Agric. Biol. Chem., 55 (9), 2375-2379, 1991 2375 Effect of Heating an Aqueous Suspension of Chitosan on the Crystallinity and Polymorphs Kozo Ogawa Research Institute for Advanced Science and Technology, University of Osaka Prefecture, Shinke-cho, Sakai, Osaka 593, Japan Received April 5, 1991 Changes in the crystallinity and polymorph of chitosan, which may affect its functionality, by heating (up to 200 C) its water suspension were studied by X-ray diffraction measurements, using tendon chitosan prepared by 7V-deacetylation of a crab tendon chitin, and chitosan powders with various degrees of polymerization (DPv= 1,720-12,600) and 7V-acetylation (zero to 26%). It was found that the presence of hydrated polymorphsor anhydrous crystals in a chitosan sample could be examined easily by measuring the powder diffraction pattern of a sample. Chitosan with a low molecular weight or low degree of TV-acetylation was highly crystallized, especially in the anhydrous form that is considered to spoil chitosan's functionality, by heating. Chitosan is a polymer of /?-(l-*4)-linked 2-amino-2-deoxy-D-glucose and is derived from the 7V-deacetylation of chitin. It is a promising bioresource for industrial purposes and is under intensive investigation for this application. Recent works have, however, revealed that the function of chitosan depends on its polymorphs. Mochizuki et al.1] have found that, in the pervaporation separation of a H2O/ethanol mixture through a chitosan membrane, the water and ethanol molecules permeated only the amorphous region of the membrane, and they couldn't penetrate into the crystalline region. We have found that chitosan makes neither acid salts2) nor complexes with transition metal ions3) when it is crystallized in an anhydrous form, the annealed polymorph.4) Furthermore, chitosan highly crystallized in an anhydrous form cannot be dissolved with the usual solvents for chitosan such as an aqueous solution of acetic acid or hydrochloric acid. In contrast, chitosan must be highly crystallized in its thread to show a high tensile strength. The polymorph of chitosan in the case of chitosan film preparation depends on the solvent, chitosan concentration, and the temperature to remove the solvent from the chitosan solution. Heating is popular in industrial procedures. Thus, we examined how the crystallinity and polymorph of chitosan changed when its aqueous suspension was heated. The results may be useful to examine the functionality of materials made ofchitosan. Materials and Methods Tendon chitosan was prepared from a crab tendon chitin by 7V-deacetylation with a 67% sodium hydroxide solution at 110 C for 2hr under a nitrogen atmosphere. This deacetylation procedure was repeated twice more to produce completely deacetylated chitosan. The viscosity average degree of polymerization (DPv) was 10,800. Six chitosan powders with different degrees of polymerization and of TV-acetylation were supplied by Katokichi Co. Ltd., Bioshell Inc., and Nippon Kayaku (Table I). According to these companies, all the chitosans were prepared by solid-state deacetylation of chitins, and are termed "as received" chitosan. Since the polymorph of chitosan depends on the preparation procedure, all the samples "as received" were dissolved in a 0.1 M acetic acid solution (2.5g/1) and reprecipitated by adding a 0.2m sodium hydroxide solution and subsequently washing with water, methyl alcohol and ethyl ether, before drying under vacuum. The resultant chitosans are termed "regenarated." The intrinsic viscosity of each chitosan was measured with its 0.2 m acetic acid-0. 1 Msodium acetate solution (ph =4.3) at 30 C by using a Ubbrohde dilution type of viscometer. The viscosity average degree of polymerization
2376 Table I. Chitosan Powder Samples _,.. Degreeof r. No. Commercial iv-acetylation DPv SupPlled 1 BDL chitosan 100 0 1,720 Katokichi 2 Chitosan 100 0 8,500 Katokichi 3 Bioshell 12 4,100 Bioshell Chitosan Inc. 4 KAYAMIC ll ll,600 Nippon 450 Kayaku 5 Chitosan 80M 16 10,100 Katokichi 6 Chitosan 70 26 12,600 Katokichi (DPv) was calculated by using the following equation5): M (cm3/g)=1.81 x l(r3m0-93 where Mis the molecular weight. Since chitosan can be dissolved in a solution in the form of an acetic acid salt, DPv is given by dividing the molecular weight of glucosamine residue-acetic acid salt into M. The degree of A^-acetylation of all the chitosans was determined by colloidal titration.6) That is, a chitosan was dissolved with a 0.5% acetic acid solution (5 g/1). One gram of the resultant chitosan solution was diluted by adding 30ml water, and was then titrated by a 1/400N potassium polyvinyl sulfate solution with an indicator of 0.1% methylene blue.7) Tendon chitosan was heated in water at various temperatures up to 240 C in a sealed vessel. Each