# Cellulose nanocrystals from native and mercerized cotton

Somia Haouache, Clara Jimenez-Saelices, Fabrice Cousin, Xavier Falourd, Bruno Pontoire, Karine Cahier, François Jérôme, Isabelle Capron

► To cite this version:

Somia Haouache, Clara Jimenez-Saelices, Fabrice Cousin, Xavier Falourd, Bruno Pontoire, et al.  
Cellulose nanocrystals from native and mercerized cotton. Cellulose, 2022, 29 (3), pp.1567-1581.  
10.1007/s10570-021-04313-8 . hal-03561543

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# Cellulose nanocrystals from native and mercerized cotton

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Somia Haouache¹,², Clara Jimenez-Saelices¹, Fabrice Cousin³, Xavier Falourd¹, Bruno

Pontoire¹, Karine Cahier¹, François Jérôme² and Isabelle Capron¹*

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¹UR1268 Biopolymères Interactions Assemblages, INRAE, 44316 Nantes, France

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²Institut de Chimie des Milieux et Matériaux de Poitiers, Université de Poitiers, 86073

Poitiers, France

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³Laboratoire Léon Brillouin, Université Paris-Saclay, CEA-Saclay, Gif-sur-Yvette, France

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*Correspondence: isabelle.capron@inrae.fr; Tel.: +33-24067-5095; orcid: 0000-0001-9145-3803

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**Abstract:** Nanocelluloses occur under various crystalline forms that are currently being selectively used for a wide variety of high performance materials. In the present study, two cellulose fibers (CF-I) were mercerized by alkaline treatment (CF-II) without molar mass variation (560,000 g/mol) and both were acid hydrolyzed, forming cellulose nanocrystals in native (CNC-I) and mercerized (CNC-II) forms. This study focuses on the detailed characterization of these two nanoparticle morphologies (light and neutron scattering, TEM, AFM), surface chemistry (zetametry and surface charge), crystallinity (XRD, ¹³C NMR), and average molar mass coupled to chromatographic techniques (SEC-MALLS-RI, A4F-MALLS-RI), revealing variations in the packing of the crystalline domains. The crystal size of CNC-II is reduced by half compared to CNC-I, with molar masses of individual chains of 41,000 g/mol and 22,000 g/mol for CNC-I and CNC-II, respectively, whereas the same charged surface chemistry is measured. This study gives an example of complementary characterization techniques as well as results to help decipher the mechanism involved in mercerization.

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**Keywords:** cellulose nanocrystals, mercerization, cellulose II, biobased nanoparticles, nanostructuration.

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## 1. Introduction

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Cellulose is a linear homopolysaccharide of D-glucopyranose units connected by β(1-4) glycosidic bonds (Habibi, Lucia, and Rojas 2010; Moon et al. 2011; Nishiyama 2009). It is stabilized by an inter- and intramolecular complex network of hydrogen bonds and van der Waals interactions.

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According to the association type, cellulose exists in six crystalline forms called cellulose I, II, III-I, III-II, IV-I and IV-II (Kroon-Batenburg, Bouma, and Kroon 1996). Cellulose I corresponds to fibrillary native cellulose with parallel oriented chains. The other forms are obtained by conversion of type I by chemical and/or thermal treatments (Atalla and VanderHart 1999; Gardner and Blackwell 1974; Nishiyama, Langan, and Chanzy 2002). Cellulose I can undergo an irreversible transition into a more thermodynamically stable crystalline form, cellulose II, by two distinct processes: regeneration or mercerization. Mercerization involves intracrystalline swelling of the cellulose in concentrated aqueous NaOH where the limit concentration depends on the temperature between 8-15%, with lower temperatures that allow transformation at lower concentrations (Duchemin 2015; Warwicker 1967) and where chains change their orientation from original parallel chains
of cellulose I to antiparallel chains (opposite polarity) (Fink and Philipp 1985; Kolpak, Weih, and Blackwell 1978; Stipanovic and Sarko 1976). The mechanism of mercerization has long been studied. An interdigitation mechanism was first proposed by Okano and Sarko (Okano and Sarko 1985). NaOH is absorbed, converting cellulose I into a swollen structure in which all contacts between adjacent chains are removed. Once NaOH has been removed by washing with water, a bi-oriented cellulose II structure is obtained (P. Langan, Nishiyama, and Chanzy 1999; Paul Langan, Nishiyama, and Chanzy 2001).

Nishiyama et al. (Nishiyama, Kuga, and Okano 2000) proposed a molecular association in Na-Cellulose where van der Waals' interaction is the driving force of the formation of cellulose II. The effect of mercerization on crystallinity was investigated for different cellulose sources (J. F. Revol, Dietrich, and Goring 1987). All cellulose II obtained had a narrow range of crystallinity and, a constant crystal size. The crystallinity index for the mercerized celluloses remained in a narrow range of 0.50-0.66, whereas it varied from 0.41 to 0.95 for the native cellulose. The crystal size was approximately constant for the mercerized celluloses, from 3.4 nm to 4.4 nm, whereas it varied from 2.9 nm to 15.4 nm in native celluloses. The result is that in the case of highly crystalline cellulose, mercerization reduces crystallinity and crystal size, whereas in the case of low crystallinity cellulose, mercerization increases crystallinity and the size of the crystal. These trends would not be expected if the conversion of cellulose I to cellulose II was simply a change in conformation of the chain or arrangement of atoms. These results are more in line with the idea that mercerization involves a complete destruction of the structure of cellulose I by separation of the molecular chains, followed by the reforming of the crystalline structure in the form of cellulose II. These results are consistent with the hypothesis that mercerization involves a mixture of adjacent and antiparallel cellulose microfibers (Okano and Sarko 1985).

