HILIC - Sugars and fructooligosaccharide analysis

Significance of the Topic

Carbohydrate profiling in food, biotechnology and pharmaceutical applications is critical for ensuring product quality, understanding functional properties and monitoring bioprocesses. Traditional sugar analysis methods often require long run times and may not simultaneously resolve monomers, dimers and higher oligomers. Hydrophilic interaction liquid chromatography (HILIC) using an amino-bonded silica phase offers a rapid, robust solution for comprehensive carbohydrate separation.

Objectives and Overview

This study investigates the capabilities of the KNAUER Eurospher II NH2 column operated in HILIC mode to separate and quantify sugars and fructooligosaccharides (FOS) with varying degrees of polymerization (DP). Both standard mixtures and enzymatically produced inulin-type FOS samples were evaluated to assess resolution, run time and applicability to complex sample matrices.

Methodology and Instrumentation

An isocratic mobile phase consisting of acetonitrile:water (60:40, v/v) was applied at a flow rate of 0.6 mL/min. The column temperature was maintained at 40 °C, with 20 µL injection volume and refractive index detection at 20 Hz.
Instrument configuration:

• Pump: Thermo SpectraSYSTEM P4000
• Degasser: Thermo SCM1000
• Autosampler: Thermo AS3000
• Detector: Shodex RI-101 refractive index
• Column: Eurospher II 100-5 NH2, 250 × 3 mm ID with precolumn

Main Results and Discussion

Standard sugar mixtures (2.5 mM each) achieved baseline separation of glucose, sucrose and FOS up to DP5 in under 8 minutes. Chicory-derived FOS samples (50 mM) were resolved from DP2 to DP7. Enzymatic inulosucrase reactions produced FOS up to DP9 within 1 hour and extended to DP17 with prolonged analysis, demonstrating the method’s capacity for high-DP profiling. A minor unidentified byproduct was consistently detected between DP grades, highlighting the sensitivity to intermediate species.

Benefits and Practical Applications

The amino HILIC approach significantly reduces analysis time (under 15 minutes) while covering a broad DP range, making it ideal for high-throughput QA/QC in food, fermentation process monitoring and bioprocess development. The simple acetonitrile–water mobile phase and RI detection facilitate straightforward implementation in routine laboratories.

Future Trends and Applications

Future developments may include coupling HILIC with mass spectrometry for detailed structural analysis, integrating automated sampling for real-time bioreactor monitoring and designing specialized stationary phases for ultra-high DP carbohydrate separation.

Conclusion

The KNAUER Eurospher II NH2 column in HILIC mode delivers rapid, high-resolution separation of mono- to oligosaccharides up to DP17, providing a versatile and efficient tool for comprehensive carbohydrate analysis in both research and industrial settings.

References

1. McCalley DV. Hydrophilic interaction chromatography. 2008.
2. University of Bonn, Group of Prof. Dr. Uwe Deppenmeier, Institute for Microbiology and Biotechnology.