Studies on cellulose nanocrystals isolated from groundnut shells

Carbohydrate Polymers 157 (2017) 1041–1049

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Studies on cellulose nanocrystals isolated from groundnut shells Saleheen Bano, Yuvraj Singh Negi ∗ Department of Polymer and Process Engineering, Indian Institute of Technology, Roorkee, Saharanpur Campus, Saharanpur 247001, India

a r t i c l e

i n f o

Article history: Received 9 August 2016 Received in revised form 29 September 2016 Accepted 22 October 2016 Available online 24 October 2016 Keywords: Groundnut shells Cellulose nanocrystals Static light scattering

a b s t r a c t Today, various renewable biomass resources are accepted as waste material and are mostly burnt or used as cattle feed. The commercial value of these wastes can be increased by utilising them in production of nanomaterials. So, the present work was conducted for isolation of cellulose nanocrystals (CNCs) from groundnut shells which are produced annually as waste in large quantity (∼7 million tons). The structural, thermal, morphological & elemental analyses were assessed through corresponding techniques. Light Scattering studies were performed to analyse more likely weight average molecular weight (Mw ) & radius of radius (Rg ). The high Mw ∼105 g/mol obtained for CNCs in lithium chloride (LiCl)/N,Ndimethylacetamide (DMAc) system, was an interesting feature which gets affected by LiCl and polymer concentrations. Solution with high polymer and low LiCl concentration was found to show higher values of Mw & Rg . © 2016 Elsevier Ltd. All rights reserved.

1. Introduction In recent years, significant effort is being given to the development of novel composite materials with potential technological applications and with improved biodegradability to mitigate environmental problems. Besides various types of synthetic fillers used, natural fibres find their place as reinforcement in composite materials. Being more economic, renewable, non-toxic, easily processable and biodegradable along with excellent mechanical properties they are gradually replacing the traditional mineral fillers (Reddy & Yang, 2009). Among natural fibres, cellulose fibres are found to be the most abundant biopolymer on earth. In last few decades, cellulose has given more importance in the field of nanotechnology due to its hierarchical structure which allows producing particles with nanometric dimensions. Among various methods applied for nanocellulose production, the widely used process is acid hydrolysis. This treatment helps in the digestion of less ordered amorphous region of cellulose micro fibrils leaving the nano crystalline particles. In the literature, these nano crystalline cellulose particles are termed in number of ways including cellulose nanocrystals (CNCs) and whiskers (Fan & Li, 2012; Habibi & Dufresne, 2008). Now-a-days, CNCs become apple of eye for the researchers and scientists in the world of material science due to their high aspect

∗ Corresponding author. E-mail address: [email protected] (Y.S. Negi). http://dx.doi.org/10.1016/j.carbpol.2016.10.069 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

ratio, high surface area, excellent modulus and tensile strength which were reported to be 150 & 10 GPa respectively (Sturcova, Davies, & Eichhorn, 2005). Besides these properties, their excellent chemical stability, biocompatibility, renewability, non-toxicity, large abundance of economic sources, low gas permeability and having surfaces for the functionality, make them a great treasure for variety of applications such as polymer nanocomposites, packaging, lithium ion batteries, drug delivery, tissue engineering, protective coatings, etc. (Moon, Martini, Nairn, Simonsen, & Youngblood, 2011). The performance of CNCs as reinforcement is effected by their structural properties which mainly depend on hydrolysis condition and the source of cellulose used for their extraction (Flauzino Neto, Silverio, Dantas, & Pasquini, 2013). Research has been growing rapidly on the use of agricultural wastes, as more economic, abundant and easily available cellulosic source, since last decades. They are produced in large quantity worldwide and a large part of these wastes are utilised as fuel. Volarisation of these agro-wastes for isolation of value added products enables a possible commercial application of these materials. Ground nut (Arachis hypogea) is a crop of global importance belongs to Fabaceae family. Its world-wide production is more than 10 million tonnes per year. India is the second largest producer of ground nut in the world. The groundnut shells are produced as waste from groundnut processing factories which are generally utilised as fuel and cattle feed (Raju & Kumarappa, 2011). The chemical composition of ground nut shells consisting of 35.7% cellulose (Raveendran, Ganesh, & Khilar, 1995), attracting it as cellulosic

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source for nanocrystals production and added a commercial value in waste management. Various studies were reported on CNCs. However the molecular weight which is an important parameter of polymeric materials in relation to their physical and chemical properties also needs to be discussed. Static light scattering (SLS) studies have been made in number of ways on the solution behaviour of cellulose samples including molecular weight determination, with LiCl/DMAc system. As a non-derivatising & non-degrading solvent, LiCl/DMAc has potential applications in cellulose chemistry ranging from analytical studies to the preparation of certain derivatives in organic synthesis (Dupont & Harrison, 2004). The formation of super or supramolecular structures such as smaller and larger aggregates have been reported in several studies (Potthast, Rosenau, Buchner, & Röder, 2002; Sjoholm, Gustafsson, Eriksson, Brown, & Colmsjo, 2000) which might responsible for higher molecular weight of different cellulose samples in LiCl/DMAc system (Röder, Morgenstern, Schelosky, & Glatter, 2001). Studies on CNCs in LiCl/DMAc solution for molecular weight determination through SLS have not yet been reported. So we made an attempt for the first time to evaluate the properties obtained from SLS studies, more likely Mw of CNCs extracted from waste groundnut shells, through direct dissolution in LiCl/DMAc system. The CNCs were also evaluated in other aspects through different characterisation techniques.

100 ◦ C and stored in air tight polybags. The final dried product is considered as chemically purified cellulose (CPC-GNS). The percentage yield of CPC-GNS was determined gravimetrically and found to be 43%.

2. Experimental

2.2.2. Isolation of cellulose nanocrystals The cellulose nanocrystals were isolated from the extracted cellulose by acid hydrolysis method as follows: For hydrolysis process, a certain amount of cellulose was treated with 65 wt% sulphuric acid (having cellulose to acid ratio 1:20) for 75 min at 45 ◦ C under vigorous stirring at 1500 rpm using mechanical stirrer. The reaction was quenched by pouring the hydrolysed cellulose in copious volume of chilled distilled water. The aqueous suspension of the hydrolysed product was centrifuged at 10,000 rpm for 10 min to remove excess acid. The aliquot obtained after centrifugation was dialysed against distilled water for several days till neutrality followed by sonication for 30 min in ice bath. The aqueous suspension thus obtained was freeze-dried to obtain cellulose nanocrystals (CNC-GNS) powder. For freeze drying, the suspension was first frozen at −40 ◦ C and then transferred to freeze dryer and dried for 72 h. The dried CNCs were stored in vacuum for further use. A percentage yield of 12% with respect to starting material was determined by gravimetric method for CNC–GNS using freeze dried samples. It was 31.3% with respect to cellulose. The characteristics values for CPC-GNS and CNC-GNS measured viscosimetrically are summarised in Supplementary materials Table S1.

2.1. Materials

2.3. Characterisations

The ground nut shells were obtained from the local supplier of the area. Sodium chlorite, sodium hydroxide and glacial acetic acid were purchased from Hi Media, India. Sulphuric acid (98%) and N,Ndimethyl acetamide (DMAc) were obtained from Merck, India. The anhydrous lithium chloride (LiCl) was procured from SRL, India.

2.3.1. Chemical composition The contents of lignin, holocellulose (cellulose + hemicellulose) and ␣-cellulose present in GNS and CPC-GNS were calculated following Technical association of Pulp and paper Industry (TAPPI) standards: T222 om-88, T19m-54 and T203 OS-74 respectively. The difference between holocellulose and ␣-cellulose determines the value of hemicellulose content. All the experiments were performed thrice and average values were reported for different contents.