chitosan powder was suspended in water and heated in a procedure similar to that for the tendon chitosan. X-Ray fiber patterns of the tendon chitosan were measured at 100% relative humidity under a helium atmosphere by using a flat film camera with Cu^a radiation generated at 40kV and 15mA. Powder patterns of the chitosan powder were recorded with a goniometer generated at 25kV and 30mA. Results and Discussion So far, the following four polymorphs have been proposed for chitosan: "tendon",8) "form II",9) "annealed",4) and "l-2".10) The single molecular chain in these polymorphs, however, has always been observed to be an extended two-fold helical structure similar to that in chitin or commoncellulose. Recently, Sakurai et al.10) have prepared a well oriented and highly crystallized chitosan film which showed a fiber pattern termed the "L-2" polymorph. They suggested that "L-2" is similar to Samuels' "form II". Although the detailed molecular structure has only been analyzed for the "L-2" polymorph,10) the difference in these four polymorphs is thought to be the packing modefor chitosan molecules in unit cells such as the numberof water molecules in the cells. The "annealed" polymorph is in an anhydrous form, whereas the other three are in a hydrated form. It must be noted that "tendon" is the most abundant polymorph of chitosan. Figure 1 shows the change in fiber pattern oftendon chitosan by heating it in the presence of water. Without heating, the fiber diagram (top left) was of a typical tendon polymorph,8) whereas that at the bottom right is a typical one for the annealed polymorph.4) A difference between tendon (hydrated) and annealed (anhydrous) polymorphs was distinguishable from their equatorial reflection spots. Tendon showed a strong (020) reflection at an angle (29) spot of 10.4 to the degree center (<i=8.5l of the A)8) (the fiber pattern closest in the top left photo), whereas the annealed chitosan showed a strong (120) reflection at 29 of 15 degree (d=5.83a)4) (the closest spot to the center in the in the bottom right photo). other photos with increasing As shown heating temperature, the anhydrous crystal appeared and increased, whereas the hydrated crystal decreased. As described later, the temperature at which the former appeared or the latter disappeared depends both on the molecular weight and on the degree of AT-acetylation of chitosan. Both reflection spots were strong and closest to each photo center; that is, they had the largest d-spacings in each fiber patterns. Furthermore, the difference of diffraction angle between them was 5 degree. Among other two hydrated polymorphs, L-2 showed a strong reflection at 29 of 10.6 and no diffraction spot at around 15 degree.10) The 1-2 polymorph, however, showed diffraction spots at both 29 of 10.7 and 15.4 degrees.n) Sakurai et al.10) have obtained chitosan film showing the 1-2 polymorph by casting a chitosan acetic acid solution and, after drying, the resultant chitosan acetate film was neutralized with a sodium hydroxide solution.
Heating Effect on Chitosan Polymorphs 2377 Fig. 1. Change in the Fiber Pattern of Tendon Chitosan by Heating in Water. Fig. 2. Changes in the Powder Pattern of"regenerated" Chitosans by Heating Their Aqueous Suspensions, a, 0% 7V-acetylated chitosans (left, sample No. 1; right, No. 2); b, approximately 10% N-acetylated (left, sample No. 3; right, No. 4); c, left 16% (sample No. 5); right 26% (No. 6). After washing with water the film was dried in air. This procedure was followed and a chitosan film resulted showed a similar diffraction pattern to that of the 1-2 polymorph. However, when heated in water, the peak at 29 of 10 decreased and that at 15 increased. It may be acceptable to consider that the 1-2 polymorph is a mixture of L-2 and the annealed polymorphs. Thus, in order to examine the presence of a hydrated or anhydrous polymorph, it is enough to measure the powder diffraction diagram of a chitosan. Figure 2a shows powder diffraction patterns of "regenerated" chitosans which were com-
2378 pletely deacetylated. The viscosity average degree of polymerization (DPv) of these chitosans was 1,720 (left patterns) and 8,500 (right). Without heating, neither chitosan showed any diffraction peak at 29 of both 10 and 15 degrees, suggesting no crystal. However, after heating their water suspensions at 100 C, they showed small peaks at both 10 and 15 degrees, indicating the presence of not only hydrated but also of anhydrous crystals, though they were small amount. With further increase of heating temperature, the anhydrous polymorph increased, whereas that of the hydrated crystal decreased. In the case of chitosan with a low molecular weight (left patterns), by 200 C heating, the peak at 15 degree (the anhydrous crystal) became very strong and no