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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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00080 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Journal of Agricultural and Food Chemistry

1

Characterization of Carboxylated Cellulose Nanocrytals Isolated

2

through Catalyst Assisted-H2O2 Oxidation in a One-Step Procedure

3 †

Roya Koshani, Theo G.M. van de Ven,

4

5 6

7 8

†

‡*

Ashkan Madadlou,

†*

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, Montreal, Quebec, Canada

9

10

11

12

13

14

15

16

17 18

*

Corresponding authors: Ashkan madadlou, Email: [email protected] and [email protected]; and Theo G.M. van de Ven, Email: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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A green and facile method was designed to isolate a type of cellulose nanocrystals

21

(CNCs) with carboxylated surfaces from native cellulose materials. Since isolation and

22

modification processes of cellulosic particles are generally performed separately using

23

harmful chemicals and multiple steps, the one-pot approach employed in this work is

24

interesting from both an economical and ecological point of view. The reaction is carried

25

out by adding hydrogen peroxide as an oxidant and copper (II) sulfate as a catalyst in

26

acidic medium under mild thermal conditions. The charge content of the carboxylated

27

CNC is about 1.0 mmol g-1 measured by conductometric titration. FTIR spectroscopy

28

also proved the presence of carboxyl groups on the CNC particles. Atomic force

29

microscopy along with optical polarized microscopy readily showed a rod shape

30

morphology for the cellulosic particles. An average length of 263 nm and width of 23 nm

31

were estimated by transmission electron microscopy. Dynamic laser scattering on

32

carboxylated CNC suspensions by adding salt confirmed that nanoparticles are

33

electrostatically stable. Carboxylated CNCs were furthermore characterized by solid

34

carbon-13 NMR and X-ray spectroscopy.

35

KEYWORDS: cellulose nanocrystal, hydrogen peroxide, catalyst, oxidation reactions


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INTRODUCTION

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Nanocelluloses derived from the most ubiquitous and abundant biological polymer in

38

nature have been receiving lots of academic and industrial attention during the 21st

39

century. Their exceptional structural properties such as nanoscale dimension, large

40

specific area and ease of surface modification (due to the large number of reactive

41

hydroxyl groups) together with low cost and non-toxicity have predestined them for

42

applications in various fields.1,2 Examples include fabrication of high-performance

43

reinforced biocomposites,3,4 medical materials,5,6 carriers for delivery systems7 and green

44

catalysts.8

45

Nanocellulosic particles are categorized in three major forms, 1) cellulose nanocrystals

46

(CNCs) which are also referred to a nanocrystalline cellulose, cellulose (nano)wiskers, or

47

rod-like cellulose microcrystals; 2) cellulose nanofibrils or nano-fibrillated cellulose, and

48

microfibrilated cellulose; and 3) hairy cellulose nanocrystalloids (HNCs) bearing a

49

crystalline body with polymer chains protruding from both ends.9 Several studies have

50

reported a structural dependency of these nanocellolusic materials on the source of

51

cellulose and the processing conditions.10,11 CNCs are conventionally isolated by acidic

52

hydrolysis of the amorphous regions12 whereas HNCs are generated by periodate

53

oxidation reaction through solubilization and cleavage of a sufficient number of chains in

54

the amorphous regions.13

55

To date, many oxidation-based methods have been used for the extraction and/or surface

56

modification of cellulosic particles. The hydroxyl groups on the C6 position of the

57

glucose units are commonly converted to the carboxyl form by using a TEMPO (2,2,6,6-

58

Tetramethylpiperidine-1-oxyl) mediated oxidation.14 Recently, spherical and rod-like 3 ACS Paragon Plus Environment


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carboxylated CNCs were prepared by ammonium persulfate (APS), an oxidizing agent

60

with low long-term toxicity, in a one-step procedure.15,16 It was reported that free

61

radicals, formed through the thermal cleavage of the peroxide bond of APS and hydrogen

62

peroxide (H2O2) produced under acidic condition, are capable of penetrating the

63

amorphous regions of cellulose to break them down to generate carboxylated CNCs.

