Acetylation of Chitin Nanofibers and their Transparent Nanocomposite

Apr 1, 2010 - Asier Salaberria , Rene H. Diaz , María Andrés , Susana Fernandes ... Wiltrud Lederle , Shinsuke Ifuku , Matthias Wessling , John G. Har...
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Acetylation of Chitin Nanofibers and their Transparent Nanocomposite Films Shinsuke Ifuku,*,† Shin Morooka,† Minoru Morimoto,‡ and Hiroyuki Saimoto† Department of Chemistry and Biotechnology, Graduate School of Engineering, and Research Center for Bioscience and Technology, Tottori University 4-101 Koyamac-cho Minami, Tottori, Japan Received January 29, 2010; Revised Manuscript Received March 19, 2010

Chitin nanofibers were acetylated to modify the fiber surface and were characterized in detail. The acetyl DS could be controlled from 0.99 to 2.96 by changing the reaction time. FT-IR spectra indicate that chitin nanofibers were acetylated completely after 50 min reaction time. X-ray diffraction profiles and TGA curves show that the chitin nanofibers were acetylated heterogeneously from the surface to the core. SEM images show that fiber shape was maintained even in the high-DS sample and that the thickness of the nanofibers increased with the introduction of bulky acetyl groups. Acetylated chitin nanofiber composites were fabricated with acrylic resin with the fiber content of approximately 25 wt %. Due to the size effect, all nanocomposites had high transparency, despite the variety of acetyl DS, and the transparency of the chitin nanofiber composite was less sensitive to acetylation. By only 1 min acetylation, the moisture absorption of the nanocomposite drastically decreased from 4.0 to 2.2%. Although the coefficient of thermal expansion (CTE) of the tricyclodecane dimethanol dimethacrylate (TCDDMA) resin was 6.4 × 10-5 °C-1, the CTE of the chitin nanofiber/TCDDMA composite decreased to 2.3 × 10-5 °C-1 by the reinforcement effect of the chitin nanofibers with low thermal expansion.

Introduction Chitin is a highly abundant biomacromolecule existing in the exoskeletons of crab, shrimp, and so on. The exoskeletons of these crustacea have a strictly hierarchical organization consisting of crystalline R-chitin nanofibers and various types of proteins and minerals.1,2 Recently, the chitin nanofibers were isolated from crab shells by a simple grinding treatment after the removal of proteins and minerals.3 The obtained nanofibers have a uniform width of approximately 10-20 nm and a high aspect ratio. We expect that these nanofibers with a uniform width and very high surface-to-volume ratio will be developed into novel green nanomaterials. To increase the number of applications for chitin nanofibers, chemical modification of the chitin nanofiber surface is very important. By the introduction of hydrophobic functional groups into hydrophilic hydroxyl groups on chitin fibers, it is expected that dispersibility in nonpolar solvents, hygroscopicity, and adhesion properties with hydrophobic matrices for fiber-reinforced composite materials are improved.4 In chemical modification, acetylation is considered to be a simple, popular, and inexpensive approach to change the surface polarity.5,6 However, there have been no reports regarding acetylation of chitin nanofibers. Hence, the reaction behavior of highly crystalline R-nanofibers and the relationships between acetyl DS values and the various properties of the nanofibers remain unclear. Yano et al. have recently reported that bacterial cellulose nanofibers have very promising characteristics as a reinforcing material.7 Due to the size effect, the nanocomposites are optically transparent even with a high fiber content of approximately 25%. We consider that the chitin nanofibers and their acetyl derivatives would also be available as a filler to reinforce plastics. It is therefore desirable to * To whom correspondence should be addressed. Phone and Fax: +81857-31-5592. E-mail: [email protected]. † Department of Chemistry and Biotechnology, Graduate School of Engineering. ‡ Research Center for Bioscience and Technology.

investigate acetyl DS dependency on the properties of chitin nanofiber composites, including transparency and hygroscopicity. In the present study, we prepared chitin nanofibers with different acetyl DS values and their nanocomposites and characterized them in detail.