Type II cellulose nanocrystals have already been obtained from acid hydrolysis (Sebe, Ham-Pichavant et al. 2012) or after mercerization of fibers (Neto et al. 2016). Sèbe et al. (Sèbe et al. 2012) prepared CNC samples using nine different conditions involving $H_2SO_4$ at concentrations varying from 62 to 66% and up to 120 min. One of them led to CNC-II only (not a mixture of CNC-I and CNC-II). The resulting nanocrystals (CNC-II) were found to be smaller than CNC-I and were ribbon-shaped with rounded tips and larger crystallites, whereas Neto et al. (Neto et al. 2016) described CNC-II as being shorter (from 240 nm to 132 nm) and broader (from 15 nm to 19 nm), with identical thickness (around 4 nm), and with an increased crystallinity from 56% to 68%. For Li et al. (Li et al. 2018), the mercerized CNCs were even much smaller (19 nm in length and 11 nm in width) with ellipsoid shapes.

CNCs are predicted to have a major impact in the coming years, and variability will be a key of this development. Recent reviews show the interest of the selective modification of the reducing end (Heise et al. 2021; Tao et al. 2020) of CNC-I. A growing interest is now focused on CNC-II with the hemiacetal form at the two extremities. A precise control of their various forms is therefore of great importance but the transition mechanism is still a matter of debate. In order to better understand the mechanism involved in mercerization, it is consequently of interest to compare different packs of data produced using both different and similar hydrolyses. But also to compare the results obtained from different technics. For example, the ratio of crystalline regions to total fibrils of cellulose (the crystallinity index) is usually investigated by X-ray diffraction and solid-state 13C-NMR experiments (Park et al. 2010; Zugenmaier 2008). The first one is based on the detection of a diffraction plane, which considers structuration of several glucose residues. The second one is based on the variation of the chemical shift associated with the angles of the glycosidic bond. These two techniques that observe cellulose at two different scales are quite complementary. In the present study, native (CF-I) and mercerized (CF-II) cotton fibers are both hydrolyzed using the same sulfuric acid hydrolysis process, leading to CNC-I and CNC-II. A full set of complementary
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techniques is described and used to precisely characterize the morphology, molar mass, structure, surface charge and degree of polymerization of both nanocrystals.

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## 2. Materials and Methods

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**Materials:** The native cotton cellulose fibers were obtained from Buckeye Technology Inc., USA. All reactants had a purity of above 95% and were acquired from Sigma Aldrich and used without further purification. Ultrapure water was produced with the Milli-Q reagent system (18.2 MΩ cm, Millipore Milli-Q purification system).

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**Cellulose sample preparation:** Native cotton cellulose fiber (CF-I) was mercerized (CF-II) according to a protocol similar to that described by Neto et al. (Neto et al. 2016). Ten grams of CF-I were introduced into 300 mL of 20 wt% NaOH and mechanically stirred for 5 h at 25°C. The mixture was washed several times with distilled water in order to remove the NaOH solution, and then dried at 40°C for 48 h. This conversion was carried out with a yield of 100%.

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**Preparation of cellulose nanocrystals (CNC-I and CNC-II):** Both CNCs were prepared by hydrolysis with sulfuric acid according to the method of Revol et al. (J.-F. Revol et al. 1992) with minor modifications. Briefly, cellulose nanocrystals (CNC-I and CNC-II) were prepared under the same conditions from fibers (CF-I and CF-II, respectively) using sulfuric acid hydrolysis at 64% at 68°C under stirring for 20 min. After hydrolysis, the suspensions were washed by centrifugation, dialyzed to neutrality against Milli-Q water for 2 weeks, and deionized using mixed bed resin (TMD-8). The final dispersion was sonicated for 10 min, filtered and stored at 4°C. The yield was 64% and 40% for CNC-I and CNC-II, respectively.

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### Cellulose sample characterization

**X-ray Diffraction.** The determination of crystalline type, crystallinity index and crystal size of the different samples was performed by X-ray Diffraction (XRD) analysis using a Bruker D8 Discover diffractometer (Karlsruhe, Germany) equipped with a VANTEC 500 2D detector. X-ray radiation, CuKα1 (λ = 0.15406 nm), produced in a sealed tube at 40 kV and 40 mA, was selected and parallelized using crossed Göbel mirrors and collimated to produce a beam of 300 or 500 µm in diameter. The suspensions of nanocrystals were freeze-dried and then pressed at room temperature to obtain dense pellets, while the fibers were used as such. The diffraction patterns were recorded for 10 min over a range from 3° to 40° (2θ). The recorded intensity was normalized by the total peak area to eliminate the influence of the thickness variation and the absorption coefficient of the samples. The X-ray crystallinity index (CI<sub>XRD</sub>) was estimated from the crystalline to amorphous areas using Origin (v8.0891) software.

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**Solid-state NMR CP-MAS.** The NMR experiments were carried out on an Avance III-400 MHz spectrometer (Bruker; France) operating at 100.62 MHz for 13C, equipped with a double-resonance H/X CP-MAS 4-mm probe for CP-MAS (Cross-Polarization Magic Angle Spinning) solid-state experiments. The samples were wetted and spun at 12,000 Hz at room temperature.

CP-MAS spectra were acquired with a contact time of 1.5 ms and over an accumulation of 2048 scans separated by a recycling delay of 10 s. The carbonyl carbon was set to 176.03 ppm through external glycine calibration. NMR spectra deconvolution was performed using PeakFit® (v.4.11) software (Systat Software, Inc., USA). Peak chemical shifts were assigned according to (Larsson et al. 1999; Newman and Davidson 2004). The
146 NMR crystallinity index of CF and CNC was calculated according to (Larsson et al. 1999;
147 Zuckerstätter et al. 2013).

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149 **Conductometry.** The hydrolysis of the cellulose with sulfuric acid makes it possible to
150 obtain a colloidal suspension of the nanometric-sized crystals with SO3- charges on their
151 surface. The measurement of the quantity of charges on the CNC surface charge was
152 performed by conductometric titration with a 0.001 M NaOH solution using a TIM900
153 titration manager and a CDM230 conductimeter equipped with a CDC749 conductivity
154 cell.

