Preparative and analytical size-exclusion chromatography of chitosans

Two chitosan samples (fraction of acetylated units (F_A) 0.15 and 0.52) were fractionated by preparative size exclusion chromatography (SEC). The molecular weights and molecular weight distributions of the fractions were analyzed by analytical size exclusion chromatography coupled to an on-line low angle laser light scattering detector and a differential refractive index detector (SEC-LALLS-DRI), and their intrinsic viscosities were determined. The exponent (a) of the Mark-Houwink-Kuhn-Sakurada (MHKS) equation was found to be 0.92 ± 0.07 and 1.1 ± 0.1, respectively, at I = 0.1 and pH 4.5. No variation in F_A related to molecular weight was found. Reversible interaction between chitosans and different column packings strongly influenced the log M-V relationships. This interaction was generally most pronounced for the low-F_A chitosan, suggesting that the protonated amino groups are involved. Ammonium acetate buffer reduced this effect and the use of a new type of SEC-packing seemed to eliminate it. The more highly acetylated chitosan also had a more pronounced tendency towards concentration dependent self-association, which most probably involve intermolecular hydrophobic interactions between the acetyl groups.