peak was observed at 10 degree (the hydrated crystal). In constract, high molecular weight chitosan still had a small amount of the hydrated polymorph (right patterns) because of its lower molecular mobility. It must be noted that 100 C heating caused the occurrence of crystals and especially of the anhydrous crystal. Such a temperature is normal in an industrial process, and these small amounts of crystal may affect chitosan's functionality. Chitosan with a degree of 7V-acetylation of approximately 10% showed similar behavior to that of the previous 0% VV-acetylated chitosans (Fig. 2b). But unlike the previous chitosans, no anhydrous crystal was detected after 100 C heating. Figure 2c shows the powder patterns of chitosans with a higher degree of N-acetylation: left, 16% and right, 26%. In the case of 16% 7V-acetylated chitosan, heating to more than 160 C was required to produce an anhydrous crystal, and 26% 7V-acetylated chitosan did not show the occurrence of an an anhydrous crystal. Both chitosans showed broad peaks at 29 of around 9. These are considered to be the (020) reflection ofa-chitin (at 20=9.27, d=9.54a)12) remaining in the samples, since "as received" chitosans which are origins of these "regenerated" chitosans showed strong reflections at 20=9.3. Fig. 3. Change in the Powder Pattern of an "as Received" Chitosan (Sample No. 2, the origin of the "Regenerated" Chitosan Presented in Fig. 2a Right) by Heating Its AqueousSuspension. Interestingly, the 26% 7V-acetylated chitosan showed broad powder patterns as a whole when heated at 160 C or more (Fig. 2c, right) indicating that they lost crystallinity and became rigid gels. Such gels could obtained when a water suspension of the "regenerated" chitosan (that is, noncrystalline chitosan) was heated. The same sample "as received", which had been prepared by solid-state deacetylation of chitin and was highly crystallized, did not give such a rigid gel. A detailed study of the gelation procedure will be published elsewhere. Figure 3 shows powder patterns of an "as received" chitosan called Chitosan 100 (Table I), which had been prepared by solid-state deacetylation. Without heating, it shows a typical tendon polymorph with high crystallinity (the bottom pattern). With an increase of heating temperature, an anhydrous crystal (annealed polymorph) occurred and increased. However, unlike "regenerated" chitosan (see the right-hand patterns of Fig. 2a), a marked amount of the hydrated crystal still remained by heating to 200 C. When compared with the right-hand patterns of Figure 2a, it is clear that the "as received" chitosan was much more highly crystallized than the "regenerated" type. Similar findings were obtained with all the other "as received" chitosans. To summarize, hydrated and anhydrous crystals of chitosan were easily distinguishable
Heating Effect on Chitosan Polymorphs 2379 from each other by measuring a powder diffraction pattern. Chitosan with a relatively low molecular weight or low degree of N- acetylation can be crystallized, especially to the anhydrous form, more easily by heating its aqueous suspension. Solid-state deacetylation of chitin gave highly crystallized chitosan as a hydrated polymorph, but when the resultant chitosan was reprecipitated, it lost its crystallinity. Chitosan's functionality is affected not only by its crystallinity but also by many other factors. However, the molecular structure is the most fundamental factor. X-ray diffraction measurement is useful to check the functionality of a chitosan material. Acknowledgments. We thank Katokichi Co., Ltd. of Kagawa, Japan, Bioshell Inc. of Oregon, U.S.A., and Nippon Kayaku oftokyo, Japan for suppling the chitosan powders. References 1) A. Mochizuki, Y. Sato, H. Ogawara and S. Yamashita, J. and Appl. Polym. ScL, 37, S. Inukai, Carbohyr. 3375 Res., (1989). 160, 425 (1987)., K. Oka, T. Miyanishi and S. Hirano, in "Chitin, Chitosan, and Related Enzymes," P. Zikakis, Academic Press, Orlando, FL. ed. by J. 1984, pp. 327-345., S. Hirano, T. Miyanishi, T. Yui and T. Watanabe, Macromolecules, 17, 973 (1984). M. Rinaudo and A. Domard, in "Chitin and Chitosan; Sources, Chemistry, Biochemistry, Physical Properties and Applications," ed. by G. Skjak-Braek, T. Anthonsen and P. Sand ford, Applied Science, London, 1989, pp. 71-86. Elsevier H. Terayama, J. Polym. ScL, 8, 243 (1952). M. Miya, Report of the Government Industrial Research Institute, Osaka, No. 369, p. 1 (1986). G. L. Clark and A. F. Smith, /. Phys. Chem., 40, 863 (1937). R. J. Samuels, /. Polym. ScL, Polym. Phys. Ed., 19, 1081 (1981). K. Sakurai, T. Shibano, K. Kimura and T. Takahashi, Sen-i Gakkaishi, 41, T361 (1985). K. Sakurai, M. Takagi and T. Takahashi, Sen-i Gakkaishi, 40, T246 (1984). D. Carlstrom, /. Biophys. Biochem. Cytol., 3, 669 (1957).