64

In our laboratory, several studies have been performed on the production and

65

characterization of cellulose-based particles via periodate and chlorite oxidations.13,17-19 A

66

reaction of periodate with cellulose fibers, followed by heating at 80 °C, yields rod-like

67

nanocrystals with amorphous regions attached to both ends, which are examples of

68

HNCs. The periodate oxidation reaction causes the conversion of C2-C3 hydroxyls to

69

aldehyde groups, and at the same time it cleaves cellulose bonds.9 Introducing ionic

70

charges facilitate the break-up of the cellulose fibers, resulting in the formation of

71

electrosterically stabilized nanofibrils cellulose or CNCs. The unique physicochemical

72

properties particularly high colloidal stability and being a useful platform for site-specific

73

conjugations suggest that electrosterically stabilized CNCs an example of HNCs have a

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wider range of applications than conventional CNCs.

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Because of the trend to develop facile, safe and eco-efficient processes to diminish

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harmful by-products, this project has focused on a one-pot fabrication and

77

characterization of a type of functionalized cellulose-based nanoparticles through Cu-

78

catalyzed oxidation of softwood pulp by H2O2. This environmentally friendly oxidizing

79

agent is extensively used in the bleaching of pulp fibers20 and in the modification of food

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polymers particularly starch.21,22 H2O2 is safe to use and is approved by the Joint

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FAO/WHO Expert Committee on Food Additives (JECFA) as a multipurpose food


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additive. It creates no harmful by-product and decomposes inevitably to oxygen and

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water.23 In general, H2O2 converts the hydroxyl groups on the polymer chains into

84

carbonyl and carboxyl functional groups which is almost always accompanied by

85

degradation of macromolecules.24 The decomposition of H2O2, in the presence of

86

transition metal catalysts such as copper, iron or tungstate, can lead to the generation of

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intermediate radical species such as HO•·(hydroxyl) and HOO• (hydroperoxyl).25 The

88

emerged free radicals can eventually oxidize the alcohol groups and cause the scission of

89

glycoside bonds in the polysaccharide chains.24,26 The nanocellulose particles produced

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with the present method can be functionalized with antibacterial agents and used for

91

stabilizing food emulsions or incorporated in films for food packaging applications. The

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stabilization of food emulsions by the novel cellulose nanocrystals is being investigated

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and will be the topic of a future publication.

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MATERIALS AND METHODS

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Materials

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For producing cellulose particles, a softwood pulp sheet (Domtar Inc. Canada) chopped

97

into small pieces was used as raw cellulose material. Hydrogen peroxide (30 wt %),

98

hydrochloric acid, sodium hydroxide and poly-L-Lysine standard solutions were

99

purchased from Sigma-Aldrich. Other chemicals supplied were copper (II) sulfate

100

pentahydrate (Fisher Scientific), uranyl acetate (SPI chemicals Inc.) and sodium chloride

101

(ACP Chemicals Inc.). All solutions were prepared with deionized water.

102

Oxidative Preparation of Carboxylated Cellulose Nanocrystals

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Dry softwood pulp pieces (2 g) were soaked in water and vigorously dispersed by a

104

magnetic stirrer for 1 day, filtered to remove the fines and extra water from the pulp. 5 ACS Paragon Plus Environment


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Then, 40 mL of 30% H2O2, 0.4 mL of 0.1 M copper (II) sulfate pentahydrate

106

(CuSO4.5H2O) solution and 80 mL distilled water were added to the swollen pulp. The

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pH of the reaction mixture was maintained in the range of 1-2 by 1 M HCl addition. The

108

reaction vessel was covered with aluminum foil and stirred (160 rpm) for 72 hr at a

109

temperature of 60 ºC. Temperature was set by a thermometer inside the reaction beaker,

110

and controlled by a magnetic stirrer with hot plate. To stabilize the temperature of the

111

sample, an oil bath was placed between the beaker and the hot plate. After completing the

112

process, the final volume of the suspension was adjusted to 200 mL with cold deionized

113

water to stop the reaction and the suspension was washed 3-4 times by centrifugation

114

until the pH reached 3-4. The supernatants were entirely water-soluble and consisted of

115

dissolved oligosaccharides, resulting from cellulose degradation through catalyst

116

mediated-H2O2 oxidation. Moreover, the supernatant contains most of the copper and

117

sulfate ions. These ions are undesirable in food applications, for which their

118

concentration must be kept below threshold values. This can be achieved by additional

119

centrifugation or washing or by ion-exchange resins.

