One of the most fundamental parameters characterizing a macromolecule, whether it is naturally occurring or synthetically produced, is its molecular weight or relative molecular mass. The knowledge of the molecular weights of polysaccharides is in general of fundamental importance for the understanding of their biotechnological applications and their role in living systems. Such knowledge is also important in a large number of medical and commercial applications.
Despite the fundamental nature of molecular weight, it has proved very difficult to determine. In the case of polysaccharide this is s principally because of several basic properties: they are polydisperse, which consist of species of different molecular weight; they can have a conformation in solution that is difficult to define or determine with any precision; they have high thermodynamic exclusion volumes resulting from large asymmetry or high solvent affinity; some polysaccharides selfassociate particularly at higher concentrations in solution.
Size exclusion chromatography (SEC) is used routinely to study the molecular weight and structural characteristics of macromolecules and is useful for polysaccharide characterization. It is a sophisticated technique available to provide information about the whole molecular weight distribution. SEC separates molecules by their hydrodynamic volumes and thus the separation is thus based on size exclusion. When the molecules migrate through the column matrix, the molecules are separated on the basis of their exclusion from pores in the column packing material. Larger molecules will elute first, while the smaller molecules interact more with the stationary phase and will elute later.
Figure 1. Schematic representation of three populations of branched polysaccharides within a SEC chromatogram and the corresponding local dispersity
SEC remains a popular molecular determination and separation method due to its versatility, the wide range of matrices commercially available and the mild conditions of operation. One of the major advantages of SEC is that separation can be performed under conditions specifically designed to maintain the stability and activity of the molecule of interest without compromising resolution. SEC typically uses an Refractive Index (RI) or UV detector, and the elution volume is calibrated against known standards. SEC coupled with light scattering can be used to determine the absolute molecular weight.
There are other approaches used for determination of molecular weight other than SEC, such as osmotic pressure measurement for the determination of the number-average molecular weight, analytical ultracentrifugation, and light scattering.
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