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class="content-sidebar-wrap"><main class="content"><article class="post-4730 post type-post status-publish format-standard has-post-thumbnail category-food-beverage category-hpae-pad tag-food-beverage tag-hpae-pad entry" aria-label="Reliable, Reproducible Determination of Trans-Galactooligosaccharides Using Dual EGC Mode HPAE-PAD"><header class="entry-header"><h1 class="entry-title">Reliable, Reproducible Determination of Trans-Galactooligosaccharides Using Dual EGC Mode HPAE-PAD</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/analyteguru/tf-author/analyteguru-staff/" title="Posts by AnalyteGuru Staff" rel="author">AnalyteGuru Staff, </a> </span> <span class="post-info-date"> 09.02.2022 </span> </header><div class="entry-content"><div class="lia-message-body-content"> <p><img decoding="async" class="alignright wp-image-4732 size-medium" src="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/15143-og-300x200.jpg" alt="" width="300" height="200" srcset="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/15143-og-300x200.jpg 300w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/15143-og-768x512.jpg 768w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/15143-og-96x65.jpg 96w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/15143-og.jpg 999w" sizes="(max-width: 300px) 100vw, 300px" /></p> <p>Trans-galactooligosaccharides (TGOS) are an important family of soluble polysaccharides that are resistant to digestion in the small intestine. As dietary prebiotics, TGOS can improve health by modulating the intestinal microbiome and immune system. To ensure the nutritional benefits of food and support a healthy gut, precise and reproducible methods for determining TGOS content in products are essential.</p> <h3 id="toc-hId-1154317037">Determining TGOS in food using HPAE-PAD</h3> <p>High-performance anion-exchange (HPAE) chromatography coupled with pulsed amperometric detection (PAD) is a well-established technique for oligosaccharide analysis. Given the reliability and widespread use of this analytical approach the AOAC, a standards-setting organization approved a HPAE-PAD-based protocol (AOAC Method 2001.02) for the determination of TGOS content in food and feed products. In this method, TGOS present in a buffered sample extracts are first hydrolyzed to galactose and glucose using β-galactosidase, before being separated by HPAE using a <a href="https://www.thermofisher.com/order/catalog/product/043096?ce=E.22CMD.PM109.05966.01&cid=E.22CMD.PM109.05966.01" target="_blank" rel="nofollow noopener">Thermo Scientific™ Dionex™ CarboPac™ PA1-4mm column</a> and subsequently detected by PAD.</p> <h3 id="toc-hId-2041820718">Dual eluent generation cartridge mode HPAE-PAD: a more reliable and convenient approach for TGOS determination</h3> <p>HPAE-PAD methods for TGOS analysis typically require high-purity sodium hydroxide/sodium acetate eluents. These can be labor-intensive and time-consuming to prepare, and variations in mobile phase composition can be a source of measurement inconsistency.</p> <p>Dual eluent generation cartridge (dual EGC) mode is a new operating approach for HPAE-PAD that replaces manual preparation of sodium hydroxide/sodium acetate eluent gradients with electrolytically generated potassium hydroxide (KOH)/potassium methanesulfonate acid (KMSA) eluents. As well as saving time and improving efficiency, dual EGC mode operation ensures consistent production of high-purity mobile phases, thereby reducing system contamination and improving method reproducibility.</p> <h3 id="toc-hId--1365642897">An improved HPAE-PAD method for TGOS analysis</h3> <p><a href="https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-73986-hpae-pad-trans-galactooligosaccharides-food-an73986-en.pdf" target="_blank" rel="nofollow noopener"><span class="lia-inline-image-display-wrapper lia-image-align-right" style="width: 400px"><img decoding="async" class="size-full wp-image-13813 alignleft" src="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2022/09/App-Note-73986.jpg" alt="" width="400" height="349" srcset="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2022/09/App-Note-73986.jpg 400w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2022/09/App-Note-73986-300x262.jpg 300w" sizes="(max-width: 400px) 100vw, 400px" /></span></a>To demonstrate the potential of dual EGC mode HPAE-PAD systems for TGOS determination, we performed AOAC Method 2001.02 using a <a href="https://www.thermofisher.com/us/en/home/products-and-services/promotions/industrial/freedom-explore-ion-chromatography.html?ce=E.22CMD.PM109.05966.01&cid=E.22CMD.PM109.05966.01" target="_blank" rel="nofollow noopener">Thermo Scientific™ Dionex™ ICS-6000 HPIC™</a> system, as reported in this <a href="https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-73986-hpae-pad-trans-galactooligosaccharides-food-an73986-en.pdf" target="_blank" rel="nofollow noopener">application note</a>. We also employed a <a href="https://www.thermofisher.com/order/catalog/product/057178?ce=E.22CMD.PM109.05966.01&cid=E.22CMD.PM109.05966.01" target="_blank" rel="nofollow noopener">Dionex CarboPac PA1 (250 mm × 1 mm) column</a>, which uses a flow rate of approximately 16 times less than the 4 mm column used in Method 2001.02, reducing eluent consumption substantially.