Triethyl Citrate (TEC) as a Dispersing Aid in Polylactic Acid/Chitin Nanocomposites Prepared via Liquid-Assisted Extrusion

The production of fully bio-based and biodegradable nanocomposites has gained attention during recent years due to environmental reasons; however, the production of these nanocomposites on the large-scale is challenging. Polylactic acid/chitin nanocrystal (PLA/ChNC) nanocomposites with triethyl citrate (TEC) at varied concentrations (2.5, 5.0, and 7.5 wt %) were prepared using liquid-assisted extrusion. The goal was to find the minimum amount of the TEC plasticizer needed to enhance the ChNC dispersion. The microscopy study showed that the dispersion and distribution of the ChNC into PLA improved with the increasing TEC content. Hence, the nanocomposite with the highest plasticizer content (7.5 wt %) showed the highest optical transparency and improved thermal and mechanical properties compared with its counterpart without the ChNC. Gel permeation chromatography confirmed that the water and ethanol used during the extrusion did not degrade PLA. Further, Fourier transform infrared spectroscopy showed improved interaction between PLA and ChNC through hydrogen bonding when TEC was added. All results confirmed that the plasticizer plays an important role as a dispersing aid in the processing of PLA/ChNC nanocomposites.


Introduction
Polylactic acid (PLA) is an attractive biopolymer for packaging and biomedical applications because of its biodegradability, non-toxicity, good mechanical properties, high optical transparency, and its commercial availability. However, PLA is brittle, and it exhibits low thermal stability, low melt strength, moderate barrier properties, and a slow crystallization rate. It is, therefore, necessary to modify the PLA to improve these properties to make PLA competitive among the common polymers used in industry [1][2][3]. PLA has been mixed with plasticizers [4], polymers [5], layered silicates [6], carbonaceous nanomaterials [7], cellulose [8], chitin [9], or a combination of these materials resulting in hybrid composites [10].
The development of nanocomposites based on PLA and chitin can be a good approach to improve the properties of PLA and to produce fully bio-based and biodegradable materials. Chitin nanofibers and nanocrystals have been recently used as additives to enhance thermal and mechanical properties of extrusion and that the plasticizer content should be at least 7.5 wt % to achieve well-dispersed and distributed nanocrystals.

Materials
Polylactic acid (PLA) (Ingeo 4043D grade) from NatureWorks LLC (Minnetonka, MN, USA) in pellet form was used as the matrix. Chitin powder from yellow lobster shell waste, purified at Pontifical Catholic University of Chile following the process reported in our earlier study [17], was used as the starting material for isolation of chitin nanocrystals (ChNC). These nanocrystals were used as to reinforce the PLA with and without the addition of a plasticizer. Liquid triethyl citrate (TEC) with a Mw of 276.3 g/mol (≥99% Alfa Aesar GmbH & Co KG, Karlsruhe, Germany) and ethanol (99.5%) was purchased from Solveco (Stockholm, Sweden). TEC was used to enhance the ChNC dispersion in the PLA matrix, and ethanol was used as a solvent for TEC, since it is partially soluble in water and to control the flowability of the suspensions for the liquid feeding. In addition, plasticizer and ethanol were the liquid media for feeding ChNC into the extruder.

Preparation of Chitin Nanocrystals and Suspensions for Liquid Feeding
Chitin nanocrystals (ChNC) were isolated via the acid-hydrolysis treatment according to the procedure reported earlier by Salaberria et al. [14]. Briefly, the chitin flakes were hydrolyzed with 3 M HCl Panreac (Barcelona, Spain) at 100 ± 5 • C under stirring for 90 min. After hydrolysis, the suspension was diluted with distilled water, washed via centrifugation and transferred to dialysis membranes for 3 days. Finally, the suspension was subjected to ultrasonic treatment for 10 min to disintegrate the remaining larger particles and then vacuum filtered using a polyamide filter Sartorious Biolab Products (Göttingen, Germany) with a 0.2 µm pore size to obtain a ChNC gel with a solid content of 19.5 wt %. Figure 1a shows an optical microscopy image of well-dispersed ChNC in water and a photograph of chitin nanocrystals displaying flow birefringence due to good dispersion. The AFM image in Figure 1b displays the typical rod-shaped ChNC with diameters in the range of 2-24 nm, which are shown as height distribution in Figure 1c, and with lengths in the range of 114-831 nm, which are shown as length distribution in Figure 1d. The width and length were measured using the Nanoscope V software Veeco (Santa Barbara, CA, USA) and the "FibreApp" (Zurich, Switzerland) respectively.

Materials
Polylactic acid (PLA) (Ingeo 4043D grade) from NatureWorks LLC (Minnetonka, MN, USA) in pellet form was used as the matrix. Chitin powder from yellow lobster shell waste, purified at Pontifical Catholic University of Chile following the process reported in our earlier study [17], was used as the starting material for isolation of chitin nanocrystals (ChNC). These nanocrystals were used as to reinforce the PLA with and without the addition of a plasticizer. Liquid triethyl citrate (TEC) with a Mw of 276.3 g/mol (≥99% Alfa Aesar GmbH & Co KG, Karlsruhe, Germany) and ethanol (99.5%) was purchased from Solveco (Stockholm, Sweden). TEC was used to enhance the ChNC dispersion in the PLA matrix, and ethanol was used as a solvent for TEC, since it is partially soluble in water and to control the flowability of the suspensions for the liquid feeding. In addition, plasticizer and ethanol were the liquid media for feeding ChNC into the extruder.

