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Industrial Crops and Products xxx (2014) xxx–xxx

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he influence of the cellulose hydrolysis process on the structure ofellulose nanocrystals extracted from capim mombac a (Panicumaximum)

ouglas Ferreira Martins, Alexandre Bernaldino de Souza, Mariana Alves Henrique,udson Alves Silvério, Wilson Pires Flauzino Neto, Daniel Pasquini ∗

nstituto de Química, Universidade Federal de Uberlândia, Campus Santa Mônica, Av. João Naves de Ávila, 2121, 38400-902 Uberlândia, Minas Gerais, Brazil

r t i c l e i n f o

rticle history:eceived 9 August 2014eceived in revised form 10 October 2014ccepted 18 October 2014vailable online xxx

eywords:apim mombac a fibrescid hydrolysisellulose nanocrystals

a b s t r a c t

This work presents studies on the isolation of cellulose nanocrystals from cellulose extracted from capimmombac a (Panicum maximum) leaves through variations in the acid hydrolysis process, and their con-sequent impact on their chemical, physical and thermal properties. The hydrolysis was performed at40 ◦C for 10 min, 20 min, 30 min or 40 min under vigorous and constant stirring. For each gram of CM,30 mL of H2SO4 (11.22 M) was used. The variations occurred in the subsequent step, which consists ofadding cold water to stop the hydrolysis. This step was carried out using two different methodologies.The resulting samples were characterized by thermogravimetic analysis (TGA), elemental analysis, X-ray diffraction (XRD) and atomic force microscopy (AFM) in order to assess changes in the physical andchemical structure of the nanocrystals obtained and in their thermal stability. It was observed that the

ellulose polymorphshermal stability

procedures adopted influenced the dimensions and physical aspect of the nanocrystals, the type of cel-lulose crystalline structure present (cellulose type I or cellulose type II) and also their thermal stability.It was possible to conclude that the changes in the thermal stability were associated with the poly-morph of cellulose that predominated, and not with the presence of sulphate groups on the surface ofthe nanocrystals.

. Introduction

The application of cellulose nanocrystals (CNCs) as a nanosizedeinforcement in polymer matrixes has attracted considerablettention, since it offers a unique combination of desirable phys-cal properties and environmental benefits (Habibi et al., 2010;eng et al., 2011). CNCs-based nanocomposites generally exhibitignificant improvements in thermal, mechanical, and barrier prop-rties compared to the neat polymer or conventional compositesAzeredo et al., 2009). CNCs are needle-shaped cellulose particlesith at least one dimension equal to or less than 100 nm and have

highly crystalline nature (Flauzino Neto et al., 2012). The maineatures driving the development of CNCs as polymer reinforce-

ent agents are their large specific surface area (estimated to be

Please cite this article in press as: Martins, D.F., et al., Tstructure of cellulose nanocrystals extracted from capim mhttp://dx.doi.org/10.1016/j.indcrop.2014.10.035

everal hundreds of m2 g−1), their very high modulus of elasticityapproximately 150 GPa), their large aspect ratio, and their abil-ty to act as a significant reinforcement at low filler loading levels.

∗ Corresponding author. Tel.: +55 34 3239 4143; fax: +55 34 3239 4208.E-mail address: pasquini@iqufu.ufu.br (D. Pasquini).

Other attractive advantages of CNCs are their low density (about1.59 g cm−3) (O’Sullivan, 1997), nonabrasive nature, nontoxic char-acter, biocompatibility, and biodegradability (Azizi Samir et al.,2005). Additionally, CNCs come from renewable natural sourcesthat are very abundant and therefore low in cost, so it is not nec-essary to synthesize them, they allow the production of compositefilms with excellent visible light transmittance, and they can beeasily modified chemically (their structure includes a reactive sur-face of OH side groups that facilitate grafting chemical speciesin order to achieve different surface properties) (Brinchi et al.,2013; Domingues et al., 2014; Fortunati et al., 2012; Silvério et al.,2013; Peng et al., 2011; Flauzino Neto et al., 2012; Moon et al.,2011).

Among the several methods of preparing CNCs, acid hydrolysisis the most well-known and widely used. This process is based onthe fact that crystalline regions are acid-insoluble under the con-ditions used in the extractions (Peng et al., 2011). The morphology

he influence of the cellulose hydrolysis process on theombac a (Panicum maximum). Ind. Crops Prod. (2014),

and properties of CNCs influence their performance as a reinforcingagent. Moreover, the morphology and properties of CNCs dependon the source of the original cellulose and on the extraction process.Thus, the development of CNCs from different sources of cellulose

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s relevant and, in the same way, the choice of hydrolysis conditionss very important too (Flauzino Neto et al., 2012).

