Carbohydrate determinations by HPAE-PAD using a PdH reference electrode

Significance of the Topic

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) enables direct, high-resolution analysis of carbohydrates without derivatization. Reliable reference electrodes are critical to maintain stable detector potential, minimize baseline drift and ensure reproducible quantification in food, pharmaceutical and biotechnology applications.

Objectives and Study Overview

This work compares the performance of a solid-state palladium hydrogen (PdH) reference electrode against a conventional silver/silver chloride (Ag/AgCl) electrode in two representative HPAE-PAD assays: rapid profiling of mono-, di- and oligosaccharides in honey, and quantitative determination of monosaccharides released from bovine fetuin glycoprotein hydrolysates.

Methodology and Instrumentation

The studies were performed on a Thermo Scientific Dionex ICS-5000+ HPIC system equipped with:

• CarboPac PA210-Fast-4 µm analytical (2 × 150 mm) and guard (2 × 30 mm) columns for honey sugars;
• CarboPac PA20-Fast-4 µm column (2 × 100 mm) for monosaccharides;
• EGC 500 KOH eluent generator delivering 10–100 mM hydroxide gradients;
• Pulsed amperometric detector with disposable gold working electrodes and interchangeable Ag/AgCl or PdH reference electrodes;
• Autosampler with cooling tray and Chromeleon CDS control for waveform configuration and RE offset calculation.

Honey sample preparation involved direct injection of mixed sugar standards and commercial honeys. Glycoprotein hydrolysates were generated by TFA or HCl digestion of fetuin, followed by SpeedVac drying and reconstitution.

Results and Discussion

Honey sugars separated within 20–45 min showed comparable baseline noise (~35 pC) and background (~30–35 nC) for both REs. The PdH electrode yielded slightly higher peak areas (1–5% increase) and equivalent retention times. Calibration curves for 12–14 sugars exhibited r2 > 0.999, with precision RSD < 2%. Quantified fructose/glucose ratios and total reducing sugars met Codex standards. Long-term testing (1 500 injections, 6 months) demonstrated stable response (≤5% variation).

Glycoprotein monosaccharide analysis resolved six analytes in 8 min with similar backgrounds (~31 nC). PdH-based peak areas varied by <5% relative to Ag/AgCl. Linearity (r2 > 0.993) and precision (RSD < 3%) matched previous results. Fetuin hydrolysates yielded expected composition: amino sugars higher after HCl digestion, neutral sugars after TFA treatment.

Benefits and Practical Applications

• PdH solid-state RE eliminates liquid junctions and aqueous filling solutions, reducing maintenance and extending lifespan.
• Stable potential ensures consistent sensitivity and low background in high-pH eluents.
• Seamless integration with existing HPAE-PAD systems supports food quality control and glycoprotein therapeutic analysis.

Future Trends and Applications

• Expansion of solid-state RE technology to multiplexed and miniaturized IC platforms.
• Application to complex carbohydrate matrices such as plant oligosaccharides and biofuel feedstocks.
• Integration with hyphenated techniques (IC-MS, IC-NMR) for structural elucidation.

Conclusion

The PdH reference electrode is a robust alternative to Ag/AgCl in HPAE-PAD, delivering equivalent or improved stability, reproducibility and ease-of-use for carbohydrate assays in diverse sectors.

Instrumentation

• Thermo Scientific Dionex ICS-5000+ HPIC with EGC 500 and ED modules
• CarboPac PA210-Fast-4 µm and PA20-Fast-4 µm columns with guard cartridges
• Pulsed amperometric detector with gold working electrodes and PdH/Ag/AgCl reference electrodes
• Chromeleon CDS v7.2.9 software, autosampler and SpeedVac concentrator

Reference

1. Thermo Scientific Technical Note 71: Eluent Preparation for HPAE-PAD.
2. Thermo Scientific Dionex ICS-5000 Installation Instructions, Doc 065343, Rev 05.
3. Thermo Scientific Electrochemical Detection User’s Compendium, P/N 065340-02.
4. Thermo Fisher Application Note 1158: HPAE-PAD Determination of Carbohydrates in Honey.
5. Codex Alimentarius Commission, Codex Standard 12: Honey, 2001.
6. White J.W. and Doner L.W., Agriculture Handbook 335: Honey Composition and Properties, 1980.
7. Bogdanov S. et al., J. Am. Coll. Nutr. 27(6):677–689, 2008.
8. Thermo Fisher Application Note 72580: Improved HPAE-PAD Method for Glycoprotein Monosaccharide Determination.