Characterization of Uniaxially Aligned Chitin Film by 2D FT-IR

Biomacromolecules 2005, 6, 1941-1947

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Characterization of Uniaxially Aligned Chitin Film by 2D FT-IR Spectroscopy Yoshimi Yamaguchi,*,† Thi Thi Nge,‡ Akio Takemura,*,† Naruhito Hori,† and Hirokuni Ono† Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, and Research Institute for Sustainable Humanosphere, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, Japan Received December 10, 2004; Revised Manuscript Received March 16, 2005

Two-dimensional FT-IR spectroscopy (2D FT-IR) was applied to the investigations of crystalline chitin structure. From this study, new information about spectral bands which are not observed in conventional 1D FT-IR was obtained. In 2D spectra, three specific bands were differentiated at 3482, 3421, and 3380 cm-1 in the OH region. They could be assigned as C(6)OH groups which are hydrogen-bonded to the next C(6)OH; C(3)OH groups hydrogen-bonded to O(5); and C(6)OH groups bifurcated hydrogen-bonded to C(6)OH as well as CdO, respectively. Two pairs of bands appeared in the amide region, indicating the two types of hydrogen-bonded states of CdO groups. This is in good agreement with the results in the OH region; half of the C(6)OH groups are hydrogen-bonded to CdO as well as the next C(6)OH. The results accurately confirmed the Blackwell model which was established by an X-ray diffraction study. The temperature-dependency of hydrogen-bonds was also revealed by 2D FT-IR. Interchain hydrogen-bonds [C(6)OH‚‚‚O(6)] first respond to a temperature followed by intrachain [C(6)OH‚‚‚OdC] and [C(3)OH‚‚‚ O(5)] with increasing temperature. Interchain hydrogen-bonds [NH‚‚‚OdC] are relatively stable in the temperature range of 40-180 °C. 1. Introduction Chitin is one of the most abundant natural polysaccharide next to cellulose, and its applications have garnered a lot of attention in various fields. However, the inert nature and the poor solubility in most common organic solvents and dilute inorganic solvents have limited its processability, which results in less developed applications compared to cellulose. Therefore, the liquid crystalline chitin, which can be obtained as a suspension in water by acid hydrolysis,1 is proposed for use in fabricating chitin-based materials. Another important advantage of liquid crystalline chitin is its unique optical property. The acid-hydrolyzed chitin spontaneously displays a chiral nematic (cholesteric) order under certain concentrations,2 and it uniaxialy aligns by manual shearing. It has a potential to be applied as an optical material because of its special optical property. In our previous studies,3-5 the optically anisotropic composite material composed of liquid crystalline chitin and a synthetic polymer was developed, and its optical properties were investigated. To expand the possibility for application of crystalline chitin, however, it is necessary to gain more information about the structure of chitin, which is an important factor in determining the optical property. The crystalline structure of chitin has been studied mainly by use of X-ray diffraction6-10 and infrared spectroscopy.8,11-13 * To whom correspondence should be addressed. Tel: +81-3-5841-5268. Fax: -81-3-5684-0299. E-mail: [email protected] (Y.Y.); [email protected] (A.T.). † The University of Tokyo. ‡ Kyoto University.

According to the literature,14,15 C(3)OH groups are intrachain hydrogen-bonded to O(5); C(6)OH groups are interchain hydrogen-bonded to next the C(6)OH group; and half of them are intrachain hydrogen-bonded to CdO at the same time. All of the CdO groups are hydrogen-bonded to NH in the next chain. In the present work, we applied 2D FT-IR for further investigation of the crystalline chitin structure. The 2D FT-IR method analyzes spectral signals that change as functions of not only time but also any other kinds of physical variables, such as temperature, pressure, concentration, and composition. Since 2D correlation spectra emphasize spectral features not readily observable in conventional onedimensional spectra, more detailed information about chitin structure is expected to be obtained. In this study, the thermal perturbation is carried out in the 40-180 °C range. Thermal perturbation causes significant changes in the hydrogen bonds which determine the fundamental structure of crystalline chitin. Therefore, the purpose of this study is to describe the hydrogen-bonded structure and its temperature-dependent changes of crystalline chitin. 2. Experimental Section 2.1. Materials. Chitin powder (R-chitin from crab), concentrated hydrochloric acid(HCl), and poly(ethylene glycol) (Mr: 20 000) were purchased from Wako Pure Chemical Industries Ltd., Tokyo, Japan. Seamless cellulose tubing (US-36-32-100) with a cutoff molecular weight of 14 000, purchased from Sanko-Junyaku Co., Tokyo, Japan, was used for dialysis. CaF2 (calcium fluoride crystal) windows (25.4 Å to 1 mm), purchased from Pier Optics Co.,

