HPAE-PAD analysis of galactosyl-oligosaccharide- containing samples using dual eluent generation cartridges

Importance of the topic

Galactosyl oligosaccharides (GOS) are prebiotic carbohydrates widely used in infant formula and dietary supplements. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) enables direct analysis of such complex carbohydrates without derivatization, supporting quality control and research in food and health sciences.

Objectives and study overview

This study evaluates a Dual Eluent Generation Cartridge (EGC) system on a Thermo Scientific Dionex ICS-6000 Reagent-Free Ion Chromatography platform to profile GOS. The aim is to demonstrate automated gradient eluent preparation using methanesulfonic acid and potassium hydroxide cartridges in series, and to compare performance to conventional sodium hydroxide/sodium acetate gradients.

Methodology and instrumentation

An RFIC system equipped with a Dual EGC 400 MSA cartridge and an EGC 400 KOH cartridge in series generated KOH/KMSA gradients on a CarboPac PA200 1 mm guard and analytical column. Key components included:

• ICS-6000 HPIC system with DP isocratic pump and eluent generation module
• Pulsed amperometric detector with gold/PTFE electrode and Ag/AgCl reference
• Chromeleon CDS software controlling Dual EGC mode
• Autosampler with a 0.4 µL injection loop
• Bimuno GOS standard (5 mg/mL) and an organic milk-based infant formula sample

Gradient conditions: 20–70 mM KMSA in 70 mM KOH over 45 min, followed by a high-strength wash and re-equilibration, at 0.063 mL/min and 30 °C.

Main results and discussion

The Dual EGC system produced precise and reproducible KOH/KMSA gradients. Analysis of Bimuno GOS standards achieved baseline separation of DP1–DP13 oligosaccharides, matching the resolution reported for manually prepared sodium hydroxide/sodium acetate methods. Application to infant formula samples likewise resolved individual GOS peaks with minimal noise and stable retention. Automated gradient control reduced preparation errors and baseline drift.

Benefits and practical applications

• Eliminates manual eluent preparation, reducing labor and reagent handling risks
• Ensures high-purity eluents and consistent gradient profiles for improved reproducibility
• Maximizes instrument uptime by using an isocratic pump to deliver only water
• Minimizes system contamination and maintenance by generating KOH and MSA on demand
• Applicable to routine quality control of prebiotics in food, infant nutrition, and research laboratories

Future trends and opportunities

Integration of Dual EGC technology with smaller column formats and faster methods can further increase throughput. Coupling HPAE-PAD with mass spectrometry may enhance structural characterization of oligosaccharides. Expanded applications to other complex carbohydrates, dietary fibers, and glycan profiling in biopharmaceuticals are anticipated.

Conclusion

The Dual EGC approach on the Dionex ICS-6000 RFIC system offers a robust, reproducible, and automated solution for gradient eluent generation in HPAE-PAD analysis of GOS. It streamlines workflows, improves data quality, and supports routine carbohydrate profiling in food and life sciences applications.

Reference

1. Huang B, Chen L, Hu J, Rohrer J. HPAE-PAD analysis of galactosyl-oligosaccharide-containing samples using dual eluent generation cartridges. Thermo Fisher Scientific Application Note 72714 (2018).
2. Thermo Fisher Scientific. Profiling Galactosyloligosaccharide-Containing Samples by HPAE-PAD. Application Note 11511 (2012).
3. Thermo Fisher Scientific. Dionex ICS-6000 Dual EGC Installation Guide, Doc No. 065760-01 (2018).
4. Thermo Fisher Scientific. Dionex ICS-6000 Ion Chromatography System Operator’s Manual, Doc No. 22181-97002 (2018).
5. Thermo Fisher Scientific. Technical Note 110: Carbohydrate Determination by HPAE-PAD with Disposable Au/PTFE Working Electrodes (2011).