Sensitive Analysis of Sugars with HPLC-Electrochemical Detection

insights from industryDr, Kristin FolmertApplication & AcademyKnauer

An interview with Dr. Kristin Folmert, Application & Academy, Knauer

Please give an overview of sugar analysis with HPLC-electrochemical detection.

Carbohydrates such as sugars are an important class of substances for the metabolism, structural biology and energy production of cells. Therefore, they also play a central role in pharmaceutical, biological and food science. Furthermore, a progressive way of science is oriented towards the energy production in nature from sugar and tries to produce modern, green fuels from biological raw materials. With our dedicated AZURA HPAEC system we develop methods to support these important branches of science with ultra-high resolution and reliable analytics.

Image credits: qoppi | Shutterstock

Carbohydrates are weak acids with pKa values between 12 and 14. Consequently, they can be completely or partially ionized under basic conditions with pH >12. Due to these harsh conditions, only polymeric anion exchange columns are suitable for the carbohydrate analysis. The retention time with AZURA HPAEC is inversely correlated with pKa value and increases significantly with the molecular weight of the carbohydrate.

How does electrochemical detection differ from other detection methods? What are the advantages and limitations of using electrochemical detection?

The most common detection methods in liquid chromatography are UV, DAD, fluorescence and MS detection. However, most carbohydrates do not have chromophore groups and therefore cannot be detected by absorption or emission spectroscopy methods. To use one of these popular techniques a derivatization step before or after the column would be necessary. This would require special equipment and method development. Often this derivatization also worsens the resolution of the separation. MS detection, on the other hand, is very cost-intensive. In addition, the solvents used for sugar LC separation are often not easy to evaporate and thus make high-resolution detection difficult.

A Refractive Index (RI) detector is an alternative to the methods mentioned, which is also very easy to use and inexpensive to purchase. However, RI detectors are non-specific in their detection and have a significantly lower sensitivity and thus worse limits of detection (LOD) compared to the other detection methods.

Another alternative is detection with an Evaporative Light Scattering Detector (ELSD). The ELSD is more sensitive than the RID, but still less sensitive than electrochemical detection (ECD). Method development with an ELSD is quite simple as long as the eluent is easier to evaporate than the analytes. However, for ELSD a constant, highly pure nitrogen stream is necessary, which makes the application quite costly.

ECD takes advantage of the fact that carbohydrates are present in solution in a redox equilibrium. Amperometry uses this fact for highly sensitive quantitative detection. An electrolysis current is measured at a working electrode under pulsed electrochemical potential. The electrolysis current is proportional to the concentration of the oxidized/reduced analyte. Depending on the analyte, the sensitivity of ECD can be 1,000 to 5,000 times higher compared to RI detection. On the other hand, RI detection is much easier to use, does not require such long equilibration times and the measuring cell is less sensitive to changes in the method. However, the sensitive SenCell in the ECD has the advantage that its volume can be individually adapted to the analytical and up to preparative conditions of the LC method.

Please give an overview of Knauer’s HPLC-ECD system. What features does this system offer to researchers?

The AZURA HPAEC system is composed of bioinert modules that have no metal or glass surfaces in contact with the eluent to prevent the formation of interfering ions. Our carbohydrate applications were performed with an autosampler that can cool the samples and an LPG (low pressure gradient, quaternary) pump. However, the user can choose between different bioinert pumps and injection methods according to his needs. The heart of the system is the AZURA ECD 2.1 electrochemical detector. The detector can temper the measuring cell to guarantee the robustness of the method. At the same time the column can be clamped into the detector. Thus, the detector is also a column thermostat. This reduces the dead volume and prevents temperature gradients. Different flow cell types can be used, which is especially important if you want to switch between pulsed and constant amperometry as a detection method for different applications. The SenCell HyRef gold flow cell suites best for carbohydrates. The volume of this highly sensitive flow cell is very easy variable and allows the user to perfectly optimize his method. Other advantages of this flow cell are its longevity and easy cleaning, which takes only a few minutes. Here KNAUER has clear advantages towards comparable competitive systems.

What makes Knauer’s HPLC-ECD system so sensitive to the detection and selection of oxidizable and reducible substances?

First, any analyte that can be oxidized or reduced is a candidate for electrochemical detection. Classical amperometry works discontinuously: filling the reactor, measurement or titration, emptying reactor, cleaning, regenerating, next measurement and so on. That means it can’t be used in combination with HPLC separation. The sugar analysis chromatography system from KNAUER uses pulsed amperometric detection (PAD) including regeneration of the electrode, required for sugar detection. Using a very small measuring cell volume, this technique enables rapid measurements in only 500 milliseconds. To realize the highest detection sensitivity, a three-electrode configuration is used consisting of a working electrode, a reference electrode and an auxiliary electrode. The auxiliary electrode is kept at precisely the same potential as the reference electrode via a voltage clamp, compensating for any polarization effects that might occur at the electrodes. Together with the extremely small cell volume of the SenCell, which can be variably adjusted between 0 to 300 nL, the AZURA ECD 2.1 is very well suited even for (U)HPLC applications.  

4-step PAD potential waveform for the detection of monosaccharides and other carbohydrates. The sample detection occurs during the highlighted time period tsample.

4-step PAD potential waveform for the detection of monosaccharides and other carbohydrates. The sample detection occurs during the highlighted time period tsample.

Where is carbohydrate analysis used within industry and why is it important to have known sugar levels in the products that are produced?