Preparation of Chitin Nanocrystals and Suspensions for Liquid Feeding
Chitin nanocrystals (ChNC) were isolated via the acid-hydrolysis treatment according to the procedure reported earlier by Salaberria et al. [14]. Briefly, the chitin flakes were hydrolyzed with 3 M HCl Panreac (Barcelona, Spain) at 100 ± 5 °C under stirring for 90 min. After hydrolysis, the suspension was diluted with distilled water, washed via centrifugation and transferred to dialysis membranes for 3 days. Finally, the suspension was subjected to ultrasonic treatment for 10 min to disintegrate the remaining larger particles and then vacuum filtered using a polyamide filter Sartorious Biolab Products (Göttingen, Germany) with a 0.2 µm pore size to obtain a ChNC gel with a solid content of 19.5 wt %. Figure 1a shows an optical microscopy image of well-dispersed ChNC in water and a photograph of chitin nanocrystals displaying flow birefringence due to good dispersion. The AFM image in Figure 1b displays the typical rod-shaped ChNC with diameters in the range of 2-24 nm, which are shown as height distribution in Figure 1c, and with lengths in the range of 114-831 nm, which are shown as length distribution in Figure 1d. The width and length were measured using the Nanoscope V software Veeco (Santa Barbara, CA, USA) and the "FibreApp" (Zurich, Switzerland) respectively. To feed the nanocrystals in liquid form, suspensions containing ChNC in water, TEC plasticizer and ethanol were prepared as follows: ChNC gel in water (19.5 wt %) was pre-dispersed in ethanol at a ratio of 1:5 water to ethanol for 2 h using magnetic stirring, and then mixed with TEC for 2 h. The same amount of the ChCN gel was added to all suspensions to prepare nanocomposites with a 3 wt % of ChNC, and the TEC content was varied in each suspension such that the final amount of plasticizer in the nanocomposites would be 2.5, 5.0, and 7.5 wt %. A suspension without a plasticizer was prepared for the extrusion of the unplasticized nanocomposite. Each suspension was ultrasonicated UP400S, Hielscher (Teltow, Germany) for 2 min in an ice bath prior to the extrusion and then pumped into the extruder. Mixtures of water, ethanol and TEC with the same proportions were prepared for To feed the nanocrystals in liquid form, suspensions containing ChNC in water, TEC plasticizer and ethanol were prepared as follows: ChNC gel in water (19.5 wt %) was pre-dispersed in ethanol at a ratio of 1:5 water to ethanol for 2 h using magnetic stirring, and then mixed with TEC for 2 h. The same amount of the ChCN gel was added to all suspensions to prepare nanocomposites with a 3 wt % of ChNC, and the TEC content was varied in each suspension such that the final amount of plasticizer in Polymers 2017, 9,406 4 of 16 the nanocomposites would be 2.5, 5.0, and 7.5 wt %. A suspension without a plasticizer was prepared for the extrusion of the unplasticized nanocomposite. Each suspension was ultrasonicated UP400S, Hielscher (Teltow, Germany) for 2 min in an ice bath prior to the extrusion and then pumped into the extruder. Mixtures of water, ethanol and TEC with the same proportions were prepared for the extrusion of plasticized PLA materials (control samples), as well as a mixture of only water and ethanol for the extrusion of PLA (control sample for unplasticized composite).
The prepared nanocomposites are coded as PLA-TEC (the number indicates the amount of plasticizer)-ChNC, the unplasticized composite is named as PLA-ChNC, and PLA always makes reference to extruded PLA under the presence of water and ethanol, and it will be indicated otherwise.

Extrusion of Nanocomposites
PLA, plasticized PLA materials (PLA-TEC), unplasticized nanocomposite (PLA-ChNC) and plasticized nanocomposites (PLA-TEC-ChNC) were prepared using a co-rotating twin-screw extruder ZSK-18 MEGALab, Coperion W&P (Stuttgart, Germany) with a liquid-assisted feeding of suspensions with a slight modification of the process described by Herrera and co-workers [9]. A K-tron gravimetric feeder (Niederlenz, Switzerland) was used to feed PLA, and a high-pressure syringe pump 500D, Teledyne Isco (Lincoln, NE, USA) was used for the liquid feeding of suspensions with ChNC and solutions without ChNC. A schematic representation of the process with the parameters and the screw configuration used are shown in Figure 2. The total throughput of the process was 2 kg/h, the screw speed was set to 300 rpm, and the temperature profile was ranging from 185 to 200 • C. The PLA pellets and suspensions were fed at the main feeding zone with a specific feeding rate for each particular material according to the final composition, as shown in Table 1. The prepared nanocomposites are coded as PLA-TEC (the number indicates the amount of plasticizer)-ChNC, the unplasticized composite is named as PLA-ChNC, and PLA always makes reference to extruded PLA under the presence of water and ethanol, and it will be indicated otherwise.

