Characterization of Carboxylated Cellulose Nanocrytals Isolated

Article Cite This: J. Agric. Food Chem. 2018, 66, 7692−7700

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Characterization of Carboxylated Cellulose Nanocrytals Isolated through Catalyst-Assisted H2O2 Oxidation in a One-Step Procedure Roya Koshani,† Theo G. M. van de Ven,*,‡ and Ashkan Madadlou*,† †

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Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran ‡ Department of Chemistry, Quebec Centre for Advanced Materials, Pulp and Paper Research Centre, McGill University, Montréal, Québec H3A 0B8, Canada ABSTRACT: A green and facile method was designed to isolate a type of cellulose nanocrystal (CNC) with carboxylated surfaces from native cellulose materials. Because isolation and modification processes of cellulosic particles are generally performed separately using harmful chemicals and multiple steps, the one-pot approach employed in this work is interesting from both an economical and ecological point of view. The reaction is carried out by adding hydrogen peroxide as an oxidant and copper(II) sulfate as a catalyst in acidic medium under mild thermal conditions. The charge content of the carboxylated CNC is about 1.0 mmol g−1, measured by a conductometric titration. Fourier transform infrared spectroscopy also proved the presence of carboxyl groups on the CNC particles. Atomic force microscopy along with optical polarized microscopy readily showed a rod shape morphology for the cellulosic particles. An average length of 263 nm and width of 23 nm were estimated by transmission electron microscopy. Dynamic laser scattering on carboxylated CNC suspensions by adding salt confirmed that nanoparticles are electrostatically stable. Carboxylated CNCs were furthermore characterized by solid carbon-13 nuclear magnetic resonance and X-ray spectroscopy. KEYWORDS: cellulose nanocrystal, hydrogen peroxide, catalyst, oxidation reactions ated oxidation.14 Recently, spherical and rod-like carboxylated CNCs were prepared by ammonium persulfate (APS), an oxidizing agent with low long-term toxicity, in a one-step procedure.15,16 It was reported that free radicals, formed through the thermal cleavage of the peroxide bond of APS and hydrogen peroxide (H2O2) produced under acidic conditions, are capable of penetrating the amorphous regions of cellulose to break them down to generate carboxylated CNCs. In our laboratory, several studies have been performed on the production and characterization of cellulose-based particles via periodate and chlorite oxidations.13,17−19 A reaction of periodate with cellulose fibers, followed by heating at 80 °C, yields rod-like nanocrystals with amorphous regions attached to both ends, which are examples of HNCs. The periodate oxidation reaction causes the conversion of C2−C3 hydroxyls to aldehyde groups, and at the same time, it cleaves cellulose bonds.9 Introducing ionic charges facilitates the breakup of the cellulose fibers, resulting in the formation of electrosterically stabilized nanofibril cellulose or CNCs. The unique physicochemical properties, particularly, high colloidal stability and being a useful platform for site-specific conjugations, suggest that electrosterically stabilized CNCs, an example of HNCs, have a wider range of applications than conventional CNCs. Because of the trend to develop facile, safe, and eco-efficient processes to diminish harmful byproducts, this project has

1. INTRODUCTION Nanocelluloses derived from the most ubiquitous and abundant biological polymer in nature have been receiving a lot of academic and industrial attention during the 21st century. Their exceptional structural properties, such as nanoscale dimension, large specific area, and ease of surface modification (as a result of the large number of reactive hydroxyl groups), together with low cost and non-toxicity, have predestined them for applications in various fields.1,2 Examples include fabrication of high-performance reinforced biocomposites,3,4 medical materials,5,6 carriers for delivery systems,7 and green catalysts.8 Nanocellulosic particles are categorized in three major forms: (1) cellulose nanocrystals (CNCs), which are also referred to as nanocrystalline cellulose, cellulose (nano)wiskers, or rod-like cellulose microcrystals, (2) cellulose nanofibrils or nanofibrillated cellulose and microfibrilated cellulose, and (3) hairy cellulose nanocrystalloids (HNCs), bearing a crystalline body with polymer chains protruding from both ends.9 Several studies have reported a structural dependency of these nanocellolusic materials upon the source of cellulose and the processing conditions.10,11 CNCs are conventionally isolated by acidic hydrolysis of the amorphous regions,12 whereas HNCs are generated by the periodate oxidation reaction through solubilization and cleavage of a sufficient number of chains in the amorphous regions.13 To date, many oxidation-based methods have been used for the extraction and/or surface modification of cellulosic particles. The hydroxyl groups on the C6 position of the glucose units are commonly converted to the carboxyl form using a 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-medi© 2018 American Chemical Society