It is well known that the use of different acids can lead toifferences in the stability of the nanocrystals in a colloidal suspen-ion due to the presence of different charges on the surface of theanoparticles (Angellier et al., 2004). In the case where sulphuriccid is employed to obtain CNCs, the presence of negative sulphateroups, introduced on the outer surface of the CNCs during theydrolysis process, is responsible for the stabilization of the CNCs

n the resulting colloidal suspension (Beck-Candanedo et al., 2005).owever, some authors have reported that the presence of sul-hate groups decreased the thermal stability of the CNCs (Romannd Winter, 2004). Usually, a higher acid sulphate group contentn cellulose leads to a lower temperature of thermal degradation ofhe cellulose. If hydrochloric acid is used instead of sulphuric acid toydrolyse the native cellulose, the thermal stability of the preparedanocrystals is improved but the nanocrystals are inclined to aggre-ate due to the absence of a force of electrostatic repulsion betweenhe crystal particles, resulting in an unstable colloidal suspensionAraki et al., 1998).

One factor often overlooked by researchers is the methodmployed in the preparation of the nanocrystals, regardless of theype of acid used. Some authors add water directly to the acid solu-ion containing the nanocrystals in order to stop the hydrolysisrior to centrifugation and dialysis (Haafiz et al., 2014; dos Santost al., 2013; Silvério et al., 2013; Henrique et al., 2013; Flauzino Netot al., 2012; Rosa et al., 2012; Belbekhouche et al., 2008; Teixeirat al., 2011). Moreover, other authors do not add water directly andust follow steps of centrifugation and dialysis after finalizing theydrolysis time (Siqueira et al., 2013; Cao et al., 2013; de Menezest al., 2009; Habibi et al., 2007; Samir et al., 2004). It is known thatt high concentrations sulphuric acid acts as a solvent for celluloseydrolysis (Xiang et al., 2003; Jayme and Lang, 1963). One hypoth-sis to be exploited in this work is that adding water to stop theydrolysis process may induce the re-precipitation of a portion ofhe cellulose solubilized on the surface of CNCs, or even the forma-ion of nanoparticles of regenerated cellulose. This precipitationnd formation of structures containing cellulose type II may be theain cause of the change in thermal stability of the nanocrystals,

nd not the presence of sulphate groups as claimed by some authorsRoman and Winter, 2004; Araki et al., 1998).

This study aimed to extract CNCs from capim mombac a (Pan-cum maximum) (CM) through experimental modifications, in ordero investigate the presence of CNCs with structures made of cellu-ose I and II. Different techniques were employed to characterize theellulose fibres and the resulting cellulose nanocrystals in order tonvestigate their chemical composition, crystallinity index, thermaltability and morphology (shape and size).

. Experimental

.1. Materials and methods

Capim mombac a (P. maximum) (CM) leaves were suppliedy São Mateus farm (Comendador Gomes, Minas Gerais, Brazil).he other reagents employed in this study were: sulphuric acid95.0–98.0 wt.%, Vetec, P.A.), sodium hydroxide (Vetec), potassiumydroxide (Vetec), sodium chlorite (NaClO2, technical grade, 80%,igma–Aldrich), glacial acetic acid (Synth), and cellulose membraneD9402, Sigma–Aldrich).

.2. Preparation of cellulose nanocrystals

.2.1. Purification of capim mombac a (CM)Initially the untreated CM was milled with a blender to pass

hrough a 14-mesh screen. After that, the CM was treated twoimes with a 2% (w/w) aqueous sodium hydroxide solution for 4 h

PRESSd Products xxx (2014) xxx–xxx

at 100 ◦C under mechanical stirring and then washed several timeswith distilled water until the alkali was completely removed (untilneutral pH), and finally dried at 40 ◦C for 24 h in an air-circulatingoven. After this, the fibres were bleached with a solution made upof equal parts (v:v) acetate buffer (27 g NaOH and 75 mL glacialacetic acid, diluted to 1 L in distilled water) and aqueous chlorite(1.7 wt% NaClO2 in water). This bleaching treatment was performedtwo times at 80 ◦C for 6 h. The bleached fibres were then washedrepeatedly in distilled water until the pH of the fibres became neu-tral and subsequently dried at 40 ◦C for 24 h in an air-circulatingoven (de Rodriguez et al., 2006; Siqueira et al., 2010a). The fibre con-tent throughout these chemical treatments was about 4–6% (w/w).The material that resulted after the purification was the purifiedcapim mombac a (CMP).

2.2.2. Extraction of cellulose nanocrystalsAfter the chemical treatment was completed, the CMP was

milled in a blender, passed through a 14-mesh screen and thenused to extract nanocrystals by acid hydrolysis. The hydrolysis wasperformed at 40 ◦C for 10 min, 20 min, 30 min or 40 min under vig-orous and constant stirring. For each gram of CMP, we used 30 mL ofH2SO4 (11.22 M). After hydrolysis, two different procedures wereemployed.

First procedure: The suspensions resulting from each time ofhydrolysis employed were diluted 10-fold with cold water to stopthe hydrolysis reaction and centrifuged for 10 min at 7500 rpm toremove the excess acid. The precipitate was then dialyzed withwater to remove acid residue, non-reactive sulphate groups, saltsand soluble sugars, until neutral pH (∼4 days) was attained. Thisprocedure was called total hydrolysis (TOT) and the nanocrystalsobtained from this procedure were named NCM10TOT, NCM20TOT,NCM30TOT and NCM40TOT.

Second procedure: In this procedure, the suspensions resultingfrom each time of hydrolysis employed were directly centrifuged,thereby obtaining two separate fractions of material. One fractioncontained the solid precipitated phase and the other the liquidsupernatant phase. Next, these fractions were also diluted sep-arately 10-fold with cold water to stop the hydrolysis reaction,centrifuged for 10 min at 7500 rpm to remove the excess acid, andsubsequently dialyzed until neutral pH was achieved (∼4 days). Thenanocrystals obtained from the solid precipitated (PPT) phase frac-tions were named NCM10PPT, NCM20PPT, NCM30PPT and NCM40PPT,and the nanocrystals obtained from the liquid supernatant (SOB)phase fractions were named NCM10SOB, NCM20SOB, NCM30SOB andNCM40SOB.

Subsequently, all the suspensions (TOT, PPT and SOB) resultingfrom the dialysis process were ultrasonicated for 10 min and storedin a refrigerator at 4 ◦C. Some drops of chloroform were added as aprotectant to the cellulose nanocrystal suspensions.

The cellulose nanocrystals of capim mombac a were labelled foreach time of hydrolysis studied as NCMTOT, NCMPPT and NCMSOB.

Fig. 1 shows the scheme of the experimental procedureemployed to obtain the NCM from the CMP.

2.3. Characterizations and measurements

2.3.1. Gravimetric analysisThe hydrolysis yield was calculated by drying an aliquot of

known volume of the NCM suspension at 105 ◦C for 12 h in anair-circulating oven.

2.3.2. Chemical composition

The chemical compositions of CM and CMP were measured asfollows: the lignin content was measured according to a standardmethod of the Technical Association of Pulp and Paper IndustryTAPPI T13M-54; the holocellulose (�-cellulose + hemicelluloses)

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NCMTOT;NCMPPT;NCMSOB

Diluted 10 times

with cold water

Diluted 10 times

with cold water

Hydrolysis (H2SO4 11.22 M, 40°C: 10, 20, 30 or 40min)

Diluted 10 times

with cold water

Capim Mombaça Purified (CMP)

Suspension of NCM

NCMTOT

NCMSOB

Dialysis until neutral pH

First Procedure

Centrifugation

Second Procedure

Centrifugation

NCMPPT

Solid Precipitated Fraction

Liquid Supernatant Fraction

CentrifugationCentrifugation

Ultrasonication 10min

rocedu

c1isttdp2s

utwpos

Fig. 1. Scheme of the experimental p

ontent was estimated by the acid chlorite method (Browning,967); briefly, the �-cellulose content was determined by treat-

ng the holocellulose with 5 and 24% (w/w) potassium hydroxideolutions, and the hemicelluloses content was found by subtrac-ing the �-cellulose portion from the total holocellulose content;he ash content was measured by considering the percentageifference between the initial weight of the dried fibre of the sam-le and that after calcination for 4 h at 800 ◦C (Trindade et al.,005). An average of three replicates was calculated for eachample.

