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The influence of the cellulose hydrolysis process on the structure of cellulose nanocrystals extracted from capim mombaça (Panicum maximum)
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The influence of the cellulose hydrolysis process on the structure of cellulose nanocrystals extracted from capim mombac¸a (Panicum maximum) Douglas Ferreira Martins, Alexandre Bernaldino de Souza, Mariana Alves Henrique, Hudson Alves Silvério, Wilson Pires Flauzino Neto, Daniel Pasquini ∗ Instituto de Química, Universidade Federal de Uberlândia, Campus Santa Mônica, Av. João Naves de Ávila, 2121, 38400-902 Uberlândia, Minas Gerais, Brazil
a r t i c l e
i n f o
Article history: Received 9 August 2014 Received in revised form 10 October 2014 Accepted 18 October 2014 Available online xxx Keywords: Capim mombac¸a fibres Acid hydrolysis Cellulose nanocrystals Cellulose polymorphs Thermal stability
a b s t r a c t This work presents studies on the isolation of cellulose nanocrystals from cellulose extracted from capim mombac¸a (Panicum maximum) leaves through variations in the acid hydrolysis process, and their consequent impact on their chemical, physical and thermal properties. The hydrolysis was performed at 40 ◦ C for 10 min, 20 min, 30 min or 40 min under vigorous and constant stirring. For each gram of CM, 30 mL of H2 SO4 (11.22 M) was used. The variations occurred in the subsequent step, which consists of adding 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, Xray diffraction (XRD) and atomic force microscopy (AFM) in order to assess changes in the physical and chemical structure of the nanocrystals obtained and in their thermal stability. It was observed that the procedures adopted influenced the dimensions and physical aspect of the nanocrystals, the type of cellulose 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 polymorph of cellulose that predominated, and not with the presence of sulphate groups on the surface of the nanocrystals. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The application of cellulose nanocrystals (CNCs) as a nanosized reinforcement in polymer matrixes has attracted considerable attention, since it offers a unique combination of desirable physical properties and environmental benefits (Habibi et al., 2010; Peng et al., 2011). CNCs-based nanocomposites generally exhibit significant improvements in thermal, mechanical, and barrier properties compared to the neat polymer or conventional composites (Azeredo et al., 2009). CNCs are needle-shaped cellulose particles with at least one dimension equal to or less than 100 nm and have a highly crystalline nature (Flauzino Neto et al., 2012). The main features driving the development of CNCs as polymer reinforcement agents are their large specific surface area (estimated to be several hundreds of m2 g−1 ), their very high modulus of elasticity (approximately 150 GPa), their large aspect ratio, and their ability 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: [email protected] (D. Pasquini).
Other attractive advantages of CNCs are their low density (about 1.59 g cm−3 ) (O’Sullivan, 1997), nonabrasive nature, nontoxic character, biocompatibility, and biodegradability (Azizi Samir et al., 2005). Additionally, CNCs come from renewable natural sources that are very abundant and therefore low in cost, so it is not necessary to synthesize them, they allow the production of composite films with excellent visible light transmittance, and they can be easily modified chemically (their structure includes a reactive surface of OH side groups that facilitate grafting chemical species in 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 hydrolysis is the most well-known and widely used. This process is based on the fact that crystalline regions are acid-insoluble under the conditions used in the extractions (Peng et al., 2011). The morphology and properties of CNCs influence their performance as a reinforcing agent. Moreover, the morphology and properties of CNCs depend on the source of the original cellulose and on the extraction process. Thus, the development of CNCs from different sources of cellulose
http://dx.doi.org/10.1016/j.indcrop.2014.10.035 0926-6690/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Martins, D.F., et al., The influence of the cellulose hydrolysis process on the structure 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
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is relevant and, in the same way, the choice of hydrolysis conditions is very important too (Flauzino Neto et al., 2012). It is well known that the use of different acids can lead to differences in the stability of the nanocrystals in a colloidal suspension due to the presence of different charges on the surface of the nanoparticles (Angellier et al., 2004). In the case where sulphuric acid is employed to obtain CNCs, the presence of negative sulphate groups, introduced on the outer surface of the CNCs during the hydrolysis process, is responsible for the stabilization of the CNCs in the resulting colloidal suspension (Beck-Candanedo et al., 2005). However, some authors have reported that the presence of sulphate groups decreased the thermal stability of the CNCs (Roman and Winter, 2004). Usually, a higher acid sulphate group content in cellulose leads to a lower temperature of thermal degradation of the cellulose. If hydrochloric acid is used instead of sulphuric acid to hydrolyse the native cellulose, the thermal stability of the prepared nanocrystals is improved but the nanocrystals are inclined to aggregate due to the absence of a force of electrostatic repulsion between the crystal particles, resulting in an unstable colloidal suspension (Araki et al., 1998). One factor often overlooked by researchers is the method employed in the preparation of the nanocrystals, regardless of the type of acid used. Some authors add water directly to the acid solution containing the nanocrystals in order to stop the hydrolysis prior to centrifugation and dialysis (Haafiz et al., 2014; dos Santos et al., 2013; Silvério et al., 2013; Henrique et al., 2013; Flauzino Neto et al., 2012; Rosa et al., 2012; Belbekhouche et al., 2008; Teixeira et al., 2011). Moreover, other authors do not add water directly and just follow steps of centrifugation and dialysis after finalizing the hydrolysis time (Siqueira et al., 2013; Cao et al., 2013; de Menezes et al., 2009; Habibi et al., 2007; Samir et al., 2004). It is known that at high concentrations sulphuric acid acts as a solvent for cellulose hydrolysis (Xiang et al., 2003; Jayme and Lang, 1963). One hypothesis to be exploited in this work is that adding water to stop the hydrolysis process may induce the re-precipitation of a portion of the cellulose solubilized on the surface of CNCs, or even the formation of nanoparticles of regenerated cellulose. This precipitation and formation of structures containing cellulose type II may be the main cause of the change in thermal stability of the nanocrystals, and not the presence of sulphate groups as claimed by some authors (Roman and Winter, 2004; Araki et al., 1998). This study aimed to extract CNCs from capim mombac¸a (Panicum maximum) (CM) through experimental modifications, in order to investigate the presence of CNCs with structures made of cellulose I and II. Different techniques were employed to characterize the cellulose fibres and the resulting cellulose nanocrystals in order to investigate their chemical composition, crystallinity index, thermal stability and morphology (shape and size). 2. Experimental 2.1. Materials and methods Capim mombac¸a (P. maximum) (CM) leaves were supplied by São Mateus farm (Comendador Gomes, Minas Gerais, Brazil). The other reagents employed in this study were: sulphuric acid (95.0–98.0 wt.%, Vetec, P.A.), sodium hydroxide (Vetec), potassium hydroxide (Vetec), sodium chlorite (NaClO2 , technical grade, 80%, Sigma–Aldrich), glacial acetic acid (Synth), and cellulose membrane (D9402, Sigma–Aldrich). 2.2. Preparation of cellulose nanocrystals 2.2.1. Purification of capim mombac¸a (CM) Initially the untreated CM was milled with a blender to pass through a 14-mesh screen. After that, the CM was treated two times with a 2% (w/w) aqueous sodium hydroxide solution for 4 h