Received: Revised: Accepted: Published: 7692

January 15, 2018 July 3, 2018 July 5, 2018 July 5, 2018 DOI: 10.1021/acs.jafc.8b00080 J. Agric. Food Chem. 2018, 66, 7692−7700

Article

Journal of Agricultural and Food Chemistry Scheme 1. Synthesis Process Diagram of Carboxylated CNCs by Catalyst-Assisted H2O2 Oxidation

raw cellulose material. Hydrogen peroxide (30 wt %), hydrochloric acid, sodium hydroxide, and poly-L-lysine standard solutions were purchased from Sigma-Aldrich. Other chemicals supplied were copper(II) sulfate pentahydrate (Fisher Scientific), uranyl acetate (SPI Chemicals, Inc.), and sodium chloride (ACP Chemicals, Inc.). All solutions were prepared with deionized water. 2.2. Oxidative Preparation of Carboxylated CNCs. Dry softwood pulp pieces (2 g) were soaked in water and vigorously dispersed by a magnetic stirrer for 1 day, filtered to remove the fines and extra water from the pulp. Then, 40 mL of 30% H2O2, 0.4 mL of 0.1 M copper(II) sulfate pentahydrate (CuSO4·5H2O) solution, and 80 mL of distilled water were added to the swollen pulp. The pH of the reaction mixture was maintained in the range of 1−2 by 1 M HCl addition. The reaction vessel was covered with aluminum foil and stirred (160 rpm) for 72 h at a temperature of 60 °C. The temperature was set by a thermometer inside the reaction beaker and controlled by a magnetic stirrer with a hot plate. To stabilize the temperature of the sample, an oil bath was placed between the beaker and the hot plate. After the process was completed, the final volume of the suspension was adjusted to 200 mL with cold deionized water to stop the reaction and the suspension was washed 3−4 times by centrifugation until the pH reached 3−4. The supernatants were entirely water-soluble and consisted of dissolved oligosaccharides, resulting from cellulose degradation through catalyst-mediated H2O2 oxidation. Moreover, the supernatant contains most of the copper and sulfate ions. These ions are undesirable in food applications, for which their concentration must be kept below threshold values. This can be achieved by additional centrifugation or washing or ion-exchange resins. Insoluble cellulose particles were suspended in 200 mL of deionized water, followed by collecting a milk-like supernatant by decantation. Then, the remaining white precipitate was added to 50 mL of distilled water and sonicated for 15 min with an ultrasonic

focused on a one-pot fabrication and characterization of a type of functionalized cellulose-based nanoparticle through Cucatalyzed oxidation of softwood pulp by H2O2. This environmentally friendly oxidizing agent is extensively used in the bleaching of pulp fibers20 and in the modification of food polymers, particularly, starch.21,22 H2O2 is safe to use and is approved by the Joint Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) as a multipurpose food additive. It creates no harmful byproduct and decomposes inevitably to oxygen and water.23 In general, H2O2 converts the hydroxyl groups on the polymer chains into carbonyl and carboxyl functional groups, which is almost always accompanied by degradation of macromolecules.24 The decomposition of H2O2, in the presence of transition metal catalysts, such as copper, iron, or tungstate, can lead to the generation of intermediate radical species, such as HO• (hydroxyl) and HOO• (hydroperoxyl).25 The emerged free radicals can eventually oxidize the alcohol groups and cause the scission of glycoside bonds in the polysaccharide chains.24,26 The nanocellulose particles produced with the present method can be functionalized with antibacterial agents and used for stabilizing food emulsions or incorporated in films for foodpackaging applications. The stabilization of food emulsions by the novel CNCs is being investigated and will be the topic of a future publication.

