polymers

Article Surface Modification of Cellulose Nanocrystals with Succinic Anhydride

Agnieszka Leszczy ´nska 1,*, Paulina Radzik 1, Ewa Szefer 1, Matej Miˇcušík 2,Mária Omastová 2 and Krzysztof Pielichowski 1

1 Department of Chemistry and Technology of Polymers, Cracow University of Technology, ul. Warszawska 24, 31-155 Kraków, Poland; [email protected] (P.R.); [email protected] (E.S.); [email protected] (K.P.) 2 Polymer Institute, Slovak Academy of Sciences, Dúbravská cesta 9, 845 41 Bratislava 45, Slovakia; [email protected] (M.M.); [email protected] (M.O.) * Correspondence: [email protected]; Tel.: +48-(12)-628-21-27 or +48-(12)-628-20-39; Fax: +48-(12)-628-20-39


Received: 24 March 2019; Accepted: 7 May 2019; Published: 13 May 2019


Abstract: The surface modification of cellulose nanocrystals (CNC) is a key intermediate step in the development of new functionalities and the tailoring of nanomaterial properties for specific applications. In the area of polymeric nanocomposites, apart from good interfacial adhesion, the high thermal stability of cellulose nanomaterial is vitally required for the stable processing and improvement of material properties. In this respect, the heterogeneous esterification of CNC with succinic anhydride was investigated in this work in order to obtain CNC with optimised surface and thermal properties. The influence of reaction parameters, such as time, temperature, and molar ratio of reagents, on the structure, morphology and thermal properties, were systematically studied over a wide range of values by DLS, FTIR, XPS, WAXD, SEM and TGA methods. It was found that the degree of surface substitution of CNC increased with the molar ratio of succinic anhydride to cellulose hydroxyl groups (SA:OH), as well as the reaction time, whilst the temperature of reaction showed a moderate effect on the degree of esterification in the range of 70–110 ◦C. The studies on the thermal stability of modified nanoparticles indicated that there is a critical extent of surface esterification below which only a slight decrease of the initial temperature of degradation was observed in pyrolytic and oxidative atmospheres. A significant reduction of CNC thermal stability was observed only for the longest reaction time (240 min) and the highest molar ratio of SA:OH. This illustrates the possibility of manufacturing thermally stable, succinylated, CNC by controlling the reaction conditions and the degree of esterification.

Keywords: cellulose nanocrystals; whiskers; surface modification; esterification; succinic anhydride

1. Introduction Cellulose nanocrystals, or CNCs, have recently emerged as a new class of versatile building blocks delivered from renewable feedstock [1]. Due to their high mechanical strength, low density, functionalisability, environmental sustainability and low cost, they have been extensively explored as a reinforcement in polymer nanocomposites [2,3]. However, the limited thermal stability of cellulose nanoparticles is a significant problem in nanocomposite manufacturing. Thus, extensive work has already been carried out to obtain thermally stable cellulose nanocrystals [4]. The most commonly used method of CNC preparation is the hydrolysis of cellulose materials in mineral acids. According to the literature, sulphuric acid has the widest application in cellulose hydrolysis [5–9]. For example, Beck-Candanedo et al. investigated the influence of reaction time

Polymers 2019, 11, 866; doi:10.3390/polym11050866 www.mdpi.com/journal/polymers Polymers 2019, 11, 866 2 of 24 and sulphuric acid-to-pulp ratio on nanocrystal properties, and showed that longer hydrolysis times produced shorter, less polydisperse cellulose nanocrystals, and furthermore, increasing the acid-to-pulp ratio caused a reduction of CNC dimensions [10]. Since cellulose nanocrystals obtained from sulphuric acid are characterised by a relatively low thermal stability, other mineral and organic acids have been used to produce CNCs. Amongst mineral acids, the application of phosphoric acid allowed for the production of nanocrystalline cellulose with higher thermal stability in comparison to sulphuric acid hydrolysis [4,11]. Other approaches employed in cellulose hydrolysis included, for example, application of hydrochloric acid [12,13], a mixture of acetic and nitric acid [14], and formic or hydrobromic acid [15–17]. One of the main applications predicted for CNCs is the manufacturing of polymeric nanocomposites. CNCs often require previous surface modification in order to achieve good compatibility with a polymer matrix and a better individualisation of nanoparticles. Alongside methods such as silylation or acylation, the carboxylation of cellulose nanocrystals leads to their better compatibility with hydrophobic thermoplastic matrices, and moreover it was shown that the yield of nanoparticles increases with the carboxyl group content for a series of TEMPO-oxidized samples [18,19]. The introduction of charged groups and the reduction of CNC hydrophilicity, by decorating their surface with less-polar organic moieties, has been widely reported. For example, Hu et al. obtained carboxylated cellulose nanocrystals with negatively charged carboxylic groups from borer powder of bamboo by direct oxidative degradation with ammonium persulfate [20]. Surface carboxylated cellulose nanocrystals, with repelling forces between individual nanocrystals, were also prepared via 2,2,6,6-tetramethylpiperidine-1-oxyl(TEMPO)-mediated oxidation of cotton linters by Montanari et al. [21]. Another approach for preparing carboxycellulose was described by Sharma et al. who used nitric acid or nitric acid-sodium nitrite mixtures for this purpose, and this method was shown to be less energy and water intensive compared to the TEMPO oxidation of cellulose [22]. The most common chemical methods of CNC surface functionalisation with organic substituents are esterification, etherification and grafting [1]. Chemical derivatisation of cellulose nanomaterial is likely to diminish its thermal stability, which is another critical factor for the processing of polymeric nanocomposites. However, the improved thermal stability of nanocellulose was reported, for example, by Yin et al. as a result of triazine derivative grafting on the cellulose surface to replace hydroxyl groups [23,24]. Furthermore, the decorating of nanocellulose with functional organic moieties provides nanoparticles with the desired reactivity in further processing steps. Amine functionalised CNCs were obtained by Yue et al. using amino trimethoxy silane. Functionalisation of the filler allowed a further reaction with a bio-based epoxy resin, derived from diphenolic acid, to manufacture bio-based epoxy hybrid nanocomposites [25]. Besides using CNCs as a reinforcement in polymer nanocomposites, other applications of modified cellulose nanocrystals are demonstrated in the literature. Hydrophobisation of cellulose nanocrystals allows them to be used as coating agents for marine vehicles, biomedical devices, windows, textiles or paints [26]. Another possibility is to use CNCs as antimicrobial agents, which is of great interest to the food packaging industry. For example, antimicrobial activity against E. coli was reported by Tang et al. and Drogat et al. for cellulose nanoparticles with polyrhodanine, or composites of silver nanoparticles and CNCs, respectively [19,26–28]. Also Sharma and Varma showed that agricultural residue derived cellulose and cotton cellulose can be used for producing quasi-spherical shaped nanoparticles with improved anti-microbial properties for E. coli [29]. Cellulose nanocrystals have also been recently investigated for their use in water treatment technologies, e.g., phosphorylated CNC has promoted heavy metal adsorption, such as Ag+, Cu2+ and Fe3+ from industrial effluents [30–32]. Other applications of nanocrystalline cellulose include support for catalysts, emulsion stabilisers and flocculating agents [19]. Succinic anhydride can be considered a promising modifying agent for nanocellulose since it enables the introduction of aliphatic chains along with carboxylic end groups. Thus, its chemical Polymers 2019, 11, 866 3 of 24 structure is useful in the engineering of physical and chemical surface properties of cellulose nanomaterial. Carboxylic functional groups provide surface charges and reactivity in further derivatization of the nanoparticles. Moreover, significant progress has been reported for the production of succinic acid from renewable resources as a platform intermediate of high significance [33]. In the bio-economy era, the application of renewable chemical feedstock with good availability is critical for the successful application of bionanomaterials. In this respect, succinic anhydride is advantageous in specific applications over, e.g., acid chlorides or common silylation agents, despite the high reactivity of the latter. The application of succinic anhydride in the chemical derivatisation of carbohydrates has been reported for starch, chitosan and cellulose [34]. However, there are few reports on the functionalisation of cellulose nanoparticles with succinic acid or anhydride and its derivatives, and they are rather focused on studying the functional properties of modified nanoparticles and composite materials. Esterification of raw cellulose with succinic anhydride has been demonstrated as a very effective method of surface charging that facilitated defibrillation in a microfluidizer [35]. Succinic acid was applied by Castro et al. as a bridging agent to attach β-cyclodextrin to the surface of cellulose nanocrystals. As a result, new materials were produced with the ability to release antibacterial molecules over a prolonged period of time [36]. Cellulose nanocrystals, decorated with succinate groups, also work as an effective nanoadsorbent for Pb2+ and Cd2+ heavy metal cations [37]. Moreover, carboxycellulose nanoparticles or zinc oxide nanocrystal-decorated microfibrillated cellulose scaffolds were used for water treatment applications, to remove, e.g., lead, arsenic or cadmium(II) [38–41]. Elsewhere, cellulose nanocrystals have been modified with alkenyl succinic anhydride (ASA) and changes in surface energies, and its influence on CNC dispersibility in polypropylene and polylactide matrices were studied [42]. Miao et al. discovered a high increase of storage modulus (487-fold increase) when ASA-modified cellulose nanocrystals were applied as a reinforcement to the polyurethane system [43]. Surface modification of cellulose nanocrystals has been intensively investigated to reach better compatibility of CNC with polymer matrix, and, in consequence, to achieve better properties of the composite materials. Although there are reports on CNC modification by succinic anhydride, we aimed at an elaboration of the optimal reaction conditions to achieve thermally stable succinylated CNC that is vital for its further application as a modifier for polymers processed at higher temperatures, by e.g., extrusion. In the previous work we found that at lower concentrations of phosphoric acid (in the range of 60–74 wt.%) and at a lower temperature (90 ◦C), the main product of hydrolysis of UFC 100 cellulose was fine microcrystalline cellulose that was further used as a substrate for the production of CNCs in the presence of succinic anhydride without the use of esterification catalyst [4]. In such conditions, further hydrolysis of amorphous regions of cellulose as a dominating process and slight surface esterification were simultaneously running. In the current work we have optimised the phosphoric acid concentration and hydrolysis temperature at higher values for the production of thermally stable cellulose nanocrystals from different raw material, and then produced CNCs were subjected to surface modification with succinic anhydride (SA) in the presence of pyridine catalyst. We report the systematic study on the effects of various reaction parameters in the heterogeneous esterification of CNCs, in particular, the influence of reaction time, temperature and molar ratio of SA to hydroxyl groups of cellulose, on the structure, morphology and thermal properties of modified cellulose nanocrystals. The influence of phosphoric acid concentration on the properties of CNCs was discussed in reference to crystallinity changes as well as its thermal and thermo-oxidative stability.