.3.3. Fourier transform infrared spectroscopy (FTIR)A Shimadzu IR Prestige-21 Infrared spectrophotometer was

sed to obtain spectra for the CM and CMP. The KBr disc (ultra-

hin pellet) method was used in taking the IR spectra. Samplesere ground and mixed with KBr (sample/KBr ratio, 1/100) torepare pastilles. The experiments were carried out in the rangef 500–4000 cm−1 with a resolution of 4 cm−1 and a total of 32cans for each sample.

re to obtain the NCM from the CMP.

2.3.4. X-ray diffraction (XRD)The X-ray diffractograms of CMP and all NCM samples were

obtained at room temperature within a 2� ranging from 5◦ to 40◦

and a scan rate of 2◦ min−1. The equipment used was a ShimadzuLabX XRD-6000 diffractometer operating at 40 kV with a current of30 mA and Cu K� radiation (1.5406 A). Before performing the XRD,all samples were dried at 50 ◦C for 12 h in an air-circulating oven.The crystallinity index (CrI) of the CMP and NCM samples weredetermined by the Segal (1959) and Revol et al. (1987) methods, asshown in Eqs. (1) and (2), respectively.

CrI = [(I002 − IAM)/I002] × 100 (1)

CrI = [(I110 − I15.0◦ )/I110] × 100 (2)

In Eqs. (1) and (2), CrI expresses the relative degree of crys-

tallinity, I0 0 2 and I1 1 0 are the maximum intensities of the 0 0 2 and1 1 0 lattice diffractions at 2� = 23◦ and 20◦, respectively, and IAMand I15

◦ are the intensities of diffraction at 2� = 18◦ and 15◦, respec-tively. I0 0 2 and I1 1 0 represent both crystalline and amorphous

4 D.F. Martins et al. / Industrial Crops and Products xxx (2014) xxx–xxx

eodowsttsrnaCwaoa

Fig. 2. Photographs of CM pasture (a); dried and milled CM

egions, while IAM and I15◦ represent only the amorphous portion,

espectively.

.3.5. Atomic force microscopy (AFM)AFM measurements were performed with Shimadzu SPM-9600

quipment for evaluating the morphologies of all NCM samplesbtained with hydrolysis times of 20 min, 30 min and 40 min. Arop of a diluted nanocrystal aqueous suspension was depositednto a freshly cleaved mica surface and air-dried. AFM imagesere obtained at room temperature in the dynamic mode with a

can rate of 1 Hz and using Si tips with a curvature radius of lesshan 10 nm and a spring constant of 42 N m−1. The dimensions ofhe nanocrystals were determined using Vector Scan software (theoftware for Shimadzu’s SPM-9600). To eliminate the effect of tipadius on width measurements, we measured the heights of theanocrystals, which are not subject to peak broadening artefacts,nd assumed the nanocrystals to be cylindrical in shape (Beck-andanedo et al., 2005). One hundred and twenty nanocrystalsere randomly selected to determine the average length, width

nd aspect ratio (length/width). For each nanocrystal, one measuref length and two measures of diameter were performed and thespect ratio calculated.

.3.6. Thermal characterizationThe thermal stabilities of CMP and NCM samples were evalu-

ted by thermogravimetic analysis (TGA) using Shimadzu DTG-60H

s (b); CMP fibres obtained after the purification process (c).

equipment. The analysis conditions were: a nitrogen atmospherewith a 30 mL min−1 flow, a heating rate of 10 ◦C min−1, a temper-ature range from 25 to 600 ◦C, a sample mass between 5 and 7 mgand aluminium pans.

2.3.7. Elemental analysisElemental analysis of all NCM samples was performed, mainly

to determine the total sulphur content after the extraction ofnanocrystals. This was carried out with an EA1110-CHNS/O ele-mental analyser from CE Instruments.

3. Results and discussion

3.1. Purification and chemical composition of CM and CMP

Fig. 2 shows photographs of a CM pasture (a), dried and milledCM leaves (b), and CMP fibres obtained after the purification pro-cess (c). The white colour of the purified fibres is also evidence thatthe removal of non-cellulosic components was successful.

