic interferences.

• HPAE-PAD system (e.g., Thermo Scientific Dionex ICS-6000)
• CarboPac anion-exchange columns (PA, MA, SA series)
• Electrochemical detector with PAD waveform
• Electrolytic eluent generator modules (EGC-KOH, Dual EGC)
• OnGuard sample preparation cartridges

Main Results and Discussion

Column design: pellicular and latex-coated resins provide high capacity and rapid mass transfer for sharp peaks and high resolution.
Retention correlates with sugar acidity (pKa) and ring structure; derivatization (alditols or methylation) shifts pKa and alters retention requirements.
Common side reactions (epimerization, de-acetylation, peeling) are minimal under typical HPAE-PAD conditions due to rapid separations and mild gradients.
PAD waveform optimization ensures broad detection of sugar alcohols, monosaccharides, disaccharides, and complex oligosaccharides with high signal-to-noise ratios.
Eluent generation and reference electrode advances (PdH vs Ag/AgCl) enhance stability, reduce contamination, and support high-throughput operation.

Practical Benefits and Applications

• Monosaccharide and sialic acid compositional analysis of glycoproteins
• Oligo- and polysaccharide profiling in foods, biofuels, and plant materials
• Sugar quantification in beverages such as coffee, juices, and molasses
• Detection of sugar alcohols, sugar acids, and anhydrosugars in environmental and industrial samples
• Glycan mapping for N- and O-linked structures with PAD or PAD-MS interfaces

Future Trends and Applications

Integration of HPAE-PAD with mass spectrometry via electrolytic desalting will expand structural glycomics.
Novel column chemistries (e.g., PA300 for O-glycans) and enhanced eluent generation will enable faster, more selective separations.
Advances in reference electrodes and disposable working electrodes will improve stability and reduce maintenance.
Automation and high-throughput workflows will support large-scale analyses in quality control and research.

Conclusion

HPAE-PAD has matured into a versatile, robust technique for native carbohydrate analysis across diverse sectors.
Ongoing innovations in columns, detection, and eluent management continue to broaden the method’s capabilities and applications.

References

1. Lee YC. Anal Biochem. 189 (1990) 151–162.
2. Townsend RR, Hardy MR. Glycobiology. 1 (1991) 139–147.
3. Cataldi RI, Campa C, De Benedette GE. Fresenius J Anal Chem. 368 (2000) 739–758.
4. Rohrer JS. Anal Biochem. 283 (2000) 3–9.
5. Chen Y et al. Anal Chem. 90 (2018) 10910–10916.