at 100 ◦ C under mechanical stirring and then washed several times with distilled water until the alkali was completely removed (until neutral pH), and finally dried at 40 ◦ C for 24 h in an air-circulating oven. After this, the fibres were bleached with a solution made up of equal parts (v:v) acetate buffer (27 g NaOH and 75 mL glacial acetic acid, diluted to 1 L in distilled water) and aqueous chlorite (1.7 wt% NaClO2 in water). This bleaching treatment was performed two times at 80 ◦ C for 6 h. The bleached fibres were then washed repeatedly in distilled water until the pH of the fibres became neutral and subsequently dried at 40 ◦ C for 24 h in an air-circulating oven (de Rodriguez et al., 2006; Siqueira et al., 2010a). The fibre content throughout these chemical treatments was about 4–6% (w/w). The material that resulted after the purification was the purified capim mombac¸a (CMP). 2.2.2. Extraction of cellulose nanocrystals After the chemical treatment was completed, the CMP was milled in a blender, passed through a 14-mesh screen and then used to extract nanocrystals by acid hydrolysis. The hydrolysis was performed at 40 ◦ C for 10 min, 20 min, 30 min or 40 min under vigorous and constant stirring. For each gram of CMP, we used 30 mL of H2 SO4 (11.22 M). After hydrolysis, two different procedures were employed. First procedure: The suspensions resulting from each time of hydrolysis employed were diluted 10-fold with cold water to stop the hydrolysis reaction and centrifuged for 10 min at 7500 rpm to remove the excess acid. The precipitate was then dialyzed with water to remove acid residue, non-reactive sulphate groups, salts and soluble sugars, until neutral pH (∼4 days) was attained. This procedure was called total hydrolysis (TOT) and the nanocrystals obtained from this procedure were named NCM10TOT , NCM20TOT , NCM30TOT and NCM40TOT . Second procedure: In this procedure, the suspensions resulting from each time of hydrolysis employed were directly centrifuged, thereby obtaining two separate fractions of material. One fraction contained the solid precipitated phase and the other the liquid supernatant phase. Next, these fractions were also diluted separately 10-fold with cold water to stop the hydrolysis reaction, centrifuged for 10 min at 7500 rpm to remove the excess acid, and subsequently dialyzed until neutral pH was achieved (∼4 days). The nanocrystals obtained from the solid precipitated (PPT) phase fractions were named NCM10PPT , NCM20PPT , NCM30PPT and NCM40PPT , and the nanocrystals obtained from the liquid supernatant (SOB) phase fractions were named NCM10SOB , NCM20SOB , NCM30SOB and NCM40SOB . Subsequently, all the suspensions (TOT, PPT and SOB) resulting from the dialysis process were ultrasonicated for 10 min and stored in a refrigerator at 4 ◦ C. Some drops of chloroform were added as a protectant to the cellulose nanocrystal suspensions. The cellulose nanocrystals of capim mombac¸a were labelled for each time of hydrolysis studied as NCMTOT , NCMPPT and NCMSOB . Fig. 1 shows the scheme of the experimental procedure employed to obtain the NCM from the CMP. 2.3. Characterizations and measurements 2.3.1. Gravimetric analysis The hydrolysis yield was calculated by drying an aliquot of known volume of the NCM suspension at 105 ◦ C for 12 h in an air-circulating oven. 2.3.2. Chemical composition The chemical compositions of CM and CMP were measured as follows: the lignin content was measured according to a standard method of the Technical Association of Pulp and Paper Industry TAPPI T13M-54; the holocellulose (␣-cellulose + hemicelluloses)
Please cite this article in press as: Martins, D.F., et al., The influence of the cellulose hydrolysis process on the structure 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
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Capim Mombaça Purified (CMP)
Hydrolysis (H2SO4 11.22 M, 40°C: 10, 20, 30 or 40min)
Suspension of NCM
First Procedure
Second Procedure
Diluted 10 times
Centrifugation
with cold water
Liquid Supernatant Fraction
Solid Precipitated Fraction
NCMTOT
Centrifugation
Diluted 10 times
Diluted 10 times
with cold water
with cold water
NCMPPT
NCMSOB
Centrifugation
Centrifugation
Dialysis until neutral pH
Ultrasonication 10min
NCMTOT; NCMPPT; NCMSOB Fig. 1. Scheme of the experimental procedure to obtain the NCM from the CMP.
content was estimated by the acid chlorite method (Browning, 1967); briefly, the ␣-cellulose content was determined by treating the holocellulose with 5 and 24% (w/w) potassium hydroxide solutions, and the hemicelluloses content was found by subtracting the ␣-cellulose portion from the total holocellulose content; the ash content was measured by considering the percentage difference between the initial weight of the dried fibre of the sample and that after calcination for 4 h at 800 ◦ C (Trindade et al., 2005). An average of three replicates was calculated for each sample. 2.3.3. Fourier transform infrared spectroscopy (FTIR) A Shimadzu IR Prestige-21 Infrared spectrophotometer was used to obtain spectra for the CM and CMP. The KBr disc (ultrathin pellet) method was used in taking the IR spectra. Samples were ground and mixed with KBr (sample/KBr ratio, 1/100) to prepare pastilles. The experiments were carried out in the range of 500–4000 cm−1 with a resolution of 4 cm−1 and a total of 32 scans for each sample.
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 Shimadzu LabX XRD-6000 diffractometer operating at 40 kV with a current of ˚ Before performing the XRD, 30 mA and Cu K␣ radiation (1.5406 A). 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 were determined by the Segal (1959) and Revol et al. (1987) methods, as shown 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 crystallinity, I0 0 2 and I1 1 0 are the maximum intensities of the 0 0 2 and 1 1 0 lattice diffractions at 2 = 23◦ and 20◦ , respectively, and IAM and I15◦ are the intensities of diffraction at 2 = 18◦ and 15◦ , respectively. I0 0 2 and I1 1 0 represent both crystalline and amorphous
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Fig. 2. Photographs of CM pasture (a); dried and milled CM leaves (b); CMP fibres obtained after the purification process (c).
regions, while IAM and I15◦ represent only the amorphous portion, respectively. 2.3.5. Atomic force microscopy (AFM) AFM measurements were performed with Shimadzu SPM-9600 equipment for evaluating the morphologies of all NCM samples obtained with hydrolysis times of 20 min, 30 min and 40 min. A drop of a diluted nanocrystal aqueous suspension was deposited onto a freshly cleaved mica surface and air-dried. AFM images were obtained at room temperature in the dynamic mode with a scan rate of 1 Hz and using Si tips with a curvature radius of less than 10 nm and a spring constant of 42 N m−1 . The dimensions of the nanocrystals were determined using Vector Scan software (the software for Shimadzu’s SPM-9600). To eliminate the effect of tip radius on width measurements, we measured the heights of the nanocrystals, which are not subject to peak broadening artefacts, and assumed the nanocrystals to be cylindrical in shape (BeckCandanedo et al., 2005). One hundred and twenty nanocrystals were randomly selected to determine the average length, width and aspect ratio (length/width). For each nanocrystal, one measure of length and two measures of diameter were performed and the aspect ratio calculated. 2.3.6. Thermal characterization The thermal stabilities of CMP and NCM samples were evaluated by thermogravimetic analysis (TGA) using Shimadzu DTG-60H
equipment. The analysis conditions were: a nitrogen atmosphere with a 30 mL min−1 flow, a heating rate of 10 ◦ C min−1 , a temperature range from 25 to 600 ◦ C, a sample mass between 5 and 7 mg and aluminium pans. 2.3.7. Elemental analysis Elemental analysis of all NCM samples was performed, mainly to determine the total sulphur content after the extraction of nanocrystals. This was carried out with an EA1110-CHNS/O elemental 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 milled CM leaves (b), and CMP fibres obtained after the purification process (c). The white colour of the purified fibres is also evidence that the removal of non-cellulosic components was successful. The cellulose content was 33.98 ± 3.6% and 51.97 ± 2.8%, hemicelluloses 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 constituents of CM are in accordance with the literature (Coan et al., 2005). After purification, the cellulose content increased, the hemicelluloses and ash contents decreased slightly and the lignin content
Please cite this article in press as: Martins, D.F., et al., The influence of the cellulose hydrolysis process on the structure 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
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Absorbance (a.u)
5
1064 896
1242
1734
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CMP Fig. 4. Resulting colloidal suspensions of NCMTOT samples obtained after hydrolysis times of 10 min, 20 min, 30 min and 40 min.