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156 **Zeta Potential** (ζ-potential). ζ-potential experiments were performed with a Malvern
157 NanoZS instrument. All measurements were made at a temperature of 20°C with a
158 detection angle of 12.8°. CNC dispersions of 1 g/L at pH = 7 were prepared at 20°C and
159 filtered by 5 µm. Each sample was measured a total of five times. The confidence
160 interval (error) presented is the standard deviation of samples measured in triplicate.
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162 **Asymmetrical flow field-flow fractionation coupled to Multi-Angle Laser Light Scattering and Refractive Index** (A4F-MALLS-RI) detection. An AF4 instrument was 163 coupled with two online detectors: a MALLS instrument (DAWN Heleos II) fitted with a 164 K5 flow cell and a GaAs laser (λ = 663 nm), and a refractometric detector operating at 165 the same wavelength (Optilab T-rEX) from Wyatt Technology (Santa Barbara, CA, USA). 166 The AF4 instrument consisted of an AF4 channel (275 mm-long), a 350-µm-thick spacer 167 and a regenerated cellulose membrane with a nominal cut-off of 10 kDa (Millipore, 168 Bedford, MA, USA). The refractive index increment dn/dc was 0.146 mL/g, a value 169 classically used for glucans in water (Paschall and Foster 1952). The AF4 channel flow, 170 cross flow, sample injection and focus flow were controlled with a Wyatt Eclipse AF4 171 flow chassis, a pump and an autosampler from ThermoFisher Scientific (Waltham, MA, 172 USA). CNC dispersions of 0.5 g/L in water were prepared at 20°C and systematically 173 freshly sonicated (amplitude 5, 8 s, 2 on/1 off) before being injected. Each sample was 174 measured a total of two times. The weight and number-average molar masses (M̄<sub>w</sub>, M̄<sub>n</sub>) 175 and the polydispersity (M̄<sub>w</sub>/M̄<sub>n</sub>) of CNCs were determined with Wyatt ASTRA® software 176 (v. 6.1.4) with Zimm extrapolation of order 1. 177

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179 **Size Exclusion Chromatography coupled to Multi-Angle Laser Light Scattering and Refractive Index** (SEC-MALLS-RI) detection. The determination of molar mass 180 distribution of chains of cellulose in DMAc/LiCl was carried out at room temperature 181 using an OMNISEC system (Malvern). The size exclusion chromatography (SEC) 182 (OMNISEC Resolve, Malvern) system was coupled with a multi-angle laser light 183 scattering detector (MALLS, Malvern) and OMNISEC Reveal devices (Malvern). The SEC 184 columns used were Viscoteck Tguard, LT4000L, LT5000L and LT7000L. The mobile phase 185 used for SEC was N,N-dimethylacetamide (DMAc) (HPLC grade) containing lithium 186 chloride (LiCl) (0.9% v/w), that had been filtered through 0.6-µm polypropylene 187 prefilters. This eluant was chosen because it solubilizes cellulose without significant 188 depolymerization during the dissolution process or during storage at room temperature 189 for long periods (Dupont and Harrison 2004; Yanagisawa and Isogai 2005). Calculation of 190 weight- and number-average molar masses (M̄<sub>w</sub>, M̄<sub>n</sub>) and polydispersity (M̄<sub>w</sub>/M̄<sub>n</sub>) of 191 samples were performed with a dn/dc value of 0.136 mL/g (Hasani et al. 2013) and 192 determined with OMNISEC software (v.10.30) with Zimm extrapolation of order 2. 193
194 Cellulose was solubilized in the DMAc/LiCl (9% v/w) (Dawsey and McCormick 1990; 195 Medronho and Lindman 2015) via solvent exchange steps H2O/Met-OH/DMAc CF-I and 196 CF-II and H2O/Et-OH/DMAc for CNC-I and CNC-II.
197 For fibers, 100 mg (dry content) of CF-I and CF-II were washed with 30 mL methanol, and 198 the excess of methanol was removed by filtration on fritter n° 3. This step was repeated
199 three times. The recovered pellet was washed three times with 30 mL of DMAc for
200 solvent exchange, and the excess of DMAc was removed by filtration on fritter n° 3.

201 After solvent exchange steps, 10 mL of D MAc/LiCl (9% v/w) were added to the vial
202 containing the sample and allowed to stir magnetically at 4°C for dissolution.

203 For CNCs, the samples in the form of aqueous suspensions were freeze-dried. The dry
204 extract obtained (approximately 20 mg) was washed with ethanol, and the excess of
205 ethanol was removed by centrifugation (2220 g for 15 min at 20 °C) (Hasani et al. 2013).

206 This step was repeated twice and the material was then put in DMAc for solvent
207 exchange under magnetic stirring at room temperature overnight. The excess of DMAc
208 was removed by centrifugation (2220 g for 15 min at 20°C). After the solvent exchange
209 steps, 2 mL of DMAc/LiCl 9% (v/w) were added to the vial containing the sample and
210 allowed to stir magnetically at 4°C for dissolution.

211 The final concentration of the samples was 10 g/L. The dissolution was stopped by the
212 addition of pure DMAc. The final concentration of samples in DMAc/LiCl (0.9% v/w) was
213 1 g/L. Before injection, the samples were filtered through a 0.45-µm
214 polytetrafluoroethylene (PTFE) membrane filter.

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216 Transmission Electron Microscopy (TEM). Droplets of CNC suspensions at 0.8 g/L were
217 deposited on freshly glow-discharged carbon-coated microscope grids (200 mesh, Dalta

218 Microscopies, France) for 2 min. The excess liquid was removed by filter paper,
219 negatively stained with an aqueous solution of phosphotungstic acid at 10 g/L for 2 min
220 and dried just before TEM observation. We used a JEOL type transmission electron
221 microscope (JEM-1230) operating at a voltage of 80 keV. The average dimensions
222 (length and width) of the CNCs were determined from TEM image analysis of
223 approximately 350 particles using ImageJ software.

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227 Atomic Force Microscopy (AFM). To determine the average thicknesses of the
228 nanocrystals, the suspensions were diluted to 0.05 g/L and then deposited on mica
229 substrates. The measurements were carried out at room temperature by an Innova AFM

230 (Bruker) using a monolithic silicon tip (TESPA, Bruker, spring constant k = 42 N/m, 231 frequency f0 = 320 kHz). Image processing was performed with WSxM 5.0 software.

