Biomacromolecules 2003, 4, 896-899
896
Alkali-Induced Conversion of β-Chitin to r-Chitin Yasutomo Noishiki,*,† Hiroko Takami,† Yoshiharu Nishiyama,† Masahisa Wada,† Shigeru Okada,‡ and Shigenori Kuga† Department of Biomaterials Science and Laboratory of Marine Biochemistry, Graduate School of Agricultural & Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Received December 1, 2002; Revised Manuscript Received April 7, 2003
Crystal conversion of β-chitin to R-chitin by aq. NaOH treatment was studied for a highly crystalline β-chitin sample from diatom spine. The minimum NaOH concentration to cause swelling was between 25% and 30% w/w. The alkali-swollen material was poorly crystalline and was regenerated as R-chitin on washing with water. This conversion caused total collapse of the original microfibrillar morphology. These features are similar to those of 7 N-8 N HCl treatment reported earlier, but alkali treatment was free from depolymerization or deacetylation. Introduction Chitin is a structural polysaccharide similar to cellulose, forming two types of crystals: R-chitin with antiparallel and β-chitin with parallel chain packing, the same as cellulose II and cellulose I, respectively.1 Similarly to transformation of cellulose I to cellulose II by crystal swelling in alkali solution (“mercerization”) or dissolution, β-chitin is known to transform to R-chitin. As such swelling agents for chitin, HNO3,2 6 N HCl,3 and 50% NaOH4 have been studied. Although the action of HCl was elucidated for highly crystalline β-chitin of tube worm,5,6 that of NaOH has been studied for a poorly crystalline sample from squid pen and for NaOH 50% concentration only.4 Although most chitin arises in biological composites with protein or minerals, some species of diatom produce an extracellular spine made of essentially pure β-chitin.7 This material, therefore, is potentially useful as raw material for manufacturing chitin/chitosan-based materials and ingredients for advanced biomedical applications. To better understand the chemical properties of this material, we examined the action of alkali in detail. The results showed basic similarity of the process to mercerization of cellulose, but with certain differences. Experimental Section Diatom β-Chitin. A centric diatom, Thalassiosira weissflogii, was obtained from Provasoli-Guillard National Center for Culture of Marine Phytoplankton (ccmp 1051). The alga was cultured in 10 L of f/2 medium8,9 supplemented with L1 medium9 under artificial illumination and CO2 enriched aeration. After about one month, the suspended solid in the medium was collected by centrifugation. The precipitate was successively treated with 5% KOH (room temperature, * To whom correspondence should be addressed. E-mail: noishiki@ sbp.fp.a.u-tokyo.ac.jp. Phone: +81-3-5841-5247. Fax: +81-3-5684-0299. † Department of Biomaterials Science. ‡ Laboratory of Marine Biochemistry.
overnight), methanol (80 °C, 2 h), 0.34% NaClO2 (buffered to pH 4.0, 70 °C, 6 h), 0.1 N HCl (boiling, 1 h), and finally 1% hydrofluoric acid (room temperature, overnight), with water rinsing after each step. The purified chitin sample was freeze-dried and kept in a desiccator. Yield from a 10 L culture was between 50 and 100 mg, depending on temperature and illumination conditions. Alkali Treatment. Approximately 10 mg of β-chitin sample was soaked in 20-40% NaOH at room temperature for 1 h, washed with water, and neutralized by dilute HCl. For comparison, an 8 N HCl treatment was applied in the same manner. The treated samples were soaked in methanol and vacuum-dried at 80 °C. X-ray Diffractometry. An X-ray diffraction diagram was obtained with a rotating anode X-ray generator (Rigaku RotaFlex RU-200BH, nickel-filtered Cu KR radiation (λ ) 0.15418 nm) operated at 100 mA and 50 kV). A diffraction diagram was recorded on imaging plates (FUJIX BAS300UR, Fuji Film) and read with RAXIS DS3 (Rigaku). FT-IR. The chitin sample was dispersed in water and cast on a Teflon plate to form a thin film. The film was subjected to NaOH or HCl treatment as above. FT-IR spectra were measured in transmission mode using Nicolet Magna 860 with 4 cm-1 resolution and accumulation of 64 scans between 4000 and 400 cm-1. The transmittance was converted to absorbance for display. Scanning Electron Microscopy (SEM). The samples were coated with Pt-Pd using ion sputter and examined by a Hitachi S-4000. Differential Scanning Calorimetry (DSC) for Detecting β-Chitin Hydrates. About 5 mg of dry chitin sample was swollen in water and the wet mass was sealed in a stainless steel pan after removing excess water by filter paper. The specimen was subjected to differential scanning calorimetry using Pyris 1 (Perkin-Elmer). The thermogram was recorded for repeated cycles between -20 and +160 °C with a heating/cooling rate of 20 °C/min.
10.1021/bm0257513 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/14/2003
Conversion of β-Chitin to R-Chitin
Biomacromolecules, Vol. 4, No. 4, 2003 897
Figure 1. X-ray diffraction patterns of chitin. (a) Diatom β-chitin; (b) 20% NaOH treated, washed, and dried; (c) 25% NaOH treated (do.); (d) 30% NaOH treated (do.); (e) 8 N HCl (do.); (f) crab shell R-chitin.
