Sugars in Honey Using HPAE-PAD: What Is the Best Column?

Significance of the Topic

The detailed profiling of individual sugars in honey serves multiple practical purposes in food quality control and consumer protection. Accurate quantification of major and minor carbohydrates helps verify product labeling, detect adulteration, distinguish blossom honey from honeydew honey, and characterize unifloral varieties. Compliance with Codex Alimentarius and regional regulations on minimum fructose-glucose levels and maximum sucrose content also relies on robust analytical methods.

Objectives and Study Overview

This study aimed to develop and compare high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) methods for the separation and quantification of ten sugars in honey: fructose, glucose, sucrose, turanose, maltose, trehalose, isomaltose, erlose, raffinose, and melezitose. Three Thermo Scientific™ Dionex™ CarboPac™ columns (PA10, PA200, PA100) were evaluated under various sodium hydroxide eluent gradients to identify the column providing optimal resolution, run time, and robustness for routine honey analysis.

Methodology and Used Instrumentation

Sample Preparation:

• Dissolve 5 g of honey in 50 mL deionized water, dilute to appropriate concentration, and filter through 0.45 µm.
• Prepare individual sugar stock solutions and a series of working standards (Table 3) stored at 4 °C.

Chromatographic System:

• Quaternary SP pump with degas and EG eluent generator for NaOH eluent (700 mM).
• Pulsed amperometric detection (ED) with gold working electrode and Ag/AgCl reference.
• Columns tested: CarboPac PA10 (3 × 250 mm), PA200 (3 × 250 mm), PA100 (2 × 250 mm), each with guard column.
• Thermo Scientific™ Dionex™ Chromeleon™ CDS v6.8 for data acquisition.

Eluent and Detection Conditions:

• Sodium hydroxide gradient from ~3 % to 36 % over 25 min; flow rate 0.34–0.40 mL/min; column at 25–32 °C; detector at 15 °C; injection 10 µL.
• Carbohydrate waveform for amperometric detection, integration interval optimized for target sugars.

Main Results and Discussion

CarboPac PA10 (Condition I) achieved rapid separation of monosaccharides but failed to resolve raffinose and turanose. On-column turanose degradation produced negligible interference (<0.05 % glucose, <0.01 % fructose).

CarboPac PA200 (Condition II/IIa) improved overall resolution in ~20 min but coeluted melezitose with isomaltose. Adjusting the NaOH gradient (Condition IIa) freed turanose but still did not fully separate maltose and erlose.

CarboPac PA100 (Condition III) provided baseline separation of all ten target sugars within a 30 min run and 38 min cycle time. The advanced pellicular resin delivered higher anion-exchange capacity and solvent compatibility, yielding clear chromatographic fingerprints even in complex matrices.

Representative honey samples (acacia and Swiss blossom) were analyzed at multiple dilutions (up to 1:25000) to quantify high-abundance fructose/glucose and lower-level oligosaccharides. An example Swiss honey showed elevated sucrose (144 g/kg) consistent with feeding or adulteration.

Benefits and Practical Applications

• Minimal sample preparation and compatibility with high-throughput QA/QC workflows.
• Compliance with international honey standards for sugar composition.
• Differentiation of honey types and detection of adulteration based on comprehensive sugar fingerprint.
• Wide dynamic range supports quantification from trace oligosaccharides to major monosaccharides.

Future Trends and Potential Applications

Integration with mass spectrometry or isotope ratio measurement may enhance authentication and origin tracing. Online sample cleanup and micro-bore HPAE-PAD could reduce run times and solvent consumption. Development of tailored columns or novel stationary phases may further improve resolution of structurally related sugars. Data analytics and chemometrics applied to sugar profiles could allow automated classification of honey varieties and detection of sophisticated adulteration.

Conclusion

The CarboPac PA100 column combined with HPAE-PAD offers a reliable, fully resolved separation of ten honey sugars in a single run. This approach meets regulatory requirements, supports honey provenance studies, and enhances quality control practices in the food industry.

References

1. Codex Alimentarius Standard for Honey, CODEX STAN 12-1981.
2. Council Directive 2001/110/EC of 20 December 2001 relating to honey.
3. Swiss Honey Information, www.swisshoney.ch (October 2015).
4. Persano Oddo, L., Piro, R. et al., Main European Unifloral Honeys: Descriptive Sheets. Apidologie 35 (2004), 38–81.
5. Honigtauhonig – Der etwas andere Sortenhonig, LAVES Niedersachsen (accessed 2016).
6. Thermo Scientific Technical Note 71: Eluent Preparation for High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection, 2016.