Determination of Plant-Derived Neutral Oligo- and Polysaccharides

Importance of the Topic

The analysis of plant-derived neutral oligo- and polysaccharides is essential for food quality control, authenticating botanical sources, and monitoring industrial bioprocesses. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) provides sensitive, derivatization-free detection and high resolving power for carbohydrates ranging from mono- to high-degree polymerization products.

Objectives and Study Overview

This application note outlines the development of robust HPAE-PAD methods for separating and detecting neutral oligo- and polysaccharides such as amylopectin (glucose polymers) and fructans (inulins). Key goals include optimizing column choice, eluent composition, gradient profile, and detection parameters to achieve reproducible, high-resolution separations.

Methodology and Instrumentation

• Strong anion-exchange CarboPac PA1 and PA100 columns with matching guard columns.
• Binary eluents: sodium hydroxide (100 or 150 mM) and sodium acetate (0.5 M to 1 M) gradients.
• Flow rate 1.0 mL/min, column temperature 30 °C, injection volumes 10–25 µL.
• Pulsed amperometric detection using a gold working electrode and Ag/AgCl reference with Waveform A.

Used Instrumentation

• Thermo Scientific™ Dionex™ BioLC chromatography system: GP50 pump with vacuum degas, ED50 electrochemical detector, E01 eluent organizer, AS50 autosampler with thermal compartment.
• Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System (CDS) v6.

Main Results and Discussion

• Gradient Profiles: A slightly concave gradient improved resolution of high-DP peaks compared to linear profiles in Maltrin M040 separations.
• Column Comparison: PA100 offered ~10% greater peak capacity (209 vs 191), higher efficiency, and enabled detection of three additional oligomers versus PA1. Reduced column capacity led to shorter retention of large polymers.
• Sodium Hydroxide Concentration: Using 150 mM NaOH slightly increased retention for larger oligosaccharides due to stronger ionization; no additional peaks were observed but solubility improved.
• Inulin Fingerprinting: Distinct polymer distributions for chicory versus dahlia inulins demonstrated HPAE-PAD’s capability to profile and compare different sources or production lots.
• Method Robustness: Controlled column reequilibration (15 min), temperature stability, optimized injection modes (full-loop, partial-loop, limited-sample) and periodic column washing maintained retention time reproducibility and analyte recovery.
• Sample Overload and Carryover: Excessive injection volumes led to peak distortion and retention shifts; bound high-molecular-weight material required intermittent column cleaning to restore capacity.

Benefits and Practical Applications

HPAE-PAD methods enable rapid, high-resolution profiling of plant-based carbohydrate polymers for quality assurance in food and beverage industries, detection of adulteration, origin authentication of honey and juices, and monitoring of bioprocess hydrolysis or extraction processes.

Future Trends and Perspectives

• Exploration of sodium nitrate eluents to enhance resolution between linear and branched polymers.
• Miniaturized 2 × 250 mm columns for reduced solvent consumption and waste.
• Automated sample cleanup and fraction collection systems coupled with HPAE-PAD for preparative separations.
• Integration of advanced data analytics and machine learning for rapid fingerprint matching and source tracking.

Conclusion

Optimized HPAE-PAD protocols using CarboPac PA100 columns, tailored gradient shapes, and controlled detection settings deliver reproducible, sensitive separations of neutral oligo- and polysaccharides. These methods support comprehensive carbohydrate profiling for research and industry.

References

1. Rocklin RD, Pohl CA. J. Liq. Chromatogr. 1983, 6, 1577–1590.
2. Lee YC. J. Chromatogr. A 1996, 720, 137–149.
3. Koizumi K, Kubota Y, Tanimoto T, Okada Y. J. Chromatogr. 1989, 464, 365–373.
4. Hanashiro I, Abe J, Hizukuri S. Carbohydr. Res. 1996, 283, 151–159.
5. Koizumi K, Fukuda M, Hizukuri S. J. Chromatogr. 1991, 585, 233–238.
6. Thermo Fisher Scientific. Application Note 82; Sunnyvale, CA.
7. Swallow KW, Low NH. J. Agric. Food Chem. 1990, 38, 828–832.
8. van der Hoeven RAM, Niessen WMA, Schols HA, Bruggink C, Voragen AGJ, van der Greef J. J. Chromatogr. 1992, 627, 63–73.
9. Campbell JM, Bauer LL, Fahey GC Jr, Hogarth AJCL, Wolf BW, Hunter DE. J. Agric. Food Chem. 1997, 45, 3076–3082.
10. Chatterton NJ, Harrison PA. New Phytol. 1997, 136, 3–10.
11. Schols HA, Lucas-Lokhorst G, Voragen AGJ, Niessen WMA. Carbohydrates in the Netherlands 1993, 9, 11–13.
12. Verbruggen MA, Spronk BA, Schols HA, Beldman G, Voragen AGJ, Thomas JR, Kamerling JP, Vliegenthart JFG. Carbohydr. Res. 1998, 306, 265–274.
13. Torto N, Buttler T, Gorton L, Marko-Varga G, Stalbrand H, Tjerneld F. Anal. Chim. Acta 1995, 313, 15–24.
14. Mou S, Sun Q, Lu D. J. Chromatogr. 1991, 546, 289–295.
15. McDougall GJ, Fry SC. Carbohydr. Res. 1991, 219, 123–132.
16. Thermo Fisher Scientific. Technical Note 21; Sunnyvale, CA.
17. Cooper GA, Rohrer JS. Anal. Biochem. 1995, 226, 182–184.
18. Thayer J, Rohrer JS, Avdalovic N, Gearing RP. Anal. Biochem. 1998, 256, 207–216.
19. Rocklin RD, Clarke AP, Weitzhandler M. Anal. Chem. 1998, 70, 1496–1501.
20. Ammeraal RN, Delgado GA, Tenbarge FL, Friedman RB. Carbohydr. Res. 1991, 215, 179–192.
21. Koch K, Andersson R, Aman P. J. Chromatogr. A 1998, 800, 199–206.
22. Wong KS, Jane JJ. J. Liq. Chromatogr. 1995, 18, 63–80.
23. Giddings JC. United Separation Science; John Wiley & Sons: New York, 1991, p. 105.