2. Materials and Methods

2.1. Materials Microcrystalline cellulose powder Avicel PH-101 from Sigma Aldrich (Darmstadt, Germany) was used as a starting material for further hydrolysis and modification. The average particle size of Polymers 2019, 11, 866 4 of 24 this material is 50 µm. Reagent grades of phosphoric acid solution (85 wt.%), dimethylformamide, pyridine and succinic anhydride were also purchased from Sigma Aldrich and used in the as received condition. Analytical grade sodium hydroxide supplied by Chempur (Piekary Sl´ ˛askie,Poland) was used to prepare the solution for alkali treatment of the starting material.

2.2. Preparation of Cellulose Nanocrystals A total of 10 g of microcrystalline cellulose was added to a round-bottomed flask containing 200 mL of sodium hydroxide solution with a concentration of 4 wt.% and the suspension was stirred for 2 h at 80 ◦C. This process was repeated twice, with subsequent washing of the residue with distilled water to pH 7. Alkali treatment of cellulose was performed to remove non-cellulosic components present in the starting material [44]. The hydrolysis conditions were selected on the basis of the work by Espinosa et al. [45]. After alkali treatment, 1 g of dried microcrystalline cellulose was added to a round-bottomed flask and mixed with distilled water. The mixture was cooled in an ice bath and phosphoric acid (85 wt.%) was slowly added via a dropping funnel, keeping the temperature below 30 ◦C. The acid concentrations used in hydrolysis were 70, 74, 76 and 78 wt.%. The hydrolysis was carried out at 100 ◦C for 90 min under magnetic stirring. Subsequently, suspensions were diluted one-fold with distilled water to stop the hydrolysis and cooled down to room temperature in an ice bath. The residue of the prepared cellulose nanocrystals (CNC_P series) was separated from the acid solution by thrice repeated centrifugation (at 5200 rpm for 15 min), the supernatant being replaced with an equal volume of distilled water before undergoing ultrasonic homogenisation (using Sonic Ruptor 250 (Omni International, Kennesaw, GA, USA) ultrasonic homogeniser working at 50% of maximum amplitude, in a pulse mode) after each centrifugation step. After the third cycle of centrifugation and sonication, the suspension was placed in a dialysis membrane (MWCO 14 000) and dialysed against distilled water for five days. When the pH value of the suspension reached 7, it was sonicated for 15 min and five drops of ethanol were added to reduce the crystallisation of water and agglomeration of CNCs during drying. Then the dispersion was frozen with liquid nitrogen and lyophilised using FreeZone Plus 2.5 freeze-dryer (Labconoco, Kansas City, MO, USA). Cellulose nanocrystals obtained from hydrolysis in phosphoric acid at a concentration of 76 wt.% (CNC_P76) were then modified with succinic anhydride (SA) under a range of reaction conditions. The influence of the reaction time (60, 120, 180 and 240 min), temperature (70, 80, 90, 100 and 110 ◦C) as well as molar ratio of succinic anhydride to total cellulose hydroxyl groups (SA:OH 2:1, 3:1, 5:1 and 10:1) on the properties of modified CNCs was investigated. In each reaction, 0.5 g of CNC_P76 was dispersed in 16 mL of dimethylformamide (DMF) and after the reaction temperature was achieved, the SA solution in 10 mL of DMF and 0.026 mL of pyridine catalyst were added into the CNC dispersion. The catalyst type and amount was determined according to the work by Sehaqui et al. [35]. Reactions were carried out under nitrogen flow. After the reaction, the mixture was cooled down in an ice bath, centrifuged twice (5200 rpm, 15 min) and sonicated (15 min) with distilled water. To purify the CNC suspension from the remaining reagents, it was dialysed against distilled water for three days. In order to reduce the crystallisation of water and aggregation of CNC during freeze-drying, tert-butanol was added to the water dispersion of CNCs to achieve t-but:H2O 5:25 (v/v) concentration [46]. Finally, the CNCs dispersion was frozen in liquid nitrogen and freeze-dried. The preparation route of modified CNCs is presented in Figure1 and denotations of all samples are shown in Table1. If not indicated in the sample name as a varying parameter, the following standard parameters were applied: 110 ◦C, 120 min and 5:1, respectively for the reaction temperature, time and molar ratio of succinic anhydride to hydroxyl groups of cellulose (SA:OH). Polymers 2019, 11, x FOR PEER REVIEW 4 of 24

2.2. Preparation of Cellulose Nanocrystals A total of 10 g of microcrystalline cellulose was added to a round-bottomed flask containing 200 mL of sodium hydroxide solution with a concentration of 4 wt.% and the suspension was stirred for 2 h at 80 °C. This process was repeated twice, with subsequent washing of the residue with distilled water to pH 7. Alkali treatment of cellulose was performed to remove non-cellulosic components present in the starting material [44]. The hydrolysis conditions were selected on the basis of the work by Espinosa et al. [45]. After alkali treatment, 1 g of dried microcrystalline cellulose was added to a round-bottomed flask and mixed with distilled water. The mixture was cooled in an ice bath and phosphoric acid (85 wt.%) was slowly added via a dropping funnel, keeping the temperature below 30 °C. The acid concentrations used in hydrolysis were 70, 74, 76 and 78 wt.%. The hydrolysis was carried out at 100 °C for 90 min under magnetic stirring. Subsequently, suspensions were diluted one-fold with distilled water to stop the hydrolysis and cooled down to room temperature in an ice bath. The residue of the prepared cellulose nanocrystals (CNC_P series) was separated from the acid solution by thrice repeated centrifugation (at 5200 rpm for 15 min), the supernatant being replaced with an equal volume of distilled water before undergoing ultrasonic homogenisation (using Sonic Ruptor 250 (Omni International, Kennesaw, GA, USA) ultrasonic homogeniser working at 50% of maximum amplitude, in a pulse mode) after each centrifugation step. After the third cycle of centrifugation and sonication, the suspension was placed in a dialysis membrane (MWCO 14 000) and dialysed against distilled water for five days. When the pH value of the suspension reached 7, it was sonicated for 15 min and five drops of ethanol were added to reduce the crystallisation of water and agglomeration of CNCs during drying. Then the dispersion was frozen with liquid nitrogen and lyophilised using FreeZone Plus 2.5 freeze-dryer (Labconoco, Kansas City, MO, USA). Cellulose nanocrystals obtained from hydrolysis in phosphoric acid at a concentration of 76 wt.% (CNC_P76) were then modified with succinic anhydride (SA) under a range of reaction conditions. The influence of the reaction time (60, 120, 180 and 240 min), temperature (70, 80, 90, 100 and 110 °C) as well as molar ratio of succinic anhydride to total cellulose hydroxyl groups (SA:OH 2:1, 3:1, 5:1 and 10:1) on the properties of modified CNCs was investigated. In each reaction, 0.5 g of CNC_P76 was dispersed in 16 mL of dimethylformamide (DMF) and after the reaction temperature was achieved, the SA solution in 10 mL of DMF and 0.026 mL of pyridine catalyst were added into the CNC dispersion. The catalyst type and amount was determined according to the work by Sehaqui et al. [35]. Reactions were carried out under nitrogen flow. After the reaction, the mixture was cooled down in an ice bath, centrifuged twice (5200 rpm, 15 min) and sonicated (15 min) with distilled water. To purify the CNC suspension from the remaining reagents, it was dialysed against distilled water for three days. In order to reduce the crystallisation of water and aggregation of CNC during freeze- drying, tert-butanol was added to the water dispersion of CNCs to achieve t-but:H2O 5:25 (v/v) concentration [46]. Finally, the CNCs dispersion was frozen in liquid nitrogen and freeze-dried. The preparation route of modified CNCs is presented in Figure 1 and denotations of all samples are shown in Table 1. If not indicated in the sample name as a varying parameter, the following standard Polymersparameters2019, 11, 866were applied: 110 °C, 120 min and 5:1, respectively for the reaction temperature, time5 of 24 and molar ratio of succinic anhydride to hydroxyl groups of cellulose (SA:OH).

Alkali 4 CNC_P70 CNC_P74 CNC_P76 CNC_P78 treatment PO Avicel_Raw H 3

Avicel_NaOH Succinic acid anhydride

CNC_SA

Figure 1. Schematic illustration of the preparation route of modified cellulose nanocrystals. Figure 1. Schematic illustration of the preparation route of modified cellulose nanocrystals. Table 1. Description of prepared samples.

Sample Hydrolysis Concentration of Acid Solution, Time, Temperature, Denotation Catalyst wt. % min ◦C Avicel _Raw - - - - CNC_P70 70.0 CNC_P74 74.0 H PO 90 100 CNC_P763 4 76.0 CNC_P78 78.0 Sample Time, Temperature, Modifier Molar Ratio of SA:OH Denotation min ◦C CNC_SA2:1 2:1 CNC_SA3:1 3:1 Succinic anhydride 120 110 CNC_SA5:1 5:1 CNC_SA10:1 10:1

CNC_ 70 ◦C 70 CNC_80 ◦C 80 CNC_90 ◦C Succinic anhydride 5:1 120 90 CNC_100 ◦C 100 CNC_110 ◦C 110 CNC_60 min 60 CNC_120 min 120 Succinic anhydride 5:1 110 CNC_180 min 180 CNC_240 min 240

2.3. Dynamic Laser Scattering (DLS) Dynamic laser scattering (DLS) measurements were undertaken to determine particle-size distributions by intensities and numbers. A Malvern Zeta Sizer Nano ZS was used (Malvern, UK), and analyses were carried out in disposable sizing cuvettes, at a scattering angle of 173◦ and room temperature. Concentration of each dispersion was 0.1 mg mL 1. The hydrodynamic radius, r , · − h derived from Stokes-Einstein equation for spherical particles, was determined by DLS method and was considered only as an indicative measure of the ‘apparent’ size of the dynamic hydrated/solvated CNCs. The intensity size distributions were obtained from analysis of the correlation function using the CONTIN algorithm in the instrument software.

