Biomacromolecules 2004, 5, 992-1001
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Comparative Study of the First Heterogeneous Deacetylation of r- and β-Chitins in a Multistep Process Guillaume Lamarque, Christophe Viton, and Alain Domard* Laboratoire des Mate´ riaux Polyme` res et Biomate´ riaux - UMR CNRS 5627, Domaine scientifique de la Doua, Baˆ timent ISTIL, 15, Bd. A. Latarjet, 69622 Villeurbanne Cedex, France Received November 27, 2003; Revised Manuscript Received February 19, 2004
Heterogeneous deacetylation of R- and β-chitins from shrimp shells and squid pens were comparatively studied. Each deacetylated sample, recovered after neutralization, was fractionated according to a watersoluble and insoluble fraction (pH 8.5). The systematic study of DAs, crystallinity changes, and distribution of N-acetylglucosamine residues were performed on the two kinds of fractions. For the two fractions resulting from a deacetylation in the presence of 50% (w/v) NaOH, for temperatures ranging from 80 to 110 °C, the activation energies of the reactions were found close to 39.9 ( 1.0 and 42.8 ( 1.8 kJ mol-1 and the frequency factors of collision were of 7.2 ( 2.4 and 54.4 ( 18.5 103 min-1, for R- and β-chitins, respectively. Deacetylations of water-soluble and insoluble fractions were compared, and the major role played by the crystallinity level during deacetylation was evidenced and, thereafter, the role of the nature of the starting chitins on the chemical behavior. Thus, in some conditions, we observed critical values of DDA where the structures were becoming fully amorphous. Introduction Chitin and chitosan are linear copolymers constituted of (1f4)-linked-2-acetamido-2-deoxy-β-D-glucan (GlcNAc) and 2-amino-2-deoxy-β-D-glucan (GlcN). As long as this copolymer is soluble in dilute acidic media, it is termed chitosan, whereas chitin is fully insoluble in aqueous or usual organic solvents. The only reported solubilizing conditions for high molecular weight chitins are N,N-dimethylacetamide (DMAc) and N-methylpyrrolidone (NMP) containing 5 and 7% (w/v) lithium chloride,1,2 respectively. Chitin is the most abundant polysaccharide in biomass with cellulose thanks to a yearly production of approximately 1010-1012 T 3, about 1.56 × 109 T being produced by the only crustaceans.4 Chitin exists according to two main polymorphic forms named R and β. In the R-crystallographic structure (arranged according to an orthorhombic cell with a P212121 space group),5 if b represents the fiber axis, the chain segments are antiparallel inside a polymer sheet, along the c axis, although they are parallel between two successive sheets of chains, packed along the a axis. The packing structure is strongly stabilized by intrachain, intra-sheet and inter-sheet hydrogen bonds in the three unit cell directions. In contrast, β-chitin crystallizes in a monoclinic cell with a P212121 space group.6,7 Because chain segments are all parallel along the a and c axes (b is taken as the fiber axis), there are no hydrogen bonds between two successive chain segments along the c axis. Consequently, β-chitin exhibits a better reactivity,8 swelling,9,10 and solubility11 than R-chitin. Despite a lower reactivity, R-chitin is preferentially used by industrials due to its higher relative abundance.12 Chitin is also present in several fungi,13 * To whom correspondence should be addressed. E-mail: alain.domard@ univ-lyon1.fr.