The cellulose content was 33.98 ± 3.6% and 51.97 ± 2.8%, hemi-celluloses was 29.56 ± 4.1% and 26.29 ± 2.3%, lignin was 18.6 ± 3.6%and 2.20 ± 0.26%, and ash was 2.21 ± 0.28% and 1.42 ± 0.13% for CM

and CMP, respectively. These values found for the main constituentsof CM are in accordance with the literature (Coan et al., 2005).After purification, the cellulose content increased, the hemicellu-loses and ash contents decreased slightly and the lignin content

4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers (cm-1)

Fig. 3. FTIR spectra of raw capim mombac a (CM) and purified capim mombac a(CMP).

Table 1The yield and crystallinity index (CrI) of different NCM samples.

Hydrolysis time (min) Yield (%) CrI (%)

TOT SOB PPT TOT SOB PPT

10 72.44 20.62 55.86 70.6 60.7 74.120 67.04 17.14 43.15 75.1 69.5 80.030 51.86 12.15 20.3 73.5 28.6 77.2

ittisa

eTCSbc

40 35.15 7.62 11.98 62.5 29.7 68.9

MP: CrI = 77.4%.

as significantly reduced by 88.17%. The purification process wasffective because CMP with a low lignin content is suitable for thextraction of cellulose nanocrystals (low lignin content and highellulose content).

The efficiency of the purification method was also analyzed bynfrared spectroscopy. Fig. 3 shows FTIR spectra of CM and CMP. Inhe spectrum of CM, the band near 1734 cm−1 is assigned mainlyo the C O stretching vibration of the carbonyl and acetyl groupsn the xylan component of hemicelluloses and in the lignin. In theame spectrum the band near 1242 cm−1 corresponds to the axialsymmetric strain of C O C, which is commonly observed whenC O are present, e.g. in ether, ester, and phenol groups (Siqueirat al., 2010b). These peaks almost disappeared in the spectra of CMP.he peaks at 1064 and 896 cm−1 are associated with cellulose, the

O stretching and C H rock vibrations of cellulose (Alemdar andain, 2008), which appeared in all of the spectra. The differencesetween the spectra indicate that the CMP sample had a higherellulose content, suggesting that it is almost pure cellulose.

.2. Extraction of cellulose nanocrystals and suspension analysis

The yields of NCM obtained in the CMP hydrolysis processes areresented in Table 1. The results presented in Table 1 demonstrate,s expected, that for each procedure (TOT, PPT and SOB), increasinghe reaction time produced a decrease in the yield values.

It was observed that the TOT procedure, for a specific time ofydrolysis, produced a higher yield than the PPT and SOB proce-ures. It is also possible also verify that the sum of the yields fromhe PPT and SOB fractions are approximately equal to or less than

he yield of the TOT fraction, indicating that the TOT fraction is theesult of separate contributions from the two fractions.

It was possible to obtain stable colloidal suspensions of NCMith all the procedures and times of hydrolysis used in this work.

Fig. 4. Resulting colloidal suspensions of NCMTOT samples obtained after hydrolysistimes of 10 min, 20 min, 30 min and 40 min.

Examples can be seen in Fig. 4, which shows the colloidal suspen-sions of NCM10TOT, NCM20TOT, NCM30TOT, and NCM40TOT.

Sulphuric acid hydrolysis leads to homogeneous stable aqueoussuspensions of cellulose nanocrystals which are negatively chargedand, thus, do not tend to aggregate. During the hydrolysis pro-cess, esterification of surface hydroxyl groups in the cellulose takesplace and, as a consequence, sulphate groups are introduced (Beck-Candanedo et al., 2005; Lima and Borsali, 2004; Silva and D’Almeida,2009).

The results of elemental analyses allowed us to verify the pres-ence of sulphate groups in all the NCM samples by determiningthe sulphur content. The results, presented in Table 2, confirm theincorporation of sulphate groups in all the NCM samples after treat-ment with sulphuric acid.

3.3. X-ray diffraction

Diffraction patterns of the CMP and NCM samples are shownin Fig. 5. Based on the XRD diffractograms the CrI were calculatedand the values are presented in Table 1. It is possible verify that thevalues of CrI obtained for NCMTOT and NCMPPT were similar to theCrI of CMP (77.4%). Additionally, the values of CrI for NCMSOB weremuch lower than those attributed to CMP, NCMTOT and NCMPPT.