CM
4000
3500
3000
2500
2000
1500
1000
500
-1 Wavenumbers (cm ) Fig. 3. FTIR spectra of raw capim mombac¸a (CM) and purified capim mombac¸a (CMP). Table 1 The yield and crystallinity index (CrI) of different NCM samples. Hydrolysis time (min)
10 20 30 40
Yield (%)
CrI (%)
TOT
SOB
PPT
TOT
SOB
PPT
72.44 67.04 51.86 35.15
20.62 17.14 12.15 7.62
55.86 43.15 20.3 11.98
70.6 75.1 73.5 62.5
60.7 69.5 28.6 29.7
74.1 80.0 77.2 68.9
CMP: CrI = 77.4%.
was significantly reduced by 88.17%. The purification process was effective because CMP with a low lignin content is suitable for the extraction of cellulose nanocrystals (low lignin content and high cellulose content). The efficiency of the purification method was also analyzed by infrared spectroscopy. Fig. 3 shows FTIR spectra of CM and CMP. In the spectrum of CM, the band near 1734 cm−1 is assigned mainly to the C O stretching vibration of the carbonyl and acetyl groups in the xylan component of hemicelluloses and in the lignin. In the same spectrum the band near 1242 cm−1 corresponds to the axial asymmetric strain of C O C, which is commonly observed when C O are present, e.g. in ether, ester, and phenol groups (Siqueira et al., 2010b). These peaks almost disappeared in the spectra of CMP. The peaks at 1064 and 896 cm−1 are associated with cellulose, the C O stretching and C H rock vibrations of cellulose (Alemdar and Sain, 2008), which appeared in all of the spectra. The differences between the spectra indicate that the CMP sample had a higher cellulose content, suggesting that it is almost pure cellulose. 3.2. Extraction of cellulose nanocrystals and suspension analysis The yields of NCM obtained in the CMP hydrolysis processes are presented in Table 1. The results presented in Table 1 demonstrate, as expected, that for each procedure (TOT, PPT and SOB), increasing the reaction time produced a decrease in the yield values. It was observed that the TOT procedure, for a specific time of hydrolysis, produced a higher yield than the PPT and SOB procedures. It is also possible also verify that the sum of the yields from the PPT and SOB fractions are approximately equal to or less than the yield of the TOT fraction, indicating that the TOT fraction is the result of separate contributions from the two fractions. It was possible to obtain stable colloidal suspensions of NCM with all the procedures and times of hydrolysis used in this work.
Examples can be seen in Fig. 4, which shows the colloidal suspensions of NCM10TOT , NCM20TOT , NCM30TOT , and NCM40TOT . Sulphuric acid hydrolysis leads to homogeneous stable aqueous suspensions of cellulose nanocrystals which are negatively charged and, thus, do not tend to aggregate. During the hydrolysis process, esterification of surface hydroxyl groups in the cellulose takes place and, as a consequence, sulphate groups are introduced (BeckCandanedo et al., 2005; Lima and Borsali, 2004; Silva and D’Almeida, 2009). The results of elemental analyses allowed us to verify the presence of sulphate groups in all the NCM samples by determining the sulphur content. The results, presented in Table 2, confirm the incorporation of sulphate groups in all the NCM samples after treatment with sulphuric acid. 3.3. X-ray diffraction Diffraction patterns of the CMP and NCM samples are shown in Fig. 5. Based on the XRD diffractograms the CrI were calculated and the values are presented in Table 1. It is possible verify that the values of CrI obtained for NCMTOT and NCMPPT were similar to the CrI of CMP (77.4%). Additionally, the values of CrI for NCMSOB were much lower than those attributed to CMP, NCMTOT and NCMPPT . The reduction in the CrI observed for NCMSOB samples can be attributed to the dissolution of cellulose upon hydrolysis, which remained in the supernatant solution and was then reprecipitated with a low crystalline order. This can also be verified by its XRD pattern shown in Fig. 5(b) that shows the predominance of cellulose ¯ type 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 rich in non-hydrolysed solid particles and had few cellulose molecules solubilized during the hydrolysis; therefore, they have higher crystallinities because little cellulose re-precipitated on the surface of the nanocrystals. As a consequence, this sample is predominantly composed of nanocrystals that were not dissolved during hydrolysis, namely cellulose type I. This can be confirmed by the XRD patterns (Fig. 5(c)), where the pattern for NCMPPT is similar to that of CMP, with a profile of cellulose type I, due to the presence of ¯ 21◦ (plane 0 2 1) peaks at 2 = 15◦ (plane 1 0 1), 17◦ (plane 101), and 23◦ (plane 0 0 2). The XRD pattern of the NCMTOT samples (Fig. 5(a)), apparently presents the profile of cellulose type I, but it is possible to verify the presence of a shoulder around 2 = 22◦ attributed to cellulose type II, confirming the possible reprecipitation of solubilized cellulose molecules on the surface of the resulting nanocrystals (Sèbe et al., 2012; O’Sullivan, 1997). This is reflected in the CrI values of the NCMTOT samples (Table 1), which are lower than those obtained for the NCMPPT samples. Therefore, it can be concluded that the presence of cellulose type II is associated with recrystallization and reprecipitation after the cellulose hydrolysis, since, as we know, 11.22 M sulphuric acid can be a solvent for cellulose (Xiang et al., 2003; Jayme and Lang, 1963). In summary, we can conclude that the NCMPPT samples were formed predominantly of cellulose type I, the NCMSOB samples
Please cite this article in press as: Martins, D.F., et al., The influence of the cellulose hydrolysis process on the structure 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
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Table 2 Sulphur content of the NCM samples obtained by elemental analyses. NCM10
S (%)
NCM20
NCM30
PPT
SOB
TOT
PPT
SOB
TOT
PPT
SOB
TOT
PPT
SOB
0.67
0.42
0.65
0.79
0.26
0.42
0.66
1.06
1.15
0.41
1.43
1.56
NCM40TOT
were formed predominantly of cellulose type II, and the NCMTOT samples were a mixture of two cellulose types (I and II). In the latter case (NCMTOT ), the profile of the XRD diffractogram seems to match that of cellulose type I but, apparently, the presence of cellulose type II is masked by the presence of cellulose type I.
NCM30TOT
3.4. Atomic force microscopy (AFM)
Intensity (a.u)
(A)
NCM20TOT
NCM10TOT CMP
5
10
15
20
25
30
35
40
2θ (degree)
(B) NCM40SOB
Intensity (a.u)
NCM30SOB NCM20SOB
NCM10SOB
CMP
5
10
15
20
25
30
35
40
2θ (degree)
(C)
Intensity (a.u)
NCM40PPT
NCM30PPT
NCM20PPT
NCM10PPT CMP
5
NCM40
TOT
10
15
20
25
30
35
40
2θ (degree) Fig. 5. X-ray diffractograms of (a) NCMTOT and CMP; (b) NCMSOB and CMP; (c) NCMPPT and CMP.
AFM topography measurements were performed in order to precisely characterize the dimensions of the individual crystallites. Determining the exact dimensions of CNCs is complicated by the specific limitations of the different analytical methods used. In the case of AFM, tip/sample broadening represents the main limitation, resulting in an overestimation of CNCs dimensions. Since the CNCs are assumed to be cylindrical in shape, the height of the CNCs was taken to be equivalent to its diameter, to compensate for image widening due to convolution of the tip and the particle (BeckCandanedo et al., 2005; Kvien et al., 2005). Tip broadening effects still cause errors in length measurements, but this is unavoidable (Beck-Candanedo et al., 2005). Fig. 6 shows AFM images of NCMTOT , NCMPPT and NCMSOB obtained with 20 min, 30 min and 40 min hydrolysis times. For samples NCMTOT and NCMPPT , the AFM images presented needleshaped nanoparticles throughout, confirming that the extraction of CNCs from CMP was successful. The AFM images of samples NCMSOB also showed the presence of CNCs; however, the images predominantly show nanoparticles with a circular shape. Therefore, this behaviour is related to the structure of the CNCs and, as shown by the XRD patterns, the NCMPPT and NCMTOT samples presented cellulose I profiles for all hydrolysis times, whereas the XRD patterns for samples of NCMSOB presented cellulose II profiles. In a study carried out by Sèbe et al. (2012), it was also verified that the shape of nanocrystals is directly related to the type of polymorph of cellulose present (I or II). Values for the lengths, widths and aspect ratios obtained from several AFM images of NCMTOT , NCMPPT and NCMSOB are shown in Table 3. Increasing the extraction time resulted in slightly shorter lengths for NCMTOT and NCMPPT . This was expected, since longer extraction times partially destroyed areas with crystalline domains. According to Table 3 it can be seen that the increase in extraction time did not result in a change in the size of the NCMSOB , which was characteristic of cellulose type II. By studying Table 3 it is clear that increasing the hydrolysis time produced no significant difference in width of the NCMTOT , NCMPPT and NCMSOB when the standard deviation of each value was taken into account. Under the experimental conditions studied (NCMTOT , NCMPPT and NCMSOB ), no significant reductions in aspect ratio were observed with increasing hydrolysis time except in the case of the NCM40TOT sample. However, comparing each condition (NCMTOT , NCMPPT and NCMSOB ) at the same hydrolysis time, a variation in the aspect ratio can be observed, where the NCMPPT samples have the highest values while the NCMSOB samples have the lowest. The average aspect ratios for the CNCs found in this work are close to the values reported in the literature; therefore, these particles have great potential to be used as reinforcing agents in nanocomposites (Kalia et al., 2011; Silvério et al., 2013). The results of the morphological investigation by AFM are consistent with other reports in the literature where CNCs were extracted from different sources (Bai
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Fig. 6. AFM images of NCMTOT , NCMPPT and NCMSOB samples obtained after different hydrolysis times.
