Degree of acetylation of chitosans determined by size exclusion chromatography coupled to UV and refractive index detection

Cord-Landwehr, Stefan; Hellmann, Margareta J.; Moerschbacher, Bruno M.

Abstract

Chitin is one of the most common biopolymers whose partial deacetylation leads to chitosans consisting of the two β-1-4-linked units N-acetyl-D-glucosamine and glucosamine. These vary in their relative occurrence defined by the degree of acetylation (DA) which has a huge impact on the physico-chemical properties and bioactivities of chitosans. Hence, simple but robust approaches for DA determination are essential. In this study, we aimed to develop a fast and easy method for DA analysis by combining ultra high pressure size exclusion chromatography separation with UV and refractive index detection (UHPSEC-UV-RI). The UV signal is used to quantify the amount of acetyl groups as a measure of DA, and the RI signal serves to assess the total amount of chitosan analyzed. Using commercial and in-house produced chitosan polymers of different molecular weight (60–650 kDa), DA (0–55 %), and production methods (heterogenous deacetylation, homogenous deacetylation, and chemical N-acetylation), we confirmed that UHPSEC-UV-RI gives reproducible DA values on par with those obtained using 1H NMR or enzymatic mass spectrometric fingerprinting. In contrast to the latter two, UHPSEC-UV-RI requires no special sample preparation and only microgram amounts of sample, allowing fast routine analyses of chitosan samples with high accuracy even for limited amounts of material.