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

Significance of the Topic

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) has evolved over four decades into a versatile technique for resolving and quantifying native carbohydrates at low picomole levels. Its ability to separate neutral and acidic sugars without derivatization, combined with sensitive, direct electrochemical detection, makes HPAE-PAD a cornerstone method in fields ranging from biopharmaceutical quality control to food and biofuel analysis.

Objectives and Study Overview

This Technical Note updates Dionex Technical Note 20 by summarizing the fundamental principles of HPAE-PAD, describing the current portfolio of anion-exchange columns and eluents, reviewing best practices, and presenting representative applications. It aims to guide analysts in selecting appropriate columns, eluents, and operating parameters for diverse carbohydrate analyses and highlights advances such as electrolytic eluent generation and modern reference electrodes.

Methodology and Instrumentation

Carbohydrate separation relies on polymeric anion-exchange columns that withstand pH >11. Key column types include:

• CarboPac PA1/PA20/PA210 series for mono- and oligosaccharides and sialic acids
• CarboPac MA1 for sugar alcohols and alditols
• CarboPac SA10 for plant-derived monosaccharides and biofuel samples
• CarboPac PA200/PA300 for neutral and O-linked oligosaccharides

Pulsed amperometric detection employs a gold working electrode, Ag/AgCl or PdH reference electrode, and integrated cleaning waveform (four-potential cycle) to oxidize carbohydrates and remove surface fouling. Eluent systems use sodium hydroxide and sodium acetate or methanesulfonate; eluent purity, deionized water resistivity, and proper filtration are critical. Electrolytic eluent generators can deliver carbonate-free KOH up to 100 mM or Dual EGC Mode eluents (KOH/KMSA) up to 200 mM.

Main Results and Discussion

The Note details dissociation constants of common sugars (pKa ≈ 12–13) and resin architectures that enable sharp peaks via pellicular or macroporous latex beads. It addresses sample pretreatment strategies—protein precipitation, cartridge cleanup for humic acids, halides, or sulfate removal—and stability concerns such as epimerization or de-O-acetylation at high pH. Representative applications illustrate monosaccharide and sialic acid compositional analyses of glycoproteins, profiling of N- and O-linked glycans (including HPAE-PAD/MS interfacing with electrolytic desalting), quantification of sugars in coffee, honey authenticity testing, biofuel feedstock hydrolysates, and airborne anhydrosugars from wood smoke.

Benefits and Practical Applications of the Method

• Direct analysis of underivatized carbohydrates with high sensitivity (low picomole detection)
• Broad scope: neutrals, acids, sugar alcohols, oligosaccharides, and glycan profiling
• Robust column chemistries resistant to high-pH eluents
• Flexible eluent delivery: manual vs. electrolytic generation
• Compatibility with mass spectrometry via electrochemical desalting
• Minimal sample preparation for transparent or hydrolyzed samples
• Standardized methods recognized by AOAC, ICUMSA, and ISO

Future Trends and Opportunities

Emerging developments include expanded use of Dual EGC Mode for stronger elution anions, disposable PTFE-based electrodes for extended lifetimes, PdH reference electrodes for enhanced stability, and miniaturized column formats for high-throughput analysis. Integration with high-resolution mass spectrometry and automated sample cleanup cartridges will further broaden applications in glycomics, bioprocess monitoring, clinical diagnostics, and environmental aerosol analysis.

Conclusion

HPAE-PAD remains a gold standard for comprehensive carbohydrate analysis. Ongoing innovations in column design, eluent generation, and electrochemical detection continue to enhance resolution, sensitivity, and throughput, supporting a wide range of research and quality-control applications.

References

1. Lee YC. High-performance anion-exchange chromatography for carbohydrate analysis. Anal Biochem. 1990;189:151–162.
2. Townsend RR, Hardy MR. Analysis of glycoprotein using high-pH anion exchange chromatography. Glycobiology. 1991;1:139–147.
3. Lee YC. Carbohydrate analyses with high-performance anion-exchange chromatography. J Chromatogr A. 1996;720:137–149.
4. Cataldi RI, Campa C, De Benedette GE. Carbohydrate analysis by HPAE-PAD. Fresenius J Anal Chem. 2000;368:739–758.
5. Rohrer JS. Analyzing sialic acids using HPAE-PAD. Anal Biochem. 2000;283:3–9.
6. Behan JL, Smith KD. The analysis of glycosylation: a continued need for high pH anion exchange chromatography. Biomed Chromatogr. 2011;25:39–46.
7. Rohrer JS, Basumallick L, Hurum D. HPAE-PAD for carbohydrate analysis of glycoproteins. Biochemistry (Mosk). 2013;78:697–709.
8. Paskach TJ, Lieker HP, Reilly PJ, Thielecke K. HPAEC of sugars and sugar alcohols. Carbohydr Res. 1991;215:1–14.
9. Rendleman JA Jr. Ionization of carbohydrates in presence of metal hydroxides. In: Carbohydrates in Solution. ACS Advances in Chemistry Series. 1973;51–69.
10. Olechno JD, Carter SR, Edwards WT, Gillen DG. Developments in chromatographic determination of carbohydrates. Am Biotechnol Lab. 1987;5:38–50.
11. Anderson R, Sorenson A. Separation and determination of alditols and sugars by HPAE-PAD. J Chromatogr A. 2000;897:195–204.
12. Hotchkiss AT, Hicks KB. Analysis of oligogalacturonic acids by HPAE-PAD. Anal Biochem. 1990;184:200–206.
13. Rocklin RD, Clarke AP, Weitzhandler M. Improved reproducibility for PAD of carbohydrates via new waveform. Anal Chem. 1998;70:1496–1501.
14. Jensen MB, Johnson DC. Electrochemical detection of carbohydrates: waveform development. Anal Chem. 1997;69:1776–1781.
15. Rohrer JS. Optimal settings for pulsed amperometric detection of carbohydrates. Thermo Fisher Sci Tech Note TN21. 2013.
16. Huang B, Rohrer JS. Effect of working electrode gasket thickness on sensitivity and linearity of carbohydrate response by PAD. Thermo Fisher Sci Tech Note 186. 2016.
17. Rohrer JS. Eluent preparation for HPAE-PAD. Thermo Fisher Sci Tech Note 71. 2017.
18. Chen Y, Barreto V, Woodruff A, Lu Z, Liu Y, Pohl C. Dual electrolytic eluent generation for oligosaccharides analysis by HPAE. Anal Chem. 2018;90:10910–10916.