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231 Small Angle Neutron Scattering (SANS) experiments. SANS experiments were carried
232 out at room temperature using the small-angle PA20 and PAXY diffractometers at the
233 Laboratoire Léon Brillouin (CEA/CNRS) in Saclay (France). Three configurations were

234 used for PA20, covering a Q range from 0.0006 and 0.44 Å⁻¹ (6 Å at 1.1 m, 6 Å at 8 m,
235 and 15 Å at 17.5 m), where Q is the wave vector (Q = 4π sin θ/2, where θ is the
236 scattering angle and λ is the neutron wavelength), and four configurations for PAXY,
237 covering a Q range from 0.002 and 0.5 Å⁻¹ (5 Å at 1 m, 5 Å at 3 m, 8.5 Å at 5 m and 15 Å
238 at 6.7 m). CNC dispersions of 2 g/L in 2 mM NaCl were prepared at 20°C and then
239 extensively dialyzed against D2O to obtain the best possible contrast as well as to
240 reduce the incoherent scattering as much as possible, and then systematically freshly
241 sonicated for 10 s and loaded in quartz cells (Hellma) with small path lengths (1 and 2
242 mm). To determine the CNC dimensions, the data were fitted with Sasview software.

243 Several fitting models were tried using the form factor of a parallelepiped with a
244 rectangular section, averaged over all space orientations, and constituting a perfectly
245 fitting model of the rod-like CNCs (Cherhal, Cousin, and Capron 2015). Aggregation
246 experiments in solution were performed on suspensions at 2 g/L of CNC-I and CNC-II in
247 2, 50 and 100 mM NaCl. The suspensions were measured after sonication.

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# 3. Results

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## 3.1 Structural description

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The XRD patterns of native, mercerized and hydrolyzed cotton samples are shown in Fig. 1.

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<figure><img src="image_2.png" /></figure>

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**Figure 1.** X-ray diffraction patterns of cotton fibers in native (CF-I) and mercerized CF-II forms and their respective hydrolyzed cellulose nanocrystals in the native (CNC-I) and mercerized (CNC-II) forms, and cross-sections of elementary crystallites deduced from the analysis of peak broadening (the indexation of corresponding lattice planes is described in Supporting Information).

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The diffraction patterns of CF-I and CNC-I are typical of cellulose I with the presence of diffraction peaks at 15.1°, 16.9°, 20.7° and 22.8°, corresponding to (1-10), (110), (012/102) and (200) crystallographic planes, respectively. After mercerization, the crystallinity index ($CI_{XRD}$) of CF-II decreased. For the mercerized sample, CF-II and CNC-II at 12.3°, 20.0° and 21.7° corresponded to the (1-10), (110) and (020) reflections, respectively (Duchemin 2015; Isogai et al. 1989; Nishiyama, Kuga, and Okano 2000), whereas traces of cellulose I residuals can be recognized at 15.1° and 16.9° (Fig. 1). This allomorphic modification was achieved without loss in mass (Table 1). XRD peak analysis (see values in SI) allowed representation of the crystals (Fig. 1). The (1-10) and (110) crystalline planes have interplane dimensions of 0.61 nm and 0.54 nm, respectively (Goussé et al. 2002; Sugiyama, Vuong, and Chanzy 1991). Similarly, for CNC-II, the distances for (1-10) and (110) are 0.72 nm and 0.44 nm, respectively (Kolpak, Weih, and Blackwell 1978; P. Langan, Nishiyama, and Chanzy 1999; Sèbe et al. 2012).
After sulfuric acid hydrolysis of the fibers, the XRD results showed an increase of the crystallinity index ($CI_{XRD}$). For the native form, 64% of the cellulosic material was recovered after hydrolysis, whereas the $CI_{XRD}$ only increase by 5% (from CF-I to CNC-I). The hydrolysis then affects amorphous as well as crystalline domains. Considering fibers, all the material was recovered after mercerization (yield of 100%). However, after acid hydrolysis, only 40% of the initial material was recovered, while the $CI_{XRD}$ increased by 30% (from CF-II to CNC-II). Mercerization leads to fibers that are more susceptible to acid hydrolysis, probably due to lower organization. Moreover, as can be observed in other studies (French 2014; Neto et al. 2016), mercerization drastically reduces crystallinity as well as the crystal dimensions of the cotton.

<table><caption>**Table 1:** Weight fraction (yield) recovered after treatment, crystallinity index (CI) calculated from XRD (CIXRD), Mean CI calculated from solid-state NMR (13C CP-MAS) spectra (CINMR). Deconvolution of the C4 region of 13C CP-MAS spectra.</caption><thead><tr><th rowspan="2">Samples</th><th rowspan="2">Yield (%)</th><th rowspan="2">CI<sub>XRD</sub> (%)</th><th rowspan="2">CI<sub>NMR</sub> (%)</th><th colspan="3">Deconvolution of the C4 region</th></tr><tr><th>crystalline</th><th>paracrystalline intermediary domain</th><th>amorphous</th></tr></thead><tbody><tr><td>CF-I</td><td>-</td><td>60</td><td>67</td><td>25%</td><td>42%</td><td>26% Acc + 7% inAcc</td></tr><tr><td>CNC-I</td><td>64</td><td>65</td><td>75</td><td>36%</td><td>39%</td><td>25%</td></tr><tr><td>CF-II</td><td>100</td><td>40</td><td>72</td><td>58%</td><td>14%</td><td>28%</td></tr><tr><td>CNC-II</td><td>40</td><td>70</td><td>85</td><td>74%</td><td>11%</td><td>15%</td></tr></tbody></table>

Figure 2 shows the 13C CP-MAS NMR spectra of CF-I and CF-II and confirms the mercerization process with the two peaks at 88.1 and 86.9 ppm in the CF-II spectrum that are characteristic of type II cellulose (Ibbett, Domvoglou, and Fasching 2007; Newman and Davidson 2004). CF-I had a $CI_{NMR}$ of 67%, and this crystallinity increased after acid hydrolysis. For CF-II, this $CI_{NMR}$ increased up to 72% after mercerization and up to 85% after subsequent hydrolysis (CNC-II preparation).

The signals in the 86-92 ppm region that refer to crystalline domains were further decomposed. This deconvolution analysis discriminates an "in-core" ordered region from a "paracrystalline" organization described as having an intermediate order between amorphous and crystalline cellulose (Zuckerstätter et al. 2013) (Fig. 2). According to this analysis, original CF-I is characterized by a $CI_{NMR}$ of 67% composed of 25% of a pure crystalline domain and 42% of a so-called paracrystalline domain (Table 1). The remaining 33% are divided into 26% of accessible and 7% of inaccessible amorphous domains.

After acid hydrolysis, an increase in the relative area of crystalline peaks at 86-92 ppm is observed, and the $CI_{NMR}$ increased in accordance with XRD results. However, the selective analysis of crystalline and paracrystalline structures shows that the paracrystalline organization is only slightly decreased. The increase in crystallinity between CF-I and CNC-I is then correlated with a loss of the amorphous part, since the paracrystalline domains are much less affected. According to the model proposed by Larsson and also used by Wickholm, paracrystalline domains are structures surrounding nanocrystals in the nanofibers and are less accessible than amorphous domains. After
316 hydrolysis, the amorphous domain, visible in the 80-86 ppm region, shows only one
317 remaining peak.

318 After mercerization, a typical spectrum of cellulose II revealed the allomorphic
319 transition. However, the 13C NMR spectrum of CF-II shows a signal characteristic of
320 crystalline C6 of cellulose I representing about 4% of the total C6 signal. This residual
321 crystalline cellulose I-type conformation results from an ineffective penetration of NaOH
322 in crystalline domains; they are potentially dispersed in a random way, as proposed by
323 Kim et al. (Kim et al. 2006).

324 The mercerization process of the nanofibers results in a slight increase of Cl<sub>NMR</sub>
325 from CF-I to CF-II (Table 1), which is contradictory with XRD results. Simultaneously, a
326 slight decrease of the amorphous contribution from 33% to 28% is observed, and only
327 one peak is observed that refers to only one amorphous type domain. Compared to this,
328 the so-called paracrystalline region, which usually refers to structures surrounding
329 cellulose I nanocrystals, undergoes a sharp decrease from 42% to 14%. The origin and
330 structure of such a state is still not clear (Bregado et al. 2019; Larsson et al. 1999),
331 except that it is intermediate (in terms of mechanical properties, hydrogen bonding and
332 chain ordering) between crystalline and amorphous cellulose. After mercerization, a
333 peak is clearly visible at 85.5 ppm (Fig. 2), referring to that imperfect crystalline region
334 (or, similarly, to an ordered amorphous region). Such a peak was previously observed
335 and attributed to partially ordered cellulose (Ibbett, Domvoglou, and Fasching 2007).
336 The result is that only one type of the amorphous structure remains in a slightly reduced
337 amount, whereas a large part of the paracrystalline-I structure that presumably
338 surrounds the crystalline domains formed by mercerization is lost.

339 Acid hydrolysis of the mercerized cellulose occurs with a loss of mass (yield: 40%)
340 but without much change in the peak attributed to the intermediate structure. The
341 same trend is then observed for both CNC-I and CNC-II. This was already reported by
342 (Wickholm et al. 2001). It implies that acid hydrolysis removes amorphous regions,
343 contrary to the mercerization process that strongly impacts paracrystalline/intermediate
344 domains. The same fraction of 4% of cellulose I observed in CF-II was recovered in the
345 CNC-II sample.
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 2.** (A) <sup>13</sup>C CP-MAS NMR spectra of CF-I and CF-II, and (B) deconvolution of the C4 region of CF-I, CF-II, CNC-I and CNC-II NMR spectra with crystalline forms (black), paracrystalline (gray) and amorphous (green).</figcaption></figure>

However, the results obtained by XRD and NMR are controversial. The loss of crystallinity observed by XRD after mercerization is not observed by NMR (Table 1). In solid-state NMR, considering only the C4 region, chemical shifts are influenced by the conformation of carbon atoms in glycosidic chains bonds, which may be involved in a crystalline, paracrystalline or amorphous structure. For XRD analysis, beyond crystallite orientation, it is the crystal lattice that is directly identified. It is therefore easy to imagine that parts of chains may have conformations related to those of crystal lattices without having a dimension that allows XRD to identify them as such, explaining a higher value of CI by NMR. The variations observed can then be linked to the ability of each technique to detect imperfect organizations. NMR assumes that all the carbons involved are in crystalline structure which considers very short-scale. It analyzes crystalline and
362 paracrystalline organizations in the so-called $Cl_{NMR}$, and distinguishes these forms from 363 the amorphous domain with signals shifted to lower ppm values. In contrast, XRD 364 analysis requires longer scale organization since the presence of paracrystalline 365 organizations is included in the widening peaks attributed to amorphous domains. 366

As a result, a major modification during mercerization comes from this 367 intermediate state that is reformed in smaller amounts after swelling in NaOH and the 368 recrystallization process. In addition, mercerization leads to more crystalline domains 369 that seem to be more discontinuous than the former. Such structures are not fully 370 detected by XRD analysis but assumed by NMR to be globally crystalline. Furthermore, 371 only one amorphous peak is visible after mercerization by NMR, implying only one type 372 of amorphous area. This might reveal a more homogeneous but less organized system, 373 with more imperfections, which is also in accordance with the increased susceptibility to 374 acid hydrolysis of CF-II. After hydrolysis, imperfections are removed and highly 375 crystalline particles are recovered, as detected by both XRD and NMR analyses. 376

## 3.2 Molar mass characterization

In order to follow the process at a molecular level, the native and mercerized fibers 378 were dissolved in DMAc/LiCl and injected into a SEC-MALLS-DRI device. This experiment 379 made it possible to determine the molar mass (Mw) distribution of individual cellulosic 380 chains. It may also determine whether the process that involved NaOH at a high 381 concentration had an impact on the glucosidic chain length. The size exclusion 382 fractionation mode implies that larger molecules are the first to elute. Both fibers were 383 found to have an average molar mass of 560,000 g/mol with a low polydispersity (Table 384 2). Even just a slight shift to higher retention volumes seems to indicate more flexibility 385 of CF-I. However, it is demonstrated here that mercerization treatment of native 386 cellulose fibers through NaOH swelling does not induce any molecular disruption. 387

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Dissolution profiles of samples obtained by SEC-MALLS-DRI. The two nanofibers (CF-I in 389 purple and CF-II in red) are eluted at low retention volumes, whereas the nanocrystals are eluted 390 at higher elution volumes (CNC-I in green and CNC-II in black). 391</figcaption></figure>

**Table 2.** Weight-average molar masses ($\bar{M}_w$), polydispersity ($\bar{M}_w/\bar{M}_n$) and degree of 393 polymerization (DP) of individual chains of cellulosic fibers (CF-I and CF-II) and cellulose 394 nanocrystals (CNC-I and CNC-II) solubilized in DMAc/0.9% LiCl. 395

<table><thead><tr><td>Samples</td><td>$M_w$ (g/mol)</td><td>$M_w/M_n$</td><td>DP<sub>w</sub></td><td>DP<sub>n</sub></td></tr></thead></table>
<table><tr><td>CF-I</td><td>565,000 ± 47,000</td><td>1.3</td><td>3487</td><td>2683</td></tr><tr><td>CF-II</td><td>556,000 ± 43,000</td><td>1.3</td><td>3432</td><td>2640</td></tr><tr><td>CNC-I</td><td>41,000 ± 1,000</td><td>1.2</td><td>253</td><td>210</td></tr><tr><td>CNC-II</td><td>22,000 ± 1,000</td><td>1.2</td><td>135</td><td>112</td></tr></table>

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

Similarly, both CNCs were solubilized in DMAc/9% LiCl (v/w) for Mw distribution determination. They logically appear to have a larger retention volume compared to the fibers (Fig. 3), indicating a significant decrease in the hydrodynamic volume of the chains. The acid hydrolysis of the fibers led to a clear decrease of the Mw, from 560,000 g/mol for both fibers, down to 41,000 g/mol for CNC-I and to 22,000 g/mol for CNC-II (Table 2). Contrary to mercerization that did not affect the chain length, the degree of polymerization (DP) of CNC-II is about half as low as CNC-I after the hydrolysis. Furthermore, the Mw distribution curves of CNC-II were shifted to lower retention volumes but superimposed on a large domain, illustrating the same proportion in occupied volume. In other words, CNC-II is similar in conformation but smaller.

Simultaneously, Mw distributions of the CNCs directly in suspension in water (without a solubilization step) were obtained using A4F-MALLS-DRI analysis (Fig. 4). Since the fractionation is carried out by a cross-flow device, the smaller molecules were the first to elute. The shift to a lower elution time for CNC-II compared to CNC-I confirmed the lower hydrodynamic volumes of CNC-II. The Mw measured were also much lower (Table 3), with 36.106 g/mol and 11.106 g/mol for CNC-I and CNC-II, respectively. These values are in agreement with the results found by the SEC-MALLS-DRI device.

<figure><img src="image_7.png" /></figure>

418

419

420

<caption>**Figure 4.** Distribution of molar masses of suspensions of CNC-I (blue) and CNC-II (red) in water, and RI signal (dotted curves).</caption>

421

422

423

424

425

When dividing the molar mass obtained in crystalline form from both CNCs (Table 3) to that of their individual chains (Table 2), the packing appeared to decrease from 878 to 500 chains for CNC-I and CNC-II, respectively. This is a very high value compared to the dimensions of the elementary CNCs, revealing that some aggregation still remains.
However, it clearly appears that the mercerized CNCs are two to three times smaller in length and packing. The result is that the crystalline domains in NF-II are shorter, with a DP of less than half of those in NF-I.

## 3.3 Characterization of cellulose nanocrystal morphology

The morphology of native and mercerized CNCs was characterized and compared by TEM, AFM and SANS. Figure 5 shows TEM and AFM images of native and mercerized CNCs. Both CNCs are in the form of rigid rods with shorter CNC-II. The average lengths of 118 ± 65 nm and 65 ± 22 nm were determined for CNC-I and CNC-II, respectively (Table 3). This is in accordance with previous results (Neto et al. 2016). When selecting individual CNCs, in order to measure elemental nanocrystals, it was found that CNC-I and CNC-II have the same individual width of 7 ± 3 nm. More surprisingly and differently from what was previously reported by (Neto et al. 2016), the average thicknesses found by AFM were 6.0 ± 2.4 nm and 3.4 ± 1.5 nm for CNC-I and CNC-II, respectively (Table 3). The thickness reduced by half of its value is clearly observable.

<figure><img src="image_8.png" /><figcaption>Figure 5. TEM images of CNC-I (a,b) and CNC-II (c,d) and AFM images of CNC-I (e) and CNC-II (f).</figcaption></figure>

**Table 3.** Weight-average molar masses (Mw) and polydispersity (Mw/Mn) of CNC-I and CNC-II dispersed in water determined by A4F-MALLS-DRI; average dimensions determined from the SANS curve, TEM images and AFM images.