2. MATERIALS AND METHODS 2.1. Materials. To produce cellulose particles, a softwood pulp sheet (Domtar, Inc., Canada) chopped into small pieces was used as 7693

DOI: 10.1021/acs.jafc.8b00080 J. Agric. Food Chem. 2018, 66, 7692−7700

Article

Journal of Agricultural and Food Chemistry

Figure 1. Conductometric titration result of 20 mg of carboxylated CNCs obtained by catalyst-assisted H2O2 oxidation. processor (Hielscher UP200H, Germany) at a frequency of 50−60 Hz under continuous magnetic stirring. The concentration was determined by drying a certain volume of the sample for at least 7 h at 50 °C. Scheme 1 illustrates the preparation steps of cellulosic particles. 2.3. Charge Analysis of Carboxylated CNCs. Conductometric titration was carried out using an 836 Titrando titrator (Metrohm, Switzerland) to measure the charge content, as an indication of the presence of carboxyl functional groups on the surface of the nanoparticles. A certain volume of suspension containing 20 mg of carboxylated CNC particles was mixed with 2 mL of 20 mM NaCl solution and 140 mL of Milli-Q water under vigorous stirring. Then, the pH of the well-dispersed suspension was adjusted to around 3.5 by dropwise addition of 0.05 M HCl. Subsequently, a 5 mM NaOH solution was gradually added at a rate of 0.1 mL min−1 to the dispersion up to a pH of around 11. The carboxyl content in millimoles per gram of carboxylated CNC was calculated from that part of the conductivity curve representing the volume of the weak acid, as indicated by the two vertical lines in Figure 1. A Malvern Zetasizer (ZEN 3600, U.K.) was also used to investigate the magnitude of the charge of the particles. The sample was diluted to 0.1% carboxylated CNC particles in Milli-Q water. 2.4. Measurement of Particle Size Distribution by Dynamic Light Scattering (DLS). The effective diameter and polydispersity of carboxylated CNC particles was determined by a Brookhaven light scattering instrument BI9000 AT digital correlator. All experiments were performed by monitoring the scattered light intensity at 90° scattering angle at 25 °C. First, suspensions (0.1 wt %) were filtered through a 0.45 μm syringe filter (Acrodisc, Pall), and then, 100 μL of sample was transferred to a low-volume microcuvette containing 900 μL of deionized water. The different concentrations of NaCl ranging from 0 to 2 M were added to a number of cellulosic suspensions to probe the elecrostatic interactions among nanoparticles. 2.5. Morphological Studies of Cellulose Particles. 2.5.1. Polarized Light Optical Microscopy (PLOM). Droplets of suspensions containing cellulose particles were sandwiched between a glass slide and a glass coverslip, and color images were taken by a Nikon Eclipse LV100POL microscope. 2.5.2. Atomic Force Microscopy (AFM). A Multimode atomic force microscope with a Nanoscope IIIa controller (Digital Instruments/ Veeco, Santa Barbara, CA, U.S.A.) was used to study the effect of

hydrogen peroxide oxidation on cellulose nanoparticle morphology. Sample preparation was performed by depositing a drop of poly-Llysine onto freshly cleaved mica attached to silicon wafers, rinsed off by deionized water after 5 min, and air-dried. Next, a 5 μL droplet of the nanoparticle suspension (0.001 wt %) was dropped onto the treated mica surface, followed by a final rinse. The samples were allowed to dry at ambient air. AFM images were obtained in tapping mode using silicon cantilevers with a force constant of 37 N/m, a frequency range of 100−500 kHz,and a nominal tip radius of 6 nm. 2.5.3. Transmission Electron Microscopy (TEM). The size of cellulose nanoparticles was measured using recorded images of a Philips Tecnai 12, 120 kV, electron microscope equipped with a Gatan 792 Bioscan 1000 × 1000 wide angle multiscan charge-coupled device (CCD) camera. A 5 μL drop of suspension diluted to 0.05 wt % was placed on a copper grid coated by a thin carbon film for 5 min and negatively stained using a drop of 2% uranyl acetate solution for 30 s, which enhances the contrast. Excess sample was carefully blotted away from the edge of the grid with filter paper (Whatman, Inc., Canada). A total of 25 images were captured from each sample, and the average sizes of 50−70 isolated particles were calculated in each image with ImageJ software. 2.6. Solid-State 13C Nuclear Magnetic Resonance (NMR) Spectroscopy. Solid-state carbon-13 NMR spectra were recorded on a Varian/Agilent VNMRS instrument at a frequency of 100.5 MHz. Powdered original pulp and carboxylated CNC were compressed uniformly in a 7.5 mm zirconium rotor and spun at 5500 Hz. Spinning sidebands were suppressed by the total suppression of spinning sidebands (TOSS) sequence. A total of 6000 transients at a contact time of 2 ms and a recycle delay of 2 s were averaged to obtain each spectrum. 2.7. Attenuated Total Reflectance (ATR)−Fourier Transform Infrared (FTIR) Spectroscopy. FTIR spectra of original pulp and carboxylated CNCs were acquired by a FTIR spectrometer (PerkinElmer, Inc., Waltham, MA, U.S.A.) with a single bounce diamond ATR accessory. All dried samples were put directly on the ATR crystal, and maximum pressure was applied by lowering the tip of the pressure clamp using a rachet-type clutch mechanism. The spectra were averaged from 32 scans at transmission mode from 400 to 4000 cm−1 with a resolution of 4 cm−1. 2.8. X-ray Diffraction (XRD). The crystallinity pattern of dried softwood pulp and carboxylated CNC was obtained through XRD to 7694


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Figure 2. (A) Particle size distribution of carboxylated CNCs obtained by catalyst-assisted H2O2 oxidation and (B) changes of the equivalent spherical diameter versus salt (NaCl) concentration. investigate the effects of catalyst-assisted H2O2 oxidation on the crystalline properties of the cellulose. Both the cellulosic samples were pressed into a cylindrical sample holder that was 25 mm in diameter and 2 mm high. The measurements were carried out by a Bruker D8 DISCOVER two-dimensional (2D) diffractometer with VANTEC 2D detector and Cu Kα radiation (k = 1.54 Å). The X-ray diffractograms were acquired with a 2θ range of 10−40° at a scan rate of 0.005° s−1.

the charge content could be due to protruding amorphous chains, a possibility investigated further by DLS (see section 3.2). Another explanation could be related to the self-assembly of CNCs into cylinder-shaped aggregates, resulting from longitudinal alignment of individual CNCs. The fact that the CNC that we produced by catalyst-mediated H2O2 oxidation has a much larger diameter (∼25 nm) than individual CNC crystals (∼5 nm) implies that the particles that we produce are bundles of aligned nanorods. Because it is unlikely that all rods inside the bundle are of the same length, it is possible that an additional surface area is exposed at both ends of the rod. The ζ potential of the carboxylated CNCs prepared by H2O2 oxidation was found to be −20.8 mV, which was close to the value of carboxylated spherical CNC (−24 mV) isolated by the APS procedure.15 It shows that the surface of CNC particles has been decorated with negatively charged carboxyl groups, creating the electrostatic repulsive forces that prevent the aggregation of the colloidal particles. Therefore, the high stability of the obtained carboxylated CNC suspension over time, as depicted in Scheme 1 (fractions 1 and 1′), can be ascribed to the electrostatic repulsion of negatively charged nanoparticles. It is well-known that metal catalysts used in H2O2 oxidation can drive the reaction to form hydroxyl and other free radicals and hydroxide ions. Equations 1−3 display the possible paths of the H2O2 reaction in the presence of copper ions, as proposed by Carvalho do Lago et al.29

