Kinetics of de-N-acetylation of the chitin disaccharide in aqueous sodium hydroxide solution Thang Trung Khong a,b , Finn L. Aachmann a , Kjell M. Vårum a,⇑ a Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway b Nha Trang University, Nha Trang, Viet Nam article info Article history: Received 30 November 2011 Received in revised form 26 January 2012 Accepted 31 January 2012 Available online 10 February 2012 Keywords: Chitin oligomer Chitosan Deacetylation Reaction rate abstract Chitosan is prepared from chitin, a process which is carried out at highly alkaline conditions, and that can be performed either on chitin in solution (homogeneous deacetylation) or heterogeneously with the chitin as a solid throughout the reaction. We report here a study of the de-N-acetylation reaction of the chitin dimer (GlcNAc–GlcNAc) in solution. The reaction was followed by 1 H NMR spectroscopy in deuterated aqueous sodium hydroxide solution as a function of time, sodium-hydroxide concentration and temperature. The 1 H NMR spectrum of GlcNAc–GlcNAc in 2.77 M deuterated aqueous sodium hydroxide solution was assigned. The interpretation of the 1 H NMR spectra allowed us to determine the rates of de-N-acetylation of the reducing and non-reducing ends, showing that the reaction rate at the reducing end is twice the rate at the non-reducing end. The total deacetylation reaction rate was determined as a function of the hydroxide ion concentration, showing for the first time that this de-N-acetylation reaction is second order with respect to hydroxide ion concentration. No significant difference in the deacetylation rates in deuterated water compared to water was observed. The activation energy for the reaction (26–54 °C) was determined to 114.4 and 98.6 kJ/mol at 2.77 and 5.5 M in deuterated aqueous sodium hydroxide solution, respectively. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Generic chitosans are prepared from chitin, and this process is currently performed by chemical de-N-acetylation in concentrated sodium-hydroxide at high temperatures in a heterogeneous process where the chitin/chitosan remains in the solid state during the pro- cess. However, the same process may also be performed in a homo- geneous process with the chitin solubilized in the alkaline solution. 1 The homogeneous de-N-acetylation of chitin has been studied as a function of temperature, and the activation energy was determined to 22 kcal/mol (92 kJ/mol). The same authors assumed that the reac- tion was first order with respect to hydroxide ion (at 10% aqueous NaOH-concentration), and found that the reaction was pseudo-first order with respect to acetamido groups. However, their attempt to study the de-N-acetylation of the monomer GlcNAc was unsuccess- ful, which they attributed to decomposition. 2 There is considerable interest in the heterogeneous deacetyla- tion process, as this is a cost-effective process for producing chito- sans from chitin and the functionality of chitosans has been found to depend on their degree of acetylation. 3 Studies of this process are more complicated due to the insolubility and crystalline nature of chitin. However, activation energies in the range from 40 to 45 kJ/mol have been reported, 4 which is less half the reported va- lue in the homogeneous process mentioned above. It is, however, more difficult to interpret results from the heterogeneous pro- cesses, as the local concentrations of for example, hydroxyl ions at the solved exposed surface where the reaction occurs may be quite different from known concentration in solution. In order to study the de-N-acetylation reaction in more detail, we decided to investigate the kinetics of the deacetylation reaction by directly following changes in time-resolved 1 H NMR spectra (in alkaline solutions) with the chitin dimer as a model substance. The results reveal that one of the Lobry de Bruyn-Alberda van Eken- stein (LdB-AvE) reactions occurs, that is, the epimerisation reaction as demonstrated by the disappearance of the H-2 proton as it was replaced by deuterium in the NMR spectra. Moreover, we demon- strate that the de-N-acetylation reaction is in second order with re- spect to the (deuterated) hydroxide ion, and that the reaction rate at the reducing end is twice the rate at the non-reducing end. 2. Experimental 2.1. NMR of the chitin dimer in deuterated aqueous sodium hydroxide solution Chitin dimer (AA) was purchased from Seikagaku, Japan. (99.5%) D 2 O and (99.5% D) NaOD 40% w/w were purchased from CDN isotopes, Canada. Stock solution was prepared by dissolving 30 mg/mL chitin dimer in D 2 O and aliquant 100 lL into eppendorf tube and stored at 35 °C. Prior to dissolving the oligomers, the NaOD was saturated with nitrogen gas to remove oxygen in order to prevent side-reactions. 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2012.01.028 ⇑ Corresponding author. Tel.: +47 73593324; fax: +47 73591283. E-mail address: Kjell.Morten.Vaarum@biotech.ntnu.no (K.M. Vårum). Carbohydrate Research 352 (2012) 82–87 Contents lists available at SciVerse ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres