Triple Detection Analysis of Beta-Glucan by GPC


Beta-glucans are chains of D-glucose polysaccharides, linked by beta type glycosidic bonds. This is a major component of water soluble cereal fibre and an important part of oat dietary fibre. Unlike starch, they can be composed of repeating D-glucose units but with branching glucose side-chains. Beta-glucans are mainly used in the food, cosmetic, and pharmaceutical industry. They are also used in the medical industry and research involving infection prevention, cancer, and other disease related research. Properties relevant to the polymer’s applications, such as solubility, vary with configuration and molecular weight which ranges between 50 and 3000 kDa.

Manufacture The various types of beta-glucans are extracted from the cell walls of many different sources in nature such as oats, barley, rye, wheat, some types of seaweed, and different species of mushrooms.

Chemical Formula: C18H32O16

Chemical Structure: Unbranched Polysaccharides of Linked B-(1,3) and B-(1,4)-D-Glucose Units

Figure 1. of linear unbranched polysaccharides of linked B-(1,3)- and B-(1,4)-D-glucose units


Two beta-glucan samples were dissolved in water at a concentration of 2 mg/mL and heated to 50oC for 10 minutes then filtered through 0.22µm nylon filters. Analysis was completed on a Viscotek Triple Detector Array (TDA) 302 equipped with a Refractive Index (RI) detector, Viscometer, Right Angle (90o) Light Scattering (RALS) and Low Angle (7o) Light Scattering (LALS). Separation was performed using two PolyAnalytik AquaGelTM columns (2 x PAA 206M) connected in series. Samples were injected at a volume of 100 µL and eluted through the system at flow rate of 1 mL/min in 0.1M NaNO3. A temperature of 30oC was maintained during separation and detection.

Results and Discussion

An overlay of the RI signal for two beta-glucan samples can be seen in Figure 2. A good signal to noise ratio was obtained for each injection as well as a symmetric mono-modal distribution. Using the information from the RI detector along with the light scattering detector and viscometer, molecular weight parameters and size data was derived using triple detection. The data in Table 1 is an average value obtained from 2 injections

Graph of the Overlay of Refractive Index Signal Versus Retention Volume of Beta-Glucan

Figure 2. An overlay plot of the RI chromatograms of two Beta-glucan samples.

Overlay Plot of Refractive Index Chromatograms of Beta-Glucan

Figure 3. An overlay of the triple-detector response signals of a Beta-glucan sample

Table 1. Molecular weight parameters and size data derived from triple detection using Omni-Sec software.

Table of the Molecular Weight and Size of Beta-Glucan Samples

A standard deviation of less than one percent was obtained for the average molecular weight for all samples indicating reproducibility of triple detection analysis. A Mark-Houwink plot was constructed as shown in Figure 4, using the intrinsic viscosity determined by the viscometer and the molecular weight determined by the RI and light scattering detectors. Branching information can be obtained from the Mark-Houwink plot; a decrease in the slope on the plot indicates branching is present. The samples are different with respect to branching because they do not completely overlap. A known linear sample would be needed in order to generate the branching ratio for each sample.

Graph of the Log Molecular Weight Versus Log Intrinsic Viscosity of Beta-Glucan

Figure 4. An overlay of the Mark-Houwink plots of two beta-glucan samples


Triple detection GPC can be used as a quality control tool for determination of molecular weight of beta-glucans. Accurate MW characterization of polyacrylamide is essential as different molecular weights dictate different properties appropriate to particular applications. Advanced GPC multi-detection method provides a simple procedure to determine molecular weight parameters and degree of branching which can assist in predicting properties such as solubility.

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