2.4. Fourier Transform Infrared (FTIR) Fourier transform infrared (FTIR) spectra were collected in the Spectrum 65 spectrophotometer (Perkin-Elmer, Waltham, MA, USA) in ATR mode with a C/ZnSe crystal. The range of each measurement 1 1 was 4000–450 cm− in 4 cm− intervals. Eight scans were averaged. Polymers 2019, 11, 866 6 of 24

2.5. X-ray Photoelectron Spectroscopy (XPS) X-ray photoelectron spectroscopy (XPS) analysis was performed using a K-Alpha XPS system (Thermo Scientific, Waltham, MA, USA) equipped with a micro-focused, monochromatic Al Kα X-ray source (1486.6 eV). An X-ray beam of 400 µm size was used at 6 mA 12 kV. The spectra were acquired × in the mode of constant analyser energy and the pass energy was 200 eV for the survey. The fractional concentration of a respective element A (in atomic%) was calculated according to Equation (1):

IA/sA %A = P 100% (1) (In/sn) · where In is the integrated peak area of the detected atoms and Sn is the Scofield sensitivity factor corrected for the analyser transmission. The determined surface compositions were used to calculate the surface substitution degree (SSD) of cellulose. The method proposed by Rodionova et al. was adapted to perform the calculations [47]. It was considered that for each cellulose unit there are five carbons in the (C–O) molecular environment. Therefore, 1/5 of the (C–O) percentage peak contribution (C%C–O) corresponds to the contribution of a single carbon atom in the (C–O) environment. Since the cellulose was esterified with dicarboxylic acid anhydride, 1/2 of the percentage concentration of carbons in the (O–C=O) environment (C%O–C=O) was proportional to the number of ester bonds. The surface substitution degree (SSD) was calculated using Equation (2), as a concentration of ester bonds related to the concentration of a single (C–O) group:

1 1 SSD = C = / C (2) 2 %O–C O 5 %C–O

2.6. Wide-Angle X-ray Diffraction (WAXD) X-ray diffraction measurements were performed using a D2 Phaser (Bruker, Billerica, MA, USA) diffractometer at room temperature in the reflection mode (kCu = 1.54 Å). Patterns were collected in the range of 5◦–40◦ using an increment of 0.02◦. The degree of crystallinity – χc, was determined as the ratio of the total area of the crystalline peaks (ΣAcrystal) to the sum of the areas of the crystalline peaks and the amorphous profile (ΣAamorphous). Deconvolution procedure was done using a MagicPlot Student 2.7.1. Five crystalline peaks were found at 14.3◦, 16.4◦, 20.0◦, 22.5◦ and 34.5◦ which correspond to the 101, 10¯ı, 021, 002 and 040 Miller indices of cellulose crystals respectively [48–50].

2.7. Scanning Electron Microscopy (SEM) Morphology of the samples was examined using a JSM-6010LA Jeol Scanning Electron Microscope (Jeol, Tokyo, Japan). The samples were metalized with gold-palladium using a Sputtering System Hummer 6.2 (Anatech USA, Hayward, CA, USA) with the thickness of the layer being approximately 3 nm.

2.8. Atomic Force Microscopy (AFM) The AFM imaging of CNCs dried on air was carried out in tapping mode on AFM INNOVA (Veeco, New York, NY, USA). The Bruker’s antimony (n) doped Si AFM probe model FESPA-V2 with k value of 22.8 N/m and resonance frequency (f 0) of 75 kHz was used at scanning rate of 0.300 µm/s. Typical topography and phase images were acquired. The CNC diameters were evaluated in the Bruker NanoScope Analysis 1.40 software by the determination of horizontal distances between valleys on the cross-sectional profiles taken perpendicular to the cellulose crystals. Thirty values of CNC diameters were averaged for each analysed sample. Polymers 2019, 11, 866 7 of 24

2.9. Thermogravimetric Analyses (TGA) Thermogravimetric analyses (TGA) were carried out using a TG F1 Libra analyser (Netzsch, Selb, Germany), and were collected in a temperature range of 30–600 C, at a heating rate of 10 C min 1, ◦ ◦ · − under a synthetic air and argon atmosphere.

3. Results and Discussion

3.1. Particle Size Distribution by DLS Measurements The dynamic light scattering (DLS) method was applied as a supporting technique for the evaluation of particle size distribution of CNC_P76 and succinylated nanocrystals. The investigated materials are polydisperse nanoparticles with a high aspect ratio. Therefore, the determined hydrodynamic radius was considered as a tentative measure of an ‘apparent’ size of the dynamic hydrated/solvated rod-like particles. Recently, Fraschini et al. demonstrated that a slight change in the length of CNC directly affected the measured DLS size, while changes in the cross-section showed hardly no effect on the diffusion velocity [51]. Also, according to Du et al., hydrodynamic diameters based on DLS measurements represents the length rather than the width of CNC samples [15]. Moreover, the DLS measurements show significantly higher sensitivity to bigger particles when intensity is plotted against size since a small number of bigger particles may cause significant scattering. Therefore, analysis of particle number versus size is more informative of the actual trends in size distribution in the modified samples. It has to be stressed that the fundamental size distribution generated by DLS is an intensity distribution, which can be converted, using Mie theory, to a volume distribution, and further to a number distribution. However, number distributions are of minor use since small errors in gathering data for the correlation function will lead to significant errors in distribution by number. Thus, due to limitations in the application of the DLS techniques for both optically and geometrically anisotropic nanoparticles presenting non-homogeneous size distribution, and the influence of larger particles, DLS measurements of cellulose nanocrystals were applied for assessing the relative size trends among the prepared series, not the exact sizes and size distribution. On the other hand, due to fast measurements providing qualitative information, DLS was considered as a convenient method providing useful technological information. DLS analysis of untreated and modified CNCs water suspensions showed that both the CNC_P76 and modified cellulose nanocrystals showed a bimodal distribution of particle sizes by intensity. CNC_P76 was composed of small nanoparticles with apparent hydrodynamic diameter in the range of 100–150 nm and the second fraction in the submicronic range of c.a. 0.6–1.4 µm. Modified CNCs showed a small fraction at around 5–6 µm in addition to nanometric fraction. With the increasing molar ratio of succinic anhydride to the hydroxyl groups of cellulose, the sizes of cellulose nanocrystals decreased. The same trend was observed for increasing the time of reaction. However, no clear dependence of the reaction temperature on the particles sizes was found, which can be seen in Figure2. All modified samples, except the one modified at 90 ◦C, had smaller particle sizes of the nanometric fraction, with the hydrodynamic diameters in the range of 50–100 nm, compared to the sample after hydrolysis in the phosphoric acid, in which most of the particles had hydrodynamic diameters in the range of 100–150 nm. Such a result indicates that during the modification reaction of cellulose nanocrystals with succinic anhydride, further hydrolysis proceeds parallel to the esterification reaction. The microparticles were hardly visible when size distribution by a number of particles was considered. This can indicate a presence of a small amount of agglomerates or microparticles in the samples. The possible origin of the agglomerated particles is the freeze-drying of the produced CNC_P76 sample before succinylation. The proper drying was necessary for the exact control of cellulose mass, and thus, molar ratios during the esterification. A chemical crosslinking of particles after modification with the bifunctional succinic acid molecule is another considered effect that could lead to formation of bigger particles. The average length of nanoparticles gradually diminished with the increase of SA:OH molar ratio. When analysing the changes in size distribution versus reaction time, one can observe that CNC kept its original length after the first Polymers 2019, 11, 866 8 of 24

60 min of esterification and underwent the most significant variation in sizes after 120 min. A further

Polymersincrease 2019 in,esterification 11, x FOR PEER timeREVIEW had little effect on the CNC dimensions. 8 of 24

Figure 2. 2. ParticleParticle size size distribution distribution for forCNC_SA CNC_SA obtained obtained at (a at,b) ( adifferent,b) different SA:OH SA:OH molar molar ratios; ratios; (c,d) temperatures;(c,d) temperatures; and ( ande,f) time (e,f) timeof reaction of reaction by intensity by intensity and number, and number, respectively. respectively.

3.2. Structure Analysis by FTIR Figure 3 represents FTIR spectra of samples modified in various reaction conditions in comparison to the spectra of cellulose, hydrolysed in phosphoric acid solution. All spectra showed a characteristic broad band in the range of 3650–3000 cm−1, which is assigned to different O–H