10.1021/bm0492172 CCC: $30.25 © 2005 American Chemical Society Published on Web 05/03/2005

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Ltd., Tatebayashi-shi, Japan, were used as a substrate for casting films. All of the chemicals were used without further purification. Deionized water was used for all experiments. 2.2. Sample Preparation. A liquid-crystalline chitin suspension was prepared by hydrolyzing the R-chitin powder from crab. Typically, 10 g of dried chitin powder was treated with 100 mL of 3 N HCl at the boil (104 °C) for 1.5 h. After acid hydrolysis, 100 mL of distilled water was added and then cooled in ice water for 30 min. Next the sample was washed with distilled water by successive centrifugation (at 1600 g, 5 min) dilution cycles until the supernatant reached a pH of about 3. At this pH, the coarse dispersion began to convert spontaneously into a colloidal suspension2. The turbid supernatant was then collected at each cycle. The liquid crystalline suspension was dialyzed against distilled water for 2-3 days until the dialysis water remained close to neutrality. After that, the suspension was brought to a concentration of about 14-16% (w/w) by dialysis against a poly(ethylene glycol) aqueous solution. The resulting liquidcrystalline suspension was treated by ultrasonication (Nissei US-150 sonicator) for 1 min to promote dispersion. The liquid-crystalline chitin film was prepared on a CaF2 substrate by manual shearing. The specimens were dried in a vacuum oven for 24 h at room temperature. 2.3. 1D FT-IR Spectroscopy. FT-IR measurements were performed on a Nicolet model Manga IR 860 spectrometer equipped with a DTGS detector at a resolution of 4 cm-1, and 32 scans were accumulated. Polarized FT-IR spectra were obtained by using a ZnSe wire grid polarizer positioned before the specimen. The infrared beam was polarized parallel or perpendicular to shearing direction of the specimens. The position of the specimen was kept constant throughout the measurement. The temperature-dependent spectra were measured in a Heated Transmission Cell HT-32 (Spectra Tech, USA) ranging from 40 to 180 °C with a step of 20 °C, controlled by a Programmable Temperature Controller 0019-109 (Spectra Tech, USA). The sample was covered by an another CaF2 plate, and heating continued at each temperature for 7 h to achieve equilibrium. The IR spectra obtained at each temperature were subjected to preliminarily baseline correction and smoothing. 2.4. 2D FT-IR Spectroscopy. Synchronous and asynchronous correlation spectra16,17 were constructed by means of the software provided by Kwansei Gakuin University. The synchronous spectrum represents the simultaneous or coincidental changes of spectral intensities measured at µ1 and µ2. In the synchronous spectrum, the peaks at diagonal positions are referred to as autopeaks. The intensity of autopeaks represents the overall extent of dynamic fluctuations of spectral intensity. Cross-peaks located at the offdiagonal positions of a synchronous spectrum represent the simultaneous changes of spectral signals at two different wavenumbers. The sign of synchronous cross-peaks becomes positive if the spectral intensities at corresponding wavenumbers are either increasing or decreasing together as functions of time. On the other hand, a negative sign of the cross-peaks indicates that one of the spectral intensities is increasing while the other is decreasing.

Yamaguchi et al.