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Insoluble cellulose particles were suspended in 200 mL deionized water, followed by

121

collecting a milk-like supernatant by decantation. Then, the remaining white precipitate

122

was added to 50 mL distilled water and sonicated for 15 min with an ultrasonic processor

123

(Hielscher UP200H, Germany) at a frequency of 50-60 Hz under continuous magnetic

124

stirring. The concentration was determined by drying a certain volume of the sample for

125

at least 7 hr at 50 °C. Scheme 1 illustrates the preparation steps of cellulosic particles.

126

[Scheme 1]


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Charge Analysis of Carboxylated Cellulose Nanocrystals

128

Conductometric titration was carried out using an 836 Titrando titrator (Metrohm,

129

Switzerland) to measure the charge content, as an indication of the presence of carboxyl

130

functional groups on the surface of the nanoparticles. A certain volume of suspension

131

containing 20 mg carboxylated CNC particles was mixed with 2 mL of 20 mM NaCl

132

solution and 140 mL milli-Q water under vigorous stirring. Then the pH of the well-

133

dispersed suspension was adjusted to around 3.5 by dropwise addition of 0.05 M HCl.

134

Subsequently, a 5 mM NaOH solution was gradually added at a rate of 0.1 mL min-1 to

135

the dispersion up to a pH around 11. The carboxyl content in mmol per gram

136

carboxylated CNC was calculated from that part of the conductivity curve representing

137

the volume of the weak acid, as indicated by the two vertical lines in Figure 1.

138

A Malvern Zetasizer (ZEN 3600, Made in UK) was also used to investigate the

139

magnitude of the charge of the particles. The sample was diluted to 0.1 % of carboxylated

140

CNC particles in Milli-Q water.

141

Measurement of Particle Size Distribution by Dynamic Light Scattering (DLS)

142

The effective diameter and polydispersity of carboxylated CNC particles was determined

143

by a Brookhaven light scattering instrument BI9000 AT digital correlator. All

144

experiments were performed by monitoring the scattered light intensity at 90° scattering

145

angle at 25 °C. Firstly, suspensions (0.1 wt %) were filtered through a 0.45 µm syringe

146

filter (Acrodisc, PALL) and then 100 µL of sample was transferred to a low-volume

147

microcuvette containing 900 µL deionized water. The different concentrations of NaCl

148

ranging from 0 to 2 M were added to a number of cellulosic suspensions to probe the

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elecrostatic interactions among nanoparticles. 7 ACS Paragon Plus Environment


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Morphological Studies of Cellulose Particles

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Polarized Light Optical Microscopy (PLOM)

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Droplets of suspensions containing cellulose particles were sandwiched between a glass

153

slide and a glass coverslip and color images were taken by a Nikon Eclipse LV100POL

154

microscope.

155

Atomic Force Microscopy (AFM)

156

A Multimode atomic force microscope with Nanoscope IIIa controller (Digital

157

Instruments/Veeco, Santa Barbara, CA, USA) was used to study the effect of hydrogen

158

peroxide oxidation on cellulose nanoparticles morphology. Sample preparation was done

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by depositing a drop of poly-L-Lysine onto freshly cleaved mica attached to silicon

160

wafers, rinsed off by deionized water after 5 min and air-dried. Next a 5 µL droplet of

161

nanoparticles suspension (0.001 wt %) was dropped onto the treated mica surface,

162

followed by a final rinse. The samples were allowed to dry at ambient air. AFM images

163

were obtained in tapping mode using silicon cantilevers with a force constant of 37 N/m,

164

a frequency range of 100 kHz to 500 kHz and a nominal tip radius of 6 nm.

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Transmittance Electron Microscopy (TEM)

166

The size of cellulose nanoparticles was measured using recorded images of a Philips

167

Tecnai 12 120 kV electron microscope equipped with a Gatan 792 Bioscan 1k × 1k Wide

168

Angle Multiscan CCD camera. A 5 µL drop of suspension diluted to 0.05 wt% was

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placed on a copper grid coated by a thin carbon film for 5 min and negatively stained

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using a drop of 2% uranyl acetate solution for 30 s, which enhances the contrast. Excess

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sample was carefully blotted away from the edge of the grid with filter paper (Whatman


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Inc., Canada). 25 images were captured from each sample and the average sizes of 50 to

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70 isolated particles were calculated in each image with Image J software.

174

Solid-State 13C NMR Spectroscopy

175

Solid-state carbon-13 NMR spectra were recorded on a Varian/Agilent VNMRS

176

instrument at a frequency of 100.5 MHz. Powdered original pulp and carboxylated CNC

177

were compressed uniformly in 7.5 mm zirconium rotor and spun at 5,500 Hz. Spinning

178

sidebands were suppressed by the TOSS sequence. A total of 6,000 transients at a contact

179

time of 2 ms and a recycle delay of 2 s were averaged to obtain each spectrum.

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ATR-FTIR Spectroscopy

181

Fourier transform infrared (FTIR) spectra of original pulp and carboxylated CNCs was

182

acquired by a FTIR spectrometer (Perkin Elmer, Inc., USA) with single bounce diamond

183

attenuated total reflectance (ATR) accessory. All dried samples were put directly on the

184

ATR crystal and maximum pressure was applied by lowering the tip of the pressure

185

clamp using a rachet-type clutch mechanism. The spectra were averaged from 32 scans at

186

transmission mode from 400 to 4000 cm- with a resolution of 4 cm-1.

187

X-Ray Diffraction (XRD)

188

The crystallinity pattern of dried softwood pulp and carboxylated CNC were obtained

189

through X-ray diffraction (XRD) to investigate the effects of catalyst-assisted H2O2

190

oxidation on the crystalline properties of the cellulose. Both the cellulosic samples were

191

pressed into a cylindrical sample holder that was 25 mm in diameter and 2 mm high. The

192

measurements were carried out by a Bruker Discover D8 Discover two dimensional

193

diffractometer with VANTEC 2D detector and CuKa radiation (k = 1.54 A°). The X-ray

194

diffractograms were acquired with a 2θ range of 10°–40° at a scan rate of 0.005° s-1. 9 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION

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Charge Content of Carboxylated CNCs

197

Conductivity and pH changes of carboxylated CNC suspension versus the volume of

198

NaOH added have been plotted in Figure 1. Carboxyl groups, rendering the particle

199

electrically charged, play an important role in the colloidal stability of the particles and in

200

minimizing aggregation.27 The amount of carboxyl groups calculated from this curve is

201

1.0 mmol carboxylic acid per gram dried carboxylated CNC particles. A 0.1 mmol g-1

202

variation was obtained in three repeat experiments. Hence, the carboxyl content is 1.0 ±

203

0.1 mmol g-1. This is lower than the carboxyl content of HNC produced by periodate-

204

chlorite oxidation (up to 6.6 mmol g-1).18 Fujisawa et al.27 reported a value of 1.74 mmol

205

g−1 for the carboxyl content of TEMPO-oxidized cellulose nanofibrils. Although carboxyl

206

and carbonyl groups have been formed on the cleaved C2-C3 of an anhydroglucose ring

207

(secondary alcohols) through H2O2 oxidation, carboxyl groups are generally introduced

208

on the C-6 position (primary alcohol), similar as in TEMPO-mediated oxidation. It

209

should be noted that the number of oxidized hydroxyl groups placed on carbon atoms in a

210

glycopyranose ring of cellulose molecules depends strongly on the type of oxidant used.21

211

The maximum charge content of conventional CNC particles has been theoretically

212

calculated about to be 0.8 mmol carboxylic acid per gram of a 10 nm by 10 nm crystal

213

cross-section

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catalytic system is slightly greater than this theoretical maximum. The amorphous

215

domains of cellulose are more sensitive to chemicals, because of easier accessibility, and

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can be dissolved during the oxidation reactions. However, the crystalline part is only

217

attacked at the surface by diverse reactions. It has been also proposed that most of the

18

whereas the charge content of carboxylated CNC produced by our


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carboxyl groups are placed on amorphous chains protruding from the crystalline

219

segments.28 Therefore, the observed difference in charge content could be due to

220

protruding amorphous chains, a possibility investigated further by DLS (see section 3.2).

221

Another explanation could be related to the self-assembly of CNCs into cylinder-shaped

222

aggregates, resulting from longitudinal alignment of individual CNCs. The fact that the

223

CNC we produce by catalyst mediated-H2O2 oxidation has a much larger diameter (~ 25

224

nm) than individual CNC crystals (~ 5 nm) implies that the particles we produce are

225

bundles of aligned nanorods. As it is unlikely that all rods inside the bundle are of the

226

same length, it is possible that additional surface area is exposed at both ends of the rod.

227

The zeta potential of the carboxylated CNCs prepared by H2O2 oxidation was found to be

228

-20.8 mV which was close to that value of carboxylated spherical CNC (-24 mV) isolated

229

by the APS procedure.15 It shows that the surface of CNC particles has been decorated

230

with negatively charged carboxyl groups, creating the electrostatic repulsive forces that

231

prevent the aggregation of the colloidal particles. Therefore, high stability of obtained

232

carboxylated CNCs suspension over time as depicted in Scheme 1 (fractions 1 and 1՜ )

233

can be ascribed to the electrostatic repulsion of negatively charged nanoparticles.

234

[Figure 1]

235

It is well known that metal catalysts used in H2O2 oxidation can drive the reaction to form

236

hydroxyl and other free radicals and hydroxide ions. Equations 1 - 3 display the possible

237

paths of the H2O2 reaction in the presence of copper ions, as proposed by Carvalho do

238

Lago et al. 29



Cu2+ + H2O2 → Cu1++ •O2- + 2H+

(1)

Cu1+ + H2O2 → Cu2++ •OH +OH-

(2)

2•O2- + 2H+ → H2O2 + O2

(3)

239

240

The free radicals produced in the above reactions can react with cellulose and introduce

241

aldehyde and carboxyl groups. A possible pathway is: RCH2OH + •OH

→

R•CHOH + H2O

(4)

R•CHOH + H2O2

→

RCHO + •OH + H2O2

(5)

RCHO + 2 •OH → RCOOH + H2O

(6)

242

243

In an acidic (pH=1-2) environment, protons (H+) can protonate the oxygen involved in

244

glycosidic bond, resulting in a cleavage of these linkages in the cellulose structure. Dias

245

et al.24 found that depolymerisation of oxidized starch under acidic conditions was more

246

extreme compared with that under alkaline conditions during starch modification.

247

Particle Size Distribution

248

The size distribution of the carboxylated CNC particles was examined by DLS as

249

presented in Figure 2A. The equivalent hydrodynamic diameter of the nanoparticles

250

prepared by H2O2 oxidation reaction is around 298 nm. The average diffusion coefficient

251

of rod-shape nanocrystals is obtained by averaging over all orientations.18 The

252

polydispersity index of suspension containing carboxylated CNCs is about 0.27, 12 ACS Paragon Plus Environment

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calculated by dividing the standard deviation of the particles size distribution by the

254

average diameter (Figure 2A). In comparison, spherical CNCs obtained by in the H2O2

255

oxidation APS had higher polydispersity (0.43).15 This implies that the initial the

256

cellulose substrate was more degraded than the CNCs produced by APS process which

257

would result in the production of more homogeneous particles. The uniformity of particle

258

size has a significant impact on the performance of CNC as nanofillers or in food

259

applications.

260

The effect of various NaCl concentrations, ranging from 0 to 1000 mM, on the

261

hydrodynamic diameter of carboxylated CNCs was monitored as another proof to support

262

the electrostatic stability of the nanoparticles. According to Figure 2B, the equivalent

263

hydrodynamic diameter of carboxylated CNC nanoparticles showed an almost constant

264

trend as the salt concentration increase to about 300 mM, well beyond which sulfate-

265

bearing CNC dispersions become unstable (above 25 mmol g-1).28 This finding implies

266

that carboxylated CNCs prepared via catalyst assisted-H2O2 oxidation consist of only

267

individual crystalline segments with few attached solubilized amorphous regions.

268

Combined with the results of charge content, carboxylated CNCs produced by catalyst

269

assisted-H2O2 oxidation cannot be classified into the HNCs category, albeit the presence

270

of short amorphous hairs particularly at the poles of CNCs remains a possibility. As can

271

be seen in Figure 2B, the equivalent hydrodynamic diameter of carboxylated CNC

272

particles remarkably increased at salt concentrations higher than 300 mM. According to

273

classic theory, increasing the electrolyte concentration results in reducing/eliminating the

274

electrostatic repulsions between particles, leading to the coagulation of the charged

275

nanoparticles. 13 ACS Paragon Plus Environment


[Figure 2]

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277 278

Microscopic Observations of Carboxylated CNCs

279

The morphological features of the products resulting from H2O2 oxidation process were

280

examined by polarized optical micrographs as illustrated in Figure 3. Images of milky

281

suspension containing carboxylated CNCs (Figure 3A) exhibit some particles that are

282

hardly visible. The submicron particles, which cannot be seen in an optical microscope,

283

were imaged by AFM. Figure 4A,B clearly shows the typical rod-like structure of

284

nanoparticles after H2O2 oxidation. Images of precipitated particles (Figure 3B) under

285

polarized light show microfibers with a length about 230 µm and a width around 25 µm.

286

When the suspension is sonicated for 15 min with a low frequency (fraction 3), most of

287

the microfibrils were completely broken up into particles in the range of nanometer as

288

displayed in Figure 3,4C. This suspension also remained stable for up to one month

289

(fraction 1՜ ). The yield of cellulosic nanoparticles extracted from softwood pulp by

290

H2O2 reaction was 54 % whereas it reached up to 81 % in combination with ultrasound. It

291

seems that H2O2-induced oxidation has caused less intense disruptions in some regions of

292

cellulose structure, allowing glycosidic bonds within the glucan chains to be easily

293

cleaved by using sonication, to give uniform particles in the nanometer scale. In

294

agreement with this finding, Yang & van de Ven,30 reported a decrease in the mechanical

295

energy required for breaking fibers down into nanosize particles by increasing the charge

296

content.

297

[Figure 3]

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[Figure 4] 14 ACS Paragon Plus Environment

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[Figure 5]

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The dimensions of rod-like CNCs prepared with H2O2 oxidation were determined by

301

TEM images as shown in Figure 5. The average length and diameter of nanoparticles in

302

the milky suspension from fraction 1 (Figure 5A) were 263 ± 28 nm and 23 ± 5 nm,

303

respectively, which is slightly lower than the value obtained from DLS measurements.

304

Note that DLS overestimates the size of particle even for standard silica nanoparticles.28

305

The sizes for ultrasonically treated sample from fraction 3 (Figure 5B) were around 305 ±

306

90 nm and 30 ± 9 nm, values close to those of fraction 1. These dimensions are in the

307

ranges of CNCs produced by other oxidation processes but with a greater diameter.16,18

308

Solid-State 13C NMR

309

The NMR spectra of the original softwood pulp and carboxylated CNC prepared by H2O2

310

oxidation are presented in Figure 6. Typical signals characteristic of functional groups

311

can be observed in the spectra. The peak between 100 and 110 ppm is assigned to the

312

anomeric carbon C1 and that between 80 and 95 ppm is for C4. The next peaks in 70-80

313

ppm region are associated with C2, C3 and C5 carbons. The region between 60 and 70

314

ppm is attributed to C6 of the primary alcohol group. In the cellulose spectra, the narrow

315

cluster at 89 ppm (C4) corresponds to anhydroglucose units in the crystalline parts. The

316

broad peak located in 84 ppm (C4’) is characteristic of the anhydroglucoses with less

317

order in cellulose structural arrangements.31

318

The NMR spectra of the original softwood fiber and carboxylated CNCs were almost

319

identical, implying that no significant changes happened in the chain conformations of

320

the cellulose. However, the intensity of the peak around 65 ppm decreased up to 32 %

321

compared to the original cellulose fiber. This change can be a sign of –CH2OH oxidation 15 ACS Paragon Plus Environment


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in the C6 position to form –COOH groups on the CNC surface, rather than other

323

hydroxyl groups. The C1 signal intensity of the cellulose backbone decreased, indicating

324

that cleaving glycoside bonds of cellulose chains occurred. The multiple peaks located

325

between 70-80 ppm are attributed to the oxidation of hydroxyl groups and bond cleavage

326

of C2-C3 during H2O2 oxidation. Observed changes in the C4 and C4՜ signals can be

327

taken as proof for the formation of highly crystalline nanoparticles, in good agreement

328

with the NMR spectra of cellulose nanowiskers produced with sulfuric acid by Sèbe et

329

al.32 [Figure. 6]

330

331

ATR-FTIR Spectroscopy

332

The original softwood pulp and carboxylated CNC particles were further characterized by

333

FTIR spectroscopy as seen in Figure 7. Normalization of FTIR spectra of two cellulosic

334

samples was done to make a meaningful qualitative comparison. The broad band at 3330

335

cm-1 is due to the stretching vibration of –OH groups which was affected by the inter-

336

molecular or intra-molecular hydrogen bonds of the cellulose molecules. Higher intensity

337

of this broad peak in carboxylated CNCs arises from hydrogen bonds breaking during

338

H2O2 oxidation reactions and consequently more stretching vibration of –OH groups

339

compare to the intact softwood structure. The absorption peaks at around 2905, 1425 and

340

1021 cm-1 are attributed to C–H stretching vibrations, –CH2 scissoring and CH2–O–CH2

341

stretching, respectively.33 The peak located at 1738 cm-1 corresponds to C=O stretching

342

while the absorption at 1630 is related to the asymmetric stretching vibration of the

343

carboxyl groups of the oxidized CNCs.34 These emerged peaks support the formation of

344

carboxyl and carbonyl groups during H2O2 oxidation which is in accordance with the


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conductometric results. The possibility that these bands were due to aldehyde groups was

346

disproved by performing a titration with hydroxylamine hydrochloride, a standard

347

method to determine aldehyde groups18. No aldehyde groups were found, proving the

348

bands cannot be due to aldehyde groups, but must be due to carboxyl groups. Of

349

importance to note is that most of carbonyl groups formed through the oxidation of the

350

hydroxyl groups have been converted to carboxyl groups. The carboxyl groups can be

351

employed as active sites in surface modifications, especially for immobilizing proteins

352

and enzymes. [Figure 7]

353 354

Investigation of the Crystalline Structure by XRD

355

Figure 8 presents X-ray diffraction patterns of softwood pulp and carboxylated CNCs

356

produced by H2O2 oxidation, confirming the NMR results. The diffraction patterns of two

357

cellulosic samples show a sharp peak at 2θ angle of 22.6° for the (200) peak and two

358

weak peaks at 2θ=15.1° for the (11ത0) peak and 2θ=16.5° for the (110) peak. These peaks

359

correspond to the main crystalline region of the cellulose structure35 which are almost

360

identical in original pulp and carboxylated CNCs. It indicates that the original crystalline

361

structure of cellulose fibrils was well maintained during H2O2 oxidation reactions. This

362

finding is consistent with the reports about the crystallinity changes of cellulose caused

363

by APS16 or sulfuric acid.32

364

The crystallinity indices (C.I.) was calculated according to Segal et al.: 36

365

C. I. ሺ%ሻ =

ூమబబ ିூೌ೘ ூమబబ

×100



366

where Iଶ଴଴ is the intensity obtained from the (200) plane reflection, Iୟ୫ is the minimum

367

intensity between the (100) and (200) peaks. The C.I. of the original pulp and

368

carboxylated CNC particles were 77.5 % and 70.5 %, respectively which exhibit a slight

369

decrease after the reaction of cellulose pulp with H2O2 and catalyst for 3 days. It is known

370

that the amorphous regions and the surface of crystalline regions in cellulose are

371

kinetically more accessible to chemical reactions.9 Therefore, the decrease in crystallinity

372

could be explained by the fact that H2O2 oxidation-induced hydrolysis does not only

373

happen at the amorphous regions but also at the surface of the crystalline parts of

374

cellulose. In addition, some extensive surface modifications have possibly caused

375

destructive changes in the crystalline structure of cellulose. Interestingly, these results are

376

in good agreement with those of Wang et al.37 who studied CNCs produced from

377

microcrystalline cellulose using a mixture of sulfuric acid and hydrochloric acid.

378

Similarly, Yang and van de Ven,30 reported that crystalline index of softwood cellulose

379

pulp (75 %) decreased to 49 % in dialdehyde modified cellulose (DAMC) obtained by

380

periodate oxidation. This is related to opening of the glucopyranose rings of cellulose

381

from C2-C3 bond during oxidation, resulting in detrimental lowering of the C.I, which

382

possibly occurs also in carboxylated CNCs produced by H2O2 oxidation. In contrast, the

383

carboxylated CNCs extracted by using APS under thermal conditions showed a higher

384

crystallinity than that of the initial cellulose.15

385

It is worth noting that the degree of crystallinity of carboxylated CNCs prepared by H2O2

386

oxidation is high and close to that of CNCs generated with acid hydrolysis from bleached

387

soft wood kraft37 and kenaf core woods38. Thus, the absence of significant changes in the


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Page 19 of 37


388

total crystallinity of cellulose and high-crystalline CNCs show that the catalyst assisted-

389

H2O2 oxidation method has great potential in the production of CNC nanoparticles. [Figure 8]

390

391

In conclusion, a one-step procedure for producing carboxylated cellulose nanocrystals

392

from softwood cellulose pulp was designed by applying H2O2 as an oxidant and

393

CuSO4.5H2O catalyst. The use of H2O2 has significant benefits over other chemicals

394

because of its easily removing and/or rapidly decomposing to water and oxygen after the

395

hydrolysis processes. The mechanism of the reaction is based on penetration of free

396

radical ions formed by catalyst-assisted H2O2 oxidation to the outer layer of the

397

crystalline regions and all amorphous regions. Since the proposed method uses only non-

398

expensive and environmentally friendly chemicals, it is capable of large-scale production

399

of CNCs decorated with negatively charged carboxyl groups. Carboxylated CNCs

400

displayed a rod-shaped morphology with high size uniformity and almost the same

401

dimensions to other cellulose nanoparticles produced by oxidative methods, albeit with a

402

larger width. The presence of carboxyl groups on the surface of CNCs was proved by

403

conductometric titration and FTIR spectroscopy. DLS measurements with NaCl addition

404

showed colloidal stability of carboxylated CNC suspension at salt concentrations less

405

than 400 mM, arising from the dominating electrostatic repulsions between the particles.

406

The NMR and XRD results showed that original structure of cellulose fibrils is, to a large

407

extent, maintained during H2O2 oxidation. More detailed studies are required to optimize

408

parameters such as amount of H2O2, different catalysts, reaction time, temperature and

409

pH and characterize carboxylated CNCs produced by H2O2 oxidation to tailor their

410

applicability to different fields. 19 ACS Paragon Plus Environment


411

ACKNOWLEDGEMENT

412

The authors thank the financial support of the Iranian Ministry of Science, Research and

413

Technology and the Natural Science and Engineering Research Council of Canada

414

(NSERC Discovery grant 42686-13). They also acknowledge the access to facilities and

415

instrumentation supported by the Pulp and Paper Research Centre and Department of

416

Chemistry, McGill University.

417

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Figure and Scheme captions

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Scheme 1. Synthesis process diagram of carboxylated cellulose nanocrystals by catalyst

549

assisted-H2O2 oxidation.

550

Figure 1. Conductometric titration result of 20 mg carboxylated cellulose nanocrystals

551

obtained by catalyst assisted-H2O2 oxidation.

552

Figure 2. Particle size distribution of carboxylated cellulose nanocrystals obtained by

553

catalyst assisted-H2O2 oxidation (A) and changes of the equivalent spherical diameter

554

versus salt (NaCl) concentration (B).

555

Figure 3. Optical and polarized micrographs of (A) carboxylated cellulose nanocrystals

556

from fraction 1 prepared by catalyst assisted-H2O2 oxidation; (B) precipitate from

557

fraction 2; and (C) ultrasonically treated precipitate from fraction 3. Scale bars of images

558

(A), (B) and (C) are 10 µm.

559

Figure 4. AFM height (A) and 3 D (B) images of carboxylated cellulose nanocrystals

560

from fraction 1 prepared by catalyst assisted-H2O2 oxidation; (C) cellulose nanocrystals

561

from fraction 3 after sonication.

562

Figure 5. TEM image of carboxylated cellulose nanocrystals prepared from softwood

563

fibers (A) by catalyst assisted-H2O2 oxidation from fraction 1 and (B) after sonication

564

from fraction 3.

565

Figure 6. Solid state carbon-13 NMR spectra of softwood pulp and carboxylated

566

cellulose nanocrystals obtained by catalyst assisted-H2O2 oxidation.


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Page 27 of 37


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Figure 7. FTIR spectra of original softwood pulp and carboxylated cellulose nanocrystals

568


569

Figure 8. XRD profiles of original softwood pulp and carboxylated cellulose nanocrystals

570




571

Page 28 of 37

Figure graphics

572

573

Soaked softwood pulp

574 Copper (II) sulfate pentahydrate-mediated H2O2 oxidation 575 Cellulosic products 576 Washing by centrifugation 577 White precipitate

578

Supernatant (Dissolved oligosaccharides)

579

Suspended in deionized water

580

Decantation after 5 min

581

Precipitate Milk-like suspension

Addition of water

582

583

584

585

Fraction 3 Stabilized by sonication

Fraction 2 Precipitate Fraction 1 Freshly prepared

586


Fraction 1 After 1 month

Page 29 of 37


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189x103mm (120 x 120 DPI)

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