Polymers 2019, 11, 866 9 of 24

3.2. Structure Analysis by FTIR Figure3 represents FTIR spectra of samples modified in various reaction conditions in comparison to the spectra of cellulose, hydrolysed in phosphoric acid solution. All spectra showed a characteristic 1 Polymersbroad band2019, 11 in, x theFOR rangePEER REVIEW of 3650–3000 cm− , which is assigned to different O–H stretching modes. 9 of 24 In cellulose I, a secondary hydroxyl group at the C3 and C2 position form a H-bond with an O5 and O6 1 stretchingatom of the modes. contiguous In cellulose ring, respectively. I, a secondary The firsthydroxyl one appears group at aroundthe C3 3342and C2 cm −position, while form the second a H- 1 1 bondone at with 3432 an cm O5− .and The O6 band atom at of 3277 the cmcontiguous− is characteristic ring, respectively. for cellulose The Ifirstβ and one is appears proportional at around to its −1 −1 −1 1 3342amount cm [52, while,53]. Thethe absorbancesecond one ofat methylene3432 cm . stretchingThe band bands,at 3277 visible cm is in characteristic the range of 2945–2857for cellulose cm Iβ− , andwas is shown proportional to slightly to increase its amount after [52,53]. modification The absorbance of cellulose of with methylene succinic acidstretching anhydride, bands, and visible thus can in thesuggest range a higher of 2945–2857 methylene cm−1 group, was content shown afterto slightly the esterification increase after reaction modification [54]. All spectraof cellulose displayed with 1 succinictypical peaks acid at anhydride, 1057 and 1162 and cm thus− corresponding can suggest to a C–O higher and C–O–C methylene stretching group vibration content in after cellulose, the 1 −1 esterificationrespectively [reaction55,56], and[54]. a All broad spectra peak displayed at 1643 cmtypical− can peaks be assigned at 1057 and to the 1162 bending cm corresponding vibrations of toadsorbed C–O and water. C–O–C stretching vibration in cellulose, respectively [55,56], and a broad peak at 1643 cm−1 canThe be partial assigned esterification to the bending of cellulose vibrations hydroxyl of adsorbed groups water. was evidenced on FTIR spectra, for all 1 modifiedThe partial samples, esterification by the presence of cellulose of the newhydroxyl band atgroups 1725 cmwas− evidenced. This absorption on FTIR band spectra, is related for all to modifiedC=O stretching samples, vibrations by the presence in ester bondsof the [new57,58 band]. Its at intensity 1725 cm increased−1. This absorption with the increasing band is related value to of C=Oall three stretching parameters, vibrations namely: in ester SA:OH bonds molar [57,58]. ratio, Its reaction intensity time increased and reaction with temperature,the increasing indicating value of alla higher three parameters, extent of CNC namely: esterification. SA:OH molar However, ratio, thereaction molar time ratio and of reagentsreaction temperature, and reaction timeindicating had a aprevailing higher extent effect of on CNC the esterification. esterification whilstHowever, the reactionthe molar temperature ratio of reagents showed and a reaction moderate time influence had a 1 prevailingon the intensity effect on of Cthe=O esterification stretching absorption whilst the band.reaction The temperature absence of showed bands at a 1860moderate and 1790 influence cm− , oncharacteristic the intensity for of succinic C=O stretching anhydride, absorption confirms thatband. the The product absence is free of frombands unreacted at 1860 and modifier 1790 [cm59].−1, characteristic for succinic anhydride, confirms that the product is free from unreacted modifier [59].

Figure 3. Cont.

Polymers 2019, 11, 866 10 of 24 Polymers 2019, 11, x FOR PEER REVIEW 10 of 24 Polymers 2019, 11, x FOR PEER REVIEW 10 of 24

FigureFigure 3. 3. FTIRFTIR spectra spectra of of CNCs CNCs modified modified with with succinic succinic anhydride anhydride at at various various (a (a,b,b) )SA:OH SA:OH molar molar ratios; ratios; ((cFigurec,,dd)) temperatures temperatures 3. FTIR spectra and and of( (ee, ,CNCsff)) times times modified of of reaction. reaction. with succinic anhydride at various (a,b) SA:OH molar ratios; (c,d) temperatures and (e,f) times of reaction. 3.3.3.3. XPS XPS Analysis Analysis of of Surface Surface Chemical Chemical Composition Composition of of CNCs CNCs 3.3. XPS Analysis of Surface Chemical Composition of CNCs XPSXPS spectra spectra of of CNC_P76 CNC_P76 after after hydrolysis hydrolysis revealed revealed peaks for peaks carbon for and carbon oxygen and but oxygen no phosphorous but no phosphorouswas detectedXPS spectra was (Figure detected of 4 CNC_P76). For (Figure this sample, after 4). For hydrolysis hydrolysisthis sample, revealed was hydrolysis carried peaks out was for under carried carbon conditions out and under oxygen (phosphoric conditions but no (phosphoricacidphosphorous concentration acid was concentrationdetected and temperature) (Figure and 4). that For temperature) werethis sample, suffi cient that hydrolysis for were the sufficient hydrolysis was carried for of out amorphous the under hydrolysis conditions regions of amorphouswithout(phosphoric the regions activation acid concentration without of cellulose the activation andphosphorylation. temperature) of cellulose The that phosphorylation. lack were of phosphate sufficient The groups for lack the bearing of hydrolysis phosphate surface of groupscharges,amorphous bearing on the regions surface as received without charges, nanocrystals, the on activationthe as may received alsoof cellulose explainnanocrystals, a phosphorylation. slight may tendency also explain to The form lacka aggregatesslight of phosphatetendency upon tofreeze-drying.groups form aggregatesbearing On surface theupon other charges, freeze-drying. hand, on the the absence asOn received the ofother phosphate nanocrystals, hand, the groups absence may was also of advantageous explain phosphate a slight groups in tendency terms was of advantageouspreservedto form aggregates thermal in terms stability upon of preserved freeze-drying. of CNC_P76, thermal asOn discussed stability the other of later. hand,CNC_P76, the absence as discussed of phosphate later. groups was 8 50 advantageous in terms of preservedO1s thermal stability of CNC_P76, as discussed later. 8 a Survey 50 b P2p Snap 7 O1s

) 45 5 67 a Survey b P2p Snap ) 10 45 5

 C1s 56 40 10

 C1s 45 O KL1 40 O KL2 35 34 O KL1

O KL3 Counts/s 3 C KL1 O KL2 35 Counts/s ( Counts/s 2

C KL1 O KL3 O2s 30Counts/s

Counts/s ( Counts/s 2 1 O2s 30 01 25 14000 1200 1000 800 600 400 200 0 25148 146 144 142 140 138 136 134 132 130 128 126 124 1400 1200 1000Binding800 energy/eV600 400 200 0 148 146 144 142Binding140 138 energy/eV136 134 132 130 128 126 124 Binding energy/eV Binding energy/eV Figure 4. XPS spectra of CNC_P76: (a) XPS wide scan patterns; (b) XPS spectra in the range of binding energiesFigureFigure 4.4. of XPS P 2p spectra peak. of CNC_P76: (a)) XPSXPS widewide scanscan patterns;patterns; ((bb)) XPSXPS spectraspectra inin thethe rangerange ofof bindingbinding energiesenergies ofof PP 2p2p peak.peak. The high degree of crystallinity, as determined from WAXD analysis and discussed later, indicatedTheThe highhigh that degree degreenanoparticles of crystallinity,of crystallinity, retained as determined as intact determined crystalline from WAXD from core. WAXD analysis Therefore, analysis and discussed changes and discussed later, in chemicalindicated later, composition,thatindicated nanoparticles that due nanoparticlesto retained succinylation intact retained crystalline of CNCs, intact core.are expected crystalline Therefore, to changesoccur core. at Therefore, intheir chemical surface. changes composition, XPS, as in a chemicalsurface due to sensitivesuccinylationcomposition, technique, ofdue CNCs, to was succinylation areapplied expected for ofevaluation toCNCs, occur are atof theirexpectedthe degree surface. to of occur esterification. XPS, at as their a surface surface. For sensitivecellulosic XPS, as technique,materials a surface itwassensitive collects applied technique, information for evaluation was from applied of the andegree for external evaluation of esterification. layer of withthe degree a For maximum cellulosic of esterification. materials width of For it 10 collects cellulosic nm [60],information materials while approximatelyfromit collects an external information 2/3 layer of the with fromsignal a maximum anoriginates external width from layer ofthe 10 with first nm a3 [ 60nm maximum], whileon the approximately surface width [61]. of 10 The 2 nm/3 applied of [60], the signal whileXPS method,originatesapproximately as from a surface 2/3 the of first sensitivethe 3 signal nm on technique, originates the surface allowedfrom [61 ].the The for first the applied 3 nm changes on XPS the in method, surfacechemical as[61]. constitution a surfaceThe applied sensitive of XPS an externaltechnique,method, layer as allowed a of surface nanocrystals for sensitivethe changes to be technique, determined. in chemical allowed However, constitution for the it has of changes an limitations external in chemical layerin terms of nanocrystalsconstitution of the evaluation toof be an ofdetermined.external molecular layer changes However, of nanocrystals in itthe has core limitationsto ofbe nanoparticles,determined. in terms However, e.g., of the exposure evaluation it has oflimitations further of molecular –OH in terms groups changes of the as inaevaluation result the core of disruption/delaminationofof nanoparticles,molecular changes e.g., exposurein theof the core crystalline of of further nanoparticles, –OHstructure. groups e.g., Carbon exposure as a result atoms, of of further disruptionlocated –OH in / differentdelaminationgroups as molecular a result of the of environments,crystallinedisruption/delamination structure. display Carbon varied of the atoms,C 1s crystalline binding located energies, structure. in different which Carbon molecular can atoms,be resolved environments, located from in the displaydifferent high variedresolution molecular C 1s XPSbindingenvironments, spectra energies, to give display which qualitative varied can be and C resolved 1s quantitative binding from energies, the data. high The which resolution binding can be energy XPS resolved spectra of the from tocarbon give the qualitativeatomhigh resolutionis shifted and toquantitativeXPS higher spectra values data.to give as Thethe qualitative bindingnumber energyandof bonds quantitative of with the carbon oxygen data. atom Theatoms binding is shiftedincreases. energy tohigher The of high-resolution the values carbon as theatom number carbon is shifted C of 1sto signal higher showed values asfour the constituent number of bondspeaks forwith all oxygen CNC samplesatoms increases. (Figure 5).The The high-resolution peak at ∼284.8 carbon eV is C connected1s signal showedto carbon four atoms constituent without peaksoxygen for bonds all CNC (in samplesCC or C(FigureH environment). 5). The peak The at ∼peaks284.8 witheV is connected to carbon atoms without oxygen bonds (in CC or CH environment). The peaks with

Polymers 2019, 11, x FOR PEER REVIEW 11 of 24 growing binding energy refer to carbon atoms with one (CO, at ∼286.5 eV), two (OCO/C=O, at ∼288.6 eV), and three oxygen bonds (OC=O, at ∼289.3 eV), respectively [62,63]. The C/O ratio was not influenced by the succinylation since the modifier introduces the same number of carbon and oxygen atoms (Table 2).