Extrusion of Nanocomposites
PLA, plasticized PLA materials (PLA-TEC), unplasticized nanocomposite (PLA-ChNC) and plasticized nanocomposites (PLA-TEC-ChNC) were prepared using a co-rotating twin-screw extruder ZSK-18 MEGALab, Coperion W&P (Stuttgart, Germany) with a liquid-assisted feeding of suspensions with a slight modification of the process described by Herrera and co-workers [9]. A Ktron gravimetric feeder (Niederlenz, Switzerland) was used to feed PLA, and a high-pressure syringe pump 500D, Teledyne Isco (Lincoln, NE, USA) was used for the liquid feeding of suspensions with ChNC and solutions without ChNC. A schematic representation of the process with the parameters and the screw configuration used are shown in Figure 2. The total throughput of the process was 2 kg/h, the screw speed was set to 300 rpm, and the temperature profile was ranging from 185 to 200 °C. The PLA pellets and suspensions were fed at the main feeding zone with a specific feeding rate for each particular material according to the final composition, as shown in Table 1. Two atmospheric venting and vacuum venting along the extruder were used to remove water and ethanol, as well as the trapped air. The extruded materials were cooled down in a water bath and then pelletized and dried at 55 °C overnight. The pelletized materials were compression molded using a hot press LPC-300 Fontijne Grotnes (Vlaardingen, Netherlands) to prepare films of approximately 200 µm thickness for further characterization. The pellets were placed inside metal plates covered with Mylar ® films Lohmann Technologies Ltd (Milton Keynes, UK) and compression molded at 190 °C for 210 s at contact pressure and then for 30 s at 4 MPa. The films were immediately removed from the metal plates and air-cooled to room temperature (~2-5 min) to avoid crystallization. Two atmospheric venting and vacuum venting along the extruder were used to remove water and ethanol, as well as the trapped air. The extruded materials were cooled down in a water bath and then pelletized and dried at 55 • C overnight. The pelletized materials were compression molded using a hot press LPC-300 Fontijne Grotnes (Vlaardingen, Netherlands) to prepare films of approximately 200 µm thickness for further characterization. The pellets were placed inside metal plates covered with Mylar ® films Lohmann Technologies Ltd (Milton Keynes, UK) and compression molded at 190 • C for 210 s at contact pressure and then for 30 s at 4 MPa. The films were immediately removed from the metal plates and air-cooled to room temperature (~2-5 min) to avoid crystallization.

Weight
The effect of water, ethanol, TEC plasticizer and ChNC on the molecular weight of PLA was evaluated via gel permeation chromatography (GPC) using an Ultimate 3000 HPLC system (Thermo Scientific, Germering, Germany). The columns use are as follows: four Phenogel GPC columns, from Phenomenex, with a 5 µm particle size and 1E5, 1E3, 100, and 50 Å porosities, respectively. Tetrahydrofuran at a flow rate of 1 mL/min was chosen as the mobile phase, and mono-disperse polystyrene standards were used for the universal calibration.

Melt Flow
The melt flow index of the prepared materials was measured using a melt indexer MI-1 Göttfert (Buchen, Germany). The measurements of the pelletized compounds were performed at least three times at 190 • C with a 2.16 kg load, and the average value in grams per 10 min is reported.

Transparency
Light transmittance of the materials was measured using a Perkin Elmer UV/VIS Spectrometer Lambda 2S (Überlingen, Germany). The scan was carried out in duplicated from 200 to 800 nm with a scan speed of 240 nm/min.

Dispersion and Morphology
The overview of the dispersion and distribution of ChNC in liquid feeding suspensions, as well as nanocomposite films were studied using a Nikon Eclipse LV100NPOL polarizing optical microscope (Shanghai, China). In the case of the nanocomposite films, cryogenic fracture surfaces were also analyzed using a FEI Magellan 400 XHR-SEM (Hillsboro, OR, USA). A thin layer (~10 nm) of tungsten was sputter-coated on the surfaces to avoid charging.

Chemical Characterization
Fourier transform infrared spectroscopy (FT-IR) studies were performed to determine the interaction between the PLA matrix and chitin nanocrystals and the effect of further addition of TEC. The samples were ground and mixed with KBr to prepare pellets. The spectra were collected

Thermal Properties and Crystallinity
The thermal properties of materials were measured using a differential scanning calorimeter DSC 821e, Mettler Toledo (Schwerzenbach, Switzerland). Approximately 3 mg of the material was heated in a semi-hermetic pan from −20 to 200 • C. The tests were performed with a heating rate of 10 • C/min under nitrogen atmosphere. The degree of crystallinity (Xc) of the films was calculated following the equation [28]: where ∆Hm is the enthalpy of melting (pre-melt crystallization was subtracted from the melting enthalpy), ∆Hcc is the enthalpy of cold crystallization, ∆Hm 0 is the enthalpy of melting for a 100% crystalline PLA sample, which is assumed to be 93 J/g [29], and w is the weight fraction of PLA in the sample.