Experimental Section Materials. Dried crab shell powder of Paralithodes camtschaticus (red king crab) was purchased from Kawai Hiryo Co. The other chemicals were purchased from Aldrich or Kanto Chemical and used without further purification. Preparation of Chitin Nanofibers. Chitin nanofibers were prepared by a procedure described previously.3,8 First, crab shell powder was refluxed in 5 wt % of potassium hydroxide for 6 h under vigorous stirring to remove most of the proteins. This suspension was cooled to room temperature, then filtered and washed with distilled water. Next, the chitin samples were treated with 7% hydrochloric acid solution for 2 days at room temperature to remove the mineral salts. After filtration and rinsing with distilled water, the treated samples were dispersed and boiled in a 5% KOH solution for 2 days to completely remove any residual proteins. The pigment was then removed from the samples using 1.7 wt % of sodium chlorite in 0.3 M sodium acetate buffer for 6 h at 80 °C, followed by filtration and washing with distilled water. The purified wet chitin from dry crab shell was dispersed in water at 1 wt %, and acetic acid was added to adjust the pH value to approximately 3 to facilitate fibrillation. The suspension was treated with a domestic blender. Finally, the slurry of 1 wt % purified chitin was passed through a grinder (MKCA6-3; Masuko Sangyo Co., Ltd.) at 1500 rpm. Grinder treatment was performed with a clearance gauge of -1.5 (corresponding to a 0.15 mm shift) from the zero position, which was determined as the point of slight contact between the grinding stones. Acetylation of Chitin Nanofibers. Fibrillated chitin nanofibers were dispersed in water at a fiber content of 0.1 wt %. The suspension was vacuum-filtered using a hydrophilic polytetrafluoroethylene membrane filter (Millipore, pore size: 0.2 µm). The obtained chitin nanofiber sheets were dried in the oven at 65 °C overnight and were cut into 3 × 4 cm

10.1021/bm100109a  2010 American Chemical Society Published on Web 04/01/2010

Acetylation of Chitin Nanofibers sheets 60 µm thick, with a weight of 55 mg. The sheets were placed in a Petri dish containing a mixture of 5.0 mL of acetic anhydride and 0.1 mL of 60% perchloric acid. The mixture was stirred for the desired time at room temperature. After acetylation, the chitin sheets were washed by Soxhlet extraction with methanol overnight. Fabrication of Chitin Nanofiber Composites. Acetylated chitin nanofiber sheets were impregnated with neat acrylic resin (tricyclodecane dimethanol dimethacrylate (TCDDMA), Shin-Nakamura Chemical Co., Ltd.) with a 2-hydroxy-2-methylpropiophenone photoinitiator under reduced pressure overnight. The refractive index of the TCDDMA resin was 1.532. The resin-impregnated sheets were cured using UV curing equipment (SPOT CURE SP-7, Ushio Inc.) for 8 min at 40 mW/cm2. The chitin nanofiber composite sheets thus obtained were approximately 150 µm thick, and the fiber content was approximately 25%. Elemental Analysis. The DS values of the acetyl groups of the acetylated chitin nanofibers were calculated from the C and N content in the elemental analysis data obtained using an elemental analyzer (Elementar Vario EL III, Elementar), and estimated according to

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Figure 1. Effect of reaction time on the acetyl DS.

C/12.01:N/14.01 ) (6 + 2x):1 where C and N are weight percents of carbon and nitrogen, obtained from elemental analysis, and x is the DS value of the acetyl groups. Fourier Transform Infrared (FT-IR) Spectroscopy. Infrared spectra of the chitin samples were recorded using potassium bromide pellets with an FT-IR spectrometer (FTIR 8300, Shimadzu). All the spectra were obtained by an accumulation of 20 scans, with a resolution of 2 cm-1 at 400-4000 cm-1. X-ray Diffraction. Equatorial diffraction profiles were obtained with Ni-filtered Cu KR from an X-ray generator (Shimadzu XRD-6000) operating at 40 kV and 30 mA. Scanning Electron Microscopy (SEM). Prepared chitin nanofiber sheets were coated with an approximately 2 nm layer of platinum by an ion sputter coater and observed with a field emission scanning electron microscope (JSM-6700F; JEOL, Ltd.). Thermogravimetric Analysis (TGA). Thermogravimetric analysis was carried out on a TG/DTA6300 (Seiko Insturuments) under nitrogen. The sample mass was approximately 5 mg, and a heating rate of 10 °C min-1 was used. Regular Light Transmittances. The regular light transmittances of transparent nanocomposites were measured using a UV-vis spectrometer (JASCO V-550). Moisture Content. Moisture content was evaluated by exposing samples under a constant relative humidity. After measuring the sample weight equilibrated in 75.1% RH at 30 °C with a saturated aqueous solution of NaCl, the sample was oven-dried at 105 °C for 24 h, and the moisture content was then determined on the basis of the ovendried weight. Three samples were used to determine the moisture content, respectively. Coefficient of Thermal Expansion (CTE). The CTEs were measured with a thermomechanical analyzer (TMA/SS6000, SII Nanotechnology Inc.). Specimens were 25 mm long and 3 mm wide with a 20 mm span. The measurements were carried out from 30 to 165 °C by elevating the temperature at a rate of 5 °C min-1 in a nitrogen atmosphere in tensile mode under a load of 3 g. The CTE values were determined in the second run to dry the sample completely in the first run.

