On the polymorphic and morphological changes of cellulose nanocrystals (CNC-I) upon mercerization and conversion to CNC-II

Carbohydrate Polymers 143 (2016) 327–335

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On the polymorphic and morphological changes of cellulose nanocrystals (CNC-I) upon mercerization and conversion to CNC-II Ersuo Jin a , Jiaqi Guo b , Fang Yang a , Yangyang Zhu a , Junlong Song a,b,∗ , Yongcan Jin a , Orlando J. Rojas b,∗∗ a b

Jiangsu Provincial Key Lab. of Pulp and Paper Science and Technology, Nanjing Forestry University, Nanjing 210037, Jiangsu, Peoples Republic of China Bio-based Colloids and Materials, School of Chemical Technology, Aalto University, PO Box 16300, Aalto FIN-00076 Espoo, Finland

a r t i c l e

i n f o

Article history: Received 8 November 2015 Received in revised form 9 January 2016 Accepted 22 January 2016 Available online 25 January 2016 Keywords: Cellulose nanocrystals CNC Mercerization Polymorphs Cellulose I and II Crystallinity Crystallite size

a b s t r a c t Polymorphic and morphological transformations of cellulosic materials are strongly associated to their properties and applications, especially in the case of emerging nanocelluloses. Related changes that take place upon treatment of cellulose nanocrystals (CNC) in alkaline conditions are studied here by XRD, TEM, AFM, and other techniques. The results indicate polymorphic transformation of CNC proceeds gradually in a certain range of alkali concentrations, i.e. from about 8% to 12.5% NaOH. In such transition alkali concentration, cellulose I and II allomorphs coexists. Such value and range of the transition concentration is strongly interdependent with the crystallite size of CNCs. In addition, it is distinctively lower than that for macroscopic fibers (12–15% NaOH). Transmission electron microscopy and particle sizing reveals that after mercerization CNCs tend to associate. Furthermore, TEMPO-oxidized mercerized CNC reveals the morphology of individual nanocrystal of the cellulose II type, which is composed of some interconnected granular structures. Overall, this work reveals how the polymorphism and morphology of individual CNC change in alkali conditions and sheds light onto the polymorphic transition from cellulose I to II. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Cellulose, composed of ␤-1,4 linked glucopyranose units, is a major resource for materials, (bio)chemicals and biofuels (Mosier et al., 2005; O’Sullivan, 1997; Wyman, Dale, Elander, Holtzapple, Ladisch, & Lee, 2005). In most cases, it is associated by hydrogen bonding and other weak forces to form a semi-crystalline structure where highly ordered regions (the crystallites) are combined with less ordered (disorder or para-crystalline) domains, also known as the amorphous phase. The molecular orientation and the hydrogen-bond network that pack the cellulose chains within the crystallites can vary widely, giving rise to different polymorphs

Abbreviations: CNC, cellulose nanocrystal; CNC-I, cellulose nanocrystal, allomorph of cellulose I; CNC-II, cellulose nanocrystal, allomorph of cellulose II; CNC-concentration%, cellulose nanocrystals treated under alkaline conditions at given concentration (%) of NaOH solution. ∗ Corresponding author at: Nanjing Forestry University, Jiangsu Provincial Key Lab. of Pulp and Paper Science and Technology, Nanjing 210037, China. Tel.: +86 25 85428163; fax: +86 25 85428689 (J.S.). ∗∗ Corresponding author at: Aalto University (O.J.R.). Tel.: +358 50 5124227; fax: +358 50 5124227. E-mail addresses: [email protected] (J. Song), orlando.rojas@aalto.fi (O.J. Rojas). http://dx.doi.org/10.1016/j.carbpol.2016.01.048 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

(cellulose I−IV), the structure of which depends on the cellulose source, method of extraction or treatment. Among them, cellulose polymorphs I and II are the most widely discussed. In nature, cellulose I is produced into crystalline forms with the Iˇ allomorph being the most common in plants, while I˛ in algae and bacteria (O’Sullivan, 1997). Cellulose II is usually obtained from cellulose I by mercerization (alkali treatment) or regeneration (solubilization and recrystallization); there are a few reports suggesting the production of cellulose II from Gluconacetobacter xylinus (Kuga, Takagi, ˚ & Brown, 1993). Cellulose Iˇ has unit cell dimensions of a = 7.78 A,