worms,14 insects,15 etc., and its properties are related to its crystallinity degree, molecular weight, and proportion of GlcNAc units (i.e., the degree of acetylation (DA)). These properties depend on both the source of production16 and the extraction process.3,15,17 In contrast, chitosan, essentially produced from the deacetylation of chitin, is much less widespread in biomass where it is almost exclusively produced only in some cell walls of fungi.18 This polysaccharide finds numerous applications,12,19 for example, in agriculture,20 biomedicine,21-23 paper-making, water treatment, or food-industry.24,25 Sample properties strongly depend on its DA26 and molecular weight,23,25 influencing not only their physicochemical behaviors,27-29 but also their biological activity.30 Several ways were proposed to deacetylate chitin. If some enzymatic processes showed a good efficiency,31 in view of the high cost of extraction and low productivity32,33 of chitindeacetylases, their use was restricted at a laboratory scale to amorphous and low molecular-weight chitin.34 Then, a chemical deacetylation route remained the optimal method to transform chitin into chitosan. Kurita et al.35 reported the possibility to carry out a deacetylation under homogeneous conditions after immersion of chitin in a 40% (w/w) NaOH solution during at least 3 h at 25 °C, under reduced pressure. The solubilization was then performed below 0 °C in a large amount of solvent. It was demonstrated that the obtained deacetylated chitin exhibited an unexpected good solubility in distilled water for a DA of about 50%,36 ascribable to a random distribution of the repeating units along the polymer chains.37 To achieve an effective reaction, deacetylation of chitin under heterogeneous conditions remains the most extensively studied and used method. Highly concentrated aqueous solutions of sodium or potassium hydroxide (at least
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Heterogeneous Deacetylation of R- and β-Chitins
40% (w/w)38-40) were processed at high temperatures ranging from 50 to 130 °C, during several hours. Unfortunately, with such severe reaction conditions, for both heterogeneous and homogeneous deacetylations, chitin underwent an important alkaline hydrolysis responsible for a strong depolymerization. Several options were proposed to minimize this degradation such as changes of the reaction atmosphere (use of an inert gas such as N2 or Ar, instead of air), reduction of the amount of alkaline solution and use of a diluting agent like acetone,41 changes of the processing conditions with the use of reactive extrusion,42 sodiumthiophenolate,43 etc. Recently, the interest of successive treatments was also investigated.39,41,43,44 This last route yielded chitin and chitosan with higher molecular weights and was necessary for the preparation of fully deacetylated chitosans.43 However, no deep fundamental study allowing us to improve the process of the heterogeneous multistep deacetylation was reported in the literature. This paper focuses on the comparative study on a first chemical deacetylation of R- and β-chitin during a multistep process in heterogeneous conditions, using 50% (w/v) NaOH, at a temperature over 80 °C, under argon atmosphere. We particularly studied the kinetics of the reaction in relation with the structural changes occurring for both kinds of chitins, when DA decreased within 90 and 40%, before their transformation into chitosan. This kind of study was not previously addressed in the literature and gave us the access to a better understanding of the mechanisms involved during the reaction. Thanks to the systematic separation between water-soluble and insoluble fractions (at pH 8.5) of each deacetylated chitin, recovered after neutralization of the reaction medium, we could determine that heterogeneous deacetylation led to composition and structure heterogeneities. A first study of the differences in the structure and microstructure of the water-soluble and insoluble fractions of R- and β-chitins was performed by X-ray diffraction and 1H NMR spectroscopy. Thanks to these investigations, a general reaction scheme for the alkaline deacetylation during the first steps of the reaction (i.e., DA from 90 to 40%) was proposed for each chitin. Materials and Methods Raw Materials and Preparation. As in a previous work,17 R-chitin was extracted from marine Parapenaeopsis stylifera shrimp shells and β-chitin from