The reduction in the CrI observed for NCMSOB samples can beattributed to the dissolution of cellulose upon hydrolysis, whichremained in the supernatant solution and was then reprecipitatedwith a low crystalline order. This can also be verified by its XRD pat-tern shown in Fig. 5(b) that shows the predominance of cellulosetype II, observed by peaks at 2� = 12◦ (plane 1 0 1), 20◦ (plane 101)and 22◦ (plane 0 0 2). Additionally the NCMPPT samples were richin non-hydrolysed solid particles and had few cellulose moleculessolubilized during the hydrolysis; therefore, they have higher crys-tallinities because little cellulose re-precipitated on the surface ofthe nanocrystals. As a consequence, this sample is predominantlycomposed of nanocrystals that were not dissolved during hydrol-ysis, namely cellulose type I. This can be confirmed by the XRDpatterns (Fig. 5(c)), where the pattern for NCMPPT is similar to thatof CMP, with a profile of cellulose type I, due to the presence ofpeaks at 2� = 15◦ (plane 1 0 1), 17◦ (plane 101), 21◦ (plane 0 2 1)and 23◦ (plane 0 0 2). The XRD pattern of the NCMTOT samples(Fig. 5(a)), apparently presents the profile of cellulose type I, butit is possible to verify the presence of a shoulder around 2� = 22◦

attributed to cellulose type II, confirming the possible reprecip-itation of solubilized cellulose molecules on the surface of theresulting nanocrystals (Sèbe et al., 2012; O’Sullivan, 1997). This isreflected in the CrI values of the NCMTOT samples (Table 1), whichare lower than those obtained for the NCMPPT samples. Therefore,it can be concluded that the presence of cellulose type II is asso-ciated with recrystallization and reprecipitation after the cellulose

hydrolysis, since, as we know, 11.22 M sulphuric acid can be a sol-vent for cellulose (Xiang et al., 2003; Jayme and Lang, 1963).

In summary, we can conclude that the NCMPPT samples wereformed predominantly of cellulose type I, the NCMSOB samples

6 D.F. Martins et al. / Industrial Crops and Products xxx (2014) xxx–xxx

Table 2Sulphur content of the NCM samples obtained by elemental analyses.

NCM10 NCM20 NCM30 NCM40

TOT PPT SOB TOT PPT SOB

S (%) 0.67 0.42 0.65 0.79 0.26 0.42

5 10 15 20 25 30 35 40

NCM40TOT

NCM30TOT

NCM20TOT

NCM10TOT

2θθ ((degree )

5 10 15 20 25 30 35 40

NCM40SOB

NCM30S OB

NCM20SOB

NCM10S OB

2θθ ((de gree)

5 10 15 20 25 30 35 40

NCM40PPT

NCM30PPT

NCM20PPT

2θθ ((degree )

NCM10PPT

Fig. 5. X-ray diffractograms of (a) NCMTOT and CMP; (b) NCMSOB and CMP; (c)NCMPPT and CMP.

TOT PPT SOB TOT PPT SOB

0.66 1.06 1.15 0.41 1.43 1.56

were formed predominantly of cellulose type II, and the NCMTOTsamples were a mixture of two cellulose types (I and II). In thelatter case (NCMTOT), the profile of the XRD diffractogram seemsto match that of cellulose type I but, apparently, the presence ofcellulose type II is masked by the presence of cellulose type I.

3.4. Atomic force microscopy (AFM)

AFM topography measurements were performed in order toprecisely characterize the dimensions of the individual crystallites.Determining the exact dimensions of CNCs is complicated by thespecific limitations of the different analytical methods used. In thecase of AFM, tip/sample broadening represents the main limitation,resulting in an overestimation of CNCs dimensions. Since the CNCsare assumed to be cylindrical in shape, the height of the CNCs wastaken to be equivalent to its diameter, to compensate for imagewidening due to convolution of the tip and the particle (Beck-Candanedo et al., 2005; Kvien et al., 2005). Tip broadening effectsstill cause errors in length measurements, but this is unavoidable(Beck-Candanedo et al., 2005).

Fig. 6 shows AFM images of NCMTOT, NCMPPT and NCMSOBobtained with 20 min, 30 min and 40 min hydrolysis times. Forsamples NCMTOT and NCMPPT, the AFM images presented needle-shaped nanoparticles throughout, confirming that the extraction ofCNCs from CMP was successful. The AFM images of samples NCMSOBalso showed the presence of CNCs; however, the images predom-inantly show nanoparticles with a circular shape. Therefore, thisbehaviour is related to the structure of the CNCs and, as shown bythe XRD patterns, the NCMPPT and NCMTOT samples presented cel-lulose I profiles for all hydrolysis times, whereas the XRD patternsfor samples of NCMSOB presented cellulose II profiles. In a study car-ried out by Sèbe et al. (2012), it was also verified that the shape ofnanocrystals is directly related to the type of polymorph of cellulosepresent (I or II).