Table 2. Atomic concentrations from XPS spectra and a resolved carbon C 1s peak for alkali treated cellulose (Avicel NaOH), CNCs obtained in phosphoric acid solution with concentration of 76 wt.% (CNC_P76) and surface modified CNCs at different molar ratio of succinic anhydride to cellulose Polymershydroxyl2019, groups.11, 866 11 of 24 Resolved C 1s peak Surface C/O Sample O [%] C [%] C–O C=O/O–C–O O–C=O substitution bonds with oxygen atoms increases.[%] TheC–H high-resolutionX [%] carbon C 1s signal showed four constituent [%] [%] [%] degree (SSD) peaks for all CNC samples (Figure5). The peak at 284.8 eV is connected to carbon atoms without Avicel_NaOH 43.0 57.0 1.3 3.9 ∼ 41.2 9.9 1.9 0.12 oxygenCNC_P76 bonds (in 42.0 C–C or C–H58.0 environment). 1.4 6.3 The peaks 41.1 with growing 9.1 binding 1.4 energy refer 0.08 to carbon atomsCNC_SA2:1 with one (C–O, 41.5 at 56.6286.5 eV), 1.4 two (O–C–O 6.4 /C=O, 40.9 at 288.6 eV),8.4 and three 1.9 oxygen bonds 0.12 (O–C=O, ∼ ∼ atCNC_SA3:1289.3 eV), respectively 41.9 56.6 [62 ,63]. 1.4 The C/O 6.7 ratio was 40.2 not influenced 8.9 by the 1.6 succinylation 0.10 since the ∼ modifierCNC_SA5:1 introduces 43.0 the same 56.5 number 1.3 of carbon 3.8 and 39.9 oxygen atoms 9.2 (Table2). 3.7 0.23 CNC_SA10:1 42.6 56.7 1.3 4.4 39.7 8.0 5.1 0.32

3000 2500 Experimental data Experimental data Envelope 2500 Envelope a b C-Hx, C-C 2000 C-Hx, C-C C-O 2000 C-O O-C-O O-C-O 1500 O-C=O O-C=O 1500 1000 1000 Counts/s (Residuals x (Residuals x Counts/s 5) Counts/s (Residuals x (Residuals x Counts/s 5) 500 500

0 0 292 290 288 286 284 282 292 290 288 286 284 282 Binding energy/eV Binding energy/eV

2500 3000 Experimental data Experimental data Envelope Envelope c 2500 d 2000 C-Hx, C-C C-Hx, C-C C-O C-O O-C-O 2000 O-C-O 1500 O-C=O O-C=O 1500 1000 1000 Counts/s (Residuals x (Residuals x Counts/s 5) (Residuals x Counts/s 5) 500 500

0 0 292 290 288 286 284 282 292 290 288 286 284 282 Binding energy/eV Binding energy/eV

Figure 5. Resolved carbon C 1s peak for (a) CNC_P76 and surface modified CNCs at increasing Figure 5. Resolved carbon C 1s peak for (a) CNC_P76 and surface modified CNCs at increasing molar molar ratio of succinic anhydride to cellulose hydroxyl groups: (b) CNC_SA3:1; (c) CNC_SA5:1; ratio of succinic anhydride to cellulose hydroxyl groups: (b) CNC_SA3:1; (c) CNC_SA5:1; and (d) and (d) CNC_SA10:1. CNC_SA10:1. Table 2. Atomic concentrations from XPS spectra and a resolved carbon C 1s peak for alkali treated Thecellulose indicative (Avicel data NaOH), concerning CNCs the obtained degree in of phosphoric esterification acid solutionwas gathered with concentration from the resolved of 76 wt.% C 1s peak that(CNC_P76) displayed and an surfaceincreased modified percentage CNCs contribution at different molar of the ratioOC=O of succinic carbons anhydride in the modified to cellulose CNC due to hydroxylthe formation groups. of an ester bond. The determined surface substitution degree (SSD) tended to increase with growing molar excess of succinic anhydride. Interestingly, the trends in obtained substitution degrees were comparable to those reportedResolved by Liu C et1s Peak al. and Shang et al.Surface for the Sample O [%] C [%] C/O [%] Substitution homogeneous succinylation of cellulose in C–H ionicX liquidsC–O withoutC=O/O–C–O catalystO–C [64,65].=O Generally, [%] [%] [%] [%] Degree (SSD) Avicel_NaOH 43.0 57.0 1.3 3.9 41.2 9.9 1.9 0.12 CNC_P76 42.0 58.0 1.4 6.3 41.1 9.1 1.4 0.08 CNC_SA2:1 41.5 56.6 1.4 6.4 40.9 8.4 1.9 0.12 CNC_SA3:1 41.9 56.6 1.4 6.7 40.2 8.9 1.6 0.10 CNC_SA5:1 43.0 56.5 1.3 3.8 39.9 9.2 3.7 0.23 CNC_SA10:1 42.6 56.7 1.3 4.4 39.7 8.0 5.1 0.32

The indicative data concerning the degree of esterification was gathered from the resolved C 1s peak that displayed an increased percentage contribution of the O–C=O carbons in the modified CNC due to the formation of an ester bond. The determined surface substitution degree (SSD) Polymers 2019, 11, 866 12 of 24 tended to increase with growing molar excess of succinic anhydride. Interestingly, the trends in obtained substitution degrees were comparable to those reported by Liu et al. and Shang et al. for the homogeneous succinylation of cellulose in ionic liquids without catalyst [64,65]. Generally, homogeneousPolymers 2019, 11 esterification, x FOR PEER REVIEW of cellulose by succinic anhydride with catalyst resulted in significantly12 of 24 higher substitution degree [66]. In the case of succinylated nanocellulose, SSD values reported for homogeneous esterification of cellulose by succinic anhydride with catalyst resulted in significantly esterified cellulose nanofibers took values comparable to our results [9,67]. It is believed that cellulose higher substitution degree [66]. In the case of succinylated nanocellulose, SSD values reported for macromolecules with a high degree of substitution have reduced the possibility of hydrogen bonding in esterified cellulose nanofibers took values comparable to our results [9,67]. It is believed that cellulose ordered crystal structures and tend to dissolve in solvent. Yuan et al. modified cellulose whiskers with macromolecules with a high degree of substitution have reduced the possibility of hydrogen bonding less reactive alkenyl succinic anhydride (ASA) and the degree of substitution (DS) value, evaluated in ordered crystal structures and tend to dissolve in solvent. Yuan et al. modified cellulose whiskers onwith the lessbasis reactive of XPS alkenyl data, was succinic 0.097 asanhydride opposed (ASA) to 0.0158, and determinedthe degree of by substitution elemental analysis (DS) value, [68]. Theyevaluated demonstrated on the basis a more of XPS adequate data, was determination 0.097 as opposed of surface to 0.0158, DS by XPSdetermined due to the by localelemental concentration analysis of[68]. ester They groups demonstrated on the surface a more of nanocrystals. adequate determination of surface DS by XPS due to the local concentration of ester groups on the surface of nanocrystals. 3.4. Crystalline Structure of CNC 3.4.Figure Crystalline6 presents Structure the of WAXD CNC di ffraction patterns of the raw material and representative modified samples. All the samples showed five crystalline peaks at 14.3 , 16.4 , 20.0 , 22.5 and 34.5 which Figure 6 presents the WAXD diffraction patterns of the◦ raw◦ material◦ and◦ representative◦ corresponds to the 101, 10¯ı, 021, 002 and 040 Miller indices, characteristic for crystallographic planes modified samples. All the samples showed five crystalline peaks at 14.3°, 16.4°, 20.0°, 22.5° and 34.5° ofwhich the monoclinic corresponds cellulose to the 101, I lattice 10ī, 021, [48– 00250]. and The 040 broad Miller reflection indices, withcharacteristic the maximum for crystallographic at about 27.0 ◦ corresponds to the amorphous phase in the samples.

FigureFigure 6. 6.Representative Representative deconvoluted deconvoluted WAXDWAXD patternspatterns ofof rawraw Avicel, cellulose after alkali alkali treatment, treatment, CNCCNC obtained obtained during during hydrolysis hydrolysis in in phosphoric phosphoric acid acid solution solution (CNC_P76), (CNC_P76), and modifiedmodified CNC (CNC_SA5:1).(CNC_SA5:1).

OneOne of of the the parameters parameters whichwhich influencesinfluences thethe yieldyield ofof thethe hydrolysis,hydrolysis, apart from the time and and temperature,temperature, is is the the acid acid concentration concentration [17 [17,69].,69]. Changes Changes in the crystallinity degree degree of of the CNC-P samples (Figure 7a) were small, for low concentrations of phosphoric acid solutions. The hydrolysis of amorphous domains was promoted when optimal acid concentration was achieved. The highest content of the crystalline fraction was observed for the sample hydrolysed in phosphoric acid with a concentration of 76 wt.%. Further increasing of the acid concentration resulted in a decrease of the crystallinity index. Hydrolysis of cellulose in acid solutions with a concentration above the optimal Polymers 2019, 11, x FOR PEER REVIEW 13 of 24 value, leads to the decrease of crystalline phase content as a result of hydrolysis of 1,4-β-glycosidic bonds in crystalline domains [4]. Moreover, level-off degree of polymerisation, which is obtained after the reduction of cellulose polymerisation degree by acid hydrolysis, correlates with the periodic crystal sizes. Severe reaction conditions may cause a significant decrease in the degree of polymerisation, and thus a decrease of crystal sizes [70,71]. The dependence of the crystallinity degree on the amount of succinic acid anhydride (Figure 7b) and reaction temperature (Figure 7c) was investigated in the course of CNC esterification. With the increasing values of both parameters the content of amorphous phase in the samples decreased. Such results indicate that mild hydrolysis proceeds during the modification reaction and these observations are in accordance with DLS analysis. After exceeding the optimal amount of the modifier (SA:OH 5:1), the crystalline phase content slightly decreased. However, there is no clear dependence of the crystallinity degree and the time of the reaction (Figure 7d). This effect can result fromPolymers a 2019 high, 11 , rate 866 of hydrolysis in the initial stage of the reaction, when more easily available13 of 24 disordered domains are being attacked by the molecules of the modifier. The rate of hydrolysis decreases when the hydrolysis catalyst attacks the remaining, more resistant, crystalline domains samples (Figure7a) were small, for low concentrations of phosphoric acid solutions. The hydrolysis [17]. The highest content of the crystalline phase (around 78%) was observed for the CNC_SA5:1 of amorphous domains was promoted when optimal acid concentration was achieved. The highest sample. In the course of CNC succinylation two chemical processes can be identified that influence content of the crystalline fraction was observed for the sample hydrolysed in phosphoric acid with a the CNC crystal structure: (i) esterification of hydroxyl groups along with (ii) hydrolysis of 1,4-β- concentration of 76 wt.%. Further increasing of the acid concentration resulted in a decrease of the glycosidic bonds. As the molar ratio of SA:OH achieved the value of 10:1 the highest concentration crystallinity index. Hydrolysis of cellulose in acid solutions with a concentration above the optimal of acidic catalyst could activate more extended hydrolysis of crystalline domains, thus lowering the value, leads to the decrease of crystalline phase content as a result of hydrolysis of 1,4-β-glycosidic cellulose crystallinity. Furthermore, the decreased crystallinity at the highest molar ratio can be bonds in crystalline domains [4]. Moreover, level-off degree of polymerisation, which is obtained after correlated with the increasing esterification degree, as determined by the XPS analysis. This resulted the reduction of cellulose polymerisation degree by acid hydrolysis, correlates with the periodic crystal in higher concentration of side chains that interfered the regular packing of cellulose macromolecules sizes. Severe reaction conditions may cause a significant decrease in the degree of polymerisation, in crystals. The crystalline structure of CNC was not compromised by the extensive esterification and and thus a decrease of crystal sizes [70,71]. hydrolysis up to SA:OH molar ratio 5:1. 80 80 a b 75 75