Thermo-Mechanical Properties
The thermo-mechanical properties of prepared materials were determined using a TA Instruments Q800 DMA (New Castle, DE, USA) on the 5 mm × 30 mm specimens. The experiments were performed in tensile mode from 25 to 100 • C with a heating rate of 1 • C/min and a constant frequency of 1 Hz. The testing was performed in duplicates.

Mechanical Testing
The tensile properties of prepared materials were measured using a Shimadzu AG-X universal tensile testing machine (Kyoto, Japan) with a 1 kN load cell. The 5 mm × 80 mm specimens were cut using a rectangular press mold and then conditioned for 24 h at room conditions (25 ± 2 • C and 25% ± 2% of relative humidity). The gauge length was 20 mm, and the crosshead speed was 2 mm/min. The values for stress and elongation at break were directly obtained from the testing results, and modulus of each sample and the work of fracture were calculated from the stress-strain curves. Moreover, the properties of extruded PLA without water and ethanol were also measured and reported to analyze the effect of water and ethanol on the mechanical properties of neat PLA. The average value of five tests was reported. One-way analysis of variance (ANOVA) followed by the Tukey-HSD multiple comparison tests with a 5% significance level was used to analyze the results.

Suspensions for Liquid Feeding
Prior to the extrusion, the dispersion of the ChNC in the prepared suspensions was studied using an optical microscope and compared to the aqueous ChNC suspension (Figure 1a) to see the effect of ethanol and TEC. Figure 3 shows that ChNC dispersed in water, ethanol, and TEC at different concentrations are similar compared with the aqueous ChNC dispersion shown in Figure 1a.
This confirms that the addition of ethanol and TEC did not significantly affect the dispersion of ChNC in the suspensions. All ChNC suspensions showed good stability before the extrusion. However, it is worth noting that the viscosity of suspensions was affected by the addition of the plasticizer. Suspension with the highest TEC content (7.5 wt %) resulted in the highest viscosity. The possible reason can be the better dispersion of ChNC, which was not evident at the optical microscope scale, or more interactions between TEC and ChNC.

Suspensions for Liquid Feeding
Prior to the extrusion, the dispersion of the ChNC in the prepared suspensions was studied using an optical microscope and compared to the aqueous ChNC suspension (Figure 1a) to see the effect of ethanol and TEC. Figure 3 shows that ChNC dispersed in water, ethanol, and TEC at different concentrations are similar compared with the aqueous ChNC dispersion shown in Figure 1a. This confirms that the addition of ethanol and TEC did not significantly affect the dispersion of ChNC in the suspensions. All ChNC suspensions showed good stability before the extrusion.

Molecular Weight
The influence of water, ethanol, TEC, and ChNC, as well as of all of them together on the molecular weight of PLA was studied using GPC, and the average molecular weights (M w ) are shown in Table 2. When comparing M w of unprocessed PLA (as received) with M w of extruded PLA with and without water and ethanol, it is observed that the extrusion process affects the molecular weight of PLA more than the feeding of water and ethanol. This can be attributed to a decrease in local shear due to the plasticizer effect of water [6]. M w of the extruded PLA with water and ethanol was similar to that of the unprocessed PLA pellets (M w~1 99 kg/mol), showing that water and ethanol did not degrade PLA even if it is known that PLA is susceptible to hydrolytic degradation. When PLA was extruded with water, ethanol and TEC, the presence of TEC increased the molecular mobility of PLA, which may increase the water diffusion rate into the PLA molecules and thus, enhances the hydrolytic degradation [30], which results in a PLA-TEC5.0 material with a somewhat lower molecular weight (M w~1 96 kg/mol) but still less degraded that the extruded PLA. When comparing the molecular weight in Table 2 of unplasticized composite (PLA-ChNC) and plasticized nanocomposite (PLA-TEC5.0-ChNC), the values show that the reduction in molecular weight of PLA due to the addition of ChNC in a water ethanol suspension (from 199 to 181 kg/mol) was more than that due to the addition of ChNC in water, ethanol and TEC suspension (from 199 to 193 kg/mol). It is possible that, in general, the presence of additives, such as ChNC, may increase the thermo-mechanical degradation of PLA due to higher shear forces, as has been reported by others [6,31]. This effect may be smaller in a presence of a plasticizer. It is concluded that in this study, the polymer degradation due to chitin was hindered by the use of plasticizer and that the addition of chitin promoted the polymer degradation more than the addition of water and ethanol. Similarly, Stoclet et al. [6] reported that the processing of PLA/halloysite nanocomposites via conventional extrusion (dry method) resulted in higher degradation of PLA than the water assisted extrusion process, where the injection of water decreased the effect of the halloysite on the PLA molecular weight. In contrast, Rizvi et al. [32] reported hydrolytic degradation of PLA when it was processed with chitin in water suspension in a micro-compounder. However, the difference between that study compared with the present one is the long processing time, and the micro-compounder does not effectively remove the water and/or solvents, and the authors did not use a plasticizer. The processing time in Rizvi's study was 6 min, while the resident time in this study is less than 1 min, which may not be enough time to promote the hydrolysis of PLA. It should also be noted that the extrusion process involving liquids works better as a continuous process than as a batch process and with extruders with an appropriate degassing system.

Melt Flow
The measurement of the melt flow index (MFI) of the prepared materials gives indirect information about the dispersion and interaction between the polymer and nanocrystals since the flow behavior of polymer nanocomposites is influenced by the interfacial characteristics and the nanoscale structure [33]. The effect of the addition of varied amounts of TEC on the flow properties of PLA and PLA-ChNC was evaluated and the MFI values are listed in Table 2. The results show that the plasticized PLA exhibited higher MFI than PLA, as expected. The MFI of PLA was 3.1 g/10 min, and PLA-TEC7.5 showed the highest value of 4.9 g/10 min due to the highest amount of the plasticizer. The addition of TEC increases the polymer free volume and the polymer chains' mobility and, thus, decreases the viscosity and increases the MFI which is a typical effect of the plasticizer [34].
Opposite to the effect of the plasticizer, the addition of nanocrystals restricts the polymer chains' mobility and, thus, the MFI of the matrix decreases. It is seen from Table 2 that all nanocomposites, except for the PLA-ChNC, exhibited lower MFI than their respective materials without ChNC. The addition of ChNC to the PLA-TEC7.5 material decreased its MFI from 4.9 to 3.7 g/10 min, showing the largest effect. This result is an indication that the dispersion and interaction of the nanocrystals in the PLA-TEC7.5-ChNC nanocomposite were better than the nanocomposites with lower TEC contents. On the other hand, the PLA-ChNC composite showed a higher MFI than PLA, which indicates that the interaction of nanocrystals with the matrix was poor. In this case, the higher MFI can also be due to the lower molecular weight of the PLA-ChNC composite.

Transparency and Visual Appearance
The visual appearance of the extruded PLA with water and ethanol and its nanocomposite films as well as the optical microscopy images of the film surfaces are shown in Figure 4 (to the left). It is clear that the unplasticized composite shows visible agglomeration, which is not observed in plasticized nanocomposites. However, the optical microscopy images also show micro-sized agglomerations for the PLA-TEC2.5-ChNC nanocomposites but not for the nanocomposites with 5.0 wt % and 7.5 wt % TEC. The optical transparency of materials was measured because it can give an indication of the dispersion and distribution of ChNC in PLA. It is known that if the size of particles is smaller than the wavelength of visible light, the transparency of the matrix is affected less [35]. It was noticed during the test that the addition of TEC did not affect the PLA transparency, and the light transmittance spectra were overlapping with that of PLA. Therefore, those UV/VIS spectra are not displayed in Figure 4 (to the right), but the spectra of the extruded PLA with water and ethanol and its nanocomposites are shown. It is observed that the light transmittance of PLA decreased with the addition of chitin nanocrystals. At 550 nm of visible light, the light transmittance of PLA was 90%, whereas it was only 52%, 44%, 24%, and 30% for the PLA-TEC7.5-ChNC, PLA-TEC5.0-ChNC, PLA-TEC2.5-ChNC, and PLA-ChNC materials, respectively. These results show that the nanocomposites with the highest TEC content (7.5 wt %) had the best transparency of the nanocomposites and, thus, expected to have the best dispersion of ChNC which is in accordance with the MFI results.  Figure 5 displays the cryogenic fracture surface of the unplasticized PLA-ChNC composite and the nanocomposites with a different TEC content. These micrographs clearly show that the dispersion and distribution of ChNC gradually improved with the plasticizer content as was also seen in the transparency and MFI studies.     Figure 5 displays the cryogenic fracture surface of the unplasticized PLA-ChNC composite and the nanocomposites with a different TEC content. These micrographs clearly show that the dispersion and distribution of ChNC gradually improved with the plasticizer content as was also seen in the transparency and MFI studies.  The micrograph at higher magnification for the PLA-ChNC composite (Figure 6a) shows poor dispersion and distribution and large agglomeration (~10 µm) of ChNC, whereas the PLA-TEC7.5-ChNC nanocomposite, with the highest TEC content (Figure 6b), exhibits more even, well-dispersed and distributed chitin nanocrystals with few agglomerations which are much smaller than those in Figure 6a. These results are in agreement with our previous studies, where the addition of poly(ethylene glycol) (PEG) enhanced the dispersion of cellulose nanocrystals [23] and with the results reported by Wang et al. [24] and Qu et al. [25] who have reported that acetyl tributyl citrate (ATBC) and PEG enhanced the dispersion of carbon black and cellulose nanofibers in PLA, respectively.

Morphology of Nanocomposites and ChNC Dispersion
Polymers 2017, 9,406 10 of 16 The micrograph at higher magnification for the PLA-ChNC composite (Figure 6a) shows poor dispersion and distribution and large agglomeration (~10 µm) of ChNC, whereas the PLA-TEC7.5-ChNC nanocomposite, with the highest TEC content (Figure 6b), exhibits more even, well-dispersed and distributed chitin nanocrystals with few agglomerations which are much smaller than those in Figure 6a. These results are in agreement with our previous studies, where the addition of poly(ethylene glycol) (PEG) enhanced the dispersion of cellulose nanocrystals [23] and with the results reported by Wang et al. [24] and Qu et al. [25] who have reported that acetyl tributyl citrate (ATBC) and PEG enhanced the dispersion of carbon black and cellulose nanofibers in PLA, respectively.

Chemical Charaterization
The effect of addition of TEC in the interaction between PLA and ChNC was analyzed using FTIR. Figure 7A shows infrared spectra of extruded PLA with water and ethanol, PLA-TEC7.5, PLA-ChNC, and PLA-TEC7.5-ChNC. The characteristic peaks of PLA were observed in all analyzed materials. The peak at 1760 is attributed to the carbonyl (-C=O) stretching of PLA. The peaks between 2850 and 3000 cm −1 belong to the C-H asymmetric and symmetric stretching vibration [36]. The peak of the -C-O-bond

Chemical Charaterization
The effect of addition of TEC in the interaction between PLA and ChNC was analyzed using FTIR. Figure 7A shows infrared spectra of extruded PLA with water and ethanol, PLA-TEC7.5, PLA-ChNC, and PLA-TEC7.5-ChNC. The micrograph at higher magnification for the PLA-ChNC composite (Figure 6a) shows poor dispersion and distribution and large agglomeration (~10 µm) of ChNC, whereas the PLA-TEC7.5-ChNC nanocomposite, with the highest TEC content (Figure 6b), exhibits more even, well-dispersed and distributed chitin nanocrystals with few agglomerations which are much smaller than those in Figure 6a. These results are in agreement with our previous studies, where the addition of poly(ethylene glycol) (PEG) enhanced the dispersion of cellulose nanocrystals [23] and with the results reported by Wang et al. [24] and Qu et al. [25] who have reported that acetyl tributyl citrate (ATBC) and PEG enhanced the dispersion of carbon black and cellulose nanofibers in PLA, respectively.

Chemical Charaterization
The effect of addition of TEC in the interaction between PLA and ChNC was analyzed using FTIR. Figure 7A shows infrared spectra of extruded PLA with water and ethanol, PLA-TEC7.5, PLA-ChNC, and PLA-TEC7.5-ChNC. The characteristic peaks of PLA were observed in all analyzed materials. The peak at 1760 is attributed to the carbonyl (-C=O) stretching of PLA. The peaks between 2850 and 3000 cm −1 belong to the C-H asymmetric and symmetric stretching vibration [36]. The peak of the -C-O-bond The characteristic peaks of PLA were observed in all analyzed materials. The peak at 1760 is attributed to the carbonyl (-C=O) stretching of PLA. The peaks between 2850 and 3000 cm −1 belong to the C-H asymmetric and symmetric stretching vibration [36]. The peak of the -C-O-bond stretching in -CH-O-and in -O-C=O of PLA appear at 1182 and 1081 cm −1 , respectively [24]. The peaks at 1621 and 1656 cm −1 and at 1556 cm −1 correspond to the amide I and II [37], respectively. The peaks at 3110 and 3271 cm −1 are ascribed to the N-H stretching [38]. The above mentioned data confirmed the presence of chitin in the composites. From the PLA spectra, a peak at approximately 3510 cm −1 can be seen, which is related to the O-H bond stretching deformation. This indicates the presence of hydroxyl groups in pure PLA [39]. This peak did not change with the addition of TEC. However, this peak was broader and slightly shifted to a lower wavenumber (3506 cm −1 ) when ChNC were added to PLA, and it further broadened and shifted to 3494 cm −1 when ChNC was added together with TEC, as can be seen in Figure 7B. These results indicate the H-bonding interactions between PLA and ChNC. Rosdi and Zakaria [40] also found that the peak at 3505 cm −1 was shifted to a lower wavenumber when chitin was added to the PLA matrix, possibly due to some interaction between the hydroxyl groups of PLA and the hydroxyl groups of chitin. The results also indicate that the H-bonding interactions between PLA and ChNC were enhanced in the presence of TEC. It is believed that TEC may help the intermolecular interaction between PLA and chitin and enhances their interfacial interaction, which is in agreement with the SEM images. Similar results have been reported by Qu et al. [25], who showed that PEG improved the intermolecular interaction between PLA, PEG, and cellulose. No new peaks were detected when TEC or ChNC were added to PLA or when TEC was added to the PLA-ChNC nanocomposite.

Thermal Properties and Crystallinity
DSC thermograms and glass transition (T g ), cold crystallization (T cc ) and melt (T m ) temperatures of extruded PLA with water and ethanol, plasticized PLA materials and nanocomposites are shown in Figure 8. All presented T g , T cc , and T m for the materials indicate their semi-crystalline nature. The T g , T cc , and T m of PLA are 60, 121 and 147 • C, respectively, and these values decreased with 7.5 wt % TEC content to 47, 113, and 142 • C. The decrease is because of the plasticizing effect [41]. T g , T cc , and T m of the plasticized PLA materials remained almost the same with the addition of ChNC, and only a slight increase of T g from 47 to 49 • C was observed for the material with a 7.5 wt % of TEC. This slight improvement of the glass transition temperature may be due to a better interaction between PLA, TEC and ChNC in this nanocomposite, which hinders the polymer molecular mobility. Figure 8 shows the degree of crystallinity (X c ) where the addition of TEC and ChNC did not show any significant effect.  [24]. The peaks at 1621 and 1656 cm −1 and at 1556 cm −1 correspond to the amide I and II [37], respectively. The peaks at 3110 and 3271 cm −1 are ascribed to the N-H stretching [38]. The above mentioned data confirmed the presence of chitin in the composites. From the PLA spectra, a peak at approximately 3510 cm −1 can be seen, which is related to the O-H bond stretching deformation. This indicates the presence of hydroxyl groups in pure PLA [39]. This peak did not change with the addition of TEC. However, this peak was broader and slightly shifted to a lower wavenumber (3506 cm −1 ) when ChNC were added to PLA, and it further broadened and shifted to 3494 cm −1 when ChNC was added together with TEC, as can be seen in Figure 7B. These results indicate the H-bonding interactions between PLA and ChNC. Rosdi and Zakaria [40] also found that the peak at 3505 cm −1 was shifted to a lower wavenumber when chitin was added to the PLA matrix, possibly due to some interaction between the hydroxyl groups of PLA and the hydroxyl groups of chitin. The results also indicate that the H-bonding interactions between PLA and ChNC were enhanced in the presence of TEC. It is believed that TEC may help the intermolecular interaction between PLA and chitin and enhances their interfacial interaction, which is in agreement with the SEM images. Similar results have been reported by Qu et al. [25], who showed that PEG improved the intermolecular interaction between PLA, PEG, and cellulose. No new peaks were detected when TEC or ChNC were added to PLA or when TEC was added to the PLA-ChNC nanocomposite.

Thermal Properties and Crystallinity
DSC thermograms and glass transition (Tg), cold crystallization (Tcc) and melt (Tm) temperatures of extruded PLA with water and ethanol, plasticized PLA materials and nanocomposites are shown in Figure 8. All presented Tg, Tcc, and Tm for the materials indicate their semi-crystalline nature. The Tg, Tcc, and Tm of PLA are 60, 121 and 147 °C, respectively, and these values decreased with 7.5 wt % TEC content to 47, 113, and 142 °C. The decrease is because of the plasticizing effect [41]. Tg, Tcc, and Tm of the plasticized PLA materials remained almost the same with the addition of ChNC, and only a slight increase of Tg from 47 to 49 °C was observed for the material with a 7.5 wt % of TEC. This slight improvement of the glass transition temperature may be due to a better interaction between PLA, TEC and ChNC in this nanocomposite, which hinders the polymer molecular mobility. Figure 8 shows the degree of crystallinity (Xc) where the addition of TEC and ChNC did not show any significant effect.   Figure 9 shows storage modulus and tan delta (δ) as a function of temperature for PLA nanocomposites and their counter-parts without nanocrystals with different TEC contents as well as those for the unplasticized materials. In Figure 9a, PLA and PLA-ChNC are compared. It is observed that the addition of ChNC did not affect the storage modulus or tan delta peak position. Respectively, in Figure 9b-d, the plasticized nanocomposites with 2.5, 5.0 and 7.5 wt % TEC content are compared with their respective counterpart without ChNC. Similar to the DSC results, only the PLA-TEC7.5-ChNC nanocomposite showed a slight increase in the tan δ position. In addition, a decrease in the intensity of the peak was also observed. A positive shift in tan δ commonly indicates restricted molecule movement, and a decreased intensity of tan δ shows that lower number of polymer chains participates in the transition, which is expected because of the well-dispersed and distributed nanocrystals in the PLA-TEC7.5-ChNC nanocomposite. This better ChNC dispersion is also reflected in an improved storage modulus. These results indicate that the nanocomposites with the highest TEC content (7.5 wt %) shows better dispersed and distributed nanocrystals and, thus, slightly enhanced thermo-mechanical properties.