The asynchronous spectrum represents sequential, or unsynchronized, changes of spectral intensities measured at µ1 and µ2. An asynchronous cross-peak develops only if the intensity of two dynamic spectral intensities vary out of phase with each other. The sign of the asynchronous cross-peak becomes positive if the intensity changes at µ1 occur predominantly before µ2. It becomes negative, on the other hand, if the changes occur after µ2. 3. Results and Discussion 3.1. 1D FT-IR Spectroscopy. Figure 1A shows the temperature-dependency of FT-IR spectra for a uniaxially aligned chitin film in the 4000-1110 cm-1 region. The spectra absorption frequency and band assignment of chitin based on literature data from X-ray6-10 and infrared8,11-13 studies in the OH region and the amide region, which are combined with our assignment from 2D FT-IR, are described in Table 1. In the OH region (3700-3000 cm-1), the peaks at 3480 and 3440 cm-1 which are assigned to OH stretching mode showed an intensity decrease. The apparent higher frequency shift is observed at 3440 cm-1. The peaks at 3265 (amide A) and 3108 cm-1 (amide B) which are assigned to the NH stretching mode showed higher frequency shift and lower frequency shift, respectively. In the amide region, the peaks at 1656 and 1622 cm-1 (amide I) are due to the CdO stretching mode of singly hydrogen-bonded to NH and doubly hydrogen-bonded to both NH and C(6)OH, respectively.10 With increasing temperature, the peak at 1622 cm-1 showed significant intensity decrease with a higher frequency shift, whereas the changes of the peak at 1656 cm-1 were small. The peak at 1560 cm-1, which is assigned to a coupled mode of dNH with nCN (amide II), also showed a slightly lower frequency shift. These absorption differences indicate the temperatureinduced changes of the strength of the hydrogen-bonds in the crystalline chitin. Above 200 °C, a new carbonyl peak began to appear at around 1725 cm-1 (not shown). The absorbance intensity of the peak increased with increasing temperature. It may be attributed to the thermal degradation of chitin. Furthermore, the color of the specimen changed from transparent to dark brown in this temperature range. On the contrary, no degradation took place in the temperature range below 180 °C since the spectra measured below 180 °C are reversible during cooling. Thus, the structure of chitin that was reflected by heating is reproducible in successive cooling. Thus, we constructed the 2D correlation spectra by using the 1D spectra in this temperature range for further investigation of the hydrogen-bonded structure. Figure 2 parts A and B, shows the absorbance variations at 3265 and 3108 cm-1 (NH stretching mode) and 1656 and 1622 cm-1 (CdO stretching mode), respectively, as a function of temperature. The absorbance of each band decreases linearly in the temperature range of 40-120 °C, whereas great changes of pattern are observed above 120 °C. This might be caused by the rotations of lateral groups or the motions of main chain segments of chitin.18,19 Thus, we divided the spectra into two sets at the point of 120 °C.

Uniaxially Aligned Chitin Film

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Figure 1. Temperature-dependent FT-IR spectra (20-180 °C) of chitin in the 4000-1110 cm-1 (a), 3700-3000 cm-1 (b), and 1700-1480 cm-1 (c) region.

3.2. 2D FT-IR Spectroscopy. 3.2.1. OH Region (37003000 cm-1). A synchronous 2D correlation spectrum of 3700-3000cm-1 region constructed by the temperaturedependent IR spectra in the range of 40-120 °C is shown in Figure 3. Two major autopeaks appeared at the diagonal positions, (3380, 3380 cm-1) and (3257, 3257 cm-1), indicating great changes at their wavenumbers. Strong positive cross-peaks appeared at the off diagonal positions, (3380, 3257 cm-1) and (3380, 3118 cm-1), suggesting the simultaneous changes between the OH stretching band at 3380 cm-1 and the NH stretching bands at 3257 and 3118 cm-1. It indicates that the origin of the band at 3380 cm-1 has a strong correlation with NH of the amide group. The band at 3380 cm-1 thus seems to derive from the C(6)OH group which is hydrogen-bonded to CdO of the amide group. This C(6)OH band at 3380 cm-1 is also related to the band at 3480 cm-1 in the OH region as can be seen by the positive cross-peak at (3380, 3482 cm-1). Therefore, the band at 3380 cm-1 is believed to be due to the bifurcated C(6)OH that is interchain hydrogen-bonded to the next C(6)-