70 70

65 65

60 Crystallinitydegree/% Crystallinitydegree/% 60 68 70 72 74 76 78 80 2:1 3:1 5:1 10:1

H3PO4 concentration/%mas. Molar ratio SA:OH/mol:mol

80 80 c d 75 75

70 70

65 65 Crystallinitydegree/% 60 Crystallinitydegree/% 60 60 70 80 90 100 110 120 60 120 180 240 300 Temperature/°C Time/min

FigureFigure 7. CrystallinityCrystallinity degree degree determined determined by by deconvolution deconvolution of of WAXD WAXD patterns patterns of of ( (aa)) CNC_P CNC_P series series hydrolysedhydrolysed in in phosphoric phosphoric acid acid solutions solutions at at different different concentrations; concentrations; as as well well as as CNC succinylated at at differentdifferent (b)) SA:OH molar ratios; ratios; ( (c)) temperature and ( d) time. Dashed Dashed line line depicts depicts the the crystallinity crystallinity indexindex of of Avicel_Raw Avicel_Raw material. The dependence of the crystallinity degree on the amount of succinic acid anhydride (Figure7b) and reaction temperature (Figure 7c) was investigated in the course of CNC esterification. With the increasing values of both parameters the content of amorphous phase in the samples decreased.

Such results indicate that mild hydrolysis proceeds during the modification reaction and these observations are in accordance with DLS analysis. After exceeding the optimal amount of the modifier (SA:OH 5:1), the crystalline phase content slightly decreased. However, there is no clear dependence of the crystallinity degree and the time of the reaction (Figure7d). This e ffect can result from a high rate of hydrolysis in the initial stage of the reaction, when more easily available disordered domains are being attacked by the molecules of the modifier. The rate of hydrolysis decreases when the hydrolysis catalyst attacks the remaining, more resistant, crystalline domains [17]. The highest content of the crystalline phase (around 78%) was observed for the CNC_SA5:1 sample. In the course of CNC succinylation two chemical processes can be identified that influence the CNC crystal structure: (i) esterification of hydroxyl groups along with (ii) hydrolysis of 1,4-β-glycosidic bonds. As the molar ratio of SA:OH achieved the value of 10:1 the highest concentration of acidic catalyst could activate more extended hydrolysis of crystalline domains, thus lowering the cellulose crystallinity. Furthermore, the decreased Polymers 2019, 11, 866 14 of 24 crystallinity at the highest molar ratio can be correlated with the increasing esterification degree, as determined by the XPS analysis. This resulted in higher concentration of side chains that interfered the regular packing of cellulose macromolecules in crystals. The crystalline structure of CNC was not compromised by the extensive esterification and hydrolysis up to SA:OH molar ratio 5:1. Polymers 2019, 11, x FOR PEER REVIEW 14 of 24 3.5. Microscopic Analysis of Cellulose Nanocrystals Morphology 3.5. Microscopic Analysis of Cellulose Nanocrystals Morphology SEM photomicrographs of cellulose material obtained by hydrolysis in 76 wt.% phosphoric acid solutionSEM displayed photomicrographs microporous of cellulose aerogel material morphology. obtained The by material hydrolysis was in composed 76 wt.% phosphoric of fibrous acid and ribbon-likesolution displayed aggregates microporous marked on aerogel the photomicrograph morphology. The with material arrows was (Figure composed8a). The of ribbons fibrous andand membranesribbon-like aggregates were formed marked by agglomerated on the photomicrograph fibrous structures with witharrows nanometric (Figure 8a). diameters. The ribbons Since and no signsmembranes of esterification were formed by phosphoric by agglomerated acid were fibrous evidenced structures for this with sample nanometric by FTIR diameters. and XPS Since analysis, no thesigns produced of esterification CNC were by phosphoric not protected acid from were aggregation evidenced by for repulsive this sample interactions. by FTIR and XPS analysis, the producedAdditionally, CNC Zhang were not et al. protected have investigated from aggregation the influence by repulsive of the acid interactions. type on the morphology of celluloseAdditionally, nanocrystals, Zhang and et showed al. have that investigated during the the hydrolysis influence inof phosphoricthe acid type acid, on the less morphology hydrogen ions of attackcellulose amorphous nanocrystals, regions, and andshowed thus that hydrolysis during the in H hydrolysis3PO4 results in phosphoric in the production acid, less of largerhydrogen particles ions thanattack those amorphous from commonly regions, and used thus sulphuric hydrolysis acid [in14 H].3PO4 results in the production of larger particles than Betterthose from individualisation commonly used of the sulphuric nanoparticles acid [14]. was observed after their modification with succinic anhydrideBetter (Figureindividualisation8c–j). The presence of the nanoparticles of succinate was groups observed on the after surface their of modification nanocrystals playedwith succinic a role inanhydride structure (Figure formation 8c–j). of The aerogel, presence probably of succinate by steric groups hindrance on the and surface repulsive of nanocrystals interactions played of negatively a role chargedin structure carboxylic formation end groups of aerogel, that limited probably agglomeration. by steric hindrance and repulsive interactions of negativelyReduction charged of the carboxylic agglomeration end groups process that was limited additionally agglomeration. supported by the use of tert-butanol whileReduction freeze-drying, of the which agglomeration can provide process steric hindrance was additionally and prevent supported hydrogen by bond the use formation of tert-butanol between nanoparticles.while freeze-drying, It results which from canthe structure provide of steric the tert-butanol hindrance and molecule, prevent which hydrogen consists bond of three formation methyl andbetween one hydroxylnanoparticles. groups, It andresults can from form hydrogenthe structure bond of withthe tert-butanol only one water molecule, molecule which or one consists hydroxyl of groupthree methyl on the and surface one of hydroxyl CNC [46 groups,,72]. and can form hydrogen bond with only one water molecule or oneMorphological hydroxyl group studies on the of surface freeze-dried of CNC modified[46,72]. cellulose nanocrystals showed that in the micrometricMorphological scale they studies take of a formfreeze-dried of loose modified aerogel or cellulose powder nanocrystals with highly showed developed that surface in the areamicrometric (Figure 8scalec,e,g,i). they The take irregularly a form of loose shaped aerogel microparticles or powder were with composedhighly developed of high surface number area of sub-micrometre(Figure 8c,e,g,i). membranes, The irregularly ribbons shaped and nanofibers microparticles (Figure were8d,f,h,j). composed The flat of elongated high number agglomerates, of sub- amicrometre few micrometres membranes, in length ribbons and diameter and nanofibers on the nanoscale, (Figure 8d,f,h,j). become lessThe dominant flat elongated while agglomerates, the nanofibrillar a aggregatesfew micrometres became in prevailing length and as the diameter molar on ratio the of nanoscale, the succinic become anhydride less to dominant cellulose while hydroxyl the groupsnanofibrillar increased. aggregates The sample became CNC_SA10:1 prevailing as showed the molar basically ratio of nanofibrous the succinic morphology anhydride to (Figure cellulose8j). Suchhydroxyl results groupsare in increased. accordance The with sample the CNC CNC_SA10:1 characteristic showed dimensions basically given nanofibrous by Moon morphology et al. [73]. Moreover,(Figure 8j). theSuch presence results are of in particles accordance with with nanometric the CNC and characteristic submicronic dimensions dimensions given was by observed Moon et foral. [73].all the Moreover, samples, butthe with presence the increasing of particles molar with ratio nanometric of SA:OH, and the submicronic contribution dimensions of submicronic was particlesobserved decreased. for all the samples, but with the increasing molar ratio of SA:OH, the contribution of submicronic particles decreased. a b

CNC_P76

Figure 8. Cont.

Polymers 2019, 11, 866 15 of 24 Polymers 2019, 11, x FOR PEER REVIEW 15 of 24

c d

CNC_SA2:1 e f

CNC_SA3:1 g h

CNC_SA5:1 i j

CNC_SA10:1

Figure 8. SEM images of ( a,,b) hydrolysed; and modified modified cellulose nanocrystals nanocrystals with with different different molar ratios of SA:OH: ( c,d) CNC_SA2:1; (e,f) CNC_SA3:1; (g,h) CNC_SA5:1 andand ((ii,,jj)) CNC_SA10:1.CNC_SA10:1.

