Effect of acid-chlorite delignification on cellulose degree of polymerization Christopher A. Hubbell, Arthur J. Ragauskas * BioEnergy Science Center, Institute of Paper Science and Technology, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA article info Article history: Received 12 November 2009 Received in revised form 3 April 2010 Accepted 13 April 2010 Available online 14 May 2010 Keywords: Cellulose Degree of polymerization (DP) Delignification GPC Molecular weight abstract Two types of pure cellulose, Avicel PH-101 and Whatman filter paper, were treated with an acid-chlorite delignification procedure in the presence of varying amounts of incorporated lignin, and the molecular weight distributions and degrees of polymerization (DP) of derivatized cellulose were determined by gel permeation chromatography (GPC). Avicel samples with 0% added lignin showed a DP reduction of nearly 5% during acid-chlorite delignification, compared to a 1% drop in DP with 30% added lignin. Lig- nin-free filter paper samples showed a DP reduction of nearly 35% after hollocellulose delignification. This drop in DP was reduced to less than 12% for samples which contained 30% lignin. Thus, the presence of lignin in biomass samples minimized the DP reduction of cellulose due to acid catalyzed cleavage dur- ing acid-chlorite delignification. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Delignification is a critical process towards the successful char- acterization of cellulose and hemicelluloses from lignocellulosic biomass. In addition to lignin removal, a better understanding of the structure and morphology of cellulose and hemicelluloses is key in the effort to reduce biomass recalcitrance, thus improving access to fermentable sugars for biofuel production (Ishizawa et al., 2009; Ragauskas et al., 2006). The presence of lignin in bio- mass reduces accessibility to cellulose microfibrils. Lignin can also hinder or mask molecular details evident to many spectroscopic techniques. For these reasons, it is essential to remove all or a majority of the incorporated lignin prior to analysis. Biomass delignification can achieved by a variety of methods. Kraft pulping is the most common industrial process used for the removal of wood lignin; however, it is not preferred on a labora- tory scale for isolating hollocellulose for many reasons, including degradation of polysaccharides, extraction of hemicelluloses and incomplete lignin removal (approximately 90–95%). Alkaline per- oxide processes are effective for both delignification and removal of hemicelluloses but their use is limited due to extensive degrada- tion of cellulose caused by the peroxide radical (Fang et al., 1999; Sun et al., 2004). Perhaps the most popular and established labora- tory method for the removal of lignin from biomass is acid-chlorite delignification utilizing an aqueous solution of acetic acid and so- dium chlorite. This method effectively bleaches and then solubiliz- es lignin at moderate temperatures. Acid-chlorite delignification is selective in the removal of lignin with only trace solubilization of glucan and xylan (Ahlgren and Goring, 1971). However, Kumar et al. (2009) have recently reported that the acid-chlorite delignifi- cation process has a significant detrimental effect on cellulose chain length. In the case of pure cellulose from filter paper, a reduc- tion of nearly 75% in average degree of polymerization (DP) was found. The reduction was less, 15%, for lower molecular weight mi- cro-crystalline cellulose. It should be noted that in both cases the acid-chlorite delignification process was conducted in the com- plete absence of lignin. Acid-chlorite primarily acts on lignin in biomass, but it can also affect the polysaccharides. According to Grierer (1986), the two most likely scenarios for cellulose degradation during acid-chlorite delignification are acidic cleavage of the glycosidic bonds and/or oxidative degradation of the polysaccharides. Acid hydrolysis is a well established mechanism for the molecular weight reduction of cellulose under even mildly acidic conditions (Millett et al., 1954). The oxidation of cellulose with hypochlorite is non-specific and degradation proceeds most rapidly near a neutral pH (Lewin and Epstein, 1962). Oxidative degradation is limited under acidic conditions (Singh, 1982) and acetic acid is generally added to the delignification procedure to reduce pH. However, the addition of acetic acid increases the likelihood of chain degradation due to acid hydrolysis. Cellulose chain length, measured as degree of polymerization by a variety of methods, is an important material characteristic that factors into the conversion of the biopolymer to fermentable sugars via enzymatic digestion. The accurate characterization of native cellulose chain length is critical to the study of cellulase per- formance, particularly in the case of exo-cellulases (Gupta and Lee, 2009). If the current method of acid-chlorite delignification is dras- tically altering the measured cellulose DP then the process may not 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.04.029 * Corresponding author. Tel.: +1 404 894 9701; fax: +1 404 894 4778. E-mail address: arthur.ragauskas@chemistry.gatech.edu (A.J. Ragauskas). Bioresource Technology 101 (2010) 7410–7415 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech