The rising importance of accurately detecting oligosaccharides in biomass hydrolyzates or as ingredients in food, such as in beverages and infant milk products, demands for the availability of tools to sensitively analyze the broad range of available oligosaccharides. Over the last decades, HPAEC-PAD has been developed into one of the major technologies for this task and represents a popular alternative to state-of-the-art LC-MS oligosaccharide analysis. This work presents the first comprehensive study which gives an overview of the separation of 38 analytes as well as enzymatic hydrolyzates of six different polysaccharides focusing on oligosaccharides. The high sensitivity of the PAD comes at cost of its stability due to recession of the gold electrode. By an in-depth analysis of the sensitivity drop over time for 35 analytes, including xylo- (XOS), arabinoxylo- (AXOS), laminari- (LOS), manno- (MOS), glucomanno- (GMOS), and cellooligosaccharides (COS), we developed an analyte-specific one-phase decay model for this effect over time. Using this model resulted in significantly improved data normalization when using an internal standard. Our results thereby allow a quantification approach which takes the inevitable and analyte-specific PAD response drop into account.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995;125(6):1401–12.
Dhingra D, Michael M, Rajput H, Patil RT. Dietary fibre in foods: a review. J Food Sci Technol. 2012; https://doi.org/10.1007/s13197-011-0365-5.
Moure A, Gullón P, Domínguez H, Parajó JC. Advances in the manufacture, purification and applications of xylo-oligosaccharides as food additives and nutraceuticals. Process Biochem. 2006; https://doi.org/10.1016/j.procbio.2006.05.011.
Mussatto SI, Mancilha IM. Non-digestible oligosaccharides: a review. Carbohydr Polym. 2007; https://doi.org/10.1016/j.carbpol.2006.12.011.
Otieno DO, Ahring BK. The potential for oligosaccharide production from the hemicellulose fraction of biomasses through pretreatment processes: xylooligosaccharides (XOS), arabinooligosaccharides (AOS), and mannooligosaccharides (MOS). Carbohydr Res. 2012; https://doi.org/10.1016/j.carres.2012.07.017.
Bali V, Panesar PS, Bera MB, Panesar R. Fructo-oligosaccharides: production, purification and potential applications. Crit Rev Food Sci Nutr. 2013; https://doi.org/10.1080/10408398.2012.694084.
Spinner J. Prebiotics market to hit $4.8 billion by 2018; 2013. http://www.nutraingredients.com/Suppliers2/Prebiotics-market-to-hit-4.8-billion-by-2018. Accessed 15 Sep 2017.
Roberfroid M. Prebiotics: the concept revisited. J Nutr. 2007;137(3 Suppl 2):830.
Broekaert WF, Courtin CM, Verbeke K, Van de Wiele T, Verstraete W, Delcour JA. Prebiotic and other health-related effects of cereal-derived arabinoxylans, arabinoxylan-oligosaccharides, and xylooligosaccharides. Crit Rev Food Sci Nutr. 2011; https://doi.org/10.1080/10408390903044768.
Albrecht S, van Muiswinkel GCJ, Xu J, Schols HA, Voragen AGJ, Gruppen H. Enzymatic production and characterization of konjac glucomannan oligosaccharides. J Agric Food Chem. 2011; https://doi.org/10.1021/jf203091h.
Bhattacharyya L, Rohrer JS. Applications of ion chromatography for pharmaceutical and biological products. Hoboken: John Wiley & Sons, Inc; 2012.
McCleary BV, McKie VA, Draga A, Rooney E, Mangan D, Larkin J. Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-l-arabinofuranosidase and β-xylosidase. Carbohydr Res. 2015; https://doi.org/10.1016/j.carres.2015.01.017.
Veillon L, Huang Y, Peng W, Dong X, Cho BG, Mechref Y. Characterization of isomeric glycan structures by LC-MS/MS. Electrophoresis. 2017; https://doi.org/10.1002/elps.201700042.
Samuelsen AB, Hanne Cohen E, Smestad Paulsen B, Brüll LP, Thomas-Oates JE. Structural studies of a heteroxylan from Plantago major L. seeds by partial hydrolysis, HPAEC-PAD, methylation and GC–MS, ESMS and ESMS/MS. Carbohydr Res. 1999; https://doi.org/10.1016/S0008-6215(99)00038-5.