OH group and intrachain hydrogen-bonded to CdO at the same time. With this thinking, 3482 cm-1 can be assigned to C(6)OH which is only hydrogen-bonded to next C(6)OH. In the higher wavenumber region, there might be some OH bands at around 3560 cm-1. The autopeaks of these bands are too weak to be observed. They might be derived from free OH groups developed in the course of sample preparation since the bands above 3500 cm-1 are assigned to free OH groups in the cellulose spectrum.20,21 The proposed chitin structure14,15 had an orthorhombic unit cell with dimensions a ) 0.476 nm, b ) 1.885 nm, and c ) 1.028 nm (fiber axis); the space group is P212121, and the cell contains disaccharide sections of the two chains passing through the center and the corner of the ab projection. According to Minke and Blackwell,10 this interchain hydrogenbond [C(6)OH‚‚‚O(6)] is formed along the ab diagonal in the chitin crystals. We used polarized spectra to investigate the conformation of the hydrogen-bond against the c axis. Figure 4, parts A and B, shows synchronous spectra in the

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Table 1. Band Assignment of the OH and Amide Region 1D FT-IR absorption frequency (cm-1)

2D FT-IR absorption frequency (cm-1)

assignment

assignment

OH Region

3480

3575 3525 3482

free OH-groups free OH-groups [C(6)OH...O(6)H] interchain H-bond

3421 3380 3290

[C(3)OH...O(5)] intrachain H-bond [C(6)OH...O)C] intrachain H-bond N-H stretching (asymmetric)

3257

N-H stretching (asymmetric)

1658

amide I (singly H-bond)

1648 1641

amide I (singly H-bond) amide I (singly H-bond)

1619 1581

amide I (doubly H-bond) amide II

1538

amide II

O-H stretching O-H stretching

3269

N-H stretching (asymmetric)

3108

N-H stretching (symmetric) Amide Region

1656

1622

1560

amide I (singly H-bond)

amide I (doubly H-bond)

amide II

Figure 3. Synchronous 2D correlation spectrum in the 3700-3000 cm-1 region (40-120 °C).

Figure 2. Absorbance variations (A(T) - A(20 °C)) at 3265 and 3108 cm-1 (A), 1656 and 1620 cm-1 (B).

parallel and perpendicular polarization, respectively. As can be seen in the spectra, no remarkable autopeak appeared at (3482, 3482 cm-1) in the parallel mode, whereas in the perpendicular mode, a notable autopeak appeared. It suggests that this hydrogen-bond is relatively perpendicular orientation to the fiber axis (c axis). We can see the cross-peak at (3482, 3380 cm-1) in both correlation spectra. The sign of the cross-peak is negative in the parallel mode and positive in the perpendicular mode. Since the intensity of the band at 3380 cm-1 decreases with increasing temperature, the intensity at 3482 cm-1 increases

in the parallel mode and decreases in the perpendicular mode. This is considered to be attributed to the changes in the orientation of the hydrogen-bond from perpendicular to parallel against the fiber axis. The asynchronous 2D correlation spectrum in the temperature range of 40-120 °C is illustrated in Figure 5. The asynchronous spectrum provides the information that the band at 3380 cm-1 in the synchronous spectrum can be divided into two bands, at about 3380 and 3421 cm -1. Since the band at 3421 cm-1 shows strong asynchronicity with 3257, 3380, and 3428 cm-1, the origin of the band is less correlated or noncorrelated with the C(6)OH band and the NH band. For this reason, the band at 3421 cm-1 could be assigned to the C(3)OH stretching mode that is intrachain hydrogen-bonded to ring oxygen, O(5).


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Figure 4. Synchronous 2D correlation spectra in the 3700-3000 cm-1 region (40-120 °C), in the parallel mode (A) and perpendicular mode (B).

Figure 5. Asynchronous 2D correlation spectrum in the 3700-3000 cm-1 region (40-120 °C).