Results and Discussion Acetylation of Chitin Nanofibers. Chitin nanofiber sheets were acetylated in a mixture of acetic anhydride and perchloric acid for the desired reaction time. Figure 1 shows the effects of reaction time on the DS of acetyl groups. The DS values were estimated by C and N content in the elemental analysis data. In general, because chitin has poor solubility in typical solvents, the reaction rate of acetylation of chitin nanofibers is very small.

Figure 2. FT-IR spectra of acetylated chitin nanofibers of (a) DS 0.99, (b) DS 1.81, and (c) DS 2.96.

However, in the case of chitin nanofibers, the acetyl DS ranged from 0.99 to 2.96, after a 50 min reaction time, indicating an almost complete substitution of acetyl groups in the chitin nanofibers. The graph shows that the DS reached 1.51 from 0.99 after only 1 min of acetylation. The high reaction rate is obviously due to the very high surface-to-volume ratio, which works well for the liquid-solid phase reaction. The DS values then increased slowly and almost proportionally up to DS 2.96. The observed nonlinearity may have been caused by a heterogeneous reaction. That is, first, the chitin nanofiber surfaces were acetylated, and then the insides of the nanofibers were acetylated more gradually, as we discuss later. Thus, the DS appears to be strictly controllable by changing the reaction time. This result could be applicable to other esterifications of chitin nanofibers using the corresponding acid anhydride. Figure 2 shows the FT-IR spectra of acetylated BC with DS values of 0.99, 1.81, and 2.96. As the DS of acetyl groups increased, two major bands at 1231 and 1748 cm-1 increased, corresponding to the C-O and CdO stretching vibration modes of the acetyl group, respectively. In contrast, the O-H stretching band at 3972 cm-1 decreased with increasing acetyl DS and almost disappeared with a DS of 2.96, indicating a complete substitution of acetyl groups in the chitin nanofibers. The X-ray diffraction profiles of a series of acetylated chitin nanofibers are shown in Figure 3. In original chitin nanofiber (DS 0.99), the four diffraction peaks of chitin nanofibers observed at 9.5, 19.4, 20.9, and 23.4°, which corresponds to 020, 110, 120, and 130 planes, respectively, show the typical antiparallel crystal pattern of R-chitin.9 At DS 2.96, the diffraction pattern from R-chitin had completely disappeared, and the sample showed a well-defined uniform pattern of diO-acetylated chitin (chitin diacetate) at 2θ ) 7.4 and 17.7°. On the other hand, the diffraction from R-chitin still remained even at DS 1.81, which indicates that approximately 50% of OH

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Figure 3. X-ray diffraction profiles of acetylated chitin nanofibers of (a) DS 0.99, (b) DS 1.81, and (c) DS 2.96.

Figure 4. SEM images of acetylated chitin nanofiber samples of (a) DS 0.99, (b) DS 1.81, and (c) DS 2.96 .The length of scale bar is 200 nm.

Ifuku et al.

Figure 5. Derivative TGA curves of acetylated chitin nanofibers of (a) DS 0.99, (b) DS 1.81, and (c) DS 2.96.