3. RESULTS AND DISCUSSION 3.1. Charge Content of Carboxylated CNCs. Conductivity and pH changes of the carboxylated CNC suspension versus the volume of NaOH added have been plotted in Figure 1. Carboxyl groups, rendering the particle electrically charged, play an important role in the colloidal stability of the particles and minimizing aggregation.27 The amount of carboxyl groups calculated from this curve is 1.0 mmol of carboxylic acid/g of dried carboxylated CNC particles. A 0.1 mmol g−1 variation was obtained in three repeat experiments. Hence, the carboxyl content is 1.0 ± 0.1 mmol g−1. This is lower than the carboxyl content of HNC produced by periodate−chlorite oxidation (up to 6.6 mmol g−1).18 Fujisawa et al.27 reported a value of 1.74 mmol g−1 for the carboxyl content of TEMPO-oxidized cellulose nanofibrils. Although carboxyl and carbonyl groups have been formed on the cleaved C2−C3 of an anhydroglucose ring (secondary alcohols) through H2O2 oxidation, carboxyl groups are generally introduced on the C6 position (primary alcohol), similar to TEMPO-mediated oxidation. It should be noted that the number of oxidized hydroxyl groups placed on carbon atoms in a glycopyranose ring of cellulose molecules depends strongly upon the type of oxidant used.21 The maximum charge content of conventional CNC particles has been theoretically calculated to be about 0.8 mmol of carboxylic acid/g of a 10 × 10 nm crystal cross section,18 whereas the charge content of carboxylated CNC produced by our catalytic system is slightly greater than this theoretical maximum. The amorphous domains of cellulose are more sensitive to chemicals because of easier accessibility and can be dissolved during the oxidation reactions. However, the crystalline part is only attacked at the surface by diverse reactions. It has also been proposed that most of the carboxyl groups are placed on amorphous chains protruding from the crystalline segments.28 Therefore, the observed difference in

Cu 2 + + H 2O2 → Cu1 + + •O2− + 2H+

(1)

Cu1 + + H 2O2 → Cu 2 + + •OH + OH−

(2)

2•O2− + 2H+ → H 2O2 + O2

(3)

The free radicals produced in the above reactions can react with cellulose and introduce aldehyde and carboxyl groups. A possible pathway is

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RCH 2OH + •OH → R•CHOH + H 2O

(4)

R•CHOH + H 2O2 → RCHO + •OH + H 2O2

(5)

RCHO + 2•OH → RCOOH + H 2O

(6)


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Figure 3. Optical and polarized micrographs of (A) carboxylated CNCs from fraction 1 prepared by catalyst-assisted H2O2 oxidation, (B) precipitate from fraction 2, and (C) ultrasonically treated precipitate from fraction 3. Scale bars of images A−C are 10 μm.

Figure 4. (A) AFM height and (B) three-dimensional (3D) images of carboxylated CNCs from fraction 1 prepared by catalyst-assisted H2O2 oxidation and (C) CNCs from fraction 3 after sonication.

In an acidic (pH 1−2) environment, protons (H+) can protonate oxygen involved in the glycosidic bond, resulting in a cleavage of these linkages in the cellulose structure. Dias et al.24 found that depolymerization of oxidized starch under acidic conditions was more extreme compared to that under alkaline conditions during starch modification. 3.2. Particle Size Distribution. The size distribution of the carboxylated CNC particles was examined by DLS, as presented in Figure 2A. The equivalent hydrodynamic diameter of the nanoparticles prepared by the H2O2 oxidation reaction is around 298 nm. The average diffusion coefficient of rod-shape nanocrystals is obtained by averaging over all orientations.18 The polydispersity index of the suspension containing carboxylated CNCs is about 0.27, calculated by dividing the standard deviation of the particle size distribution by the average diameter (Figure 2A). In comparison, spherical CNCs obtained in the H2O2 oxidation by APS had higher polydispersity (0.43).15 This implies that the initial cellulose substrate was more degraded than the CNCs produced by the APS process, which would result in the production of more homogeneous particles. The uniformity of the particle size has a significant impact on the performance of CNCs as nanofillers or in food applications. The effect of various NaCl concentrations, ranging from 0 to 1000 mM, on the hydrodynamic diameter of carboxylated CNCs was monitored as another proof to support the electrostatic stability of the nanoparticles. According to Figure 2B, the equivalent hydrodynamic diameter of carboxylated CNC nanoparticles showed an almost constant trend as the salt concentration increased to about 300 mM, well beyond