Some notable bands are also observed in the region of 3500-3700 cm-1. These bands show intense asynchronous cross-peaks between other bands, which indicates that they are located in different local molecular environments. This might be due to the free OH groups as mentioned above in the synchronous study. Supplemental study by nonoriented sample with 5 °C intervals also gives the same correlations as 20 °C step 2D spectra of oriented sample. According to Noda’s rule of 2D IR spectroscopy,22,23 the sequence of band intensity changes occurring during temperature increase is 3525 cm-1 to 3482 cm-1 to 3380 cm-1 to 3421 cm-1. It means that the interchain[C(6)OH‚‚‚O(6)] first response to increased temperature, and then the strength of cooperative intrachain [C(6)OH‚‚‚OdC] changes, followed by intrachain [C(3)OH‚‚‚O(5)]. A synchronous spectrum in the higher temperature range (120-180 °C) is presented in Figure 6. The intense autopeak at (3380, 3380 cm-1) in the synchronous spectrum of the lower temperature range shifts to a higher frequency (3432, 3432 cm-1) in this temperature range. The newly appeared negative cross-peak at around (3540, 3432 cm-1) indicates

the temperature-induced decrease in the band intensity at 3432 cm-1 and increase in the band at 3540 cm-1. Therefore, the band at around 3540 cm-1 may be due to weakly hydrogen-bonded or free OH groups developed by breaking of inter- or intra-hydrogen-bonds. This is in good agreement with the results in Figures 3 and 5. 3.2.2. Amide Region (1720-1480 cm-1). Figure 7 shows a synchronous spectrum in the amide region (1720-1480 cm-1) in the temperature range of 40-120 °C. In the amide I region, an intense autopeak is observed only in the lower frequency region at (1619, 1619 cm-1) although there are two peaks in the 1D spectrum. It indicates that doubly hydrogen-bonded amide group is more sensitive to the temperature and the strength of this bond changes in this temperature range. On the contrary, singly hydrogen-bond is relatively stable in this temperature range according to no remarkable autopeak. In the amide II region, there exist at least two bands at 1581 and 1538 cm-1, because of the presence of a positive cross-peak at (1619, 1581 cm-1) and a negative cross-peak at (1619, 1538 cm-1). The positive cross-peak at (1619, 1581 cm-1) indicates the cooperative interaction between the bands

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Figure 9. Synchronous 2D correlation spectrum constructed between the 3700-3000 and 1720-1480 cm-1 region (40-120 °C).

spectrum generated from 3700 to 3000 cm-1 (µ1) and 17201480 cm-1 (µ2) region, in the temperature range of 40-120 °C. The strong cross-peaks at (3380, 1619 cm-1) and (3380, 1581 cm-1) indicate the strong synchronicity of the combination bands. It is consistent with the results above: the band at 3380 cm-1 is due to the bifurcated hydrogen-bond [C(6)OH‚‚‚OdC]/[C(6)OH‚‚‚O(6)], and the bands at 1619 and 1581 cm-1 are derived from amide groups of doubly hydrogen-bonded to C(6)OH and NH. All of the proposed assignments in our 2D FT-IR study are combined together in Table 1. 4. Conclusion


at 1619 and 1581 cm-1, which suggests the shared origin of the two bands. Therefore, the band in the higher frequency region of amide II may be due to the NH deformation mode of the doubly hydrogen-bonded amide groups. In the higher temperature range (120-180 °C), some new correlation peaks appeared in the synchronous spectrum (Figure 8). Cross-peaks at (1619, 1581 cm-1) and (1648, 1538 cm-1) are positive, on the other hand cross-peaks at (1619, 1648 cm-1) and (1581, 1538 cm-1) are negative. From the sign of the peaks, the intensity of the bands at 1619 (amide I) and 1581 cm-1 (amide II) are derived from the doubly hydrogen-bond decrease with increasing temperature, whereas the bands at 1648 (amide I) and 1538 cm-1 (amide II) increase. The pair of bands, 1648 and 1538 cm-1, thus can be assigned to the vibrational mode of the singly hydrogen-bonded groups. In this temperature range, doubly hydrogen-bonded [C(6)OH‚‚‚OdC]/[NH‚‚‚OdC] may turn into singly hydrogen-bonded [NH‚‚‚OdC] because of the breaking of the intrachain hydrogen-bond [C(6)OH‚‚‚Od C] induced by heating. 3.2.3. Correlation between OH and Amide Region. The correlation between the OH region and the amide region were investigated by 2D spectrum. Figure 9 shows a synchronous

