*3.5. The Interactions of Single and Hybrid Fillers with the TPS Matrix as Observed through FTIR and XRD Analyses*

FTIR analysis was used to interpret the chemical functionalities' presence in the biocomposite's chemical structure and analyze a potential interaction between the TPS matrix and single filler and with the hybrid filler system. FTIR is sensitive to detecting the change of TPS structure at the molecular level, such as chain conformation, crystallinity, water content, and TPS and filler interaction [40]. According to Figure 10, the FTIR spectra of all the samples indicate a broad band located at 3000 to 3700 cm−<sup>1</sup> , corresponding to free, inter-and intramolecular O-H stretching. This indicates the presence of a high amount of O-H functional group of the TPS and TPS bio-composite films' structure [41]. For the unfilled TPS film, wavelength from 800 to 1200 cm−<sup>1</sup> is the fingerprint region of the TPS, contributing to glucan ring vibration by C-OH stretching and bending vibration and the C-O-C glycoside bond vibration [42]. A single peak observed at 1640 cm−<sup>1</sup> represents the water—bound tightly in TPS film due to its hygroscopic nature [42].

The characteristic peak at 2929 cm−<sup>1</sup> was attributed to asymmetrical C-H stretching and vibration. Meanwhile, the characteristic peak at 1375 cm−<sup>1</sup> represents the –CH<sup>2</sup> bending. Furthermore, there is a small peak at 1153 and 1080 cm−<sup>1</sup> representing the C-O stretch in the C-O-H group in the TPS film, whereas 1240 cm−<sup>1</sup> shows the C-O stretch of C-O-C bond in the structure of the film [41,43]. The peaks at 927 and 862 cm−<sup>1</sup> represent the starch glycosidic linkage of glucose in starch [41]. Overall, the FTIR spectra for all the TPS bio-composite films are almost similar to the unfilled TPS films but there was a slight change in the intensity of some peaks. Furthermore, the shifting of certain bands was also noticed.

**Figure 10.** FTIR of unfilled TPS, TPS bio-composites and TPS hybrid bio-composite, in the region of (**a**) 650–4000 cm<sup>−</sup>1 and (**b**) 1450–1700 cm<sup>−</sup>1. **Figure 10.** FTIR of unfilled TPS, TPS bio-composites and TPS hybrid bio-composite, in the region of (**a**) 650–4000 cm−<sup>1</sup> and (**b**) 1450–1700 cm−<sup>1</sup> .

> Figure 11a shows the XRD pattern of the unfilled TPS, TPS bio-composite and TPS hybrid bio-composite films. TPS has experienced a reduction in peak intensity in the region of 2Ɵ = 10°–40° when added with filler (either single or hybrid fillers) due to the reduction in retrogradation rate that occurs in the matrix. Interface interactions between the matrix and fillers can slow down the retrogradation process of the starch. During the TPS matrix cooling, the amylose and amylopectin chains are starting to arrange back into an ordered structure different from native starch granules. The retrogradation process involved a few steps: extrusion of water, an increase in viscosity, gel formation and forming a crystalline structure. The typical retrogradation peaks of TPS can be seen at 17°and 22.6°. In the region of 3000 to 3700 cm−<sup>1</sup> , the band appears due to O-H stretching that provides information related to hydrogen bonding between TPS and fillers. For TPS/5B bio-composite, the disappearance of the nano-bentonite peak at 3435 cm−<sup>1</sup> is associated with the hydroxyl linkage formation within the alumino-silicate layered structure of the clay, indicating that the filler is forming new hydrogen bonding with the TPS. XRD analysis has further proved this by showing a broadening of d<sup>001</sup> peak for the nano-bentonite. For the TPS/5B sample, there is a shift of peak at 3320 cm−<sup>1</sup> to a lower wavenumber, which is 3310 cm−<sup>1</sup> , indicating new and stable hydrogen bonds formed in the TPS bio-composite films [44]. Hydrogen bonding has been developed due to compatibility between the TPS and B filler.

> Previous research showed that there is good interfacial bonding between the cellulose and TPS due to their chemical similarity and good compatibility that allow for the formation of hydrogen bonding between them [45]. Based on the FTIR spectra of TPS/5C, it can be observed that incorporation of C into the TPS matrix has slightly sharpened the peak and also shifted the peak to a lower wavenumber (3315 cm−<sup>1</sup> ). This can be associated with the O-H vibration of the high crystalline structure of the C filler. This outcome was in accordance with the study of Zhang et al. They have concluded that the O-H stretching

vibration shifts to a lower wavenumber in the FTIR spectrum due to new hydrogen bonding between TPS and nanocellulose [46]. For the analysis of the TPS/4B1C hybrid bio-composite film, interactions between the TPS and hybrid fillers can also be realized through the FTIR data. Since both of the single fillers showed interaction with TPS films by forming hydrogen bonding, hybrid fillers are expected to interact with TPS films in the same ways. Peaks at 2929 (C-H) and 3320 cm−<sup>1</sup> of the TPS were shifted to 2020 and 3316 cm−<sup>1</sup> due to new hydrogen forming between the TPS and hybrid fillers (B and C). This indicates that hybrid fillers have good compatibility with the TPS matrix by forming strong polar hydroxyl interactions. *Polymers* **2021**, *13*, x FOR PEER REVIEW 2 of 22