2.2. Preparation of cellulose nanocrystals This process consists of following two steps and shown in Fig. 1. 2.2.1. Separation of chemically purified cellulose from ground nut shells Before chemical treatment the obtained groundnut shells (GNS) were cleaned by washing with water for several times and then dried. Dried & cleaned shells thence milled in a laboratory mill and were sieved over 40–80 mess screen to maintain the size uniformity of powdered shells. These powdered shells were finally subjected to chemical treatment for purification and isolation of cellulose as per previous reports (Kumar, Negi, Choudhary, & Bhardwaj, 2014 ; Rhim, Reddy, & Luo, 2015). The dried and powdered shells were put under soxhlet extraction for 8 h using benzene: methanol (2:1 ratio) as solvent. This process helped in dewaxing of raw material. The de waxed shells were subsequently bleached to remove lignin, by treating at 70 ◦ C for 2 h with 1.5% (w/v) sodium chlorite solution having pH 3–4 adjusted by 5% glacial acetic acid. The material was filtered after 2 h and washed with distilled water, this process of bleaching was repeated for 5–6 times untill white product obtained. The obtained product was washed with distilled water upto neutrality. The resulting material is holocellulose composed of hemicelluloses and cellulose. The holocellulose obtained was treated with 1 M NaOH solution at 65 ◦ C for 2 h to remove hemicelluloses. Finally, the solubilised hemicelluloses were filtered off leaving the insoluble content which was thoroughly washed with distilled water till neutrality. The extracted product obtained was dried for 24 h at

2.3.2. Fourier transformed infrared (FTIR) spectroscopy FTIR spectroscopic study was made to study the structural properties of isolated materials. The spectra were recorded in Transmittance mode using Perkin Elmer spectrophotometer with 16 scans and at resolution of 4 cm−1 over 4000–400 cm−1 . To obtain the spectra, thin transparent pellets were prepared by grinding and mixing the dried samples with KBr in 1:100(w/w) ratio followed by pressing under vacuum. 2.3.3. Wide-angle X- ray diffraction (WAXD) The XRD pattern were collected over 2␪ = 5–60◦ at scan rate of 4◦ /min, for evaluation of crystalline phases of samples, by using RIGAKU ULTIMA IV X ray diffractometer operating at 40 kV & 30 mA equipped with Nickel filtered CuK␣ radiation source (␭ = 1.5406 Å). The crystallinity percentage was calculated by amorphous subtraction method using following equation: XC = (AC /AC + Am ) × 100 Where XC is percentage crystallinity, AC is area under crystalline region and Am is area of amorphous region. The average crystallite thickness of cellulose I structure was determined using Sherrer’s equation: Crystalthickness(t) = K␭/␤Cos␪

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Fig. 1. Schematic representation of (A) Separation of CPC–GNS and (B) Isolation of CNC-GNS.

Where ␭ is radiation wavelength (=1.5406 Å), K is correction factor having value of 0.91, ␪ and ␤ are diffraction angle and full width at half maximum of the peak angle corresponding to (002) crystalline plane respectively. 2.3.4. Thermo gravimetric analysis (TGA) A Netzsch TG 209 F3 Thermogravimetric Analyser was used to study the thermal stability of samples. The thermograms were recorded for approximately 5 mg of each sample from 30 ◦ C to 600 ◦ C at 10 ◦ C/min in nitrogen atmosphere with 60 ml/min gas flow rate. 2.3.5. Differential Scanning Calorimetry (DSC) DSC measurements were made to study the pyrolysis behaviour of cellulosic samples by using Netzsch DSC 200 F3 Differential Scanning Calorimeter. The data was obtained by heating approximately 5 mg of samples from 30 ◦ C to 500 ◦ C at heating rate of 10 ◦ C/min under nitrogen atmosphere with gas flow rate of 60 ml/min. 2.3.6. Hot stage microscopy (HSM) The hot stage microscope was performed by Leica DM2500 optical microscope equipped with LTS 420E Hot Stage. It helps to study the morphological changes that take place with temperature. The freeze dried sample of CNC-GNS was used for the study