<table><thead><tr><td>Samples</td><td>M&#773;<sub>w</sub></td><td>M&#773;<sub>w</sub>/M&#773;<sub>n</sub></td><td>Length (nm)</td><td>Width (nm)</td><td>Thickness (nm)</td></tr></thead></table>
<table><thead><tr><th></th><th>(10<sup>6</sup>g/mol)</th><th></th><th>SANS</th><th>TEM</th><th>SANS</th><th>TEM</th><th>SANS</th><th>AFM</th></tr></thead><tbody><tr><td>CNC-I</td><td>36 ± 1</td><td>1.5</td><td>175 ± 25</td><td>118 ± 65</td><td>21 ± 1</td><td>7 ± 3</td><td>6.5 ± 0.5</td><td>6.0 ± 2.5</td></tr><tr><td>CNC-II</td><td>11 ± 1</td><td>1.5</td><td>75 ± 25</td><td>65 ± 22</td><td>22 ± 2</td><td>7 ± 3</td><td>3.5 ± 0.5</td><td>3.4 ± 1.5</td></tr></tbody></table>

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

The validation of these results was carried out in suspensions of CNCs in water at 2 mM NaCl by the fit of the curves obtained by small angle neutron scattering (SANS) using the parallelepiped form factor (Figure 6). This measurement allows analysis in dilute suspensions without a drying step. CNC-I shows a higher intensity at low q, revealing a higher Mw, and crosses the profile of CNC-II at intermediate Q. For both samples, the best fit obtained confirmed length and thickness values obtained by microscopy. Even if some individual CNCs must be present in suspension, a best fit is obtained for an average width of 21 nm for both samples, corresponding to an average of three to four elementary laterally associated crystals, as already measured (Cherhal, Cathala, and Capron 2015; Elazzouzi-Hafraoui et al. 2007). The lateral association is then not modified during the mercerization process. The elementary cotton-based CNC-I is generally viewed with a squared cross-section. CNC-II then appears with a rectangular cross-section. The values are in agreement with the results found by A4F-MALLS-DRI and SEC-MALLS-DRI devices.

<figure><img src="image_9.png" /><figcaption>Figure 6. I = f(Q) SANS curves of suspensions of CNC-I and CNC-II in water at 2 g/L in 2 mM NaCl.</figcaption></figure>

464

465

466

467

468

## 3.4. CNC surface charge density

469

470

471

Hydrolysis with sulfuric acid is known to graft anionic sulfate half esters (OSO3−) on to the surface of the CNCs. The same surface charge density is obtained for both CNCs as indicated by the sulfate content of 0.27% and the zeta-potential values of −42 mV for
472 both CNC-I and CNC-II (Table 4). This implies the same susceptibility of both fiber surfaces to acid treatment.

474
475 **Table 4.** Sulfur content (S), surface charge density (SC) and zeta potential of CNC-I and CNC-II.

<table>
   <thead>
    <tr>
     <td>
      Samples
     </td>
     <td>
      S (%)
     </td>
     <td>
      SC (mmol/g)
     </td>
     <td>
      ζ-potential (mV)
     </td>
    </tr>
   </thead>
   <tbody>
    <tr>
     <td>
      CNC-I
     </td>
     <td>
      0.278 ± 0.09
     </td>
     <td>
      0.087 ± 0.03
     </td>
     <td>
      - 42.3 ± 2.7
     </td>
    </tr>
    <tr>
     <td>
      CNC-II
     </td>
     <td>
      0.271 ± 0.03
     </td>
     <td>
      0.085 ± 0.01
     </td>
     <td>
      - 41.9 ± 1.9
     </td>
    </tr>
   </tbody>
  </table>

476
477 **4. Discussion**

478 **Table 5:** Comparison with other studies in length (L), width (W), thickness (T) and crystallinity index (CI).

479

Comparing these results with previous ones (Table 5), Sèbe et al. (Sèbe et al. 2012) prepared CNC samples using nine different sulfuric acid conditions. This resulted in a new type of preparation of cellulose II that led to shorter CNCs with rounded tips and larger crystallites but a lower degree of order. This morphology is very different from our mercerized samples. Since we used the same process as Neto et al. (Neto et al. 2016), the results are more similar. These two studies reveal that the nanocrystals are shorter and preserve lateral associations after mercerization, known as an average of trimer associations. However, the thickness was half as much after mercerization, whereas we confirmed by several technics a decrease by half in our experiment.

490
491 On the basis of our results, we can determine the average amount of chains per elementary crystal in several ways.

493 Using the number-average molar mass (Mn) given by A4F-MALLS-RI, and dividing these values by 3, we obtain an average molar mass of 8·10⁶ g/mol for the elementary CNC-I, and 2.4·10⁶ g/mol for the elementary CNC-II (Table 6). These results, together with those obtained by SEC/MALLS, make it possible to determine the number of cellulosic chains in an elementary crystal: 235 chains per elementary CNC-I and 133 chains per elementary CNC-II. This is large compared to theoretical calculations based on crystal dimensions.

500 Another calculation considers the CNC section obtained from microscopy and the interchain dimensions. The average CNC thickness is 6.5 nm and 3.5 nm for CNC-I and CNC-II, respectively. The (1-10) and (110) interplane dimensions in CNC-I are 0.61 nm and 0.54 nm, respectively (Goussé et al. 2002; Sugiyama, Vuong, and Chanzy 1991). Similarly, interplane dimensions for CNC-II are 0.72 nm and 0.44 nm for (1-10) and (110), respectively (Kolpak, Weih, and Blackwell 1978; P. Langan, Nishiyama, and Chanzy 1999; Sèbe et al. 2012). Considering that CNC is completely crystalline, this leads to
507 7x6.5/0.61x0.54=162 cellulose chains per elementary CNC-I and 7x3.5/0.72x0.44=77
508 cellulose chains for CNC-II.

509 Calculating average crystalline dimensions from XRD analysis (see Fig. 1), we obtain
510 4.3x6.2/0.61x0.54=80 cellulose chains per elementary CNC-I and 2.9x5.5/0.72x0.44=50
511 cellulose chains for CNC-II.

512 Considering these different results, the microscopy should overestimate the crystal
513 dimension; overestimating also the number of chains per elementary crystals. On the
514 other hand XRD results taking into account the effective crystalline part may not take
515 into account defaults and surface effects underestimating the number of chains per
516 elementary crystals. The effective value should then be somewhere in between that we
517 can estimate at 120 chains/elementary crystals for CNC-I, and 60 chains/elementary
518 crystals for CNC-II.

519 We couldn't find values to compare such results with other works, regardless of the
520 calculation method, about half of the former number of chains per elementary
521 nanocrystal is recovered after mercerization. The chains are presumably mixed in the
522 global fiber by interdigitation and during crystallization rearrangement on shorter
523 distances with smaller crystals packing less chain. However, they seem more
524 homogeneously distributed along the fiber.