Values for the lengths, widths and aspect ratios obtained fromseveral AFM images of NCMTOT, NCMPPT and NCMSOB are shown inTable 3. Increasing the extraction time resulted in slightly shorterlengths for NCMTOT and NCMPPT. This was expected, since longerextraction times partially destroyed areas with crystalline domains.According to Table 3 it can be seen that the increase in extrac-tion time did not result in a change in the size of the NCMSOB,which was characteristic of cellulose type II. By studying Table 3it is clear that increasing the hydrolysis time produced no sig-nificant difference in width of the NCMTOT, NCMPPT and NCMSOBwhen the standard deviation of each value was taken into account.Under the experimental conditions studied (NCMTOT, NCMPPT andNCMSOB), no significant reductions in aspect ratio were observedwith increasing hydrolysis time except in the case of the NCM40TOTsample. However, comparing each condition (NCMTOT, NCMPPT andNCMSOB) at the same hydrolysis time, a variation in the aspectratio can be observed, where the NCMPPT samples have the high-est values while the NCMSOB samples have the lowest. The averageaspect ratios for the CNCs found in this work are close to the val-ues reported in the literature; therefore, these particles have great

potential to be used as reinforcing agents in nanocomposites (Kaliaet al., 2011; Silvério et al., 2013). The results of the morphologi-cal investigation by AFM are consistent with other reports in theliterature where CNCs were extracted from different sources (Bai

Please cite this article in press as: Martins, D.F., et al., The influence of the cellulose hydrolysis process on thestructure of cellulose nanocrystals extracted from capim mombac a (Panicum maximum). Ind. Crops Prod. (2014),http://dx.doi.org/10.1016/j.indcrop.2014.10.035

Fig. 6. AFM images of NCMTOT, NCMPPT and NCMSOB samples obtained after different hydrolysis times.

Table 3Length, width and aspect ratio for NCMTOT, NCMPPT and NCMSOB samples.

Hydrolysistime (min)

NCMTOT NCMPPT NCMSOB

Length (nm) Width (nm) Aspect ratio (nm) Length (nm) Width (nm) Aspect ratio (nm) Length (nm) Width (nm) Aspect ratio (nm)

20 301.50 ± 48.22 6.20 ± 1.77 48.67 ± 23.84 316.31 ± 59.28 6.04 ± 1.48 52.34 ± 25.54 73.39 ± 20.25 3.10 ± 0.58 23.68 ± 7.8330 255.53 ± 50.98 5.63 ± 1.50 45.39 ± 14.60 265.17 ± 32.77 4.94 ± 1.02 53.71 ± 14.90 73.11 ± 8.46 3.09 ± 1.02 23.66 ± 7.3240 181.38 ± 38.32 5.39 ± 1.74 28.06 ± 13.99 239.06 ± 59.10 4.84 ± 1.12 49.35 ± 14.04 73.16 ± 13.72 2.72 ± 0.40 26.86 ± 6.23

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Table 4Onset temperature (Tonset), degradation temperature at maximum weight-loss rate(Tmax) and char yield for CMP and NCM samples obtained from TGA.

Sample Cellulose thermaldegradation

Carbonic residuedegradation

Char yield (%)

Tonset (◦C) Tmax (◦C) Tonset (◦C) Tmax (◦C)

CMP 281 331 404 448 1.5NCM10

TOT 271 309 391 470 3PPT 277 312 398 483 1.5SOB 259 308 400 445 2

TOT 262 300 403 468 2.5PPT 265 302 396 463 0.1SOB 247 304 424 485 8

TOT 235 276 419 494 1.5PPT 260 300 396 442 5SOB 245 288 431 502 7

TOT 253 304 391 454 1.5PPT 258 295 387 462 7

t al., 2009; Beck-Candanedo et al., 2005; de Rodriguez et al., 2006;lazzouzi-Hafraoui et al., 2008; Kvien et al., 2005; Rosa et al., 2010;iqueira et al., 2010a; Teixeira et al., 2011).