Table 3 Length, width and aspect ratio for NCMTOT , NCMPPT and NCMSOB samples. Hydrolysis time (min) 20 30 40
NCMTOT
NCMPPT
NCMSOB
Length (nm)
Width (nm)
Aspect ratio (nm) Length (nm)
Width (nm)
Aspect ratio (nm) Length (nm)
Width (nm)
Aspect ratio (nm)
301.50 ± 48.22 255.53 ± 50.98 181.38 ± 38.32
6.20 ± 1.77 5.63 ± 1.50 5.39 ± 1.74
48.67 ± 23.84 45.39 ± 14.60 28.06 ± 13.99
6.04 ± 1.48 4.94 ± 1.02 4.84 ± 1.12
52.34 ± 25.54 53.71 ± 14.90 49.35 ± 14.04
3.10 ± 0.58 3.09 ± 1.02 2.72 ± 0.40
23.68 ± 7.83 23.66 ± 7.32 26.86 ± 6.23
316.31 ± 59.28 265.17 ± 32.77 239.06 ± 59.10
73.39 ± 20.25 73.11 ± 8.46 73.16 ± 13.72
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et al., 2009; Beck-Candanedo et al., 2005; de Rodriguez et al., 2006; Elazzouzi-Hafraoui et al., 2008; Kvien et al., 2005; Rosa et al., 2010; Siqueira et al., 2010a; Teixeira et al., 2011).
Table 4 Onset temperature (Tonset ), degradation temperature at maximum weight-loss rate (Tmax ) and char yield for CMP and NCM samples obtained from TGA. Sample
3.5. Thermogravimetric analysis (TGA) The thermograms obtained for CMP and NCMTOT , NCMPPT and NCMSOB isolated with different hydrolysis times are shown in Fig. 7. The values obtained for onset temperatures (Tonset ), degradation temperatures at maximum weight loss rate (Tmax ) and char yields for all samples are shown in Table 4. For all samples the thermogram profiles exhibit essentially three events. The first event is related to the evaporation of adsorbed water or compounds of low molecular weight. This occurred between 30 and 150 ◦ C and there was even a small mass loss (<10%). The second event corresponds basically to the process of cellulose degradation, which consists of several concurrent processes: depolymerization, dehydration and decomposition of glycosidic units (Araki et al., 1998). In this step, the thermal degradation temperatures of all the NCM samples were lower than that of CMP. This behaviour was expected since some studies have shown that the introduction of sulphate groups decreases the thermal stability of CNCs due to the dehydration of the cellulose (Roman and Winter, 2004). The incorporation of sulphate groups on the surface of cellulose after hydrolysis has a catalytic effect in reactions of thermal degradation. Another effect that has been reported is the replacement of OH groups on the cellulose with sulphate groups, which leads to a decreased activation energy for the degradation of the cellulose chains (Teixeira et al., 2010). In this step large differences in initial degradation temperature were also observed for all NCM samples. Such behaviour could
CMP NCM10 TOT PPT SOB NCM20 TOT PPT SOB NCM30 TOT PPT SOB NCM40 TOT PPT SOB
Cellulose thermal degradation
Carbonic residue degradation
Tonset (◦ C)
Tmax (◦ C)
Tonset (◦ C)
Tmax (◦ C)
281
331
404
448
1.5
271 277 259
309 312 308
391 398 400
470 483 445
3 1.5 2
262 265 247
300 302 304
403 396 424
468 463 485
2.5 0.1 8
235 260 245
276 300 288
419 396 431
494 442 502
1.5 5 7
253 258 236
304 295 299
391 387 403
454 462 471
1.5 7 8
Char yield (%)
not be attributed solely to the presence of sulphate groups, since the results of the elemental analysis showed that the amount of sulphate present was virtually the same for all samples studied. As shown by the XRD diffractogram profiles, it is possible verify a predominance of cellulose type I in the NCMPPT samples, a predominance of cellulose type II in the NCMSOB samples, and a mixture of cellulose I and II in the NCMTOT samples. One may also point out that the degradation temperatures for NCMPPT (mainly cellulose I) are higher than the degradation temperatures for
Fig. 7. TGA curves of the CMP and NCMTOT , NCMPPT and NCMSOB obtained after different hydrolysis times.
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NCMSOB (mainly cellulose II). In addition, the degradation temperatures for NCMTOT (a mixture of cellulose I and II) are intermediate between those found for NCMPPT and NCMSOB . Thus, the differences in the degradation temperatures (Table 4) are directly linked to the type of material (cellulose type I or II) and not to the presence of sulphate groups. The third event is attributed to the oxidation and breakdown of charred residues to form gaseous products of low molecular weight (Roman and Winter, 2004; Teixeira et al., 2010). Some authors have related that the presence of sulphate groups is responsible for an increase in the charred residue, and attribute this fact to the flame retardant effect of this group (Roman and Winter, 2004). In this step, it was observed that the NCMSOB showed, in general, an increase in char residue as well as in the initial temperature of degradation (Table 4) when compared to CMP, NCMPPT and NCMTOT . It is also possible to verify that the NCMTOT again showed intermediate values when compared with the values of NCMSOB and NCMPPT samples. This increase in the char residue can be attributed mainly to the cellulose polymorph present in the NCM samples; in this case, attributed to the presence of cellulose type II. 4. Conclusions The present work shows that CNCs can be isolated from capim mombac¸a fibres. The conditions of hydrolysis using different methodologies were effective, resulting in stable aqueous suspensions of NCM that were negatively charged due to the presence of similar quantities of sulphate groups for all the studied conditions. Through X-ray diffraction it was possible to observe that cellulose nanocrystals formed with different polymorphs of cellulose (type I and II) as a consequence of the hydrolysis conditions used and due to reprecipitation of cellulose. It was observed that the different hydrolysis processes used resulted in nanocrystals containing different polymorphs of cellulose, which directly influenced the physical aspect of the resulting nanocrystals. NCM composed predominantly of cellulose I have needle-like structures; in contrast, NCM made predominantly of cellulose II have circular structures. It was found that the thermal stability of the NCM was dependent of the kind of cellulose polymorph present in the structure of the NCM. It was concluded that a reduction in the initial thermal degradation temperature and an increase in the char residue are associated with the presence of cellulose type II and not with the presence of sulphate groups. Acknowledgements The authors thank CAPES/PROAP, CNPq and FAPEMIG for financial support. References Alemdar, A., Sain, M., 2008. Isolation and characterization of nanofibers from agricultural residues – wheat straw and soy hulls. Bioresour. Technol. 99, 1664–1671. Angellier, H., Choisnard, L., Boisseau, S.M., Ozil, P., Dufresne, A., 2004. Optimization of the preparation of aqueous suspensions of waxy maize starch nanocrystals using a response surface methodology. Biomacromolecules 5, 1545–1551. Araki, J., Wada, M., Kuga, S., Okano, T., 1998. Flow properties of microcrystalline cellulose suspension prepared by acid treatment of native cellulose. Colloids Surf. A: Physicochem. Eng. Aspects 142, 75–82. Azeredo, H.M.C., Mattoso, L.H.C., Wood, D., Williams, T.G., Avena-Bustillos, R.J., McHugh, T.H., 2009. Nanocomposite edible films from mango puree reinforced with cellulose nanofibers. J. Food Sci. 74, N31–N35. Azizi Samir, M.A.S., Alloin, F., Dufresne, A., 2005. Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field. Biomacromolecules 6, 612–626. Bai, W., Holbery, J., Li, K.C., 2009. A technique for production of nanocrystalline cellulose with a narrow size distribution. Cellulose 16, 455–465. Beck-Candanedo, S., Roman, M., Gray, D.G., 2005. Effect of reaction conditions on the properties and behavior of wood cellulose nanocrystal suspensions. Biomacromolecules 6, 1048–1054.