Figure 6. Appearance of optically transparent acetylated chitin nanofiber composite (DS 1.70).

groups were substituted. Moreover, diffraction from chitin diacetate was also observed at 7.4°, and small shoulder peak was also observed at 17.7°. This profile clearly shows that chitin nanofibers are acetylated heterogeneously from the surface to the core.5,6 Scanning electron microscope (SEM) images of acetylated chitin nanofibers with a series of DS values are shown in Figure 4. Interestingly, nanofiber shapes remain in the DS 2.96 sample, which indicates that chitin diacetate is insoluble in the reaction mixture (mainly acetic anhydride), although cellulose triacetate is soluble in acetic anhydride. All images of the chitin nanofiber sheets seem to exist separately without coagulation, and as the DS values increase, the thickness of the nanofibers is obviously increased. That is, the average thicknesses of nanofibers with DS 0.99, 1.81, and 2.96 were 21.6, 28.9, and 32.1 nm, respectively. This change in thickness is due to the introduction of bulky acetyl groups into the nanofibers. Thermogravimetric analysis (TGA) of a series of acetylated chitin nanofibers was carried out to evaluate their degradation profiles and thermal stability. Figure 5 shows the derivative TGA curves of acetylated chitin nanofibers with DS 0.99, 1.81, and 2.96. The thermal degradation temperature of the original fibers (DS 0.99) was 388 °C according to the derivative curves. The derivative TGA curves drastically changed in the DS 2.96 sample, with no evidence of the peak derived from the original chitin nanofibers, and there were two peaks at 242 and 326 °C, which are attributed to the thermal decomposition of chitin diacetate. Interestingly, at DS 1.81, the TGA curve showed two kinds of thermal decomposition behaviors derived from chitin and chitin diacetate. This result also clearly indicates that acetyl groups were introduced from the surface to the core of the nanofibers, as noted above.

Figure 7. Regular light transmittance of a series of acetylated chitin nanocomposites.

Characterization of Acetylated Chitin Nanofiber Composites. Acetylated chitin nanofiber composites were fabricated with acrylic resin of TCDDMA. Appearance of optically transparent acetylated chitin nanofiber composite with DS of 1.70 is shown in Figure 6. Because the diameters of the chitin nanofibers are sufficiently smaller than the visible light wavelength, all nanocomposites have high transparency despite a variety of acetyl DS values ranging from 0.99 to 2.96.7,10 Figure 7 shows the regular transmittance of a series of acetylated chitin nanofiber composites at a 700 nm wavelength as an example. At 0.99, the transparency of the nanocomposite was 77%. However, as the acetyl DS values increased, the transmittance decreased slightly and proportionally up to 73% in the DS 2.96 sample. This decrease can likely be attributed to the change in the refractive index of the nanofibers. The optimum refractive index of resin to obtain the highest transparency nanocomposite with chitin nanofibers is known to be approximately 1.56, which is close to that of the TCDDMA resin

Acetylation of Chitin Nanofibers

Figure 8. Moisture contents of a series of acetylated chitin nanofiber sheets (open circles) and their nanocomposites (filled circles).

(1.532).11 However, since it is known that acetylation decreases the refractive index, the gap in the refractive index between chitin nanofiber and resin became wider with acetylation, resulting in a reduction in transparency. Interestingly, the transparency of chitin nanofiber composite was less sensitive to acetylation than that of bacterial cellulose (BC) nanofiber composite.6 The optical loss of the acetylated BC nanocomposite was approximately 20% with changes in the acetyl DS from 0.74 to 1.76. The difference of the optical loss is obviously due to the size effect. That is, because the thickness of the chitin nanofiber is approximately 20 nm, which is considerably smaller than that of BC nanofibers (50 nm width), the chitin nanofibers are freer from light scattering than BC nanofibers.10 Although the chitin nanofiber reinforced composite seems to have promising characteristics as an optically functional composite, it is quite hygroscopic. Absorption of moisture causes a deformation of the composite. Acetylation seems to reduce the moisture content of the nanocomposite. We therefore investigated the effects of acetylation on the hygroscopicity of the nanocomposites. Figure 8 shows the moisture content of a series of acetylated chitin nanofiber sheets and their composites at 30 °C and 75.1% relative humidity. Interestingly, acetylation of chitin nanofiber sheet decreased its moisture content slightly. The slight difference from 7.8 to 7.3% may be due to the nanosize effect. Because the chitin nanofiber sheet, with a very high surface-to-volume ratio, was exposed to the air, water molecules were easily adsorbed onto the surface. In the case of their composites, the moisture content of the original sample with DS 0.99 was 4.0%, which is considerably higher than that of acrylic resin of 0.33% due to the filler of hygroscopic chitin nanofiber. However, the moisture absorption of the nanocomposite with DS 1.30 drastically decreased to 2.2%. Clearly, this can be attributed to the introduction of hydrophobic acetyl groups into the hydrophilic hydroxyl moiety of chitin molecules. Acetylation of the nanofibers improved the compatibility with the acrylic resin, and improvement of miscibility at the filler/matrix interface decreased water adsorption onto the interface. However, with further acetylation, the moisture content showed little change, with a range of 2.0-2.5%. This result suggests that most hydroxyl groups on the chitin nanofiber surface were acetylated after 1 min reaction time, as mentioned above in Figure 1. Because chitin nanofibers have an extended antiparallel crystalline structure, the coefficient of thermal expansion (CTE) of the nanofibers is small. We therefore thought that the chitin nanofibers act as a reinforcement agent to reduce the thermal expansion of a resin. However, the CTE of nanofibers and its

