Methods for Determination of Hyaluronan Molecular Weight

This chapter presents three types of methods for the determination of hyaluronan molecular weight viscometry, electrophoretic technique, and light scattering. Hyaluronan samples are not generally monodisperse in molecular weight. While characterizing the molecular weight, either an average molecular weight is determined, or the distribution of molecular weights is characterized. The type of average measured depends on the method used. Viscometry allows the routine determination of viscosity-average molecular weight for hyaluronan over a wide range of molecular weights. Measurement of the viscosity of a solution containing hyaluronan allows the determination of the polymer viscosity-average molecular weight. For impure preparations of hyaluronan and soluble extracts from vitreous, synovial fluid, or other tissues, the viscometric method can give a reasonable estimate of hyaluronan molecular weight because, the solution viscosity is mainly determined by the high molecular weight flexible polymer and not by the much smaller soluble globular proteins. In addition, electrophoretic techniques can be used for characterization of hyaluronan molecular weight distributions (MWDs) ranging from oligosaccharides to polymers with molecular weights up to about 6 × 106. In order to obtain a molecular weight-dependent separation of hyaluronan using electrophoresis, a gel matrix is commonly employed as a sieving and separation-stabilizing medium. Further, light scattering (LS) concerns the interaction of light with matter in the specific case with macromolecules in solution. LS gives a weight-average molecular weight, and when used in conjunction with separation by size exclusion chromatography (SEC), the complete MWD can be determined.