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The influence of the cellulose hydrolysis process on the structure of cellulose nanocrystals extracted from capim mombac¸a (Panicum maximum) Douglas Ferreira Martins, Alexandre Bernaldino de Souza, Mariana Alves Henrique, Hudson Alves Silvério, Wilson Pires Flauzino Neto, Daniel Pasquini ∗ Instituto de Química, Universidade Federal de Uberlândia, Campus Santa Mônica, Av. João Naves de Ávila, 2121, 38400-902 Uberlândia, Minas Gerais, Brazil
a r t i c l e
i n f o
Article history: Received 9 August 2014 Received in revised form 10 October 2014 Accepted 18 October 2014 Available online xxx Keywords: Capim mombac¸a fibres Acid hydrolysis Cellulose nanocrystals Cellulose polymorphs Thermal stability
a b s t r a c t This work presents studies on the isolation of cellulose nanocrystals from cellulose extracted from capim mombac¸a (Panicum maximum) leaves through variations in the acid hydrolysis process, and their consequent impact on their chemical, physical and thermal properties. The hydrolysis was performed at 40 ◦ C for 10 min, 20 min, 30 min or 40 min under vigorous and constant stirring. For each gram of CM, 30 mL of H2 SO4 (11.22 M) was used. The variations occurred in the subsequent step, which consists of adding 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, Xray diffraction (XRD) and atomic force microscopy (AFM) in order to assess changes in the physical and chemical structure of the nanocrystals obtained and in their thermal stability. It was observed that the procedures adopted influenced the dimensions and physical aspect of the nanocrystals, the type of cellulose 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 polymorph of cellulose that predominated, and not with the presence of sulphate groups on the surface of the nanocrystals. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The application of cellulose nanocrystals (CNCs) as a nanosized reinforcement in polymer matrixes has attracted considerable attention, since it offers a unique combination of desirable physical properties and environmental benefits (Habibi et al., 2010; Peng et al., 2011). CNCs-based nanocomposites generally exhibit significant improvements in thermal, mechanical, and barrier properties compared to the neat polymer or conventional composites (Azeredo et al., 2009). CNCs are needle-shaped cellulose particles with at least one dimension equal to or less than 100 nm and have a highly crystalline nature (Flauzino Neto et al., 2012). The main features driving the development of CNCs as polymer reinforcement agents are their large specific surface area (estimated to be several hundreds of m2 g−1 ), their very high modulus of elasticity (approximately 150 GPa), their large aspect ratio, and their ability 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: [email protected] (D. Pasquini).
Other attractive advantages of CNCs are their low density (about 1.59 g cm−3 ) (O’Sullivan, 1997), nonabrasive nature, nontoxic character, biocompatibility, and biodegradability (Azizi Samir et al., 2005). Additionally, CNCs come from renewable natural sources that are very abundant and therefore low in cost, so it is not necessary to synthesize them, they allow the production of composite films with excellent visible light transmittance, and they can be easily modified chemically (their structure includes a reactive surface of OH side groups that facilitate grafting chemical species in 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 hydrolysis is the most well-known and widely used. This process is based on the fact that crystalline regions are acid-insoluble under the conditions used in the extractions (Peng et al., 2011). The morphology and properties of CNCs influence their performance as a reinforcing agent. Moreover, the morphology and properties of CNCs depend on 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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is relevant and, in the same way, the choice of hydrolysis conditions is very important too (Flauzino Neto et al., 2012). It is well known that the use of different acids can lead to differences in the stability of the nanocrystals in a colloidal suspension due to the presence of different charges on the surface of the nanoparticles (Angellier et al., 2004). In the case where sulphuric acid is employed to obtain CNCs, the presence of negative sulphate groups, introduced on the outer surface of the CNCs during the hydrolysis process, is responsible for the stabilization of the CNCs in the resulting colloidal suspension (Beck-Candanedo et al., 2005). However, some authors have reported that the presence of sulphate groups decreased the thermal stability of the CNCs (Roman and Winter, 2004). Usually, a higher acid sulphate group content in cellulose leads to a lower temperature of thermal degradation of the cellulose. If hydrochloric acid is used instead of sulphuric acid to hydrolyse the native cellulose, the thermal stability of the prepared nanocrystals is improved but the nanocrystals are inclined to aggregate due to the absence of a force of electrostatic repulsion between the crystal particles, resulting in an unstable colloidal suspension (Araki et al., 1998). One factor often overlooked by researchers is the method employed in the preparation of the nanocrystals, regardless of the type of acid used. Some authors add water directly to the acid solution containing the nanocrystals in order to stop the hydrolysis prior to centrifugation and dialysis (Haafiz et al., 2014; dos Santos et al., 2013; Silvério et al., 2013; Henrique et al., 2013; Flauzino Neto et al., 2012; Rosa et al., 2012; Belbekhouche et al., 2008; Teixeira et al., 2011). Moreover, other authors do not add water directly and just follow steps of centrifugation and dialysis after finalizing the hydrolysis time (Siqueira et al., 2013; Cao et al., 2013; de Menezes et al., 2009; Habibi et al., 2007; Samir et al., 2004). It is known that at high concentrations sulphuric acid acts as a solvent for cellulose hydrolysis (Xiang et al., 2003; Jayme and Lang, 1963). One hypothesis to be exploited in this work is that adding water to stop the hydrolysis process may induce the re-precipitation of a portion of the cellulose solubilized on the surface of CNCs, or even the formation of nanoparticles of regenerated cellulose. This precipitation and formation of structures containing cellulose type II may be the main cause of the change in thermal stability of the nanocrystals, and not the presence of sulphate groups as claimed by some authors (Roman and Winter, 2004; Araki et al., 1998). This study aimed to extract CNCs from capim mombac¸a (Panicum maximum) (CM) through experimental modifications, in order to investigate the presence of CNCs with structures made of cellulose I and II. Different techniques were employed to characterize the cellulose fibres and the resulting cellulose nanocrystals in order to investigate their chemical composition, crystallinity index, thermal stability and morphology (shape and size). 2. Experimental 2.1. Materials and methods Capim mombac¸a (P. maximum) (CM) leaves were supplied by São Mateus farm (Comendador Gomes, Minas Gerais, Brazil). The other reagents employed in this study were: sulphuric acid (95.0–98.0 wt.%, Vetec, P.A.), sodium hydroxide (Vetec), potassium hydroxide (Vetec), sodium chlorite (NaClO2 , technical grade, 80%, Sigma–Aldrich), glacial acetic acid (Synth), and cellulose membrane (D9402, Sigma–Aldrich). 2.2. Preparation of cellulose nanocrystals 2.2.1. Purification of capim mombac¸a (CM) Initially the untreated CM was milled with a blender to pass through a 14-mesh screen. After that, the CM was treated two times with a 2% (w/w) aqueous sodium hydroxide solution for 4 h