.5. Thermogravimetric analysis (TGA)

The thermograms obtained for CMP and NCMTOT, NCMPPT andCMSOB isolated with different hydrolysis times are shown in Fig. 7.he values obtained for onset temperatures (Tonset), degradationemperatures at maximum weight loss rate (Tmax) and char yieldsor all samples are shown in Table 4.

For all samples the thermogram profiles exhibit essentially threevents. The first event is related to the evaporation of adsorbedater or compounds of low molecular weight. This occurred

etween 30 and 150 ◦C and there was even a small mass loss (<10%).he second event corresponds basically to the process of cellu-ose degradation, which consists of several concurrent processes:epolymerization, dehydration and decomposition of glycosidicnits (Araki et al., 1998). In this step, the thermal degradation tem-eratures of all the NCM samples were lower than that of CMP. Thisehaviour was expected since some studies have shown that the

ntroduction of sulphate groups decreases the thermal stability ofNCs due to the dehydration of the cellulose (Roman and Winter,004). The incorporation of sulphate groups on the surface of cel-

ulose after hydrolysis has a catalytic effect in reactions of thermalegradation. Another effect that has been reported is the replace-ent of OH groups on the cellulose with sulphate groups, which

eads to a decreased activation energy for the degradation of theellulose chains (Teixeira et al., 2010).

In this step large differences in initial degradation temperatureere also observed for all NCM samples. Such behaviour could

Fig. 7. TGA curves of the CMP and NCMTOT, NCMPPT and

SOB 236 299 403 471 8

not be attributed solely to the presence of sulphate groups, sincethe results of the elemental analysis showed that the amount ofsulphate present was virtually the same for all samples studied.As shown by the XRD diffractogram profiles, it is possible ver-ify a predominance of cellulose type I in the NCMPPT samples, apredominance of cellulose type II in the NCMSOB samples, and a

mixture of cellulose I and II in the NCMTOT samples. One may alsopoint out that the degradation temperatures for NCMPPT (mainlycellulose I) are higher than the degradation temperatures for

NCMSOB obtained after different hydrolysis times.

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Nabits

c(hftIaoNiNmt

mmssTnIdedpdNwtItws

CMSOB (mainly cellulose II). In addition, the degradation temper-tures for NCMTOT (a mixture of cellulose I and II) are intermediateetween those found for NCMPPT and NCMSOB. Thus, the differences

n the degradation temperatures (Table 4) are directly linked to theype of material (cellulose type I or II) and not to the presence ofulphate groups.

The third event is attributed to the oxidation and breakdown ofharred residues to form gaseous products of low molecular weightRoman and Winter, 2004; Teixeira et al., 2010). Some authorsave related that the presence of sulphate groups is responsible

or an increase in the charred residue, and attribute this fact tohe flame retardant effect of this group (Roman and Winter, 2004).n this step, it was observed that the NCMSOB showed, in general,n increase in char residue as well as in the initial temperaturef degradation (Table 4) when compared to CMP, NCMPPT andCMTOT. It is also possible to verify that the NCMTOT again showed

ntermediate values when compared with the values of NCMSOB andCMPPT samples. This increase in the char residue can be attributedainly to the cellulose polymorph present in the NCM samples; in

his case, attributed to the presence of cellulose type II.

. Conclusions

The present work shows that CNCs can be isolated from capimombac a fibres. The conditions of hydrolysis using differentethodologies were effective, resulting in stable aqueous suspen-

ions of NCM that were negatively charged due to the presence ofimilar quantities of sulphate groups for all the studied conditions.hrough X-ray diffraction it was possible to observe that celluloseanocrystals formed with different polymorphs of cellulose (type

and II) as a consequence of the hydrolysis conditions used andue to reprecipitation of cellulose. It was observed that the differ-nt hydrolysis processes used resulted in nanocrystals containingifferent polymorphs of cellulose, which directly influenced thehysical aspect of the resulting nanocrystals. NCM composed pre-ominantly of cellulose I have needle-like structures; in contrast,CM made predominantly of cellulose II have circular structures. Itas found that the thermal stability of the NCM was dependent of

he kind of cellulose polymorph present in the structure of the NCM.t was concluded that a reduction in the initial thermal degrada-ion temperature and an increase in the char residue are associatedith the presence of cellulose type II and not with the presence of

ulphate groups.

cknowledgements

The authors thank CAPES/PROAP, CNPq and FAPEMIG for finan-ial support.

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