Kailemia MJ, Ruhaak LR, Lebrilla CB, Amster IJ. Oligosaccharide analysis by mass spectrometry: a review of recent developments. Anal Chem. 2014; https://doi.org/10.1021/ac403969n.
Corradini C, Cavazza A, Bignardi C. High-performance anion-exchange chromatography coupled with pulsed electrochemical detection as a powerful tool to evaluate carbohydrates of food interest: principles and applications. Int J Carbohydr Chem. 2012; https://doi.org/10.1155/2012/487564.
Corradini C, Lantano C, Cavazza A. Innovative analytical tools to characterize prebiotic carbohydrates of functional food interest. Anal Bioanal Chem. 2013; https://doi.org/10.1007/s00216-013-6731-6.
Rohrer J. Technical Note 21: optimal settings for pulsed amperometric detection of carbohydrates using the Dionex ED40 Electrochemical Detector. Accessed 15 Sep 2017.
Otieno DO, Ahring BK. A thermochemical pretreatment process to produce xylooligosaccharides (XOS), arabinooligosaccharides (AOS) and mannooligosaccharides (MOS) from lignocellulosic biomasses. Bioresour Technol. 2012; https://doi.org/10.1016/j.biortech.2012.01.162.
Wong KS, Jane J. Effects of pushing agents on the separation and detection of debranched amylopectin by high-performance anion-exchange chromatography with pulsed amperometric detection. J Liq Chromatogr. 1995; https://doi.org/10.1080/10826079508009221.
White D, Hudson P, Adamson JT. Dextrin characterization by high-performance anion-exchange chromatography–pulsed amperometric detection and size-exclusion chromatography–multi-angle light scattering–refractive index detection. J Chromatogr A. 2003; https://doi.org/10.1016/S0021-9673(03)00626-5.
Wang Y, Vilaplana F, Brumer H, Aspeborg H. Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana. Planta. 2014; https://doi.org/10.1007/s00425-013-2005-y.
Mechelke M, Koeck DE, Broeker J, Roessler B, Krabichler F, Schwarz WH, et al. Characterization of the arabinoxylan-degrading machinery of the thermophilic bacterium Herbinix hemicellulosilytica—six new xylanases, three arabinofuranosidases and one xylosidase. J Biotechnol. 2017; https://doi.org/10.1016/j.jbiotec.2017.04.023.
Adelsberger H, Hertel C, Glawischnig E, Zverlov VV, Schwarz WH. Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes. Microbiology. 2004; https://doi.org/10.1099/mic.0.27066-0.
Kormelink FJ, Gruppen H, Viëtor RJ, Voragen AG. Mode of action of the xylan-degrading enzymes from Aspergillus awamori on alkali-extractable cereal arabinoxylans. Carbohydr Res. 1993; https://doi.org/10.1016/0008-6215(93)84100-K.
Koutaniemi S, Tenkanen M. Action of three GH51 and one GH54 alpha-arabinofuranosidases on internally and terminally located arabinofuranosyl branches. J Biotechnol. 2016; https://doi.org/10.1016/j.jbiotec.2016.04.050.
Rohrer J. Technical Note 20: analysis of carbohydrates by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Accessed 19 May 2017.
Rivière A, Eeltink S, Pierlot C, Balzarini T, Moens F, Selak M, et al. Development of an ion-exchange chromatography method for monitoring the degradation of prebiotic arabinoxylan-oligosaccharides in a complex fermentation medium. Anal Chem. 2013; https://doi.org/10.1021/ac400187f.
Paskach TJ, Lieker H-P, Reilly PJ, Thielecke K. High-performance anion-exchange chromatography of sugars and sugar alcohols on quaternary ammonium resins under alkaline conditions. Carbohydr Res. 1991; https://doi.org/10.1016/0008-6215(91)84002-V.
Fry SC, York WS, Albersheim P, Darvill A, Hayashi T, Joseleau J-P, et al. An unambiguous nomenclature for xyloglucan-derived oligosaccharides. Physiol Plant. 1993; https://doi.org/10.1111/j.1399-3054.1993.tb01778.x.
Xu Y, Fan L, Wang X, Yong Q, Yu S-Y. Simultaneous separation and quantification of linear xylo- and cello-oligosaccharides mixtures in lignocellulosics processing products on high-performance anion-exchange chromatography coupled with pulsed amperometric detection. Bioresource 2013;8(3).
