In general, many water-soluble polymers exhibit high viscosity, and concentration-dependent effects are often observed in analyses performed by size exclusion chromatography (SEC). In this application, hyaluronic acid solutions at different concentrations were analyzed by OHpak SB-806M HQ, a polymer-based aqueous SEC (GFC) column, combined with a multi-angle light scattering detector (SEC/MALS). Their separation pattern was compared. The upper figure plots the relationship between molecular size (root mean square (RMS) radius) measured by MALS and elution volume, while the chromatograms show the signals obtained with a refractive index (RI) detector. As shown in the figure, the 0.10 % sample solution elutes later than the 0.05 % sample solution, indicating a concentration-dependent shift in elution. In contrast, the lower figure plots the relationship between molecular weight and RMS radius. In this case, no differences attributable to sample concentration are observed. These results suggest that the shift in elution volume observed for samples at different concentrations is caused by differences in solution viscosity, rather than by structural changes in the hyaluronic acid molecules themselves. Thus, the use of SEC/MALS enables detailed evaluation of separation behavior and the effects of concentration dependence in water-soluble polymer analysis.

Sample: 50 µL
Hyaluronic acid

1. Blue0.10 %
2. Red0.05 %