</p> <p>Method development involved a stock standard containing galactose, glucose, fructose, sucrose, and lactose. To achieve similar separation using dual EGC mode to that achieved using Method 2001.02, a KOH/KMSA gradient method with a 12-minute column wash was developed. To confirm the column wash was sufficient for this application, retention time stability was monitored. Retention times of galactose and lactose were stable for seven days of continuous injections of samples and standards.</p> <p>Calibration curves with seven concentration levels were constructed for galactose and lactose. The coefficient of determination (r<sup>2</sup>) was greater than 0.999 for each component, highlighting the linearity of this approach. Method precision was then assessed by triplicate injection of a calibration standard. The calculated peak area precision ranged from 0.66–1.23%, with retention time precision of <0.21% for all target carbohydrates. The excellent precision of this approach is characteristic of a system operating in dual EGC mode.</p> <h3 id="toc-hId--478139216">Improving method reproducibility and convenience using dual eluent generation cartridge HPAE-PAD</h3> <p>Given its linearity, precision and robustness, this method was used to analyze three food samples for TGOS content. Two separate extracts were prepared from each food sample. The first was used to determine the initial concentrations of lactose and galactose; β-galactosidase was added to “matrix match” this extract, but the enzyme was deactivated to prevent hydrolysis of TGOS and lactose. The second extract was used to determine the final concentration of galactose after treating the sample with β-galactosidase to hydrolyze TGOS and lactose to galactose. For each sample, the two extracts were analyzed using the HPAE-PAD method.</p> <p><span class="lia-inline-image-display-wrapper lia-image-align-right" style="width: 479px"><span class="lia-inline-image-caption"><img loading="lazy" decoding="async" class="aligncenter size-full wp-image-13815" src="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2022/09/HPAE-PAD-chromatograms.png" alt="" width="478" height="486" srcset="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2022/09/HPAE-PAD-chromatograms.png 478w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2022/09/HPAE-PAD-chromatograms-295x300.png 295w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2022/09/HPAE-PAD-chromatograms-80x80.png 80w" sizes="(max-width: 478px) 100vw, 478px" />Figure 1. HPAE-PAD chromatograms of a prebiotic food supplement sample (A) before and (B) after hydrolysis with β-galactosidase, obtained using the Dionex ICS-6000 HPIC system.</span></span>Figure 1 shows the carbohydrate profile of a prebiotic food supplement sample before and after hydrolysis with β-galactosidase. Some galactooligosaccharides (GOS) eluting between 10 and 30 minutes were removed after enzymatic hydrolysis, while levels of glucose and galactose increased, indicating high GOS content in the sample. Across the three samples, TGOS content ranged from 49–80 g per 100 g, in good agreement with the values stated on the product labels. Using dual EGC greatly simplified the preparation of the eluents used in this method, while maintaining precise and robust separation conditions for the analysis.</p> <h3 id="toc-hId-409364465">Optimizing method efficiency in oligosaccharide analysis</h3> <p>The dual EGC HPAE-PAD method described here is a powerful approach for TGOS determination, achieving similar separation of carbohydrates to that of AOAC Method 2001.02, while simplifying operation and improving retention time precision by eliminating the need for manual eluent preparation.</p> <p>Read more about this method for TGOS determination in this <a href="https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-73986-hpae-pad-trans-galactooligosaccharides-food-an73986-en.pdf" target="_blank" rel="nofollow noopener">application note </a>and watch for our upcoming blog post on magnesium oxide analysis using ion chromatography.</p> <h3 id="toc-hId--2110595469"><span style="color: #ff0000"><em><strong><span style="font-size: large">Co-authored by <a href="https://www.analyteguru.com/t5/user/viewprofilepage/user-id/174" target="_blank" rel="noopener">Jingli Hu,</a> senior applications chemist for Thermo Fisher Scientific.</span></strong></em></span></h3> </div> </div></article><section class="author-box"><h4 class="author-box-title">AnalyteGuru Staff</h4><div class="author-box-content" itemprop="description"></div></section><div class="adjacent-entry-pagination pagination"><div class="pagination-previous alignleft"><i class="fa fa-icon fa-chevron-left"></i> <a href="https://www.thermofisher.com/blog/analyteguru/stay-up-to-date-with-new-seminar-series-emerging-issues-in/" rel="prev">Emerging Issues in Pesticides, PFAS Analysis: On-Demand Seminar Series</a></div><div class="pagination-next alignright"><a href="https://www.thermofisher.com/blog/analyteguru/magnesium-oxide-monograph-modernization-using-ion-chromatography/" rel="next">Magnesium Oxide Monograph Modernization Using Ion Chromatography</a> <i class="fa fa-icon fa-chevron-right"></i></div></div></main></div></div><footer class="site-footer"><div class="wrap"><div class="creds"><p class="footer-links"><a href="https://corporate.thermofisher.com/en/footer/privacy-statement.html" target="_blank" rel="noopener noreferrer">Privacy Statement</a><a href="https://corporate.thermofisher.com/en/footer/terms-and-conditions.html" target="_blank" rel="noopener noreferrer">Terms & Conditions</a><a href="/blog/locations/">Locations</a><a href="/blog/sitemap/">Sitemap</a></p><p>© 2024 <a href="https://www.thermofisher.com">Thermo Fisher Scientific</a>. 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