at 100 ◦ C under mechanical stirring and then washed several times with distilled water until the alkali was completely removed (until neutral pH), and finally dried at 40 ◦ C for 24 h in an air-circulating oven. After this, the fibres were bleached with a solution made up of equal parts (v:v) acetate buffer (27 g NaOH and 75 mL glacial acetic acid, diluted to 1 L in distilled water) and aqueous chlorite (1.7 wt% NaClO2 in water). This bleaching treatment was performed two times at 80 ◦ C for 6 h. The bleached fibres were then washed repeatedly in distilled water until the pH of the fibres became neutral and subsequently dried at 40 ◦ C for 24 h in an air-circulating oven (de Rodriguez et al., 2006; Siqueira et al., 2010a). The fibre content throughout these chemical treatments was about 4–6% (w/w). The material that resulted after the purification was the purified capim mombac¸a (CMP). 2.2.2. Extraction of cellulose nanocrystals After the chemical treatment was completed, the CMP was milled in a blender, passed through a 14-mesh screen and then used to extract nanocrystals by acid hydrolysis. The hydrolysis was performed at 40 ◦ C for 10 min, 20 min, 30 min or 40 min under vigorous and constant stirring. For each gram of CMP, we used 30 mL of H2 SO4 (11.22 M). After hydrolysis, two different procedures were employed. First procedure: The suspensions resulting from each time of hydrolysis employed were diluted 10-fold with cold water to stop the hydrolysis reaction and centrifuged for 10 min at 7500 rpm to remove the excess acid. The precipitate was then dialyzed with water to remove acid residue, non-reactive sulphate groups, salts and soluble sugars, until neutral pH (∼4 days) was attained. This procedure was called total hydrolysis (TOT) and the nanocrystals obtained from this procedure were named NCM10TOT , NCM20TOT , NCM30TOT and NCM40TOT . Second procedure: In this procedure, the suspensions resulting from each time of hydrolysis employed were directly centrifuged, thereby obtaining two separate fractions of material. One fraction contained the solid precipitated phase and the other the liquid supernatant phase. Next, these fractions were also diluted separately 10-fold with cold water to stop the hydrolysis reaction, centrifuged for 10 min at 7500 rpm to remove the excess acid, and subsequently dialyzed until neutral pH was achieved (∼4 days). The nanocrystals obtained from the solid precipitated (PPT) phase fractions were named NCM10PPT , NCM20PPT , NCM30PPT and NCM40PPT , and the nanocrystals obtained from the liquid supernatant (SOB) phase fractions were named NCM10SOB , NCM20SOB , NCM30SOB and NCM40SOB . Subsequently, all the suspensions (TOT, PPT and SOB) resulting from the dialysis process were ultrasonicated for 10 min and stored in a refrigerator at 4 ◦ C. Some drops of chloroform were added as a protectant to the cellulose nanocrystal suspensions. The cellulose nanocrystals of capim mombac¸a were labelled for each time of hydrolysis studied as NCMTOT , NCMPPT and NCMSOB . Fig. 1 shows the scheme of the experimental procedure employed to obtain the NCM from the CMP. 2.3. Characterizations and measurements 2.3.1. Gravimetric analysis The hydrolysis yield was calculated by drying an aliquot of known volume of the NCM suspension at 105 ◦ C for 12 h in an air-circulating oven. 2.3.2. Chemical composition The chemical compositions of CM and CMP were measured as follows: the lignin content was measured according to a standard method of the Technical Association of Pulp and Paper Industry TAPPI T13M-54; the holocellulose (␣-cellulose + hemicelluloses)
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Capim Mombaça Purified (CMP)
Hydrolysis (H2SO4 11.22 M, 40°C: 10, 20, 30 or 40min)
Suspension of NCM
First Procedure
Second Procedure
Diluted 10 times
Centrifugation
with cold water
Liquid Supernatant Fraction
Solid Precipitated Fraction
NCMTOT
Centrifugation
Diluted 10 times
Diluted 10 times
with cold water
with cold water
NCMPPT
NCMSOB
Centrifugation
Centrifugation
Dialysis until neutral pH
Ultrasonication 10min
NCMTOT; NCMPPT; NCMSOB Fig. 1. Scheme of the experimental procedure to obtain the NCM from the CMP.
content was estimated by the acid chlorite method (Browning, 1967); briefly, the ␣-cellulose content was determined by treating the holocellulose with 5 and 24% (w/w) potassium hydroxide solutions, and the hemicelluloses content was found by subtracting the ␣-cellulose portion from the total holocellulose content; the ash content was measured by considering the percentage difference between the initial weight of the dried fibre of the sample and that after calcination for 4 h at 800 ◦ C (Trindade et al., 2005). An average of three replicates was calculated for each sample. 2.3.3. Fourier transform infrared spectroscopy (FTIR) A Shimadzu IR Prestige-21 Infrared spectrophotometer was used to obtain spectra for the CM and CMP. The KBr disc (ultrathin pellet) method was used in taking the IR spectra. Samples were ground and mixed with KBr (sample/KBr ratio, 1/100) to prepare pastilles. The experiments were carried out in the range of 500–4000 cm−1 with a resolution of 4 cm−1 and a total of 32 scans for each sample.
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 Shimadzu LabX XRD-6000 diffractometer operating at 40 kV with a current of ˚ Before performing the XRD, 30 mA and Cu K␣ radiation (1.5406 A). 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 were determined by the Segal (1959) and Revol et al. (1987) methods, as shown 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 crystallinity, I0 0 2 and I1 1 0 are the maximum intensities of the 0 0 2 and 1 1 0 lattice diffractions at 2 = 23◦ and 20◦ , respectively, and IAM and I15◦ are the intensities of diffraction at 2 = 18◦ and 15◦ , respectively. I0 0 2 and I1 1 0 represent both crystalline and amorphous
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Fig. 2. Photographs of CM pasture (a); dried and milled CM leaves (b); CMP fibres obtained after the purification process (c).
regions, while IAM and I15◦ represent only the amorphous portion, respectively. 2.3.5. Atomic force microscopy (AFM) AFM measurements were performed with Shimadzu SPM-9600 equipment for evaluating the morphologies of all NCM samples obtained with hydrolysis times of 20 min, 30 min and 40 min. A drop of a diluted nanocrystal aqueous suspension was deposited onto a freshly cleaved mica surface and air-dried. AFM images were obtained at room temperature in the dynamic mode with a scan rate of 1 Hz and using Si tips with a curvature radius of less than 10 nm and a spring constant of 42 N m−1 . The dimensions of the nanocrystals were determined using Vector Scan software (the software for Shimadzu’s SPM-9600). To eliminate the effect of tip radius on width measurements, we measured the heights of the nanocrystals, which are not subject to peak broadening artefacts, and assumed the nanocrystals to be cylindrical in shape (BeckCandanedo et al., 2005). One hundred and twenty nanocrystals were randomly selected to determine the average length, width and aspect ratio (length/width). For each nanocrystal, one measure of length and two measures of diameter were performed and the aspect ratio calculated. 2.3.6. Thermal characterization The thermal stabilities of CMP and NCM samples were evaluated by thermogravimetic analysis (TGA) using Shimadzu DTG-60H
equipment. The analysis conditions were: a nitrogen atmosphere with a 30 mL min−1 flow, a heating rate of 10 ◦ C min−1 , a temperature range from 25 to 600 ◦ C, a sample mass between 5 and 7 mg and aluminium pans. 2.3.7. Elemental analysis Elemental analysis of all NCM samples was performed, mainly to determine the total sulphur content after the extraction of nanocrystals. This was carried out with an EA1110-CHNS/O elemental 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 milled CM leaves (b), and CMP fibres obtained after the purification process (c). The white colour of the purified fibres is also evidence that the removal of non-cellulosic components was successful. The cellulose content was 33.98 ± 3.6% and 51.97 ± 2.8%, hemicelluloses 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 constituents of CM are in accordance with the literature (Coan et al., 2005). After purification, the cellulose content increased, the hemicelluloses and ash contents decreased slightly and the lignin content
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Absorbance (a.u)
5
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1242
1734
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CMP Fig. 4. Resulting colloidal suspensions of NCMTOT samples obtained after hydrolysis times of 10 min, 20 min, 30 min and 40 min.