which sulfate-bearing CNC dispersions become unstable (above 25 mmol g−1).28 This finding implies that carboxylated CNCs prepared via catalyst-assisted H2O2 oxidation consist of only individual crystalline segments with few attached solubilized amorphous regions. In combination with the results of the charge content, carboxylated CNCs produced by catalyst-assisted H2O2 oxidation cannot be classified into the HNC category; albeit, the presence of short amorphous hairs particularly at the poles of CNCs remains a possibility. As seen in Figure 2B, the equivalent hydrodynamic diameter of carboxylated CNC particles remarkably increased at salt concentrations higher than 300 mM. According to the classic theory, increasing the electrolyte concentration results in reducing/eliminating the electrostatic repulsions between particles, leading to the coagulation of the charged nanoparticles. 3.3. Microscopic Observations of Carboxylated CNCs. The morphological features of the products resulting from the H2O2 oxidation process were examined by polarized optical micrographs, as illustrated in Figure 3. Images of a milky suspension containing carboxylated CNCs (Figure 3A) exhibit some particles that are hardly visible. The sub-micrometer particles, which cannot be seen in an optical microscope, were imaged by AFM. Panels A and B of Figure 4 clearly show the typical rod-like structure of nanoparticles after H2O2 oxidation. Images of precipitated particles (Figure 3B) under polarized light show microfibers with a length of about 230 μm and a width of around 25 μm. When the suspension is sonicated for 15 min with a low frequency (fraction 3), most of the microfibrils were completely broken up into particles in the 7696


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nanoparticles.28 The sizes for the ultrasonically treated sample from fraction 3 (Figure 5B) were around 305 ± 90 and 30 ± 9 nm, values close to those of fraction 1. These dimensions are in the range of CNCs produced by other oxidation processes but with a greater diameter.16,18 3.4. Solid-State 13C NMR. The NMR spectra of the original softwood pulp and carboxylated CNC prepared by H2O2 oxidation are presented in Figure 6. Typical signals characteristic of functional groups can be observed in the spectra. The peak between 100 and 110 ppm is assigned to anomeric carbon C1 and that between 80 and 95 ppm is for C4. The next peaks in the 70−80 ppm region are associated with C2, C3, and C5 carbons. The region between 60 and 70 ppm is attributed to C6 of the primary alcohol group. In the cellulose spectra, the narrow cluster at 89 ppm (C4) corresponds to anhydroglucose units in the crystalline parts. The broad peak located at 84 ppm (C4′) is characteristic of the anhydroglucoses with less order in cellulose structural arrangements.31 The NMR spectra of the original softwood fiber and carboxylated CNCs were almost identical, implying that no significant changes happened in the chain conformations of the cellulose. However, the intensity of the peak around 65 ppm decreased up to 32% compared to the original cellulose fiber. This change can be a sign of −CH2OH oxidation in the C6 position to form −COOH groups on the CNC surface rather than other hydroxyl groups. The C1 signal intensity of the cellulose backbone decreased, indicating that cleaving glycoside bonds of cellulose chains occurred. The multiple peaks located between 70 and 80 ppm are attributed to the oxidation of hydroxyl groups and bond cleavage of C2−C3 during H2O2 oxidation. Observed changes in the C4 and C4′ signals can be taken as proof for the formation of highly crystalline nanoparticles, in good agreement with the NMR spectra of cellulose nanowiskers produced with sulfuric acid by Sèbe et al.32

