High-performance anion-exchange (HPAE) chromatography coupled with pulsed amperometric detection (PAD) is widely used for a broad range of carbohydrate analysis applications, including the determination of carbohydrates in biofuels, air samples, food and beverages, compositional analysis of glycoconjugates, and oligosaccharide profiling of glycoproteins.
Here, we highlight three ways to optimize HPAE-PAD performance, utilizing current best practice and recent advances in anion-exchange chromatography technology.
HPAE-PAD: A powerful technique for carbohydrate analysis
HPAE-PAD is a well-established technique for carbohydrate quantification. Combining high-resolution analyte separation with sensitive, direct detection, this powerful approach can accurately determine carbohydrates at picomole-level concentrations without the need for lengthy derivatization steps.
Key to its success is its impressive selectivity and specificity. With PAD, only compounds containing functional groups oxidizable at the predetermined detection voltage are identified, enabling detection of the carbohydrates of interest with minimal interference. And, because neutral or cationic sample components elute in, or close to, the void volume of the column, even if non-anionic species are oxidizable, they do not usually interfere with the determination of key analytes.
HPAE-PAD is capable of achieving impressive results in carbohydrate analysis, but there are several ways you can optimize performance for your specific application:
1. Improve separation with innovative anion-exchange column chemistries
Advances in polymer chemistry have led to the development of anion-exchange columns optimized for specific carbohydrates and sample matrices. With such a wide variety of options now available, analysts are achieving high-resolution separations across a comprehensive range of carbohydrate analysis applications.
For example, monosaccharides, such as those in plant and biofuel samples, have traditionally proven challenging to resolve using established column chemistries. Efforts to improve separation have led to the development of high-capacity columns, such as the Thermo Scientific™ Dionex™ CarboPac™ SA10. Thanks in part to its supermacroporous polymeric resin, the Dionex™ CarboPac™ SA10 column supports fast separations, without compromising resolution.
Other columns have been designed for the high-resolution separation of sugar alcohols and anhydrosugars. Thermo Scientific™ Dionex™ CarboPac™ MA1 analytical and guard columns are packed with a surface-functionalized macroporous polymeric resin that provides 45 times more anion-exchange capacity compared with traditional latexed resin columns. This innovative chemistry enables the use of high-concentration sodium hydroxide eluents, leading to greater ionization of sugar alcohols, and improved retention and resolution of these compounds. Newer Dionex CarboPac columns, such as the Thermo Scientific™ Dionex™ CarboPac™ PA300-4µm column can improve methods initially developed using the Dionex™ CarboPac™ MA1 column.
2. Use high-quality eluents and eliminate manual eluent preparation
Improper eluent preparation is a common cause of HPAE-PAD performance issues. Failure to use high-purity eluent components can lead to problems such as high signal noise, loss of sensitivity, poor analyte retention and system contamination. To achieve optimal performance, it is important to use deionized water with 18 MΩ cm resistivity. Sodium acetate eluents should be made with high-purity sodium acetate that has been tested specifically for electrochemical applications, passed through a 0.2 μm nylon filter, and contain hydroxide to prevent microbial contamination.
Eluent preparation is often time-consuming, requiring considerable care to ensure accurate and consistent buffer concentrations. Recent years have seen the development of HPAE-PAD systems that eliminate the need for manual eluent preparation through electrolytic eluent generation. This innovative approach enables more accurate eluent concentrations to be used, thereby supporting more consistent and reproducible retention times and improved baseline stability.
The Thermo Scientific™ Dionex™ ICS-6000 HPIC system supports dual eluent generation cartridge (ECG) mode operation, allowing users to automatically produce eluents containing potassium hydroxide (KOH), methanesulfonic acid (MSA), or KOH/KMSA by connecting an eluent generator equipped with a KOH cartridge in series with a MSA cartridge. As well as saving time by reducing the number of manual workflow steps, dual EGC mode operation also minimizes downtime by extending the lifetime of pump seals and pistons, which only need to come into contact with deionized water, rather than acidic or basic eluents.
3. Apply best practices when preparing your sample
Carbohydrate samples may contain a variety of compounds such as phenols, metals, cations, anions, and hydrophobic substances that can interfere with carbohydrate analysis and make low-level carbohydrate analysis challenging. To achieve high-quality results, it is therefore important to use the best sample preparation strategies.
Recent years have seen the development of a wide range of sample pretreatment cartridges specifically designed to eliminate common interference compounds from complex sample matrices. The Thermo Scientific™ Dionex™ OnGuard™ II RP cartridge, for example, contains a macroporous resin with a high selectivity for hydrophobic substances, making it well suited for the removal of proteins from physiological fluids such as plasma and urine. For samples containing elevated halide levels, on the other hand, the Dionex™ OnGuard™ II Ag cartridge will selectively remove chloride, bromide, and iodide in preference to other anionic species. Alternative cartridge types can be used to improve the analysis of other challenging matrices, such as those containing high levels of sulfate or humic acids. Importantly, the effect of pretreatment cartridges should always be determined prior to analysis using standard solutions, as specific carbohydrates may have a strong affinity for some cartridge packing materials.
Advanced HPAE-PAD for confident carbohydrate analysis
HPAE-PAD is capable of highly selective and specific carbohydrate analyses. Thanks to ongoing improvements in HPAE-PAD technologies, this important technique is delivering impressive performance across a broad range of quantitative applications.
Read more about how to get the most from HPAE-PAD for your carbohydrate analysis workflow in this application note.
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Thermo Fisher Scientific
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