and the examination was done from 30 ◦ C to 400 ◦ C at heating rate of 10 ◦ C/min. 2.3.7. Particle size analysis The hydrodynamic size of the cellulose nanocrystals were measured using Zeta PALS Brookhaven instruments at a fixed scattering angle of 90◦ and at wavelength of 659 nm. The aqueous suspension of cellulose nanocrystals (0.005 wt%) were prepared using freeze dried samples. The samples were filtered using 0.2 ␮ Teflon membrane filter before measurement. The results were averaged over three measurements cycles of 120 s each at 25 ◦ C. 2.3.8. Zeta potential The zeta potential of CNC-GNS in aqueous suspension was measured using Zeta PALS, Zeta Potential Analyser, (Brookheaven instruments), working on electrophoretic mobility. The measurements were carried out on samples prepared for DLS at 25 ◦ C temperature using wavelength of 659 nm. 2.3.9. Field Emission Scanning Electron Microscopy (FE-SEM) A TESCAN MIRA 3 Field Emission Scanning Electron Microscope was used to analyse the surface morphology of samples at different stages of chemical treatment and the microstructure of cellulose nanocrystals, at accelerating voltage from 10 to 20 kV. For examination, the dried samples were mounted on aluminium stubs with

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Table 1 Chemical composition of GNS and CPC-GNS.

3.2. Structural and crystalline analysis of extracted cellulose and cellulose nanocrystals

Contents

GNS

CPC-GNS

Cellulose (wt%) Hemicellulose (wt%) Lignin (wt%)

38.31 27.62 21.10

82.7 6.05 0.34

carbon tapes while the dilute suspension of nanocrystals were first spread on a glass slide followed by air drying and then mounted on the stubs. Before analysis, the samples were sputtered with gold using vacuum sputter coater. 2.3.10. Energy dispersive X-Ray (EDX) spectroscopy The sulphur content of CNC-GNS was determined by elemental analysis using EDX attached with FESEM unit. 2.3.11. Atomic Force Microscopy (AFM) The AFM imaging of CNC-GNS was done by using BRUKER NANOCOPE 5 Atomic Force Microscope. Before imaging, few drops of diluted aqueous suspension were deposited on a glass slide and allowed to dry. The samples were scanned in tapping mode, using standard silicon probes, at ambient temperature. 2.3.12. Transmission Electron Microscopy (TEM) The morphology and dimensions of CNC-GNS were assessed using a FEI TECNAI G2 S-twin Transmission Electron Microscope operating at 200 kV. The samples for TEM observation were prepared by depositing a drop of diluted aqueous suspension of CNCs on copper grid (400mess) and allowed to dry. The grid was then inserted in TEM instrument to capture the images using CCD camera. The length and width of cellulose nanocrystals were determined using image J software. 2.3.13. Static light scattering (SLS) SLS studies were made in the batch mode using Brookheaven instruments, BI-200SM goniometer, which measure the intensity of scattered light at scattering angles from 30 to 135◦ in 15 increments. The solutions of CNCS were prepared in LiCl/DMAc system. Prior to measurement the solutions were filtered using 0.2 ␮ PTFE filter into clean dried sample cells. All measurements were carried out at room temperature and at wavelength of 637 nm. The molecular parameters i.e. Mw , Rg and second virial coefficient (A2 ) were obtained from Zimm Plot using inbuilt Zimm software. 3. Results and discussion 3.1. Chemical composition and purification The chemical compositions of GNS and CPC-GNS were summarized in Table 1. The data shows that GNS contains 38.31 wt% cellulose, 27.62 wt% Lignin, and 21.10 wt% hemicellulose. The compositional value obtained are in good agreement with the data published by Raveendran et al. (1995), for the ground nut shells consisting of 35.7 wt% cellulose, 18.7 wt% hemicellulose, 30.2 wt% lignin. The presence of 38.31 wt% cellulose suggests that the groundnut shells can be exploited as potential source of cellulose and thence nanocellulose. The amount of hemicellulose and lignin was lower while cellulose content was higher in CPC-GNS compared to GNS (Table 1). This indicates significant removal of lignin and other components during purification. The CPC-GNS with negligible amount of lignin and 82% cellulose is suitable for extraction of CNCs.