> Figure 11a shows the XRD pattern of the unfilled TPS, TPS bio-composite and TPS hybrid bio-composite films. TPS has experienced a reduction in peak intensity in the region of 2θ = 10–40◦ when added with filler (either single or hybrid fillers) due to the reduction in retrogradation rate that occurs in the matrix. Interface interactions between the matrix and fillers can slow down the retrogradation process of the starch. During the TPS matrix cooling, the amylose and amylopectin chains are starting to arrange back into an ordered structure different from native starch granules. The retrogradation process involved a few steps: extrusion of water, an increase in viscosity, gel formation and forming a crystalline structure. The typical retrogradation peaks of TPS can be seen at 17◦ and 22.6◦ . The XRD pattern of the TPS/5C film presents the typical peak of retrograded starch structure (type B and type Vh peak) and the signal of the C filler. However, due to the similar chemical structure of TPS and microcrystalline cellulose in the TPS/5C composite, the XRD pattern shows superimposition of both parent components balanced by the composition. This is in line with the study of Dufresne et al. [37]. Incorporation of C into the TPS has brought a reduction in the intensity of Vh peaks in the XRD spectrum. This was possibly due to the transcrystallization of amylose and amylopectin on the microcrystalline cellulose surface, leading to a reduction of the starch chain's recrystallization. The same observation was reported in the study of Fourati et al. They found that the Vh-type structure of the TPS reduced with the increase in the cellulose nanofiber content [47].

**Figure 11.** XRD diagram of TPS, TPS bio-composite and TPS hybrid bio-composite films, at the region of (**a**) 2 theta = 5°–40° and (**b**) 2 theta = 5°–10°. **Figure 11.** XRD diagram of TPS, TPS bio-composite and TPS hybrid bio-composite films, at the region of (**a**) 2θ = 5–40◦ and (**b**) 2θ = 5–10◦ .

It can also be observed that the intensity of the peak related to the Vh-type structure of the TPS is lower in the TPS/4B1C hybrid bio-composite as compared to the unfilled TPS. The Vh-type structure formed in TPS was due to amylose's recrystallization with plasti-The XRD pattern of the TPS/5C film presents the typical peak of retrograded starch structure (type B and type Vh peak) and the signal of the C filler. However, due to the

similar chemical structure of TPS and microcrystalline cellulose in the TPS/5C composite, the XRD pattern shows superimposition of both parent components balanced by the composition. This is in line with the study of Dufresne et al. [37]. Incorporation of C into the TPS has brought a reduction in the intensity of Vh peaks in the XRD spectrum. This was possibly due to the transcrystallization of amylose and amylopectin on the microcrystalline cellulose surface, leading to a reduction of the starch chain's recrystallization. The same observation was reported in the study of Fourati et al. They found that the Vh-type structure of the TPS reduced with the increase in the cellulose nanofiber content [47].

It can also be observed that the intensity of the peak related to the Vh-type structure of the TPS is lower in the TPS/4B1C hybrid bio-composite as compared to the unfilled TPS. The Vh-type structure formed in TPS was due to amylose's recrystallization with plasticizer in the helix channel. Reduction in the peak intensity of the Vh type crystalline structure indicates that the retrogradation of the TPS was hindered with the incorporation of the single B filler or hybrid B/C fillers. The anti-retrogradation of the TPS films with the nano-bentonite was due to the hydrophilicity of bentonite clay's surface that increased the interaction between the starch chain and bentonite platelets, affecting the dynamic rearrangement of the starch chain. Consequently, the ability of the TPS chains to recrystallize was reduced. This trend was also reported by Lara et al., where they found that the retrogradation of starch plasticized by water and glycerol was reduced by incorporating MMT [48]. Interestingly, the TPS/4B1C hybrid bio-composite film exhibits a smaller and broader peak of the Vh-type structure as compared to the TPS/5B bio-composite. This proved that the use of hybrid filler with a low content of C (1 wt%) may result in greater efficiency in preventing retrogradation than the use of single C or single B filler.

Next, the XRD signal from 5◦ to 10◦ was focused to have a clear comparison on the d<sup>001</sup> basal spacing of the nano-bentonite clay before and after being incorporated into the TPS matrix. As expected, there is no peak that can be observed in the XRD signal of the unfilled TPS and TPS/5C bio-composite in the low-angle region (2θ = 5–10◦ ) since TPS is amorphous. It is known that the peak that appears at a low angle value between 5–10◦ represents the d<sup>001</sup> basal spacing of bentonite clay [8]. When single B filler or hybrid B/C fillers were incorporated into the TPS, the d<sup>001</sup> peak of B had shifted to a lower angle, indicating an increase in the interlayer basal spacing of the clay due to the intercalation of TPS chains and microcrystalline cellulose into the layered silicate structure of bentonite without complete exfoliation.

The TPS/4B1C hybrid bio-composite film shows the highest increment in basal spacing in which the d<sup>001</sup> increased from 1.47 to 1.55 nm. Moreover, broadening of the d<sup>001</sup> peak can clearly be seen. It seems that the inter-platelets of the nano-bentonite have been well intercalated by the TPS chains with the presence of a small amount of the microcrystalline cellulose. This could be due to the capability of bentonite to interact with the C filler, other than the host biopolymer. As mentioned earlier, there are strong polar interactions between the TPS, C and B fillers due to the hydroxyl group composition within their structure. The interface interactions between B and C help to pull the B platelets away from its tactoid structure, making a wider space for the TPS chains intercalation. This factor explains why the TPS/4B1C performed the greatest mechanical properties compared to other TPS bio-composite films.
