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

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.