Thermo-Mechanical Properties
When comparing PLA with the plasticized PLA materials, it is observed that the increased TEC content in PLA decreases the tan delta peak position towards lower temperature from 62 • C to 53 • C with the addition of 7.5 wt % TEC, which confirms the plasticizer effect of TEC. Moreover, it is seen that the increased TEC content together with ChNC enhances cold crystallization, and higher TEC content is more effective that the lower TEC content.  Figure 9 shows storage modulus and tan delta (δ) as a function of temperature for PLA nanocomposites and their counter-parts without nanocrystals with different TEC contents as well as those for the unplasticized materials. In Figure 9a, PLA and PLA-ChNC are compared. It is observed that the addition of ChNC did not affect the storage modulus or tan delta peak position. Respectively, in Figure 9b-d, the plasticized nanocomposites with 2.5, 5.0 and 7.5 wt % TEC content are compared with their respective counterpart without ChNC. Similar to the DSC results, only the PLA-TEC7.5-ChNC nanocomposite showed a slight increase in the tan δ position. In addition, a decrease in the intensity of the peak was also observed. A positive shift in tan δ commonly indicates restricted molecule movement, and a decreased intensity of tan δ shows that lower number of polymer chains participates in the transition, which is expected because of the well-dispersed and distributed nanocrystals in the PLA-TEC7.5-ChNC nanocomposite. This better ChNC dispersion is also reflected in an improved storage modulus. These results indicate that the nanocomposites with the highest TEC content (7.5 wt %) shows better dispersed and distributed nanocrystals and, thus, slightly enhanced thermo-mechanical properties.

Thermo-Mechanical Properties
When comparing PLA with the plasticized PLA materials, it is observed that the increased TEC content in PLA decreases the tan delta peak position towards lower temperature from 62 °C to 53 °C with the addition of 7.5 wt % TEC, which confirms the plasticizer effect of TEC. Moreover, it is seen that the increased TEC content together with ChNC enhances cold crystallization, and higher TEC content is more effective that the lower TEC content.

Mechanical Properties
The mechanical properties of PLA nanocomposites and their counter-part materials without nanocrystals are reported in Table 3. In addition, the mechanical properties of the extruded PLA without water and ethanol are reported, and if comparing these properties with those from the extruded PLA in the presence of water and ethanol, no significant effect on the mechanical properties of PLA was noticed. It is possible to see in Table 3 that the addition of TEC decreased the tensile strength of PLA and did not increase the elongation at break or work of fracture, as expected. These results indicate that a higher amount of plasticizer is required to obtain a noticeable effect on the toughness. Labrecque et al. [4] reported that all citrate esters are effective in improving the elongation at break at higher concentrations (≥20%), but do not show any significant increase at lower concentration. However, both DSC and DMA results showed that the plasticizer contents used in this study were enough to plasticize PLA.
The nanocomposites with TEC ≥ 5 wt % showed higher tensile strength and ultimate strength than the respective plasticized PLA without ChNC. Moreover, these materials showed slightly improved Young's modulus based on the ANOVA test. The elongation at break and work of fracture were decreased in all cases except for the nanocomposite with 7.5 wt % of TEC. This is due to less agglomeration and better dispersion of ChNC in the PLA-TEC7.5-ChNC nanocomposite.
The decrease observed in the tensile strength of PLA-ChNC can be attributed to the hydrolysis of PLA during the processing [32], which was observed in the GPC results, and because of the presence of micro agglomerations with a poor interface, as observed in the SEM studies. These results are similar to the results reported by Hishammuddin and Zakaria [36] where the incorporation by mixing, and then casting of commercial chitin into PLA, resulted in reduced tensile strength and elongation. Salaberria et al. [20] also reported a slight decrease of mechanical properties of PLA when functionalized (acylation) chitin nanocrystals were introduced into PLA via extrusion/compression. Rizvi et al. [32] found that the stiffness of PLA increased with increasing chitin content while the strength was found to decrease. However, in this study, it was found that the addition of ChNC into PLA together with TEC showed enhanced mechanical properties when ≥5.0 wt % of plasticizer was used. This is explained because the dispersion and distribution of ChNC and their interaction with the PLA matrix was improved with increasing plasticizer content as it has been shown in the previous sections of this paper. Similarly, Li et al. [18] reported that PEG worked as a compatibilizer for chitin nanofibers and PLA when it was used as pretreatment for the nanofibers before the compounding process.

Conclusions
This study was carried out to determine a plasticizer content that has the minimum plasticizer effect on PLA, but still enhances the dispersion and distribution of ChNC in the PLA matrix and, thus, obtain a nanocomposite with improved properties. Therefore, PLA composites with 3 wt % of chitin nanocrystals and triethyl citrate with varied contents of 2.5, 5.0, and 7.5 wt % were produced via liquid-assisted extrusion.
The gel permeation chromatography confirmed that the addition of water and ethanol during the extrusion process did not significantly affect the molecular weight of PLA.
The liquid feeding of ChNC together with TEC plasticizer resulted in PLA-TEC-ChNC nanocomposites with improved dispersion and distribution of ChNC. The nanocomposite with the highest plasticizer content (PLA-TEC7.5-ChNC) showed enhanced mechanical, thermal, and thermo-mechanical properties, compared with its counter-part without ChNC (PLA-TEC7.5). The improved interaction between PLA and ChNC in the presence of TEC is attributed to hydrogen bonding, which was supported by the FTIR study.
It will be interesting to study the effect of a higher plasticizer content to determine the synergic effect of the plasticizer as a dispersing and toughening aid with a minimum impact on the properties of PLA. The presented facile process of nanocomposites using liquid-assisted extrusion with a plasticizer, which facilities nanomaterial dispersion, can be a step forward for a large-scale production of bionanocomposites.