Journal of Chromatography A, 1372 (2014) 212–220 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma A novel method to analyze the degree of acetylation in biopolymers T. Zweckmair a,1 , M. Becker a,1 , K. Ahn a , H. Hettegger a , P. Kosma b , T. Rosenau a , A. Potthast a,∗ a Department of Chemistry, Division of Chemistry of Renewable Resources and Christian-Doppler Laboratory “Advanced Cellulose Chemistry and Analytics”, University of Natural Resources and Life Sciences-Vienna, Muthgasse 18, A-1190 Vienna, Austria b Department of Chemistry, Division of Organic Chemistry, University of Natural Resources and Life Sciences-Vienna, Muthgasse 18, A-1190 Vienna, Austria a r t i c l e i n f o Article history: Received 16 July 2014 Received in revised form 20 October 2014 Accepted 25 October 2014 Available online 31 October 2014 Keywords: Carbohydrates Degree of acetylation GC–MS Methyl acetate Polysaccharides SPME Zemplén transesterification a b s t r a c t 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 devel- opment 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 sen- sitivity. By applying isotopically labeled methyl acetate released from 4-O-( 13 C 2 -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 biopoly- mers, 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. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Acetylation is a chemical reaction that replaces an acidic hydro- gen in a chemical compound by an acetyl (CH 3 CO ) group, commonly abbreviated as “Ac” [1–3]. Ester formation with acetic acid is a nearly ubiquitous feature of plant polysaccharides, most importantly hemicelluloses and pectins [4,5]. Also lignin, the sec- ond main component of woody matter besides polysaccharides, is acetylated to some low extent [6]. The partial acetylation of hemicelluloses causes some solubility in water and certain organic solvents. While a hydroxyl group can be both a potent hydrogen bond donor as well as a strong hydrogen bond acceptor, acety- lation of the hydroxyl group cancels its H-bond donor capability and switches the H-bond acceptor activity from a hydroxyl- to a carbonyl-oxygen group. Non-acetylated or deacetylated hemi- celluloses – with a higher content of free hydroxyl groups – consequently show much lower solubility in organic solvents such as acetone or chloroform than their natural, acetylated ∗ Corresponding author. Tel.: +43 1 47654 6454; fax: +43 1 47654 6059. E-mail address: antje.potthast@boku.ac.at (A. Potthast). 1 These authors contributed equally to this work. counterparts. Natural cellulose is free of acetylation, contrary to hemicelluloses. It displays a complex network of strong intra- and intermolecular hydrogen bonds that accounts for its insolubility in water and most organic solvents. When artificially acetylated – cellulose acetates of different degree of substitution are important industrial products [7] – the hydrogen bond network is weakened and solubility is improved. Solubility of cellulose acetates strongly depends on the degree of substitution. Cellulose 2.5-acetate and cellulose 3-acetate are fully soluble in organic solvents such as acetone or chloroform. In woody and annual plant hemicelluloses, most of the natu- rally present acetyl groups are lost during pulping. Those groups are esters of acetic acid with the polysaccharide as co-reacting polyalcohol, and they show the typical reactivity of esters: cleavage can be effected both under acidic and alkaline conditions. While acidic ester cleavage produces alcohol and free acid and stops at an equilibrium determined by the law of mass action, alka- line saponification yields alcohol and the salt of the acid and is thus quantitative and irreversible. According to the pulping pro- cess used, the acetates in hemicelluloses are cleaved in acidic or alkaline medium, and either free acetic acid or the respective salt (inorganic acetate) is liberated. The cleavage of acetates must not be disregarded as minor and unimportant side reaction: on the one http://dx.doi.org/10.1016/j.chroma.2014.10.082 0021-9673/© 2014 Elsevier B.V. All rights reserved.