squid Loligo pens, both provided by France Chitine. Shrimp shells and squid pens were extensively washed with distilled water until the conductivity reached that of distilled water. Shells and pens were then freeze-dried and cryo-grounded between 8,000 and 12,000 rpm under liquid nitrogen. The powders thus obtained were sieved in order to increase the surface-to-volume ratio and improve the demineralization process. For shrimp shells, the fraction below 80 µm was used, and that between 80 and 120 µm was preferred for squid pens because of its important proportion compared to the others. Deproteinisation of squid pens was directly performed without a demineralization step by adding 15 mL of 1 M NaOH per gram of dried powder in a glass flask placed under argon. A
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vigorous stirring was then maintained at ambient temperature during 24 h before washing with distilled water to remove residual NaOH and proteins. β-chitin was finally freeze-dried. Characterization of Starting Materials. Water contents of chitin and deacetylated chitins were estimated by thermogravimetric analysis (TGA) using a DuPont Instrument TGA 2000 with 10-20 mg samples and a temperature ramp of 2 °C/min up to 200 °C before an isotherm of 15 min. For the ash content, we used a temperature ramp of 2 °C/min up to 200 °C, then an isotherm of 15 min, and a temperature ramp of 10 °C/min up to 900 °C before an isotherm of 50 min. Each analysis was performed under a flow of helium. Intrinsic viscosity was measured using an automatic capillary viscometer, Viscologic TI 1 SEMATech (diameter 0.8 mm), at 25 ( 0.1 °C. The Mark-Houwink-KuhnSakurada (MHKS) parameters determined by Terbojevich et al.1,2 for chitin were used to deduce the viscosity-average molecular weights. Heterogeneous Deacetylation of Chitin. Typically, 10 mL of a 50% (w/v) NaOH solution (prepared under argon bubbling) heated at a desired temperature (80-110 °C) was added to 0.45 g of R- or β-chitin powder in a round-bottom flask. Kinetics were carried out at different temperatures for different reaction times ranging within 5 and 120 min. Reactions were stopped by immersion of the reaction medium in liquid nitrogen. The deacetylated alkali chitin solutions were neutralized by a 0.1 M HCl solution below 0 °C up to pH 8.5 in order to stop the reaction and increase the proportion of the insoluble fraction. The reaction medium was then centrifuged and the supernatant separated from the insoluble fraction. It was then dialyzed through a membrane tubing (Spectra/Por Biotech CE MWCO: 500 g/mol) against water at pH 8.5 (fixed with ammonia), whereas the insoluble part was extensively washed with deionized water at pH 8.5 until the conductivity reached that of water. Both fractions (soluble and insoluble at pH 8.5) were then lyophilised and characterized. Characterization of the Degree of N-Acetylation and Distribution of N-Acetyl Groups by NMR Spectroscopy. 1 H NMR spectroscopy was preferentially used to determine the DAs of the samples. The starting chitin was dissolved in 20% (w/w) DCl/D2O with a vigorous stirring for 8 h at 60-70 °C. These conditions were necessary to sufficiently depolymerize chitin, thus allowing the full solubilization of the polymer.45 Chitosan samples (DA < 40%) were dissolved in D2O acidified with 20% (w/w) DCl at pD 3-4 (10 mg for 1 mL of D2O and 5 µL of 20% (w/w) DCl). Deacetylated chitin samples (40% < DA < 80%) were each time dissolved in D2O (at pD 3-4) as well as in 20% DCl/D2O. The sample showing the best solubility was kept for analysis by 1H NMR spectroscopy, and the measured DA was compared to results obtained by elemental analysis or solid-state 13C CP/MAS NMR spectroscopy. Spectra were recorded on a Bruker AC 200 spectrometer (200 MHz for 1H) at 298 K or higher temperatures, when necessary. At least 250 scans were acquired. DA was calculated as proposed by Hirai et al.,46 from the ratio of the methyl proton signal of the (1f4)-2acetamido-2-deoxy-β-D-glucan residues to the whole H-2 to H-6′ proton signals. 1H chemical shifts were expressed from
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Table 1. DA, Intrinsic Viscosities and Related Molecular Weights, and Water and Ash Contents of the Starting Chitins
chitin R β
DA
(%)46 a
89.28 ( 0.07 90.30 ( 0.06
water ash content content [η]o Mw Mw b 2 c 1 d e (%) (mL/mg) (g/mol) (g/mol) (%)e 3.36 4.05
820000 1010000
1020000 1340000
6.34 10.46