525 This may indicate that all the chains of the fibril are redistributed during
526 mercerization, forming a globally more regular fiber, but composed of smaller, more
527 discontinuous and bi-oriented crystallites.

528

529

<table><caption>Table 6. Number-average molar mass (M̄ₙ) of CNCs, elementary nanocrystals and individual chains, and the number of chains per individual CNC.</caption><thead><tr><th>CNCs</th><th>M̄<sub>n</sub> of CNCs (g/mol)</th><th>M̄<sub>n</sub> of elementary nanocrystals (g/mol)</th><th>M̄<sub>n</sub> of individual chains (g/mol)</th><th>Number of chains/elementary crystals (from Mn)</th><th>Number of chains/elementary crystals (from microscopy)</th><th>Number of chains/elementary crystals (from XRD)</th></tr></thead><tbody><tr><td>CNC-I</td><td>24 ± 1·10<sup>6</sup></td><td>8·10<sup>6</sup></td><td>34,000 ± 1,000</td><td>235</td><td>162</td><td>82</td></tr><tr><td>CNC-II</td><td>7 ± 1·10<sup>6</sup></td><td>2.4·10<sup>6</sup></td><td>18,000 ± 1,000</td><td>133</td><td>77</td><td>50</td></tr></tbody></table>

532

533

534

535

536 **5. Conclusions**

537 Using identical acid hydrolysis on native and mercerized NFs, a panel of techniques
538 is used to show that the mercerization treatment does not degrade cellulosic chains
539 (Mw of 560,000 g/mol) but instead limits the resistance to acid (yield of 64% and 40%
540 for CNC-I and CNC-II, respectively) and impacts the resulting CNCs. The thickness and
541 length of nanocrystals are reduced, preserving the lateral average association
542 corresponding to a trimer (three elementary nanocrystals), and resulting in molar
543 masses of 40,000 g/mol and 11,000 g/mol for CNC-I and CNC-II, respectively. By probing
544 the internal structure, we were able to show more intermediary structures between
ordered and amorphous domains. In addition, the two distinct (accessible/inaccessible) amorphous domains that are detected in cellulose I are not detected in mercerized form, even before acid hydrolysis. This occurs with unchanged surface charge density but a reduction of the crystal thickness by half. Finally, mercerization has a major impact on crystal organization with a much lower chain packing per nanocrystal. Compared to previous works, this article includes additional values notably on molar masses and proposed various comparative techniques. We hope this analysis can further help researchers to characterize their own samples.

553

554 Acknowledgements

The authors are grateful to Nadege Beury for AFM images and Emilie Perrin for TEM images with instruments from the BIBS platform (INRAE, Nantes, France), and to Laboratoire Léon Brillouin for providing neutron radiation facilities (CEA-Saclay, Gif sur Yvette, France). They are also grateful to Benoit Duchemin and Yoshiharu Nishiyama for their very stimulating discussions.

560

561 Funding

This project received funding from the French National Research Agency (ANR) (CELLOPLASM project, N° ANR-16-CE07-0003-03), including the PhD grant of SH. The authors are also grateful to INRAE for financial support.

565

Conflicts of interest: No conflicts of interest

566

Ethics approval: No ethical approval required

567

568

569

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684

## 685 **Figure captions:**

686 **Figure 1.** X-ray diffraction patterns of cotton fibers in native (CF-I) and mercerized CF-II forms and their respective hydrolyzed cellulose nanocrystals in the native (CNC-I) and mercerized (CNC-II) forms, and cross-sections of elementary crystallites deduced from the analysis of peak broadening (the indexation of corresponding lattice planes is described in Supporting Information).

691 **Figure 2.** (A) <sup>13</sup>C CP-MAS NMR spectra of CF-I and CF-II, and (B) deconvolution of the C4 region of CF-I, CF-II, CNC-I and CNC-II NMR spectra with crystalline forms (black), paracrystalline (gray) and amorphous (green).

694 **Figure 3.** Dissolution profiles of samples obtained by SEC-MALLS-DRI. The two nanofibers (CF-I in purple and CF-II in red) are eluted at low retention volumes, whereas the nanocrystals are eluted at higher elution volumes (CNC-I in green and CNC-II in black).

697 **Figure 4.** Distribution of molar masses of suspensions of CNC-I (blue) and CNC-II (red) in water, and RI signal (dotted curves).

699 **Figure 5.** TEM images of CNC-I (a,b) and CNC-II (c,d) and AFM images of CNC-I (e) and CNC-II (f)

700 **Figure 6.** I = f(Q) SANS curves of suspensions of CNC-I and CNC-II in water at 2 g/L in NaCl 2 mM

701

702

## 703 **Table Captions:**

704 **Table 1:** Weight fraction (yield) recovered after treatment; crystallinity index (CI) calculated from XRD (CIXRD); mean CI calculated from solid-state NMR (13C CP-MAS) spectra (CINMR); and deconvolution of the C4 region of 13C CP-MAS spectra.
707 **Table 2.** Weight-average molar masses (M̄<sub>w</sub>); polydispersity (M̄<sub>w</sub>/M̄<sub>n</sub>); and degree of
708 polymerization (DP) of individual chains of cellulosic fibers (CF-I and CF-II) and cellulose
709 nanocrystals (CNC-I and CNC-II) solubilized in DMAc/0.9% LiCl.

710 **Table 3.** Weight-average molar masses (Mw) and polydispersity (Mw/Mn) of CNC-I and CNC-II
dispersed in water determined by A4F-MALLS-DRI; and average dimensions determined from the
711 SANS curve, TEM images and AFM images.
712

713 **Table 4.** Sulfur content (S), surface charge density (SC) and zeta potential of CNC-I and CNC-II.

714 **Table 5.** Number-average molar mass (M̄<sub>n</sub>) of CNCs, elementary nanocrystals and individual
715 chains, and the number of chains per individual CNC

716

717

718

719