Viscosimetry. Chitin sample was dissolved in 5% LiClDMAc solution to 0.02-0.05% (w/v). The viscosity of solution was measured with a Cannon-Fenske viscometer, and the molecular weight was determined according to Tervojevich et al.10 as follows: [η] ) (2.1 × 10-4) Mv0.88 where [η] is the limiting viscosity number (dL g-1) and Mv is the viscosity average molecular weight. Results and Discussion Figures 1 and 2 show X-ray diffraction patterns and profiles of various chitin samples. The d-spacings of R- and β-chitin agreed with reported values.11-13 Although NaOH treatment up to 20% NaOH caused no change in crystal structure, 25% treatment caused slight broadening of diffraction peaks. 30% treatment resulted in complete conversion to R-chitin. The diffraction profile of the 30% NaOH treated sample was identical with that of 8 N HCl treated sample. The diffraction peaks of the samples that underwent crystal conversion were much broader than those of the original β-chitin, indicating significant decrease in crystallite size. Still, the overall pattern is nearly identical with that of crab shell R-chitin. The lower limit of the average crystallite size was evaluated from the peak width without correction on instrument factors using the Scherrer equation: Lhkl ) Kλ/β0 cos θ (K ) 0.9; Lhkl, crystallite size in vertical direction to hkl plane; λ, wavelength; β0, full width at halfmaximum).14 L010 of initial β-chitin and 20% NaOH treated sample were both 15 nm. In contrast, L010 of the 25% NaOH treated sample was 12.5 nm, and L020 of 30% NaOH treated sample (R-chitin) was 8 nm. Thus, the lateral size of crystallite became about half by conversion to R-chitin. The change in infrared spectra (Figure 3) was consistent with the results of diffraction study. The OH stretching (3435 cm-1), NH stretching (3292 cm-1), Amide I (1628 cm-1), and Amide II (1560 cm-1) of β-chitin remained the same as the starting material up to 25% NaOH treatment, however, caused shifts of peaks to the corresponding positions of R-chitin by over 30% NaOH treatment. A characteristic feature of β-chitin (compared to R-chitin) is thermally reversible hydration-dehydration,15 which can be used for identification of the β form. The DSC thermograms for the heating-cooling cycle (Figure 4) show that β-chitin crystallinity remained up to 25% NaOH treatment but was completely lost by above 30% NaOH or 8 N HCl treatment.
Figure 2. X-ray diffraction profiles generated by digital processing of data in Figure 1. Parts a-f are the same as for Figure 1.
The crystal conversion was also accompanied by a drastic morphology change (Figure 5). Although the original diatom chitin consisted of 50-100 nm wide microfibrils (Figure 5A), the 30% NaOH-treated sample lost fibrillar morphology and became a coagulated mass with bumpy surface (Figure 5B). This change was noticed also by macroscopic shrinkage of
898
Biomacromolecules, Vol. 4, No. 4, 2003
Noishiki et al.
Figure 4. DSC thermograms of chitin. Parts a-f are the same as for Figure 1. Heating/cooling rate: 20 °C/min.
Figure 3. FT-IR spectra of chitin. Parts a-f are the same as for Figure 1.
the material in 30% NaOH and its hardening by subsequent regeneration in water. Because the crystal conversion of chitin studied here involves harsh treatments with alkali or acid, some degree of chemical decomposition is possible. Therefore, we assessed the degree of depolymerization and deacetylation after the treatments. Elemental analysis showed no change in N/C ratio either after 30% NaOH or HCl treatment at room temperature, thus ruling out occurrence of deacetylation. The molecular weight from viscometry (Table 1) showed that the alkali treatments did not cause significant depolymerization, but 8 N HCl treatment resulted in severe cleavage of molecular chains (6900-290 in DPv). The latter result is consistent with a previous report on tube worm β-chitin.6 Mercerization of cellulose involves conversion of parallelchain structure of cellulose I into antiparallel-chain structure of cellulose II and is considered to proceed by penetration of ions and water molecules into cellulose crystallites
Figure 5. SEM micrographs of chitin. (A) original β-chitin; and (B) 30% NaOH treated. Table 1. Molecular Weight of β-Chitin Samples treatment
[h] (dL/g)
Mv
DPv
initial 20% NaOH 30% NaOH 8N HCl
54.0 53.5 50.0 3.5
1410000 1390000 1290000 58700
6900 6850 6350 290
followed by intermingling of oppositely oriented chains.16,17 This mechanism requires close arrangement of oppositely oriented microfibirils, which is the case for higher plant cellulose. In contrast, highly crystalline cellulose from bacteria, algae, or tunicate occurs as isolated, single-
Conversion of β-Chitin to R-Chitin
Biomacromolecules, Vol. 4, No. 4, 2003 899
β-chitin does not swell in 50% NaOH, whereas R-chitin from crab shell does as reported by Li et al.4 This alkali resistance of the regenerated R-chitin may be due to the morphological change from microfibril to rigid mass described above. Conclusion Diatom β-chitin is a potential source of pure chitin/ chitosan. It also has the unique property of including small polar molecules to form crystallosolvates.12,15,22 The knowledge about alkali action obtained here will serve as basis for advanced chemical processing of β-chitin. Acknowledgment. We thank Ms. H. Naito, Analytical Center, Graduate School of Agriculture and Life Sciences, the University of Tokyo, for providing elemental analysis. Figure 6. X-ray diffraction patterns of alkali-chitin. (a) 30% NaOH treated; (b) and (a) after water wash, wet state.