Mono- and polysaccharides, i.e. the different classes of sugars, are extremely versatile in their properties. Due to their water solubility, they have good bioavailability and they are generally non-toxic. In the pharmaceutical industry, sugar is not only used as an active ingredient but also as a filler in tablets or as an additive in juices. In the food industry, sugars are used as a sweetener but also as a stabilizer in the form of starch. There is hardly any food that does not contain some form of sugar. Manufacturers in the pharmaceutical and food industries must provide accurate information on the nature and composition of the ingredients. For this purpose, analytical methods are important which can quantify all types of sugars very sensitively and reliable. Other substances that can also be analysed in the same mode and which are interesting for industry are for example aminoglycosides, aliphatic alcohols, amino acids and glycoproteins.

Looking specifically at the food and beverage industry, how can Knauer’s HPLC-ECD system be used to identify different sugars, for example separating glucose, fructose and lactose?

To distinguish glucose, fructose and lactose from each other is basically quite simple. The key is the right column and method. Normally ion exchange columns are used for sugar analysis. In combination with electrochemical detection, care must of course be taken to ensure that the column material is also stable in the basic state. For example, the Eurokat Na column, which is also modified with sodium hydroxide solution, is recommended here.

Food quality, like physicochemical properties of foods such as sweetness, appearance, stability and texture depend on the type and concentration of the present carbohydrates. For many questions in the food industry, however, detection with an RI detector is sufficient. In some cases, for example, the EU or FDA prescribe very low limit values which must be declared and then the AZURA ECD 2.1 detector is the right choice. One of those examples is lactose in lactose-free products. The prescribed detection limit of 10 mg/100 g is thus easily reached and, with an LOD of 1.5 µg/100 g, far below. This gives the manufacturer and the end user the necessary safety.

Calibration curve for lactose concentrations in the range of 2.18 μg/100g to 109.00 μg/100 g.

Calibration curve for lactose concentrations in the range of 2.18 μg/100g to 109.00 μg/100 g.

Why is sensitive analysis of sugars important for the pharmaceutical industry?

There are numerous effective sugar-based active ingredients. For example, novel antibiotics from sugar oligomers are interesting, or when sugar is used in combination with other molecules like lipids or proteins to obtain new classes of active ingredients. For instance, glycoproteins, glycolipids, and proteoglycans are particularly potent. The combination of proteins with sugars can, for example, increase bioavailability or improve metabolism. A high-resolution analytical method is particularly important for monitoring the low concentrations of active substances in the organism or for identifying metabolic products.

How does the sensitive sugar analysis impact biofuel production and refining?

In times of climate crisis and scarcity of raw materials, research is looking for new alternatives for green effective fuels. So far, biofuels have mostly been produced from grain or maize and are therefore in competition with food production. In addition to grass and food waste, the focus is on wood waste as an alternative raw material source for biofuel production. Regardless of the source, biomasses, rich in cellulose, are always thermally or chemically broken down and then enzymatically fermented to obtain lower or monomeric sugars. In the case of wood, the sugars fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose as well as the sugar acids galacturonic acid and glucuronic acid are produced. A quantitative evaluation of the decomposition products is crucial for the evaluation of the process in order to calculate the effectiveness of the raw material utilization. This is because the proportion of different carbohydrates is directly proportional to the bioethanol yield generated from these sugars in the next step. In addition to the reliability of the analytical method for the investigation of wood sugars with electrochemical detection, which we describe in an application (https://www.knauer.net/en/sensitive-and-selective-analysis-of-wood-sugars-and-uronic-acids-for-biofuel-research-with-electrochemical-detection/a24748), the use of aqueous eluents also speaks for the use in this very ecological field of research.

Chromatogram of a standard mixture containing 0.1 mg/mL fucose (1), rhamnose (2), arabinose (3), galactose (4), glucose (5), xylose (6), mannose (7), galacturonic acid (8) and glucuronic acid (9). And a zoom into the peaks for the uronic acids.

Chromatogram of a standard mixture containing 0.1 mg/mL fucose (1), rhamnose (2), arabinose (3), galactose (4), glucose (5), xylose (6), mannose (7), galacturonic acid (8) and glucuronic acid (9). And a zoom into the peaks for the uronic acids.

What is the future of HPLC-ECD and carbohydrate analysis systems from KNAUER?

At the moment we are testing different column types to increase the variety of methods for our AZURA HPAEC system. We are also exploring new fields of application. Glutamates, cortisones or polyphenol-based additives, for example, would be interesting for the food industry. For the pharmaceutical industry, for example, aminoglycosides are an interesting topic for antibiotics research that can be addressed well with electrochemical detection.

Where can readers find more information?

For example, we have set up a Q&A section on our homepage where users can find answers to the most frequently asked questions about the methods, amperometry and the system (https://www.knauer.net/en/Support/FAQ/Detection?utm_source=DNL&utm_medium=email&utm_campaign=DNL109_2019-12#ECD). For the system and the carbohydrate topic was also dedicated a separate page where you can read a bit more about it (https://www.knauer.net/en/Food-Analysis/HPLC_ECD_Saccharides_Applications). And of course, we have several application notes on our homepage about sugar analysis with different kinds of detectors and of course with electrochemical detection (https://www.knauer.net/en/Applications/applications-start).

About Dr. Kristin Folmert

Dr. Kristin FolmertDr. Kristin Folmert studied chemistry at the Freie Universität Berlin and received her Master of Science in 2012. She then completed her dissertation at the FU Berlin until 2017 with the focus on the analysis of aggregating peptides and photoresponsive molecules. Since 2018 she has been working for KNAUER Wissenschaftliche Geräte GmbH in the Applications and Academy Department. Her tasks include HPLC method development, feasibility studies and regular representation of KNAUER at trade fairs and conferences. She is also very active as a trainer at the Knauer Academy.

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