range of nanometers, as displayed in Figures 3C and 4C. This suspension also remained stable for up to 1 month (fraction 1′). The yield of cellulosic nanoparticles extracted from softwood pulp by the H2O2 reaction was 54%, whereas it reached up to 81% in combination with ultrasound. It seems that H2 O 2 -induced oxidation has caused less intense disruptions in some regions of the cellulose structure, allowing glycosidic bonds within the glucan chains to be easily cleaved using sonication, to give uniform particles in the nanometer scale. In agreement with this finding, Yang and van de Ven30 reported a decrease in the mechanical energy required for breaking fibers down into nanosize particles by increasing the charge content. The dimensions of rod-like CNCs prepared with H2O2 oxidation were determined by TEM images, as shown in Figure 5. The average length and diameter of nanoparticles in

Figure 5. TEM image of carboxylated CNCs prepared from softwood fibers (A) by catalyst-assisted H2O2 oxidation from fraction 1 and (B) after sonication from fraction 3.

the milky suspension from fraction 1 (Figure 5A) were 263 ± 28 and 23 ± 5 nm, respectively, which are slightly lower than the values obtained from DLS measurements. Note that DLS overestimates the size of the particle, even for standard silica

Figure 6. Solid-state carbon-13 NMR spectra of softwood pulp and carboxylated CNCs obtained by catalyst-assisted H2O2 oxidation. 7697


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Figure 7. FTIR spectra of original softwood pulp and carboxylated CNCs obtained by catalyst-assisted H2O2 oxidation.

3.5. ATR−FTIR Spectroscopy. The original softwood pulp and carboxylated CNC particles were further characterized by FTIR spectroscopy, as seen in Figure 7. Normalization of FTIR spectra of two cellulosic samples was performed to make a meaningful qualitative comparison. The broad band at 3330 cm−1 is due to the stretching vibration of −OH groups, which was affected by the inter- or intramolecular hydrogen bonds of the cellulose molecules. Higher intensity of this broad peak in carboxylated CNCs arises from hydrogen bonds breaking during H2O2 oxidation reactions and, consequently, more stretching vibration of −OH groups compared to the intact softwood structure. The absorption peaks at around 2905, 1425, and 1021 cm−1 are attributed to C−H stretching vibrations, −CH2 scissoring, and CH2−O−CH2 stretching, respectively.33 The peak located at 1738 cm−1 corresponds to CO stretching, while the absorption at 1630 cm−1 is related to the asymmetric stretching vibration of the carboxyl groups of the oxidized CNCs.34 These emerged peaks support the formation of carboxyl and carbonyl groups during H2O2 oxidation, which is in accordance with the conductometric results. The possibility that these bands were due to aldehyde groups was disproven by performing a titration with hydroxylamine hydrochloride, a standard method to determine aldehyde groups.18 No aldehyde groups were found, proving that the bands cannot be due to aldehyde groups but must be due to carboxyl groups. Of importance to note is that most carbonyl groups formed through the oxidation of the hydroxyl groups have been converted to carboxyl groups. The carboxyl groups can be employed as active sites in surface modifications, especially for immobilizing proteins and enzymes. 3.6. Investigation of the Crystalline Structure by XRD. Figure 8 presents XRD patterns of softwood pulp and carboxylated CNCs produced by H2O2 oxidation, confirming the NMR results. The diffraction patterns of two cellulosic samples show a sharp peak at the 2θ angle of 22.6° for the (200) peak and two weak peaks at 2θ = 15.1° for the (11̅0) peak and 2θ = 16.5° for the (110) peak. These peaks correspond to the main crystalline region of the cellulose structure,35 which are almost identical in original pulp and carboxylated CNCs. It indicates that the original crystalline structure of cellulose fibrils was well-maintained during H2O2 oxidation reactions. This finding is consistent with the reports about the crystallinity changes of cellulose caused by APS16 or sulfuric acid.32

Figure 8. XRD profiles of original softwood pulp and carboxylated CNCs obtained by catalyst-assisted H2O2 oxidation.