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Figure 9. Coefficient of thermal expansion of a series of acetylated chitin nanofibers (filled circles) and their nanocomposites (open circles).

composites will be changed by the acetylation. Figure 9 shows the CTE of chitin nanofiber sheets, TCDDMA resin, and transparent nanocomposites from 30 to 165 °C plotted against the acetyl DS. Although the CTE of the TCDDMA resin was 6.4 × 10-5 °C-1, the CTE of the chitin nanofiber/TCDDMA composite was measured to be 2.3 × 10-5 °C-1. It is easy to see that the chitin nanofibers with a low thermal expansion of 9.2 × 10-6 °C-1 drastically reduce the CTE of the TCDDMA by reinforcement with a fiber content of 25%. On the other hand, the CTE of chitin nanofiber samples and their composites increased proportionally and gradually with increases in the acetyl DS. This increase is due to the acetylation reducing the degree of crystallinity of the chitin nanofibers, as shown in Figure 3, which increases the CTE of chitin nanofibers and the nanocomposites.

Conclusion Chitin nanofibers were chemically modified by acetyl groups and characterized in detail. Acetylated chitin nanofibers with a variety of DS values from 1.99 to 2.96 were obtained by adjusting the reaction time. Acetyl groups were heterogeneously introduced inside the nanofibers after the surface reaction. X-ray diffraction profiles, SEM images, and TGA curves show that the acetylation of chitin nanofibers changes their crystal structure, fiber thickness, and thermal degradation temperature, respectively. Chitin nanofiber-reinforced plastics have high transparency with a variety of acetyl DS values. Acetylation reduced the moisture absorption of chitin nanocomposite by the introduction of hydrophobic acetyl groups. We expect that hydrophobic chitin nanofibers with a uniform nanofiber structure and a very high surface-to-volume ratio will be advantageous for novel green nanomaterials. Acknowledgment. This work was financially supported by KAKENHI (20559003) of JSPS and Research for Promoting Technological Seeds of JST.

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(6) Ifuku, S.; Nogi, M.; Abe, K.; Handa, K.; Nakatsubo, F.; Yano, H. Biomacromolecules 2007, 8, 1973–1978. (7) Yano, H.; Sugiyama, J.; Nakagaito, A. N.; Nogi, M.; Matsuura, T.; Hikita, M.; Handa, K. AdV. Mater. 2005, 17, 153–155. (8) Gopalan Nair, K.; Dufresne, A. Biomacromolecules 2003, 4, 657– 665. (9) Minke, R.; Blackwell, J. J. Mol. Biol. 1978, 120, 167–181.

Ifuku et al. (10) Nogi, M.; Handa, K.; Nakagaito, A. N.; Yano, H. Appl. Phys. Lett. 2005, 17, 153–155. (11) Vigneron, J. P.; Rassart, M.; Vandenbem, C.; Lousse, V.; Deparis, O.; Biro, L. P.; Dedouaire, D.; Cornet, A.; Defrance, P. Phys. ReV. E. 2006, 73, 041905.

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