Polymers 2019, 11, 866 16 of 24 Polymers 2019, 11, x FOR PEER REVIEW 16 of 24

AFM scans scans of of CNCs CNCs dried dried on air on showed air showed similar morphology similar morphology before and before after surface and after modification surface (Figuremodification9). Both (Figure esterified 9). Both and pristineesterified CNCs and hadpristine tendency CNCs to had form tendency randomly to orientedform randomly agglomerates oriented of alignedagglomerates nanoparticles. of aligned The nanoparticles. typical areas The of paralleltypical celluloseareas of parallel nanocrystals cellulose forming nanocrystals aggregates forming were markedaggregates with were rectangles marked onwith the rectangles phase images on the (Figure phase 9imagesd,g). The (Figure nanocrystals 9d,g). The showed nanocrystals a tendency showed to aligna tendency along to the align long along edges the and long pack edges tightly and when pack dried tightly on when air. The dried AFM on observations air. The AFM revealed observations strong surfacerevealed tension strong of surface CNCs leading tension to of agglomeration CNCs leading regardless to agglomeration of the surface regardless modification. of the That surface shows themodification. necessity to That apply shows special the drying necessity procedure, to apply such special as freeze-drying drying procedure, with addition such as freeze-drying of organic solvents, with inaddition order toof preserveorganic solvents, the nanoparticulate in order to morphologypreserve the ofnanoparticulate the produced CNCs.morphology The average of the produced diameter ofCNCs. the nanocrystalsThe average diameter in the referential of the nanocrystals CNC-P76 in sample the referential was 33.0 CNC-P76 nm with sample a standard was 33.0 deviation nm with of 9.4a standard nm, while deviation for succinylated of 9.4 nm, CNC while (CNC_SA5:1) for succinylated those parameters CNC (CNC_SA5:1) took values those of 29.2 parameters nm and 9.5 took nm, respectively.values of 29.2 Esterification nm and had9.5 anm, moderate respectively. influence Esterification on the nanocrystals had a diameters. moderate A influence slight decrease on the in thenanocrystals dimensions diameters. of succinylated A slight CNCs, decrease that was in evidenced the dimensions by AFM of and succinylated DLS measurements, CNCs, indicatedthat was mildevidenced hydrolysis by AFM of cellulose and DLS occurring measurements, at the surface indicated of nanoparticlesmild hydrolysis in theof cellulose course of occurring esterification. at the surface of nanoparticles in the course of esterification.

Figure 9. Cont.

Polymers 2019, 11, 866 17 of 24 Polymers 2019, 11, x FOR PEER REVIEW 17 of 24

Figure 9. 9. RepresentativeRepresentative AFM AFM topography topography and and phase phase images images of CNC-P76 of CNC-P76 at scanning at scanning areas of areas (a,b) of 2 (×a ,2b μm,) 2 and2 µm (c,, andd) 500 (c, d×) 500 500 nm,500 and nm, (f and,g) CNC-SA (f,g) CNC-SA 5:1 at 5:1scanning at scanning area areaof 500 of × 500 500 nm500 nmsamples; samples; (e) × × × (cross-sectionale) cross-sectional profiles profiles taken taken from from the the CNC-P76 CNC-P76 topography topography image. image.

3.6. Studies of Pyrolytic and Thermo-Oxidative Degradation of CNCs Obtained by Hydrolysis and Surface Surface Esterification Esterification Thermogravimetric analysis was performed in the temperature range of 30–600 C for both argon Thermogravimetric analysis was performed in the temperature range of 30–600◦ C for both and air atmospheres. Figure 10 presents TG and DTG curves for modified samples recorded in synthetic argon and air atmospheres. Figure 10 presents TG and DTG curves for modified samples recorded in air atmospheres. The TGA curves on the graphs were presented in narrower temperature range for synthetic air atmospheres. The TGA curves on the graphs were presented in narrower temperature better presentation of differences in the course of mass loss during the main steps of degradation. range for better presentation of differences in the course of mass loss during the main steps of The characteristic degradation parameters of all cellulosic materials are shown in Table3. The thermal degradation. The characteristic degradation parameters of all cellulosic materials are shown in Table stability of cellulose hydrolysed in phosphoric acid with different concentrations showed, that the acid 3. The thermal stability of cellulose hydrolysed in phosphoric acid with different concentrations concentration does not have a significant influence on the thermal stability of CNCs prepared from showed, that the acid concentration does not have a significant influence on the thermal stability of Avicel, until the optimum concentration (76 wt.%) is exceeded. CNCs prepared from Avicel, until the optimum concentration (76 wt.%) is exceeded. Table 3. Parameters of pyrolytic and thermo-oxidative degradation of cellulose materials. Table 3. Parameters of pyrolytic and thermo-oxidative degradation of cellulose materials. Pyrolytic Degradation Thermooxidative Degradation Pyrolytic degradation Thermooxidative degradation Sample T5%, ◦C Tonset, ◦C Tmax, ◦C Char % * T5%, ◦C Tonset, ◦C Tmax, ◦C Char % * Sample T5%, °C Tonset, °C Tmax, °C Char %* T5%, °C Tonset, °C Tmax, °C Char %* 0.4 ◦C 2 ◦C 2 ◦C at 600 ◦C 0.4 ◦C 2 ◦C 2 ◦C at 600 ◦C ± ± 0.4 °C ± ± 2 °C ± ± 2 °C at 600°C ± ± 0.4 °C ±± 2 °C ±± 2 °C at 600°C Avicel-Raw 300.7 314 335 8.13 293.0 303 320 2.10 Avicel-Raw 300.7 314 335 8.13 293.0 303 320 2.10 Avicel-NaOH 281.6 335 363 10.72 290.0 320 333 1.84 Avicel-NaOH 281.6 335 363 10.72 290.0 320 333 1.84 CNC_P70 285.6 338 365 10.44 294.2 315 327 2.15 CNC_P70 285.6 338 365 10.44 294.2 315 327 2.15 CNC_P74 288.9 336 357 7.30 297.0 315 327 3.46 CNC_P76CNC_P74 285.6 288.9 332 336 354 357 4.60 7.30 299.0 297.0 314 315 327 2.843.46 CNC_P78CNC_P76 233.6 285.6 300 332 338 354 14.18 4.60 217.0 299.0 247 314 308327 14.57 2.84 CNC_SA2:1CNC_P78 286.4 233.6 340 300 367 338 7.23 14.18 285.1 217.0 316 247 330308 14.57 8.70 CNC_SA3:1CNC_SA2:1 262.4 286.4 334 340 367 367 6.87 7.23 275.1 285.1 311 316 328330 4.908.70 CNC_SA5:1CNC_SA3:1 260.1 262.4 342 334 373 367 6.57 6.87 270.6 275.1 311 311 326328 2.804.90 CNC_SA10:1CNC_SA5:1 258.1 260.1 327 342 358 373 5.56 6.57 263.4 270.6 304 311 322326 2.982.80 CNC_CNC_SA10:1 70 ◦C 260.4 258.1 331 327 364 358 7.50 5.56 261.0 263.4 312 304 325322 2.052.98 CNC_CNC_ 80 70◦C °C 277.1 260.4 340 331 364 364 5.04 7.50 275.6 261.0 314 312 328325 4.462.05 CNC_ 90 C 269.1 333 361 7.26 264.9 309 324 3.04 CNC_ 80◦ °C 277.1 340 364 5.04 275.6 314 328 4.46 CNC_ 100 ◦C 282.9 340 371 6.86 269.1 316 330 3.78 CNC_CNC_ 110 90◦C °C 260.1 269.1 342 333 373 361 6.57 7.26 270.6 264.9 311 309 326324 2.803.04 CNC_CNC_ 120 100 min °C 260.1 282.9 342 340 373 371 6.57 6.86 270.6 269.1 311 316 326330 2.803.78 CNC_CNC_ 180 110 min °C 260.9 260.1 334 342 365 373 6.37 6.57 270.1 270.6 306 311 321326 1.442.80 CNC_ 240 min 212.1 330 356 7.68 220.9 300 322 0.66 CNC_ 120 min 260.1 342 373 6.57 270.6 311 326 2.80 * The relative error in the measurement of char residue is c.a. 0.003. CNC_ 180 min 260.9 334 365 6.37 270.1 306 321 1.44

CNC_ 240 min 212.1 330 356 7.68 220.9 300 322 0.66 *the relative error in the measurement of char residue is c.a. 0.003.

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FigureFigure 10. 10.TG TG and and DTG DTG curves curves of modified of modified cellulose nanocrystalscellulose nanocrystals during thermo-oxidative during thermo-oxidative degradation. degradation. It was demonstrated that CNC bore no phosphate groups when produced in phosphoric acid at concentrationIt was demonstrated of 76 wt.% (Figure that CNC4a,b) bore which no phosphate was crucial groups for preserving when produced the good in thermal phosphoric stability acid of at CNC.concentration Increasing of of76 thewt.% phosphoric (Figure 4a,b) acid which concentration was crucial from for 76 preserving wt.% to 78 the wt.% good caused thermal a tremendous stability of decreaseCNC. Increasing of the initial of the degradation phosphoric temperature acid concentration (T5%) from from 285.6 76 wt.%◦C to to 233.6 78 wt.%◦C (by caused 52 ◦C) a (Figuretremendous 11a). Thisdecrease observation of the initial allows degradation the determination temperature of hydrolysis (T5%) from parameters 285.6 °C to that 233.6 preserve °C (by the 52 thermal°C) (Figure stability 11a). ofThis cellulose observation nanocrystals allows on the the determination initial level, and of hydrolysis is crucial for parameters the foreseen that application preserve of the CNC thermal as a fillerstability for engineeringof cellulose biopolymersnanocrystals withon the processing initial level, temperature and is crucial over 230 for◦ theC. foreseen application of CNC as a filler for engineering biopolymers with processing temperature over 230 °C. The application of Avicel microcrystalline cellulose as a raw material allowed to produce thermally stable cellulose nanocrystals by the phosphoric acid catalysed hydrolysis. The outcome from our earlier work [4], where a different raw material was used, and current results demonstrate the effect of raw material on the thermal stability of hydrolysis product. When UFC 100 was used as

Polymers 2019, 11, 866 19 of 24 Polymers 2019, 11, x FOR PEER REVIEW 20 of 24

a b 310 310 290 290 C ° / C

° 270 270 5% / T 5%

T 250 250 230 230 210 210 68 70 72 74 76 78 80 2:1 3:1 5:1 10:1

H3PO4 concentration/%mas. Molar ratio SA:OH/mol:mol c d 310 310 290 290 C C

° 270 270 ° / / 5% 5%

T 250 250 T 230 230 210 210 60 70 80 90 100 110 120 60 120 180 240 300 Temperature/°C Time/min

Figure 11. 11. DependenceDependence of the of initial the initial temperature temperature of thermo-oxidative of thermo-oxidative degradation degradation (T5%) of cellulose (T5%) of nanocrystalscellulose nanocrystals (a) obtained (a) obtained during hydrolysis during hydrolysis at different at di ff concentrationserent concentrations of phosphoric of phosphoric acid; acid; and succinylatedand succinylated cellulose cellulose nanocrystals nanocrystals obtained obtained at increasing at increasing (b) ( bSA:OH) SA:OH molar molar ratio, ratio, (c (c) )temperature, temperature, and ( d) time of reaction. Dashed Dashed line depicts the T 5% valuevalue for for the the CNC_P76 CNC_P76 nanocrystals nanocrystals subjected subjected to surface esterification. esterification.