The crystallinity index (CI) was calculated according to Segal et al.36 I − Iam × 100 CI (%) = 200 I200 where I200 is the intensity obtained from the (200) plane reflection and Iam is the minimum intensity between the (100) and (200) peaks. The CIs of the original pulp and carboxylated CNC particles were 77.5 and 70.5%, respectively, which exhibit a slight decrease after the reaction of cellulose pulp with H2O2 and catalyst for 3 days. It is known that the amorphous regions and the surface of crystalline regions in cellulose are kinetically more accessible to chemical reactions.9 Therefore, the decrease in crystallinity could be explained by the fact that H2O2oxidation-induced hydrolysis does not only happen at the amorphous regions but also at the surface of the crystalline parts of cellulose. In addition, some extensive surface modifications have possibly caused destructive changes in the crystalline structure of cellulose. Interestingly, these results are in good agreement with those of Wang et al.,37 who studied CNCs produced from microcrystalline cellulose using a mixture of sulfuric and hydrochloric acids. Similarly, Yang and van de Ven30 reported that the CI of softwood cellulose 7698


Journal of Agricultural and Food Chemistry pulp (75%) decreased to 49% in dialdehyde-modified cellulose (DAMC) obtained by periodate oxidation. This is related to opening of the glucopyranose rings of cellulose from the C2− C3 bond during oxidation, resulting in detrimental lowering of the CI, which possibly also occurs in carboxylated CNCs produced by H2O2 oxidation. In contrast, the carboxylated CNCs extracted using APS under thermal conditions showed a higher crystallinity than that of the initial cellulose.15 It is worth noting that the degree of crystallinity of carboxylated CNCs prepared by H2O2 oxidation is high and close to that of CNCs generated with acid hydrolysis from bleached soft wood kraft38 and kenaf core woods.39 Thus, the absence of significant changes in the total crystallinity of cellulose and high-crystalline CNCs show that the catalystassisted H2O2 oxidation method has great potential in the production of CNC nanoparticles. In conclusion, a one-step procedure for producing carboxylated CNCs from softwood cellulose pulp was designed by applying H2O2 as an oxidant and CuSO4·5H2O as a catalyst. The use of H2O2 has significant benefits over other chemicals because of its easy removal and/or rapid decomposition to water and oxygen after the hydrolysis processes. The mechanism of the reaction is based on penetration of free radical ions formed by catalyst-assisted H2O2 oxidation to the outer layer of the crystalline regions and all amorphous regions. Because the proposed method uses only non-expensive and environmentally friendly chemicals, it is capable of large-scale production of CNCs decorated with negatively charged carboxyl groups. Carboxylated CNCs displayed a rod-shaped morphology with high size uniformity and almost the same dimensions as other cellulose nanoparticles produced by oxidative methods, albeit with a larger width. The presence of carboxyl groups on the surface of CNCs was proven by conductometric titration and FTIR spectroscopy. DLS measurements with NaCl addition showed colloidal stability of carboxylated CNC suspension at salt concentrations less than 400 mM, arising from the dominating electrostatic repulsions between the particles. The NMR and XRD results showed that the original structure of cellulose fibrils is to a large extent maintained during H2O2 oxidation. More detailed studies are required to optimize parameters, such as the amount of H2O2, different catalysts, reaction time, temperature, and pH, and characterize carboxylated CNCs produced by H2O2 oxidation to tailor their applicability to different fields.

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ACKNOWLEDGMENTS

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REFERENCES

The authors acknowledge the access to facilities and instrumentation supported by the Pulp and Paper Research Centre and Department of Chemistry, McGill University.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected] and/or [email protected]. ir. ORCID

Ashkan Madadlou: 0000-0003-4572-868X Funding

The authors thank the financial support of the Iranian Ministry of Science, Research and Technology and the Natural Science and Engineering Research Council of Canada (NSERC Discovery Grant 42686-13). Notes

The authors declare no competing financial interest. 7699


Article

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Characterization of Carboxylated Cellulose Nanocrytals Isolated

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