Qing Q, Yang B, Wyman CE. Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. Bioresour Technol. 2010; https://doi.org/10.1016/j.biortech.2010.06.137.
Koizumi K, Kubota Y, Tanimoto T, Okada Y. High-performance anion-exchange chromatography of homogeneous d-gluco-oligosaccharides and -polysaccharides (polymerization degree ⩾ 50) with pulsed amperometric detection. J Chromatogr A. 1991; https://doi.org/10.1016/S0021-9673(00)94254-7.
Borromei C, Cavazza A, Merusi C, Corradini C. Characterization and quantitation of short-chain fructooligosaccharides and inulooligosaccharides in fermented milks by high-performance anion-exchange chromatography with pulsed amperometric detection. J Sep Sci. 2009; https://doi.org/10.1002/jssc.200900322.
Wefers D, Bunzel M. Arabinan and galactan oligosaccharide profiling by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). J Agric Food Chem. 2016; https://doi.org/10.1021/acs.jafc.6b01121.
Kootstra AMJ, Beeftink HH, Scott EL, Sanders JPM. Optimization of the dilute maleic acid pretreatment of wheat straw. Biotechnol Biofuels. 2009; https://doi.org/10.1186/1754-6834-2-31.
Rohrer J. Technical note 133: HPAE-PAD peak area response of glycoprotein oligosaccharides. Accessed 19 May 2017.
Rohrer JS, Thayer J, Weitzhandler M, Avdalovic N. Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection. Glycobiology. 1998;8(1):35–43.
Schäffler KJ, Mrel du boil PG, Walford SN. HPAEC: some precautions required for the reliable analysis of carbohydrates. Proc S Afr Sug Technol Assoc. 1996;70:241–50.
Qing Q, Li H, Kumar R, Wyman CE. Xylooligosaccharides production, quantification, and characterization in context of lignocellulosic biomass pretreatment. In: Wyman CE, editor. Aqueous pretreatment of plant biomass for biological and chemical conversion to fuels and chemicals. Chichester: John Wiley & Sons, Ltd; 2013. p. 391–415.
Timmermans JW, van Leeuwen MB, Tournois H, de Wit D, Vliegenthart JFG. Quantitative analysis of the molecular weight distribution of inulin by means of anion exchange HPLC with pulsed amperometric detection. J Carbohydr Chem. 1994; https://doi.org/10.1080/07328309408011688.
Ammeraal RN, Delgado GA, Tenbarge FL, Friedman RB. High-performance anion-exchange chromatography with pulsed amperometric detection of linear and branched glucose oligosaccharides. Carbohydr Res. 1991; https://doi.org/10.1016/0008-6215(91)84017-9.
Sasagawa T, Sakamoto Y, Hirose T, Yoshida T, Kobayashi Y, Sato Y, et al. Prediction of retention times in ion-exchange chromatography. J Chromatogr A. 1989; https://doi.org/10.1016/S0021-9673(01)89160-3.
Kunz C, Rudloff S, Hintelmann A, Pohlentz G, Egge H. High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides. J Chromatogr B. 1996; https://doi.org/10.1016/S0378-4347(96)00181-8.
Abballe F, Toppazzini M, Campa C, Uggeri F, Paoletti S. Study of molar response of dextrans in electrochemical detection. J Chromatogr A. 2007; https://doi.org/10.1016/j.chroma.2006.11.086.
Anumula KR, Dhume ST. High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid. Glycobiology. 1998; https://doi.org/10.1093/glycob/8.7.685.
Financial support from the European Commission (Collaborative FP7-KBBE Project Valor Plus, contract No. 613802) and the German Federal Ministry of Education and Research (grant number FKZ: 031A556) is gratefully acknowledged.
Conflict of interest
The authors declare that they have no conflicts of interest.
Electronic supplementary material
About this article
Cite this article
Mechelke, M., Herlet, J., Benz, J.P. et al. HPAEC-PAD for oligosaccharide analysis—novel insights into analyte sensitivity and response stability. Anal Bioanal Chem 409, 7169–7181 (2017). https://doi.org/10.1007/s00216-017-0678-y
- Oligosaccharide analysis
- PAD response factor
- Data normalization