A novel method to analyze the degree of acetylation in biopolymers

• Zemplen-deacetylation and GC–MS were used to quantify the degree of acetylation.
• Liquid phase injection represents a fast and straightforward method.
• Internal standardization using 4-O-(13C2-acetyl)-vanillin is a key-point.
• SPME enables accurate and precise quantification of the degree of acetylation.

A novel approach to measure the degree of acetylation in biopolymers applying a combination of Zemplén-deacetylation by sodium methanolate and GC–MS methodology is introduced. The development focuses on very low limits of detection to cover also samples with extremely low degrees of acetylation which hitherto eluded accurate determination. Free acetic acid or inorganic acetates, often present in biopolymer samples, do not disturb the quantification. Two techniques to measure the Zemplén-released methyl acetate were comparatively assessed, direct injection of the liquid phase and a SPME-based approach, the former being more straightforward, but being inferior to the latter in sensitivity. By applying isotopically labeled methyl acetate released from 4-O-(13C2-acetyl)-vanillin as the internal standard, influences, such as varying moisture contents, are corrected, improving the overall method reliability to a large extent. The combination of Zemplén-release of acetyl groups in biopolymers as methyl acetate, in connection with its accurate quantification by SPME–GC–MS, was found to be the method of choice for routine, yet very accurate analysis of a wide range of acetylation degrees of biopolymers, showing satisfying analytical parameters along with easy handling and widest applicability. Limit of detection for acetylated cellulose samples is 0.09 nmol/mg, for hemicellulose samples 0.48 nmol/mg.