˚ c = 10.38 A, ˚ ˛ = ˇ = 90◦ , and  = 96.5◦ (Nishiyama, Langan, b = 8.20 A, & Chanzy, 2002). Cellulose II produced by the mercerization has ˚ b = 9.08 A, ˚ c = 10.36 A, ˚ ˛ = ˇ = 90◦ , unit cell dimensions of a = 8.10 A, and  = 117.3◦ (Gessler et al., 1995; Kobayashi, Kimura, Togawa, & Wada, 2011; Kolpak & Blackwell, 1976; Langan, Nishiyama, & Chanzy, 1999, 2001; Raymond, Kvick, & Chanzy, 1995; Sarko & Muggli, 1974; Stipanovic & Sarko, 1976). There is a general consensus for the parallel chain packing for cellulose I, while the chain directionality of cellulose II is antiparallel (Lee et al., 2013). A possible route how antiparallel chains of cellulose II produced from cellulose I which has parallel chains was proposed by Yamane, Miyamoto, Hayakawa, and Ueda (2013) Cellulose I and cellulose II possess different properties and advantages over the other. Compared to cellulose II, cellulose I exhibits much better mechanical

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properties, while the first offer benefits in terms of functionality (Diddens, Murphy, Krisch, & Muller, 2008; Wang, Li, Yano & Abe, 2014). Cellulose II, as indicated before, can be produced by alkali treatment of cellulose I or by regeneration from cellulose solution; however, cellulose II can be obtained from native cellulose through other approaches as well. For example, Ago, Endo, and Hirotsu (2004) demonstrated that a mechano-chemical treatment of native cellulose with a given amount of water (∼30 wt% as present in the cellulose solid state) via ball milling for several hours, caused the crystalline transformation to cellulose II polymorph, even though the crystallinity index of cellulose II was quite low, ranging from 30 to 37%. Sèbe, Ham-Pichavant, Ibarboure, Koffi, and Tingaut (2012) used a combination of extended treatment time (>50 min) and high final acid concentration (>64% H2 SO4 ) to obtain cellulose nanocrystals with some cellulose II. They also found that pure cellulose II nanowhiskers are obtained only in a narrow range of conditions (66% H2 SO4 /60 min addition). Martins et al. (2015) also produced cellulose II crystals through sulfuric hydrolysis (under a concentration of acid not reported). In principle, sulfuric acid is a swelling agent for cellulose and therefore recrystallization of cellulose may have occurred upon subsequent removal of the swelling agent or during the hydrolysis process. (Sèbe et al., 2012). Cellulose nanocrystals (CNC) display a high specific strength, modulus and aspect ratio which are main reasons for their effect in improving the mechanical properties of synthetic polymers at low loading levels (Habibi, Lucia, & Rojas, 2010). Benefits derived from the utilization of CNCs are being reported in fields of material science, electronics, catalysis and biomedicine (Gatenholm & Klemm, 2010). In most cases, CNCs are obtained by sulfuric acid hydrolysis of native cellulose, which results in purified CNCs. The properties of the nanocrystals (e.g., shape, length, and diameter) depend on the source of the cellulose and the degradation process (e.g., time and temperature for acid hydrolysis, high-pressure homogenization conditions, etc.). Thus, the properties of CNC-based materials are closely related to CNC pretreatment method and resultant nanoparticle morphology (Liu, Song, Anderson, Chang, & Hua, 2012; Lu & Hsieh, 2010). Despite the fact that Mercer introduced the “mercerization” transformation in 1850, the unraveling of its mechanism remains a question of intense debate. As for the macroscopic state of cellulose, i.e. cellulosic fibers, the general agreement indicates that at room temperature the polymorphic transformation starts at an alkali concentration of 12.5% and is completed at a concentration of 17.5% (Halonen, Larsson, & Iversen, 2013; Purz & Fink, 2003; Revol, Dietrich, & Goring, 2011). However, if the temperature or other conditions are changed, the polymorphic transformation may occur at different alkali concentrations. For instance, the transition occurs at 14–15% NaOH concentration for the mercerization of jute fibers at 85 ◦ C and 4 h (Yu et al., 2014). Polymorphic transformation from cellulose I to cellulose II for cotton linter by alkali pretreatment and urea additive was investigated by Gupta, Uniyal, and Naithani (2013). Their results showed a sudden polymorphic transformation at a concentration of 15 wt% NaOH, regardless of the presence of urea. In two-phase solvent systems, such as the mercerization experiments conducted with four different solution systems (ethanol/water, acetone, dimethyl sulphoxide and xylene), structural changes of cellulose crystallites depended primarily on the distribution and solubility of sodium hydroxide in the solvent. However, there was a critical concentration in the hydrophilic phase that must exceed 7–8% NaOH before cellulose I undergoes complete crystal change to cellulose II (Mansikkamaki, Lahtinen and Rissanen, 2005). The understanding of the polymorphic transformation of cellulose in the nanoscale, i.e. nanocellulose crystals, has found some controversies in the literature. Li, Ding, Li, and Jiang (2002) reported

Fig. 1. Schematic diagram illustrating the general process to obtaining individual cellulose nanocrystals with dominant polymorphs I or II.

that CNCs obtained by sulfuric acid hydrolysis undergo complete polymorphic transformation by reaction with 1% NaOH solution in 3 s. Liu and Hu (2008) treated bamboo nanofibers with 12% NaOH solution for 20 min to generate cellulose II with some residual cellulose I while full conversion to cellulose II was achieved at 16% NaOH concentration. Abe and Yano (2011) treated wood cellulose nanofibers and full conversion of cellulose I into II was accomplished in 15% NaOH solution. Interestingly, the reported values of NaOH concentration for mercerization of nanocellulose are very close to those needed for polymorphic transformation of macroscopic fibers. So far, investigations dealing with mercerization of cellulose have only been focused on whole cellulose fibers, i.e., organized assemblies of microfibrils. Very few investigations have dealt with the properties of cellulose I and II nanocrystals (Yue et al., 2012). Thus, it is necessary to conduct a systematic study on the transformation of individual CNCs, especially their polymorphic and morphological changes. Our results show that there are effects induced by the nano-scale size of the CNCs, which do not hold in the case of macroscopic fibers. The polymorphic transformation of CNCs upon mercerization proceeds gradually, starting below 8% NaOH concentration and is complete above 12.5% NaOH. This transition range also depends on the initial cellulose source. CNCs produced by sulfuric acid hydrolysis contain negatively-charged groups (anionic sulfate half-ester groups) that can be removed during mercerization under alkaline conditions. Additionally, individual CNCs are difficult to observe (TEM, AFM) but appear to form aggregates. Thus, moderate TEMPO-oxidation of the mercerized cellulose crystals (schematic illustration in Fig. 1) is required for better dispersion and observation. 2. Experimental 2.1. Materials Two microcrystalline cellulose grades were used in this study, from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), thereafter referred to as MCC1 and, AvicelTM PH-101, provided by Sigma-Aldrich Co., Ltd (Shanghai, China), referred to as MCC2. 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 99% (GC) purity was purchased from Jiana Chemical Co., Ltd (Changzhou, China). Sodium hypochlorite, sodium hydroxide, sulfuric acid and other reagents were analytical grade and purchased from Nanjing Chemical Reagent Co., Ltd (Nanjing, China).

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2.2. Preparation of cellulose nanocrystals (CNCs) The procedure for preparation of CNCs is described elsewhere (Beck-Candanedo, Roman, & Gray, 2005; Fu, He, Jin, Cheng, & Song, 2012; Song, Fu, Cheng, & Jin, 2014; Viet, Beck-Candanedo, & Gray, 2007). Briefly, MCC powder (10 g) was hydrolyzed with sulfuric acid (250 mL, 64 wt%) at 45 ◦ C for 1 h under continuous stirring. The hydrolysis was quenched by adding a large amount of water (∼10 times the volume of the acid solution used). The system was centrifuged (12,000 rpm) at 4 ◦ C for 10 min and the supernatant was decanted. This procedure for acid removal was repeated three times until a white suspension was obtained. Then the suspension was dialyzed with cellulose acetate membrane tubes (MWCO 14000) against deionized water, for about 5–7 days, until a constant pH was reached. A portion of the CNC aqueous dispersion (thereafter referred to as CNC-I) was stored in a bottle and the other was freeze-dried. 2.3. Mercerization of CNC-I 10 mL of NaOH solutions (1.0, 2.0, 5.0, 8.0, 10, 12.5, 15 and 17.5% concentration) were prepared initially. Then 100 mg precisely weighted CNC was added into the alkali solution under constant stirring for 30 min at ambient temperature. Upon completion of the reaction, the dispersion was neutralized with 5% sulfuric acid solution. The respective mercerized CNC dispersion was dialyzed with cellulose acetate membrane tubes (MWCO14000) against deionized water for 3–5 days, until a constant conductivity was reached. The obtained samples, herein indicated by a numeral representing the NaOH concentration used during mercerization (e.g. CNC-10%), were freeze-dried and stored in a desiccator. For comparison, the other MCC sample, MCC1, was mercerized with 17.5% NaOH for 24 h at first, and then subjected to sulfuric acid hydrolysis as described in the previous section (a shorter time was used, 20 min). This sample is thereafter denoted as CNC-II. 2.4. TEMPO-mediated oxidation of mercerized CNCs Negative charges on CNC-I are reduced to some degree after treatment with alkali, due to hydrolysis of ether bonds (Lokanathan, Uddin, Rojas, & Laine, 2014). As a consequence, CNCs tend to agglomerate after mercerization, limiting the possibility of detailed studies, for example, via imaging. Therefore mercerized CNCs were subjected to TEMPO-mediated oxidation, which adds negative charges to the surface and improves colloidal stabilization. The TEMPO-mediated oxidation followed the method described elsewhere (Habibi, Chanzy, & Vignon, 2006) except that more moderate conditions were used. Briefly, the CNC-17.5% sample (50 mL dispersion) was first sonicated for 5 min. Then TEMPO (14.75 mg, 0.094 mmol) and NaBr (162 mg, 1.57 mmol) were added to the dispersion. NaClO solution (0.4 mL, 1.24 M) was added slowly and the pH of the mixture was maintained at 10 by adding 0.5 M NaOH. The oxidation was terminated after 30 min by adding methanol (1 mL) and the pH was adjusted to 7 using 0.5 M HCl solution. The aqueous dispersion was then introduced in cellulose acetate membrane tubes (MWCO14000) for dialysis against deionized water. The obtained sample, after freeze–drying, is referred thereafter as CNC-17.5%-TEMPO. 2.5. X-ray diffraction X-ray diffraction measurements were carried out by using a Goniometer Ultima IV (Rigaku Co. Ltd., Japan) X-ray diffractometer equipped with CuK␣ radiation (0.15406 nm) generated at 40 kV and 30 mA at room temperature. Vacuum dried cellulose powder samples were placed on quartz substrates that have no background

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signal. X-ray diffraction data were collected from 2 = 5–55◦ in steps of 0.02◦ ; and were further analyzed using the MDI Jade 6.5 software (Materials Data, Inc.). XRD patterns were smoothed and baselinecorrected by a software-generated parabolic curve. Curve-fitting was performed to identify individual peaks and to calculate the crystallinity and crystallite size. The Segal method (Creely, Segal, & Ziifle, 1956) uses the ratio of the peak height at the 2 = 18◦ position, which is assumed to result from the contribution of amorphous regions of cellulose, to that of plane 200. This method is usually applied to cellulose I but not for cellulose II. Here, we used the areas of the crystalline and amorphous regions in the XRD spectra. Therefore, the crystallinity based on the ratio of crystalline region’s area to the total area was employed for convenient comparison. The Scherrer’s equation was used for estimating the crystallite size (Mittal, Katahira, Himmel, & Johnson, 2011): ˇ=

k  cos 

where  is the wavelength of the incident X-ray (0.15406 nm),  is the Bragg angles corresponding to the planes, ˇ is the full-width at half maximum (FWHM) of the X-ray peak corresponding to the planes;  is the X-ray crystallite size, and k is a constant of 0.89. 2.6. CNC imaging and sizing A drop of aqueous CNC dispersion in water (∼0.01%w/v) was deposited on a carbon-coated electron microscope grid and then negatively stained with phosphotungstic acid. A piece of tissue paper was used to absorb excess water and then allowed to dry in air. The sample on the grids was imaged with a JEM-2100 UHR (JEOL, Japan) transmission electron microscope operated at an accelerating voltage of 200 kV. AFM observation was performed using Multimode Nanoscope V controller (Bruker Corporation, USA) in air at room temperature (23 ◦ C). A drop with respective CNC dispersion was placed on a silicon wafer and spin-dried at a speed of 3000 rev/min using a spin coater (WS-650-23, Laurell Technology Co. Ltd., USA). Images from AFM were recorded in tapping mode by using silicon cantilevers. The commercial AFM probes had a spring constant of 20–80 N/m and a resonance frequency of 300–340 kHz. The relative size of mercerized CNC aggregates as well as CNCII was determined by dynamic light scattering using a Malvern Zetasizer NanoS90 at a 90◦ scattering angle. 3. Results and discussion 3.1. Untreated cellulose nanocrystals Two untreated cellulose nanocrystal samples (CNC-I1 and CNC-I2) were obtained by sulfuric acid hydrolysis from two microcrystalline cellulose sources, MCC1 and MCC2, respectively. In Fig. 2a, both XRD patterns of CNC-I1 and CNC-I2 exhibit a sharp high peak at 2 = 22.6◦ and two overlapping, weaker diffraction peaks at 2 = 14.7◦ and 2 = 16.4◦ , which are assigned to the typical pattern of cellulose I (French, 2014; French & Cintrón, 2013). The d-spacings of these planes calculated by Bragg equation are 0.39 nm, 0.60 nm and 0.54 nm, respectively. These parameters are consistent with those reported in the literature (Ishikawa, Okano, Sugiyama, Ishikawa, & Okano, 1997; Sèbe et al., 2012). However, compared to the diffraction bands of CNC-I1, the pattern for CNCI2 is smoother and overlaps more markedly. Two diffraction peaks at 14.7◦ and 16.4◦ merge into one broad band and the small peak at 2 = 20.1 (plane 012) is integrated into the most pronounced peak of plane 200. The crystallinity index (CrI) of CNC-I1 and CNC-I2 are quite similar, 77.9% and 77.5%, respectively.

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Fig. 2. X-ray diffraction patterns of CNC-I1 and CNC-I2 (a) and a representative TEM image of CNC-I1 (b) (the images for CNC-I2 are very similar, not included here).

The morphology of CNC-I1 and CNC-I2 as observed by TEM is quite similar (Fig. 2b). The crystals show a needle-like shape, 100–300 nm in length and ca. 10 nm in width, consistent with other values reported in the literature for MCC-derived CNCs (Habibi et al., 2010). 3.2. CNC polymorphic transformation from cellulose I into cellulose II and size-induced polymorphism A systematic evaluation of the process by which cellulose I nanocrystals evolve into cellulose II crystals was undertaken and the role of size-induced polymorphic transformation upon mercerization was assessed. To this end, the two CNC samples (CNC-I1 and CNC-I2) were treated with NaOH solutions of given concentrations, from 1% to 17.5%, for 30 min and at room temperature. The X-ray diffraction patterns of CNC-I1 and CNC-I2 after alkali treatment are shown in Fig. 3a and b, respectively. The XRD patterns of CNC-I1 upon treatment with dilute alkali, e.g. 1, 2, 5, and 8% NaOH solutions display similar peaks as those of untreated CNC and indicate the main contribution of cellulose I (French, 2014). However, for the sample treated by 8% NaOH, a small bump at the position of 2 = 12.0 starts emerging. When the alkali concentration is increased to 10%, two additional distinctive diffraction peaks appear at 2 = 12.0 and 2 = 20.0, which are assigned to cellulose II (French, 2014). This observation hints to the fact that at concentration of ca. 10% NaOH, both polymorphs, cellulose I and cellulose II, coexist. When the alkali concentration is increased to 12.5%, three peaks from cellulose II dominate but

two noticeable peaks at 2 = 14.7 and 16.4, assigned to cellulose I, still remain. Samples subjected to NaOH treatment at concentrations above 12.5% (i.e. 15 and 17.5%) indicate similar XRD patterns and display only the characteristic profile of cellulose II, with three peaks positioned at 2 = 12.0, 20.0 and 21.8o . The alkali-treated CNC-I2 samples produce similar trends as those discussed before for CNC-I1, except that the transition concentration is shifted from 10% to 8% (CNC-I1). Crystallinity index, d-spacing and crystallize size for each plane were determined via Jade code. The d-spacings of the ˚ (1 −1 0), (1 1 0) and (2 0 0) planes for cellulose I are 6.01 ± 0.06 A, ˚ respectively. While these values are 5.35 ± 0.05 A˚ and 3.92 ± 0.02 A, ˚ 4.43 ± 0.03 A˚ and 4.08 ± 0.04 A˚ for cellulose shifted to 7.41 ± 0.06 A, II. The crystallite size for the (1 −1 0) and (1 1 0) planes of cellulose I, and (1 1 0) and (2 0 0) for cellulose II varied extensively due to overlap. However the values of the average crystallize size of the most prominent peak(s), i.e., 2 = 22.6◦ for cellulose I and 2 = 20.0 and 21.8◦ for cellulose II, can be considered accurate. The crystallinity indices and average crystallite sizes of CNC-I1 and CNC-I2 after treatment with alkali solutions of given concentrations are listed in Tables 1 and 2, respectively. In the case of CNC-I1, CrI continuously decreases with the increase of alkali concentration used in the treatment. The fraction of cellulose I and cellulose II can be estimated by the area of fitted curves. At 10% NaOH concentration, the fraction of Cellulose I allomorph is 72.8%, indicating the presence of 27.2% cellulose II allomorph. In the case of 8% NaOH, cellulose I dominates (84.0%) and only a small portion (16.0%) of cellulose II allomorph is

Fig. 3. XRD patterns after mercerization (30 min, room temperature) of CNC-I1 (a) and CNC-I2 (b) under given concentrations of NaOH solution, from 1 to 17.5%.

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Table 1 Crystallinity indices and crystallite size of CNC-I1 after alkali treatment for 0.5 h. NaOH Conc. (%)

CrI (%)

Crystallite size (nm) Cellulose I

Cellulose II

MCC1 CNC-I1 1 2 5 8 10 12.5 15 17.5

83.7 77.9 76.5 75.9 73.8 71.4 66.6 68.2 68.2 56.7

6.9 6.6 6.8 6.8 6.8 6.5 6.3 5.1 – –

– – – – – 5.2 5.3 5.5 5.5 5.5

Table 2 Crystallinity indices and crystallite size of CNC-I2 after alkali treatment for 0.5 h. NaOH Conc. (%)

CrI (%)

Average crystallite size (nm) Cellulose I

Cellulose II

MCC2 CNC-I2 1 2 5 8 10 12.5 15 17.5

69.9 77.5 78.9 81.2 73.1 72.1 70.8 67.7 59.2 55.3

4.4 4.1 3.9 3.9 3.9 2.8 – – – –

– – – – – 4.5 5.0 4.8 5.0 4.9

Fig. 4. XRD patterns of CNC-I1 treated with alkali at the transition concentration (10%) for given periods of time.

is slightly higher than that of the produced CNCs. This is explained by the fact that in the production of CNC some molecules located in the periphery of the crystals react with sulfuric acid and become dissolved. The reason why two different MCC sources have different crystallite sizes may be due to the difference in fiber source and treatments applied in their production. This issue needs further elucidation. 3.3. Time for CNC polymorphic transformation

identified. For 12.5% NaOH, cellulose II dominates (82.4%) and a small portion (17.6%) of cellulose I allomorph is identified. These observations hint that the polymorphic transformation proceeds gradually, starting from below 8% NaOH and ending at a NaOH concentration above 12.5%. This gradual transition is also evidenced in the average crystallize size. The average crystallize size for MCC1 is 6.9 nm. This size is reduced to 6.6 nm for CNC-I1. When CNC-I1 is treated in dilute alkali, the size remains similar. When the alkali concentration increased to 8%, the average crystallize size for cellulose I crystallites are reduced slightly, to 6.5 nm. When the concentrations increased to 10% and 12.5%, the average crystallize size for cellulose I crystallites is reduced further to 6.3 and 5.1 nm, respectively. In the meantime, the average size of cellulose II crystallites grows from 5.2 nm to 5.5 nm. At an alkali concentration above 12.5%, the average size of cellulose II crystallites doesn’t change further. The CrI variation upon pretreatment of CNC-I2 with alkali follows the same trend as that for CNC-I1, i.e. CrI continuously decreases with the increase in alkali concentration used in the treatment. At 8% NaOH, the CNC mixture comprises 68.0% Cellulose I and 32.0% Cellulose II crystallites. The fraction of cellulose II is higher than that in the case of CNC-I1 for treatment with the same alkali concentration. Furthermore, negligible amounts of cellulose I or II are found at 5% and 10% NaOH. In terms of the average crystallite size of CNC-I2, it also follows the same trend as that of CNC-I1, i.e. the average crystallite size for cellulose I gradually reduces at the transition alkali concentration, while the average crystallite size for cellulose II grows. A very interesting is that the average crystallite size of cellulose I in CNC-I2, 4.0 nm, is much smaller than that of CNC-I1, 6.8 nm. The difference in crystallize size may be one of the reasons why the polymorphic transformation for CNC-I2 takes place at a lower alkali concentration and is completed within a narrow range. The difference in crystallize size for both CNC samples can be traced back to the MCC sources. The crystallite size in MCC1 is 6.9 nm, while that in MCC2 is only 4.4 nm. The crystallite size for both MCC samples

The XRD pattern of CNC pretreated at the transition NaOH concentration includes all the peaks of cellulose I and cellulose II. This facilitates determination of the extent of completion of the polymorphic transformation, i.e., via monitoring the change in XRD patterns with the pretreatment time. As such, Fig. 4 includes XRD patterns of CNC-I treated with 10% NaOH during given times, up to 24 h. In all cases, from 0.5 h and up to 24 h treatment, the same distinguishable features in the XRD profiles are observed, indicating that an extensive treatment is not conducive of further polymorphic transformation. The polymorphic transformation occurs quite rapidly, in less than 30 min, regardless of the concentration of alkali used. This result is in agreement with the conclusion of Liu and Hu (2008). 3.4. Morphological changes of cellulose nanocrystals during polymorphic transformation An AFM image of CNC-I1 after treatment with 10% NaOH is shown in Fig. 5a. Needle-like shapes are observed, with ca. 10 nm in width and 100–200 nm in length. Negligible differences in size and morphology are observed if compared to the untreated CNC-I. CNC-I1 pretreated with 17.5% NaOH resulted in important morphological changes as observed by AFM and TEM (Fig. 5b and c), respectively. Round-shaped agglomerates, about 200 nm in size, composed of granules (size of around 30 nm), can be observed in Fig. 5b; no individual crystals are identified, even if the dispersion was diluted before drying and imaging. Such agglomerates can be clearly seen under TEM (Fig. 5c) as uniform spherical aggregates of ca. 150–200 nm. The size of granules for mercerized CNC is also consistent with that of regenerated cellulose (Yamane et al., 2013). Untreated CNCs produced by sulfuric acid hydrolysis carry negatively charged groups and are dispersed easily in aqueous medium (Habibi et al., 2010). The ester bonds of negatively charged sulfatehalf ester groups break down, at least partially, upon treatment in an alkaline solution (Lokanathan et al., 2014). Therefore, the surface charge of CNCs is reduced and facilitates their agglomeration. This

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Fig. 5. AFM image of CNC-I1 upon treatment at the critical concentration of alkali, 10% (a) and corresponding images (AFM, b and TEM, C) upon CNC-I1 treatment at higher concentration (17.5% NaOH).

Fig. 6. Size of CNC and corresponding agglomerates as determined by light scattering in aqueous dispersions of nanocrystals (CNC-I1) before and after treatment at given alkali concentrations. The image of CNC-II is also included.

tendency is clearly demonstrated in Fig. 6 for size measurements that were carried out in aqueous dispersion via light scattering. Bearing in mind that the size recorded can only be taken as a relative value and cannot be compared with that determined by TEM, it is still interesting to note similar observations: the diameter of untreated CNC-I1 and that after treatment with 10% NaOH

solution is somewhat similar. For CNC-17.5%, the size shown indicates large aggregates. As a comparison, the size of CNC-II (hydrolysis product of mercerized MCC), is shown to be 20-30 nm (see Fig. 7a), which is in agreement with the granule size in AFM observations for CNC-17.5% (Fig. 5b). Since CNCs loss their charges after mercerization and tend to agglomerate, TEMPO-mediated oxidation was carried out in order to facilitate dispersion and to observe the morphology of individual particles. Oxidation strength was carefully adjusted by the molar ratio of NaClO/equivalent anhydroglucose unit (AGU). In a conventional TEMPO-mediated oxidation, this value is about 0.5, which is too severe for the intended purpose. After several tests, it was found that NaClO/AGU = 0.15 is the minimum necessary to promote colloidal stabilization of the CNCs, i.e., by oxidation of some of its C6 hydroxyl groups. A TEM image of CNC-17.5%-TEMPO is shown in Fig. 7b. The morphology of individual mercerized cellulose crystal indicates interconnected spherical features and bended kidney-like granules. The size of these granules is consistent with that observed in AFM (Figs. 5b and 7a), ca. 30–40 nm. To the best of our knowledge, this is the first report on the morphology of individual CNC with cellulose II polymorph, especially obtained from the mercerization. Sèbe et al. (2012) have imaged individual cellulose nanocrystals with cellulose II allomorph obtained from acid regeneration. Compared with that of cellulose I nanowhiskers, their morphology (AFM images) showed a ribbon-like shape with rounded tips, with a smaller size but

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Fig. 7. AFM image of CNC-II (a) and TEM image of CNC-17.5%-TEMPO (b).

expanded width. The morphology shown in AFM images demonstrate that such ribbons were composed of several granules. Hence the morphology includes several interconnected granules shared by both individual nanocellulose samples, regardless the treatment process used, either by concentrated acid or mercerization; they undergo swelling and recrystallization. Yue et al. (2012) have reported rod-like crystals for cellulose II having similar widths but reduced length, compared with pristine cellulose I crystals. However these authors obtained cellulose II crystals from mercerized macroscopic fibers, via sulfuric acid hydrolysis and followed by high-pressure homogenization. This means that the polymorphic transformation occurred at the macroscopic state and the morphological changes may have been restricted by the surrounding crystals and were not able to relax, which is in contrast to the present observations. The XRD patterns of CNC-17.5%-TEMPO, and CNC-II are shown in Fig. 8. The XRD pattern for CNC-17.5% is added for comparison. They all include the characteristic peaks of cellulose II but with different intensity. After TEMPO-mediated oxidation, mercerized CNCs still retain their crystalline structure. The crystallinity indices and crystallite size of CNC-17.5%-TEMPO and CNC-II are given in Table 3. Since CNC-II contains mainly the crystalline component (upon removal of amorphous regions), its crystallinity, 75.4%, is close to the original CNC samples, and higher than that of mercerized CNC (CNC-17.5%), 56.7%. This value is much higher than that of TEMPO-oxidized sample (CNC-17.5%-TEMPO), 37.6%. The average crystallite size of CNC-II, 5.1 nm, is slightly smaller than those

of mercerized CNCs, 5.5 nm. While the average crystallite size of TEMPO-oxidized sample (CNC-17.5%-TEMPO), is only 4.2 nm. This observation indicates that the crystallite size of CNC-II and CNC17.5%-TEMPO is reduced in their production. All the above observations provide some evidence and shed some light to better understanding how the polymorphic transformation of cellulose takes place. For a regular cellulose I crystal, with a width of several nanometers and a length of hundreds nanometers, and at a NaOH concentration above the critical value, hydroxide ions start to penetrate into the crystals. As hydroxide ions diffuse within the crystallite and swells it, the cellulose chains rearrange their orientation as hydroxide ions are removed from the lattice of the cellulose matrix. This process takes place as suggested by Yamane et al. (2013) who indicated that some six ring conformers of glucose form hair-pin turns and therefore cellulose chains fold and pack antiparallel forming a more thermodynamically stable state, allomorph cellulose II. As such, the cellulose II crystallites formed have much shorter length, ca. 30–40 nm (see TEM images), if compared with their original state, cellulose I, 100–300 nm. Our results also demonstrate that one cellulose chain may be involved in the formation of several cellulose II crystallites. The “bridge” connecting granules provides this evidence. While images of individual CNC composed of the cellulose II polymorph can be obtained, the process by which cellulose I evolves into cellulose II, and the variables that influence this process, remain unresolved subjects. Computational simulation and related approaches may shed more light on these issues. Overall, the results obtained suggest that: (1) cellulose I allomorph cannot be converted under dilute alkali treatment (<5% NaOH); (2) polymorphic transformation proceeds gradually, in the alkali transition concentration range. In the transition state, both allomorphs, cellulose I and II, coexist in the cellulose nanocrystals. The ratio of each fraction depends only on the alkali concentration used in the treatment. Below this transition, cellulose I clearly dominates the system while above the transition concentration, cellulose II dominates. (3) If compared to the polymorphic transformation of macroscopic fibers, where the polymorph conversion only occurs when alkali concentration is above 12.5%, the critical

Table 3 Crystallinity indices and crystallite size of CNC-17.5%-TEMPO and CNC-II.

Fig. 8. XRD patterns for cellulose nanocrystals with polymorph of cellulose II.

Sample

Polymorph

CrI (%)

Average crystallite size (nm)

CNC-17.5%-TEMPO CNC-II

II II

37.6 75.4

4.2 5.1

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transition concentration for CNCs is reduced, to about 8% NaOH. This indicates a size-induced polymorphic transformation, which is strongly interconnected with the CNC crystallite size. However, this effect is not as pronounced as that claimed by Li et al. (2002) who reported complete conversion upon treatment with 1% NaOH solution for three seconds. This may be attributed to the concentrated alkali that remains after mercerization during drying, which facilitates the polymorphic transformation. At the transition NaOH solution concentration of 8–12.5%, and for the polymorphic transformation of the studied CNCs, it is hypothesized that the hydrated hydroxide ions start to penetrate the cellulose crystals and undergo partial reaction with cellulose. At alkali concentrations above the transition concentration, hydroxide ions penetrate extensively within the crystals, producing CNC swelling. Upon ion removal during dialysis, the cellulose chains recrystallize into cellulose II, which is more stable thermodynamically. As a consequence, the respective XRD patterns display the contribution of cellulose II allomorph while the characteristic peaks of native cellulose I are absent. Treatment with alkali at a concentration below the transition value provides conditions that are not sufficient for hydroxide ions to penetrate into the cellulose I crystals and therefore no polymorphic transformation takes place, even if the size of the particle is in the nano-scale (Abe & Yano, 2011; Dinand, Vignon, Chanzy, & Heux, 2002; Fengel, Jakob, & Strobel, 1995). Based on the discussion of Liu and Hu (2008), the size of the hydroxide ions vary with the alkali concentrations. At a low concentration, hydroxide ions are fully hydrated and their size may prevent them from penetrating and disrupting the cellulose lattice. However, as the NaOH concentration increases, the amount of free water available for hydrating the hydroxide ions is limited. As such, the characteristic size of hydrated hydroxide ions are relatively smaller and more easily penetrate into the cellulose lattices. When their size is reduced to dimensions small enough to penetrate into the cellulose lattices, the critical alkali concentration required for polymorphic transformation is identified. At the NaOH transition concentration, some crystals of cellulose I are converted into cellulose II. The ratio of cellulose I to II is a function of alkali concentration used. There is indication that the polymorphic transformation of CNCs is actually a dynamic process. Above the NaOH transition concentration, hydroxide ions penetrate easily into all pristine crystals and convert them into crystals of cellulose II polymorph. In terms of the penetration of hydrated hydroxide ions, as for CNC particles of inherently large surface area, the penetration is facilitated. This the reason for size-induced polymorphic transformation. In regards to the CNC sample with a small crystallite size, hydroxide ions penetrate easily into the matrix and therefore shift the concentration required for polymorphic transformation to a lower value and within a narrower range.

4. Conclusions The polymorphic and morphological changes of cellulose nanocrystals were systematically investigated by treating CNCs under various concentrations of NaOH (1, 2, 5, 8, 10, 12.5, 15 and 17.5%). It is found that a transition range of alkali concentration exist for polymorphic transformation of cellulose and the mercerization process is completed in less than 30 min. The transition NaOH concentration is found to be in the range of 8–12.5%, which strongly correlates with the CNC crystallite size. Within this transition concentration range, both allomorphs of cellulose I and cellulose II coexists while below the polymorph of cellulose I dominates. Above this concentration range, cellulose I converts to cellulose II completely. The polymorphic transformation proceeds gradually, which is evidenced by the gradual decrease in crystallite size of cellulose I and the growth of cellulose II.

The nano-size induced polymorphic transformation plays a role in reducing the critical concentration required for allomorph conversion. Finally, morphological changes (TEM and AFM) of individual cellulose crystal during polymorphic transformation illustrate that CNC comprising the Cellulose II polymorph exhibits interconnected granules. This indicates that single cellulose chains may be involved in Cellulose II the formation of Cellulose II crystals.

Acknowledgements This project was financially supported by Special Fund for Forestry Scientific Research in the Public Interest (201404510), National Natural Science Foundation of China (Nos. 31270613, 31200454), Qing-Lan Project, Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resource (NJFU) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. JS acknowledges funding support from China Scholarship Council provided for his research mission in Aalto University. This work was also partially supported by the Academy of Finland Centres of Excellence Programme (2014–2019) “Molecular Engineering of Biosynthetic Hybrid Materials Research” (HYBER).

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