Fractionation and characterization of ultra-high molar mass hyaluronan: 2. On-line size exclusion chromatography methods

Present study concerns SEC fractionation of ultra-high molar mass hyaluronan. Problems in SEC of UHMM HA samples are the fractionation in the columns and the calibration of the system. We have overcome the calibration problem using absolute detectors, light scattering and viscometer, on-line to the SEC system that do not need of calibration. Shear degradation, concentration effects, viscous fingering, poor column resolution, and in general, low reproducibility are the main difficulties in SEC fractionation. A successful characterization of UHMM HA polymers requires an optimization of the experimental protocol. Each step of the experimental protocol should be performed methodically to obtain reliable results. Commercially available SEC aqueous columns have not optimized for UHMM polymers. Using an optimized SEC system shear degradation and non-ideal SEC fractionation can occur when Mw of the HA sample is approximately higher than three million. Also the dispersity index could be underestimated.