CM
4000
3500
3000
2500
2000
1500
1000
500
-1 Wavenumbers (cm ) Fig. 3. FTIR spectra of raw capim mombac¸a (CM) and purified capim mombac¸a (CMP). Table 1 The yield and crystallinity index (CrI) of different NCM samples. Hydrolysis time (min)
10 20 30 40
Yield (%)
CrI (%)
TOT
SOB
PPT
TOT
SOB
PPT
72.44 67.04 51.86 35.15
20.62 17.14 12.15 7.62
55.86 43.15 20.3 11.98
70.6 75.1 73.5 62.5
60.7 69.5 28.6 29.7
74.1 80.0 77.2 68.9
CMP: CrI = 77.4%.
was significantly reduced by 88.17%. The purification process was effective because CMP with a low lignin content is suitable for the extraction of cellulose nanocrystals (low lignin content and high cellulose content). The efficiency of the purification method was also analyzed by infrared spectroscopy. Fig. 3 shows FTIR spectra of CM and CMP. In the spectrum of CM, the band near 1734 cm−1 is assigned mainly to the C O stretching vibration of the carbonyl and acetyl groups in the xylan component of hemicelluloses and in the lignin. In the same spectrum the band near 1242 cm−1 corresponds to the axial asymmetric strain of C O C, which is commonly observed when C O are present, e.g. in ether, ester, and phenol groups (Siqueira et al., 2010b). These peaks almost disappeared in the spectra of CMP. The peaks at 1064 and 896 cm−1 are associated with cellulose, the C O stretching and C H rock vibrations of cellulose (Alemdar and Sain, 2008), which appeared in all of the spectra. The differences between the spectra indicate that the CMP sample had a higher cellulose content, suggesting that it is almost pure cellulose. 3.2. Extraction of cellulose nanocrystals and suspension analysis The yields of NCM obtained in the CMP hydrolysis processes are presented in Table 1. The results presented in Table 1 demonstrate, as expected, that for each procedure (TOT, PPT and SOB), increasing the reaction time produced a decrease in the yield values. It was observed that the TOT procedure, for a specific time of hydrolysis, produced a higher yield than the PPT and SOB procedures. It is also possible also verify that the sum of the yields from the PPT and SOB fractions are approximately equal to or less than the yield of the TOT fraction, indicating that the TOT fraction is the result of separate contributions from the two fractions. It was possible to obtain stable colloidal suspensions of NCM with all the procedures and times of hydrolysis used in this work.
Examples can be seen in Fig. 4, which shows the colloidal suspensions of NCM10TOT , NCM20TOT , NCM30TOT , and NCM40TOT . Sulphuric acid hydrolysis leads to homogeneous stable aqueous suspensions of cellulose nanocrystals which are negatively charged and, thus, do not tend to aggregate. During the hydrolysis process, esterification of surface hydroxyl groups in the cellulose takes place and, as a consequence, sulphate groups are introduced (BeckCandanedo et al., 2005; Lima and Borsali, 2004; Silva and D’Almeida, 2009). The results of elemental analyses allowed us to verify the presence of sulphate groups in all the NCM samples by determining the sulphur content. The results, presented in Table 2, confirm the incorporation of sulphate groups in all the NCM samples after treatment with sulphuric acid. 3.3. X-ray diffraction Diffraction patterns of the CMP and NCM samples are shown in Fig. 5. Based on the XRD diffractograms the CrI were calculated and the values are presented in Table 1. It is possible verify that the values of CrI obtained for NCMTOT and NCMPPT were similar to the CrI of CMP (77.4%). Additionally, the values of CrI for NCMSOB were much lower than those attributed to CMP, NCMTOT and NCMPPT . The reduction in the CrI observed for NCMSOB samples can be attributed to the dissolution of cellulose upon hydrolysis, which remained in the supernatant solution and was then reprecipitated with a low crystalline order. This can also be verified by its XRD pattern shown in Fig. 5(b) that shows the predominance of cellulose ¯ type 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 rich in non-hydrolysed solid particles and had few cellulose molecules solubilized during the hydrolysis; therefore, they have higher crystallinities because little cellulose re-precipitated on the surface of the nanocrystals. As a consequence, this sample is predominantly composed of nanocrystals that were not dissolved during hydrolysis, namely cellulose type I. This can be confirmed by the XRD patterns (Fig. 5(c)), where the pattern for NCMPPT is similar to that of CMP, with a profile of cellulose type I, due to the presence of ¯ 21◦ (plane 0 2 1) peaks at 2 = 15◦ (plane 1 0 1), 17◦ (plane 101), and 23◦ (plane 0 0 2). The XRD pattern of the NCMTOT samples (Fig. 5(a)), apparently presents the profile of cellulose type I, but it is possible to verify the presence of a shoulder around 2 = 22◦ attributed to cellulose type II, confirming the possible reprecipitation of solubilized cellulose molecules on the surface of the resulting nanocrystals (Sèbe et al., 2012; O’Sullivan, 1997). This is reflected in the CrI values of the NCMTOT samples (Table 1), which are lower than those obtained for the NCMPPT samples. Therefore, it can be concluded that the presence of cellulose type II is associated with recrystallization and reprecipitation after the cellulose hydrolysis, since, as we know, 11.22 M sulphuric acid can be a solvent for cellulose (Xiang et al., 2003; Jayme and Lang, 1963). In summary, we can conclude that the NCMPPT samples were formed predominantly of cellulose type I, the NCMSOB samples
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Table 2 Sulphur content of the NCM samples obtained by elemental analyses. NCM10
S (%)
NCM20
NCM30
PPT
SOB
TOT
PPT
SOB
TOT
PPT
SOB
TOT
PPT
SOB
0.67
0.42
0.65
0.79
0.26
0.42
0.66
1.06
1.15
0.41
1.43
1.56
NCM40TOT
were formed predominantly of cellulose type II, and the NCMTOT samples were a mixture of two cellulose types (I and II). In the latter case (NCMTOT ), the profile of the XRD diffractogram seems to match that of cellulose type I but, apparently, the presence of cellulose type II is masked by the presence of cellulose type I.
NCM30TOT
3.4. Atomic force microscopy (AFM)
Intensity (a.u)
(A)
NCM20TOT
NCM10TOT CMP
5
10
15
20
25
30
35
40
2θ (degree)
(B) NCM40SOB
Intensity (a.u)
NCM30SOB NCM20SOB
NCM10SOB
CMP
5
10
15
20
25
30
35
40
2θ (degree)
(C)
Intensity (a.u)
NCM40PPT
NCM30PPT
NCM20PPT
NCM10PPT CMP
5
NCM40
TOT
10
15
20
25
30
35
40
2θ (degree) Fig. 5. X-ray diffractograms of (a) NCMTOT and CMP; (b) NCMSOB and CMP; (c) NCMPPT and CMP.
AFM topography measurements were performed in order to precisely characterize the dimensions of the individual crystallites. Determining the exact dimensions of CNCs is complicated by the specific limitations of the different analytical methods used. In the case of AFM, tip/sample broadening represents the main limitation, resulting in an overestimation of CNCs dimensions. Since the CNCs are assumed to be cylindrical in shape, the height of the CNCs was taken to be equivalent to its diameter, to compensate for image widening due to convolution of the tip and the particle (BeckCandanedo et al., 2005; Kvien et al., 2005). Tip broadening effects still cause errors in length measurements, but this is unavoidable (Beck-Candanedo et al., 2005). Fig. 6 shows AFM images of NCMTOT , NCMPPT and NCMSOB obtained with 20 min, 30 min and 40 min hydrolysis times. For samples NCMTOT and NCMPPT , the AFM images presented needleshaped nanoparticles throughout, confirming that the extraction of CNCs from CMP was successful. The AFM images of samples NCMSOB also showed the presence of CNCs; however, the images predominantly show nanoparticles with a circular shape. Therefore, this behaviour is related to the structure of the CNCs and, as shown by the XRD patterns, the NCMPPT and NCMTOT samples presented cellulose I profiles for all hydrolysis times, whereas the XRD patterns for samples of NCMSOB presented cellulose II profiles. In a study carried out by Sèbe et al. (2012), it was also verified that the shape of nanocrystals is directly related to the type of polymorph of cellulose present (I or II). Values for the lengths, widths and aspect ratios obtained from several AFM images of NCMTOT , NCMPPT and NCMSOB are shown in Table 3. Increasing the extraction time resulted in slightly shorter lengths for NCMTOT and NCMPPT . This was expected, since longer extraction times partially destroyed areas with crystalline domains. According to Table 3 it can be seen that the increase in extraction time did not result in a change in the size of the NCMSOB , which was characteristic of cellulose type II. By studying Table 3 it is clear that increasing the hydrolysis time produced no significant difference in width of the NCMTOT , NCMPPT and NCMSOB when the standard deviation of each value was taken into account. Under the experimental conditions studied (NCMTOT , NCMPPT and NCMSOB ), no significant reductions in aspect ratio were observed with increasing hydrolysis time except in the case of the NCM40TOT sample. However, comparing each condition (NCMTOT , NCMPPT and NCMSOB ) at the same hydrolysis time, a variation in the aspect ratio can be observed, where the NCMPPT samples have the highest values while the NCMSOB samples have the lowest. The average aspect ratios for the CNCs found in this work are close to the values reported in the literature; therefore, these particles have great potential to be used as reinforcing agents in nanocomposites (Kalia et al., 2011; Silvério et al., 2013). The results of the morphological investigation by AFM are consistent with other reports in the literature where CNCs were extracted from different sources (Bai
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Fig. 6. AFM images of NCMTOT , NCMPPT and NCMSOB samples obtained after different hydrolysis times.