The 2D FT-IR analysis was carried out on crystalline chitin. According to the investigation of the OH and amide region, detailed assignments of hydrogen-bonds and their temperature-dependent changes were described. This result can be a good confirmation of the chitin structure model proposed by Minke and Blackwell.10 In the OH region, three specific bands were differentiated which are responsible for the intrachain hydrogen-bond of C(3)OH, the interchain hydrogen-bond of C(6)OH, and the bifurcated hydrogenbond. Depending on the bifurcated hydrogen-bonded structure, half of the carboxyl groups are doubly hydrogen-bonded to OH as well as NH. The rest of the CdO groups are singly hydrogen-bonded to NH. These two types of hydrogenbonded states of CdO groups could be confirmed in the amide region as two combination bands of the CdO stretching mode and NH deformation mode. With temperature increase, unstable hydrogen-bonds [C(6)OH‚‚‚O(6)] first respond in the lower temperature range, and then the strength of cooperated hydrogen-bonds [C(6)OH‚ ‚‚CdO] changes. In the higher temperature range, [C(6)OH‚‚‚OdC] start to break into free C(6)OH, and doubly hydrogen-bonded [C(6)OH‚‚‚OdC]/[NH‚‚‚OdC] may turn into singly [NH‚‚‚OdC]. Interchain hydrogen-bonds [Cd O‚‚‚NH] are relatively stable in the temperature range up to 180 °C. Acknowledgment. The authors thank Professor Y. Ozaki of Kwansei Gakuin University for providing the 2D-Pocha software.


References and Notes (1) Marchessault, R.H.; Morehead, F. F.; Walter, N. M. Nature 1959, 184, 632. (2) Revol, J. F.; Marchessault, R. H. Int. J. Biol. Macromol. 1993, 15, 329-335. (3) Nge, T. T.; Hori, N.; Takemura, A.; Ono, H.; Kimura, T. J. Appl. Polym. Sci. 2003, 90, 1932-1940. (4) Nge, T. T.; Hori, N.; Takemura, A.; Ono, H.; Kimura, T. Langmuir 2003, 19, 1390-1395. (5) Nge, T. T.; Hori, N.; Takemura, A.; Ono, H.; Kimura, T. J. Polym. Sci. Part B: Polym. Phys. 2003, 41, 711-714. (6) Darmon, S. E.; Rudall, K. M. Discuss. Faraday Soc. 1950, 9, 251. (7) Lotmar, W.; Picken, L. E. R. Experimentia 1950, 6, 58. (8) Carlstrom, D. J. Biophys. Biochem. Cytol. 1957, 3, 669. (9) Gardner, K. H.; Blackwell, J. Biopolymers 1975, 14, 1581. (10) Minke, R.; Blackwell, J. Mol. Biol. 1978, 120, 167-181. (11) Pearson, F. G.; Marchessault, R. H.; Liang, C. Y. J. Polym. Sci. 1960, 63, 101-116. (12) Nageshwar Prasad, J.; Ramakrishnan, C. Ind. J. Pure Appl. Phys. 1972, 10, 501-505.

Biomacromolecules, Vol. 6, No. 4, 2005 1947 (13) Abdul Haleem, M.; Parker, K. D. Z. Naturforsch. 1976, 31c, 383388. (14) Wood, W. A., Kellogy, S. T. Methods Enzymol. 1988, 161, 435442. (15) Roberts, G. A. F. Chitin Chemistry; Macmillan Press Ltd.: London, 1992. (16) Noda, I. Appl. Spectrosc. 1993, 47, 1329-1336. (17) Noda, I. Appl. Spectrosc. 1990, 44, 550-561. (18) Kim, S. S.; Kim, S. J.; Moon, Y. D.; Lee, Y. M. Polymer 1994, 35, 3212-3216. (19) Kittur, F. S.; Harish Prashanth, K. V.; Udaya sankar, K.; Tharanathan, R. N. Carbohydr. Polym. 2002, 49, 185-193. (20) Kokot, S.; Matusewicz, B. C.; Ozaki, Y. Biopolymers 2002, 67, 456469. (21) Kondo, T. Cellulose 1997, 4, 281-292. (22) Noda, I.; Liu, Y.; Ozaki, Y. J. Phys. Chem. 1996, 100, 8665-8673. (23) Ozaki, Y.; Liu, Y.; Noda, I. Appl. Spectrosc. 1997, 51, 526-535.

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Characterization of Uniaxially Aligned Chitin Film by 2D FT-IR

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