crystalline microfibrils. These materials usually require stronger alkali treatments for crystal swelling, and conversion to cellulose II results in collapse of microfibrils18 or formation of shish kebab.19 Therefore, the behavior of diatom β-chitin observed here seems very analogous to that of highly crystalline cellulose. The formation of cellulose II on removal of alkali is considered to result from better fitting between antiparallel pairs,20 and a similar situation is probable for β-chitin. In fact, alkali swelling of β-chitin could be detected by X-ray diffraction. The diffraction pattern of the 20% NaOHwetted sample was the same as original β-chitin (hydrate, data not shown), but the 30% NaOH-wetted sample gave an amorphous diffraction pattern (Figure 6A), which is similar to that of alkali chitin reported by Li et al.4 The diffraction pattern of regenerated, never-dried material was that of R-chitin (as in Figure 1) with the background halo from remaining water (Figure 6B). Therefore, there seems to be no “water R-chitin” analogous to “water-cellulose (Nacellulose IV)”.17,21 The only preceding report on alkali-induced conversion of β-chitin to R-chitin is that of Li et al.,4 who used poorly crystalline squid pen sample and 50% NaOH concentration only. Our preliminary examination showed that squid pen β-chitin was converted to R-chitin by 20% NaOH, probably because of small crystallite sizes. Interesting difference from the behavior of cellulose mercerization is that the R-chitin converted from the diatom
References and Notes (1) Blackwell, J. In Cellulose and other natural polymer systems; Brown, R. M., Jr., Ed.; Plenum Press: New York, 1982; pp 403-428. (2) ; Lotmer, W.; Picken L. E. R. Experientia 1950, 6, 58. (3) Rudall, K. M. AdV. Insect Physiol. 1963, 1, 257. (4) Li, J.; Revol, J. F.; Marchessault, R. H. In Biopolymers utilizeing Nature’s AdVanced Materials, ACS symposium series Vol. 723; Imam, S. I., Greene, R. V., Zaidi, B. R., Eds.; American Chemical Society, Washington, DC, 1999; pp 88-96. (5) Saito, Y.; Putaux, J.-L.; Okano, T.; Gaill F.; Chanzy H. Macromolecules 1997, 30, 3867. (6) Saito, Y.; Okano, T.; Gaill, F.; Chanzy, H.; Putaux, J.-L. Int. J. Biol. Macromol. 2000, 28, 81-88. (7) Blackwell, J.; Parker, K. D.; Rudall, K. M. J. Mol. Biol. 1967, 28, 383-385. (8) Guillard, R. R. L.; Ryther, J. H. Can. J. Microbiol. 1962, 8, 229. (9) The recipes were listed in the website of Provasoli-Guillard National Center for Culture of Marine Phytoplankton following address: http:// www.bigelow.org/. (10) Terbojevich, M.; Carraro, C.; Cosani, A. Carbohydr. Res. 1988, 180, 73. (11) Minke, R.; Blackwell, J. J. Mol. Biol. 1978, 120, 167. (12) Blackwell, J. Biopolymers 1969, 7, 281. (13) Gardner, K. H.; Blackwell, J. Biopolymers 1975, 14, 1581. (14) Klug, H. P.; Alexander, L. E. In X-ray Diffraction Procedures, 2nd ed.; Wiley: New York, 1974; Chapter 9, pp 618-708. (15) Saito, Y.; Kumagai H.; Wada M.; Kuga S. Biomacromolecules 2002, 3, 407-410. (16) Kolpak, F. J.; Blackwell, J. Polymer 1978, 19, 133. (17) Okano, T.; Sarko, A. J. Appl. Polym. Sci. 1985, 30, 325. (18) Shibazaki, H.; Kuga, S.; Okano, T. Cellulose 1997, 4, 75. (19) Chanzy, H. D.; Roche, E. J. J. Polym. Sci., Polym. Phys. Ed. 1975, 13, 1859. (20) Nishiyama, Y.; Kuga, S.; Okano T. J. Wood Sci. 2000, 46, 452. (21) Okano, T.; Sarko, A. J. Appl. Polym. Sci. 1984, 29, 4175. (22) Saito, Y.; Okano, T.; Putaux, J.-L.; Gaill, F.; Chanzy, H. In AdVances in chitin science; Domard, A., Roberts, G. A. F., Varum, K. M., Eds.; Jacques Andre Publishers: Lyon, France, 1997; Vol. II, pp 507-512.
BM0257513