The application of Avicel microcrystalline cellulose as a raw material allowed to produce thermally The overall characteristics of produced CNCs, that are considered as a potential filler for bioplastics,stable cellulose indicated nanocrystals a critical by phosphoric the phosphoric acid concentration acid catalysed during hydrolysis. hydrolysis The (76 outcome wt.%) as from well our as earlier work [4], where a different raw material was used, and current results demonstrate the effect of SA:OH molar ratio (5:1) in the course of esterification. Exceeding the critical parameters resulted in reductionraw material of thermal on the thermal stability stability required of hydrolysisfor composite product. processing. When UFCThe decrease 100 was usedin thermal as a raw stability material of the reduction of thermal stability was already observed at the phosphoric acid concentration of 74 wt.% produced CNCs could arise from both chemical and structural changes of cellulose, such as incorporationdespite the product of significant was hydrolysed amount of at less lower thermally temperature stable (90 ester◦C) groups and preserved of inorganic itsmicrocrystalline or organic acid, extensivemorphology. hydrolysis The initial as well temperature as reduction of in degradation crystallinity. (T5%) On the for other this hand material CNCs was succinylated 219.5 ◦C. On at the highestother hand, SA:OH for themolar Avicel ratio cellulose 10:1, offered used inthe this finest work nanoporous the initial temperature morphology, of as degradation revealed by took SEM, the combinedsignificantly with higher the value highest of 285.6 substitution◦C for the degree, sample evidenced hydrolysed by in XPS 76 wt.% and FTIRphosphoric analysis, acid and solution well preservedand at 100 ◦crystallinity.C. Different commercialThis material microcrystalline would fulfil cellulosesrequirements used of as functional a feedstock applications, showed variation among in others,thermal filtration stability ofmembranes, hydrolysis sorbents, products probablysensors, drug due tocarriers, different etc. natural The highest origin andapplied processing esterification history. temperatureAll modified (110 C) samples could be were recommended characterised for by the slightly synthesis lower of succinylated thermal stability CNCs. as The compared increase of to temperaturecellulose nanocrystals had no significant from 76 wt.%effect phosphoric on the thermal acid. stability The investigations of whiskers onwhile the it influence promoted of growth SA:OH ofmolar CNCs’ ratio crystallinity. on thermo-oxidative High crystallinity, and pyrolytic and thus degradation intrinsic of stiffness, cellulose along nanocrystals with high showed aspect thatratio with are desiredthe increasing features amounts of the reinforcing of succinic nanofiller. anhydride, Generally, the thermal the stability selection of samplesof manufacturing decreased parameters in both air wouldand argon depend atmospheres. strictly on The the systematicforeseen application decrease of of T 5%CNCswith and the demanded increasing essential SA:OH molar properties. ratio in the course of thermo-oxidative degradation is presented in Figure 11b. The onset temperature of pyrolytic 4.degradation Conclusions (T onset) for the CNC_SA2:1 sample was 316.3 ◦C, while for CNC_SA10:1 it decreased to 303.6 ◦C. According to Sehaqui et al., the lower thermal stability of carboxylated CNC samples may resultCellulose from their nanocrystals higher surface were area, produced which resultsby hydrolysis in a lower in resistancephosphoric to acid thermal solutions degradation at different [35]. concentrations.There was Theno clear optimal dependence acid concentration of thermal that resistance allowed of modifiedeffective hydrolysis cellulose nanocrystals of amorphous on regions, without activation of cellulose phosphorylation, was found at 76 wt.%. The nanocrystals the reaction temperature (Figure 11c), however, the highest thermal stability, with regards to Tonset, produced in optimal conditions had a high crystallinity index of 74% and preserved thermal stability. was observed for the sample modified at 100 ◦C. The onset temperature for this sample was 340.1 ◦C The influence of reaction time, temperature, and molar ratio of the reagents in the course of and 315.5 ◦C in argon and air atmospheres, respectively. Investigations of the influence of reaction time heterogeneouson the thermal behaviouresterification of succinylated of cellulose nanocrystals CNC showed by that succinic there areanhydride no significant was further differences investigated. between The surface esterification of CNC was confirmed by FTIR and XPS. The partial hydrolysis of cellulose, along with the esterification reaction, was indicated by the reduction of average particle size from the length of approximately 100 nm for CNC_P76 to 60 nm for the succinylated CNC. The surface

Polymers 2019, 11, 866 20 of 24 the thermal stability of samples modified for 120 and 180 min. However, when the reaction was carried out for 240 min, the resulting sample had apparently lower values of T5% as compared to samples modified at shorter reaction times (Figure 11d). The variation of initial temperature of degradation (T5%) indicated that succinylation generally caused a decrease of CNC thermal stability, especially in an oxidative atmosphere. However, the highest reduction of thermal stability, with substantial mass loss starting at around 200 ◦C, was observed for the longest reaction time (240 min). The stability of modified nanocrystals was higher in an inert atmosphere. This observation has a practical meaning for the application of modified CNC as a filler in polymer nanocomposites. The degradation can be prevented by feeding the nanofiller to an already melted polymer. This could reduce the exposure of CNC-SA to the detrimental oxygen action by immediate blending. Therefore, by a proper selection of succinylation parameters and processing procedure, one can achieve a narrow but stable processing window for biopolymer/succinylated CNC nanocomposites. The differences in the thermal stability of cellulosic samples may result not only from their surface area, but also from their degree of polymerisation. Samples with lower degrees of polymerisation have lower thermal stability, and therefore, with the increasing time of modification reaction, the thermal stability of CNCs decreased [35]. Furthermore, apart from the surface area and a degree of polymerisation, the thermal stability of cellulose nanocrystals is also influenced by the degree of substitution. The correlation of thermogravimetric studies with the results obtained from XPS analysis indicated that the thermal stability of nanoparticles decreases with the increasing degree of substitution. According to investigations provided by Trinh et al. and Wang et. al, this effect may be associated with the partial substitution of –OH groups in cellulose by succinic anhydride and thus, incorporation of free carboxylic group on the CNCs surface. Acidic groups can catalyse the dehydration process, and therefore, shift the beginning of the degradation towards lower temperatures [74,75]. The overall characteristics of produced CNCs, that are considered as a potential filler for bioplastics, indicated a critical phosphoric acid concentration during hydrolysis (76 wt.%) as well as SA:OH molar ratio (5:1) in the course of esterification. Exceeding the critical parameters resulted in reduction of thermal stability required for composite processing. The decrease in thermal stability of produced CNCs could arise from both chemical and structural changes of cellulose, such as incorporation of significant amount of less thermally stable ester groups of inorganic or organic acid, extensive hydrolysis as well as reduction in crystallinity. On the other hand CNCs succinylated at the highest SA:OH molar ratio 10:1, offered the finest nanoporous morphology, as revealed by SEM, combined with the highest substitution degree, evidenced by XPS and FTIR analysis, and well preserved crystallinity. This material would fulfil requirements of functional applications, among others, filtration membranes, sorbents, sensors, drug carriers, etc. The highest applied esterification temperature (110 ◦C) could be recommended for the synthesis of succinylated CNCs. The increase of temperature had no significant effect on the thermal stability of whiskers while it promoted growth of CNCs’ crystallinity. High crystallinity, and thus intrinsic stiffness, along with high aspect ratio are desired features of the reinforcing nanofiller. Generally, the selection of manufacturing parameters would depend strictly on the foreseen application of CNCs and demanded essential properties.

4. Conclusions Cellulose nanocrystals were produced by hydrolysis in phosphoric acid solutions at different concentrations. The optimal acid concentration that allowed effective hydrolysis of amorphous regions, without activation of cellulose phosphorylation, was found at 76 wt.%. The nanocrystals produced in optimal conditions had a high crystallinity index of 74% and preserved thermal stability. The influence of reaction time, temperature, and molar ratio of the reagents in the course of heterogeneous esterification of cellulose nanocrystals by succinic anhydride was further investigated. The surface esterification of CNC was confirmed by FTIR and XPS. The partial hydrolysis of cellulose, along with the esterification reaction, was indicated by the reduction of average particle size from the length of approximately 100 nm for CNC_P76 to 60 nm for the succinylated CNC. The surface substitution degree of esterified CNCs Polymers 2019, 11, 866 21 of 24 clearly increased with the increasing molar ratio of SA:OH and varied from 0.10 to 0.32. Significant reduction of thermal stability of modified CNCs was observed only for the highest substitution degree and the longest time of reaction (240 min). By the controlling of succinylation parameters, one can manufacture CNCs with desired sizes, extent of esterification or preserved thermal stability. The elaborated method of CNC functionalization by using succinic anhydride makes it possible to fabricate compatible and thermally stable cellulose nanocrystals that can be applied as a reinforcement in polymer composites. Such a study, utilizing selected polyamides, is now in progress.

Author Contributions: Conceptualization, A.L., K.P.; Methodology, A.L., P.R.; Software, P.R.; Validation, A.L., P.R. and E.S.; Formal Analysis, P.R., A.L.; Investigation, P.R., A.L., E.S., M.M.; Resources, P.R., E.S.; Writing-Original Draft Preparation, A.L. and P.R.; Writing-Review & Editing, A.L. and P.R.; Visualization, A.L. and P.R.; Supervision, A.L., K.P. and M.O.; Funding Acquisition, K.P. Funding: Funding for this work was provided by the Polish National Science Centre under Contract No. UMO/2015/19/B/ST8/01060. The PROM Programme—“International scholarship exchange of Ph.D candidates and academic staff” co-financed by the European Social Fund under the Knowledge Education Development Operational Programme, non-competitive mode entitled International scholarship exchange for Ph.D candidates and academic staff, contract number: POWR.03.03.00-00-PN13/18 is also kindly acknowledged. Acknowledgments: The authors are grateful to Edyta Hebda from Cracow University of Technology for SEM imaging of the materials. Conflicts of Interest: The authors declare no conflict of interest.

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