Table 3 Length, width and aspect ratio for NCMTOT , NCMPPT and NCMSOB samples. Hydrolysis time (min) 20 30 40
NCMTOT
NCMPPT
NCMSOB
Length (nm)
Width (nm)
Aspect ratio (nm) Length (nm)
Width (nm)
Aspect ratio (nm) Length (nm)
Width (nm)
Aspect ratio (nm)
301.50 ± 48.22 255.53 ± 50.98 181.38 ± 38.32
6.20 ± 1.77 5.63 ± 1.50 5.39 ± 1.74
48.67 ± 23.84 45.39 ± 14.60 28.06 ± 13.99
6.04 ± 1.48 4.94 ± 1.02 4.84 ± 1.12
52.34 ± 25.54 53.71 ± 14.90 49.35 ± 14.04
3.10 ± 0.58 3.09 ± 1.02 2.72 ± 0.40
23.68 ± 7.83 23.66 ± 7.32 26.86 ± 6.23
316.31 ± 59.28 265.17 ± 32.77 239.06 ± 59.10
73.39 ± 20.25 73.11 ± 8.46 73.16 ± 13.72
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et al., 2009; Beck-Candanedo et al., 2005; de Rodriguez et al., 2006; Elazzouzi-Hafraoui et al., 2008; Kvien et al., 2005; Rosa et al., 2010; Siqueira et al., 2010a; Teixeira et al., 2011).
Table 4 Onset temperature (Tonset ), degradation temperature at maximum weight-loss rate (Tmax ) and char yield for CMP and NCM samples obtained from TGA. Sample
3.5. Thermogravimetric analysis (TGA) The thermograms obtained for CMP and NCMTOT , NCMPPT and NCMSOB isolated with different hydrolysis times are shown in Fig. 7. The values obtained for onset temperatures (Tonset ), degradation temperatures at maximum weight loss rate (Tmax ) and char yields for all samples are shown in Table 4. For all samples the thermogram profiles exhibit essentially three events. The first event is related to the evaporation of adsorbed water or compounds of low molecular weight. This occurred between 30 and 150 ◦ C and there was even a small mass loss (<10%). The second event corresponds basically to the process of cellulose degradation, which consists of several concurrent processes: depolymerization, dehydration and decomposition of glycosidic units (Araki et al., 1998). In this step, the thermal degradation temperatures of all the NCM samples were lower than that of CMP. This behaviour was expected since some studies have shown that the introduction of sulphate groups decreases the thermal stability of CNCs due to the dehydration of the cellulose (Roman and Winter, 2004). The incorporation of sulphate groups on the surface of cellulose after hydrolysis has a catalytic effect in reactions of thermal degradation. Another effect that has been reported is the replacement of OH groups on the cellulose with sulphate groups, which leads to a decreased activation energy for the degradation of the cellulose chains (Teixeira et al., 2010). In this step large differences in initial degradation temperature were also observed for all NCM samples. Such behaviour could
CMP NCM10 TOT PPT SOB NCM20 TOT PPT SOB NCM30 TOT PPT SOB NCM40 TOT PPT SOB
Cellulose thermal degradation
Carbonic residue degradation
Tonset (◦ C)
Tmax (◦ C)
Tonset (◦ C)
Tmax (◦ C)
281
331
404
448
1.5
271 277 259
309 312 308
391 398 400
470 483 445
3 1.5 2
262 265 247
300 302 304
403 396 424
468 463 485
2.5 0.1 8
235 260 245
276 300 288
419 396 431
494 442 502
1.5 5 7
253 258 236
304 295 299
391 387 403
454 462 471
1.5 7 8
Char yield (%)
not be attributed solely to the presence of sulphate groups, since the results of the elemental analysis showed that the amount of sulphate present was virtually the same for all samples studied. As shown by the XRD diffractogram profiles, it is possible verify a predominance of cellulose type I in the NCMPPT samples, a predominance of cellulose type II in the NCMSOB samples, and a mixture of cellulose I and II in the NCMTOT samples. One may also point out that the degradation temperatures for NCMPPT (mainly cellulose I) are higher than the degradation temperatures for
Fig. 7. TGA curves of the CMP and NCMTOT , NCMPPT and NCMSOB obtained after different hydrolysis times.
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NCMSOB (mainly cellulose II). In addition, the degradation temperatures for NCMTOT (a mixture of cellulose I and II) are intermediate between those found for NCMPPT and NCMSOB . Thus, the differences in the degradation temperatures (Table 4) are directly linked to the type of material (cellulose type I or II) and not to the presence of sulphate groups. The third event is attributed to the oxidation and breakdown of charred residues to form gaseous products of low molecular weight (Roman and Winter, 2004; Teixeira et al., 2010). Some authors have related that the presence of sulphate groups is responsible for an increase in the charred residue, and attribute this fact to the flame retardant effect of this group (Roman and Winter, 2004). In this step, it was observed that the NCMSOB showed, in general, an increase in char residue as well as in the initial temperature of degradation (Table 4) when compared to CMP, NCMPPT and NCMTOT . It is also possible to verify that the NCMTOT again showed intermediate values when compared with the values of NCMSOB and NCMPPT samples. This increase in the char residue can be attributed mainly to the cellulose polymorph present in the NCM samples; in this case, attributed to the presence of cellulose type II. 4. Conclusions The present work shows that CNCs can be isolated from capim mombac¸a fibres. The conditions of hydrolysis using different methodologies were effective, resulting in stable aqueous suspensions of NCM that were negatively charged due to the presence of similar quantities of sulphate groups for all the studied conditions. Through X-ray diffraction it was possible to observe that cellulose nanocrystals formed with different polymorphs of cellulose (type I and II) as a consequence of the hydrolysis conditions used and due to reprecipitation of cellulose. It was observed that the different hydrolysis processes used resulted in nanocrystals containing different polymorphs of cellulose, which directly influenced the physical aspect of the resulting nanocrystals. NCM composed predominantly of cellulose I have needle-like structures; in contrast, NCM made predominantly of cellulose II have circular structures. It was found that the thermal stability of the NCM was dependent of the kind of cellulose polymorph present in the structure of the NCM. It was concluded that a reduction in the initial thermal degradation temperature and an increase in the char residue are associated with the presence of cellulose type II and not with the presence of sulphate groups. Acknowledgements The authors thank CAPES/PROAP, CNPq and FAPEMIG for financial support. References Alemdar, A., Sain, M., 2008. Isolation and characterization of nanofibers from agricultural residues – wheat straw and soy hulls. Bioresour. Technol. 99, 1664–1671. Angellier, H., Choisnard, L., Boisseau, S.M., Ozil, P., Dufresne, A., 2004. Optimization of the preparation of aqueous suspensions of waxy maize starch nanocrystals using a response surface methodology. Biomacromolecules 5, 1545–1551. Araki, J., Wada, M., Kuga, S., Okano, T., 1998. Flow properties of microcrystalline cellulose suspension prepared by acid treatment of native cellulose. Colloids Surf. A: Physicochem. Eng. Aspects 142, 75–82. Azeredo, H.M.C., Mattoso, L.H.C., Wood, D., Williams, T.G., Avena-Bustillos, R.J., McHugh, T.H., 2009. Nanocomposite edible films from mango puree reinforced with cellulose nanofibers. J. Food Sci. 74, N31–N35. Azizi Samir, M.A.S., Alloin, F., Dufresne, A., 2005. Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field. Biomacromolecules 6, 612–626. Bai, W., Holbery, J., Li, K.C., 2009. A technique for production of nanocrystalline cellulose with a narrow size distribution. Cellulose 16, 455–465. Beck-Candanedo, S., Roman, M., Gray, D.G., 2005. Effect of reaction conditions on the properties and behavior of wood cellulose nanocrystal suspensions. Biomacromolecules 6, 1048–1054.
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Please cite this article in press as: Martins, D.F., et al., The influence of the cellulose hydrolysis process on the structure 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
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Please cite this article in press as: Martins, D.F., et al., The influence of the cellulose hydrolysis process on the structure 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