High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)

HPAEC-PAD becomes a popular method that is widely used for the determination of a large variety of carbohydrates with the advantages of high-resolution separations and sensitive detection. Creative Biolabs is a world-leading custom services provider in the field of HPAEC-PAD analysis.

Glycoproteins Analysis Based on HPAEC-PAD

HPAEC-PAD is a well-established technique for glycoprotein analysis as well as for other fields of carbohydrate analysis. It was first applied to glycoprotein analysis in the late 1980s. Currently, HPAEC-PAD is widely used for determinations of monosaccharide, sialic acid, mannose-6-phosphate (M-6-P), and oligosaccharide contents of a glycoprotein. This technique can effectively separate carbohydrates that become oxyanions at high pH using anion-exchange chromatography. The separated carbohydrates can be detected by PAD which is a direct and sensitive detection technique. It allows detecting carbohydrates, with excellent signal-to-noise ratios and sensitivities down to subpicomole levels for standard bore and microbore systems and femtomole levels for capillary systems, respectively, without requiring derivatization. For these advantages, HPAEC-PAD has been used for the initial investigation of a glycoprotein and routine assays of recombinant therapeutic glycoproteins. For example, HPAEC-PAD has been used for the determination of the monosaccharide and sialic acids content of a glycoprotein. The determination of individual sialic acids content in proteins by HPAEC-PAD technique has been compared with other chromatographic methods requiring labeling of the sialic acids, showing comparable results. HPAEC-PAD is also performed to determine the relative amounts of N-acetylneuraminic acid (Neu5Ac) and Neu5Gc as well as the M-6-P content of a glycoprotein.

HPAEC-PAD Analysis Services

With the years of improvements, our HPAEC-PAD technology has become a sensitive and rapid analysis tool for Glycan Profiling. Based on HPAEC-PAD technique, the monosaccharide composition can be determined. Besides, 13 monosaccharides (such as glucose, fructose, rhamnose, arabinose, fucose, galactose, mannose, ribose, xylose, glucuronic acid, galacturonic acid, mannuronic acid, and guluronic acid), glucosamine, galactosamine, sialic acid, and M-6-P can be quantified by HPAEC-PAD. Our scientists, with extensive experience in HPAEC-PAD analysis, can assist you to design a suitable protocol for glycan profiling.

Features of Our Services Including but Not Limited to:

• Experienced expert team
• Advanced technique platforms
• Providing accurate and comprehensive analysis data
• High-quality analysis service

Creative Biolabs has accumulated rich experience in HPAEC-PAD analysis. We are very confident to offer high-quality and comprehensive services of HPAEC-PAD analysis for global clients. With our excellent service, we will offer you not only an accurate analysis result but also our professional technical guidance to facilitate the later project.

If you are interested in our HPAEC-PAD analysis services, please feel free to contact us for more details.

Published data

In Amaranthaceae, the structures of galactan and arabinan become complex and their chemical properties change due to the substitution of ferulate. Therefore, studying the environment of ferulate is particularly important to understand the relationship between the structure and functions of these polysaccharides. After enzymatic degradation, galactan and arabinan can release feruloylated oligosaccharides (FAOS) and non-ferruloylated oligosaccharides (NFAOS). In this study, the authors developed a simple analytical method to reveal the molecular environment of ferulic acid in Amaranthaceae polysaccharides by first enzymatic cleavage of galactan and arabinan, and then semi-quantitative analysis by de-esterification and HPAEC-PAD technology. HPAEC-PAD analysis results showed that FAOS showed more signals after 70 minutes, suggesting that it had a richer oligomeric structure. Compared with NFAOS, the oligosaccharide structures released from FAOS were more complex, and FAOS contained a higher proportion of A-3a, which might mean that ferulate affected the enzymatic cleavage process.

Fig.1 HPAEC-PAD analysis results of FAOS and NFAOS oligosaccharides.¹

Case Study

Background: The client commissioned Creative Biolabs to determine the composition and content of monosaccharides in polysaccharide samples.

Method: HPAEC-PAD

Table 1 Instrument parameters.

Abstract: Creative Biolabs uses HPAEC-PAD technology to efficiently and accurately determine the composition and content of each monosaccharide in polysaccharide samples, including 13 monosaccharides such as glucose (Glc), fructose (Fru), rhamnose (Rha), arabinose (Ara), fucose (Fuc), galactose (Gal), mannose (Man), ribose (Rib), xylose (Xyl), glucuronic acid (Glc-UA), galacturonic acid (Gal-UA), mannuronic acid (Man-UA) and guluronic acid (Gul-UA). The entire detection process includes the following steps:

• Preparation of monosaccharide standards

We took a clean 15mL test tube, added 8mL ddH₂O, and added 13 monosaccharide standards, 100mg each. We dissolved them and prepared them into a 10mg/mL mixed standard stock solution. Then, we diluted the above mixed standard stock solution according to different concentration requirements to prepare standard curve working solutions of different concentrations.

• Sample pretreatment

We weighed an appropriate amount of the polysaccharide sample and added a trifluoroacetic acid (TFA) solution to hydrolyze it into monosaccharides for subsequent detection.

• Sample testing

We injected the monosaccharide standard and sample solution into the HPAEC-PAD system respectively, and obtained the corresponding chromatograms through the system. Different compounds have different retention capacities on the analytical column and different elution times. We compared and calculated the monosaccharide content in the sample with the standard curve according to the retention time and peak area of each monosaccharide peak in the chromatogram to obtain the concentration and content of each monosaccharide in the sample. In addition, during the testing process, we designed QC and experimental repetitions according to the number and sample size of the client's samples to ensure the reliability of the data.

• Data processing and result

As shown in the figure below, these were the test results of the high-concentration standard curve working solution (30 μg/mL) and the sample solution. The composition and content of each monosaccharide in the polysaccharide sample were accurately determined by HPAEC-PAD. The results were as follows:

Fig.2 HPAEC-PAD analysis results.

Table 2 HPAEC-PAD content results of polysaccharide sample.

NA: This data was not available as it was below the detection limit.

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Reference

1. Junker, Florian, and Mirko Bunzel. "Chromatography based profiling of feruloylated arabinans and galactans." Carbohydrate Research 538 (2024): 109076. Distributed under Open Access license CC BY 4.0, without modification.

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