*3.5. XRD Analysis of MCC and NC*

The effects of mechanical and chemical treatments on the crystalline structure of the cellulose samples were further characterized by XRD as shown in Figure 4. Both MCC and MD-MCC exhibited characteristic crystalline peaks near 2θ = 15.1◦, 16.2◦, 21.0◦, 22.6◦, and 34.5◦; these peaks correspond to

the (1<sup>−</sup> 10), (110), (012), (200), and (004) crystallographic planes, respectively, and are characteristic of the cellulose I structure [41,42]. The AH-NC had the same crystalline peaks as MCC and MD-MCC except that the diffraction peak at 2θ = 34.5◦ was broader and flatter, and this peak disappeared after high-pressure homogenization and high-intensity ultrasonication. Generally, NC obtained via the three different preparation methods exhibited the characteristic cellulose I peaks, which indicates that acid hydrolysis, high-pressure homogenization, and high-intensity ultrasonication do not affect the main crystalline properties of cellulose.

**Figure 4.** X-ray diffraction (XRD) patterns of MCC, MD-MCC, AH-NC, HH-NC, and US-NC.

The crystallinity of NC is an important parameter as it determines its reinforcing capability and mechanical strength in composite films [43]. Highly crystalline fibers are expected to be more effective at providing reinforcement for composite materials because of their increased stiffness and rigidity, which result in a higher Young's modulus. However, the crystallinity of MD-MCC and the prepared NC each decreased to a different extent relative to MCC (Table 2). The peak at 22.6◦ was less sharp for the alkali-treated MCC, which suggests that alkali swelling might destroy part of the crystalline structure [44]. Cationic modification of MCC disrupted both intermolecular and intramolecular hydrogen bonding and facilitated the formation of amorphous regions [6]. The results of this study indicate that the NC preparation methods were non-selective as they damaged both amorphous and crystalline cellulose; therefore, the crystallinity of NC decreased [2,7,45]. As shown in Table 2, the crystallinity of AH-NC (67.6%) was the highest of the three NC, which implies that the crystalline regions of MD-MCC were more resistant to the acid treatment than the mechanical treatments. Moreover, the crystallinity of US-NC (62.8%) was slightly higher than that of HH-NC (61.8%), which suggests that high-intensity ultrasonication was less aggressive than high-pressure homogenization.


**Table 2.** Crystallinity index (CI), degradation temperature, and mass loss of MCC, MD-MCC, AH-NC, HH-NC, and US-NC.

*3.6. Fourier Transform Infrared (FTIR) Spectroscopy Analysis of MCC and NC*

Fourier transform infrared spectroscopy was used to understand the changes in the chemical structures of the MCC and NC (Figure 5). Two main absorption regions, 2800–3600 cm−<sup>1</sup> and 750–1750 cm−1, were present in the spectra. The broad band centered at approximately 3332 cm−<sup>1</sup> corresponds to O-H stretching of cellulose in the fiber [46]. The intensity of the O-H peak in the MD-MCC spectrum was considerably less than that of the MCC spectrum, which indicates that the number of hydroxyl groups in the MD-MCC was reduced by the etherification reaction [6]. The peaks near 2900 cm−<sup>1</sup> are attributed to the C-H stretching vibration of cellulose and the peaks at 1644 cm−<sup>1</sup> have been assigned to water absorption because of the strong cellulose-water interaction [47]. In addition to the characteristic peaks of the cellulose backbone, there was a small peak near 1479 cm−<sup>1</sup> in the MD-MCC and NC spectra. This peak was not present in the MCC spectrum and thus was assigned to the trimethyl quaternary ammonium groups, which implies that trimethyl quaternary ammonium groups were successfully added on to cellulose chains even though the degree of substitution was low.

**Figure 5.** Fourier transform infrared (FTIR) spectra of MCC, MD-MCC, AH-NC, HH-NC, and US-NC.

The changes to the crystal structure of cellulose resulted in the intensity reduction of partial FTIR peaks attributed to the crystalline domains of cellulose [1]. The peak at 1427 cm−<sup>1</sup> corresponds to the CH2 bending vibration of crystalline cellulose and the NC spectra exhibit reduced intensity of this crystalline band compared to those of the MCC. The reduced intensity of this peak also supports the decreased crystallinity of NC after acid hydrolysis, high-pressure homogenization, and high-intensity ultrasonication as demonstrated by the XRD results. The reduced crystallinity of NC compared to MCC implies that there is less intermolecular and intramolecular hydrogen bonding in NC, which might increase the dispersion of NC in water [1]. The peak at 1030 cm−<sup>1</sup> is related to C-O stretching of the pyranose ring skeleton [48]. The peak at 890 cm−<sup>1</sup> is corresponds to β-glycosidic linkages between the glucose units of cellulose. Based on the above analysis, there was not a significant difference between the MCC and NC spectra, which indicates that neither the mechanical nor chemical treatments changed the main chemical structure of the fibers. This result agrees with the XRD analysis.