The efficiency of purification can be analysed further by FTIR and XRD studies along with structural and crystalline nature of CPCGNS and CNC-GNS. The FTIR spectrum shown in Fig. 2A reveals the structural changes occurring during extraction. The spectral bands in GNS at 1735 cm−1 , 1512 cm−1 and 1264 cm−1 , gradually disappeared at different stages of extraction. These bands are results of characteristic C O stretching frequency of ester group and stretching vibrations of aromatic rings present in lignin, hemicelluloses and other components (Rosa et al., 2010; Viera et al., 2007). Absence of these bands in the spectrum of CPC-GNS confirms the removal of most of the lignin and other polysaccharide components. The CPC-GNS has characteristic absorption bands for cellulose I at 1432 cm−1 , 1060 cm−1 and 897 cm−1 which are results of –CH2 –(C6 )– bending, C O stretching of pyranose ring and bending vibration of ␤-glycosidic linkages respectively, including the bands at 1640 cm−1 , 3410 cm−1 and 2900 cm−1 assigned for H O H bending vibration of absorbed water, stretching frequency of hydrogen bonded OH groups present in the cellulose molecule and C H symmetric stretching frequency respectively (Le Troedec et al., 2008; Lu & Hsieh, 2012). The sharp and intense band at 3410 cm−1 for CNC-GNS compared to GNS again reveals complete removal of non-cellulosic components which are strongly associated with cellulose fibrils in GNS causing disruption of hydrogen bonding network of cellulosic chain and reflects the broadening of hydroxyl band around 3400 cm−1 compared to CNC-GNS. The XRD studies were relevant to FTIR studies as the diffraction pattern obtained showed three distinct peaks at 2␪ = 16.4◦ , 22.5◦ and 34.4◦ (Fig. 2B) which are characteristic peaks for cellulose I crystals assigned for 110, 200 and 004 planes respectively (Kargarzadeh et al., 2012; Wada, Heux, & Sugiyama, 2004). The percentage crystallinity (Xc ) for CPC-GNS (68%) was comparatively higher than GNS (56%), again revealing that lignin and hemicelluloses, responsible to enhance amorphicity in GNS, get removed. The FTIR and XRD analysis assures that the cellulosic nature was well retained in CNC-GNS (Fig. 2A and B).The highest value of Xc for CNC-GNS (74%), compared to GNS and CPC-GNS may be attributed to combined effect of complete removal of lignin and other components and the dissolution of amorphous region (caused by chain dislocation in the cellulosic structure) during acid hydrolysis leaving the well-defined crystalline domains. The crystal size or thickness of CNC-GNS with respect to (200) plane was found to be 4.2 nm and the data get supported by TEM which was made in further studies. Analysis of some of the important FTIR bands at 1432 cm−1 , 897 cm−1 , and 1372 cm−1 including hydrogen bonding pattern in cellulose were found to explore the crystalline structure in cellulosic materials and can be corroborated with XRD studies. The increase in the intensity of crystalline band at 1432 cm−1 reflects the increase in crystallinity degree and decrease in disordered region. As mentioned earlier (Ciolacu, Kovac, & Kokol, 2010; Kumar et al., 2014; Nelson & O’ Connor, 1964), the absorbance ratio of FTIR bands i.e. A1432/897 (CI1 ) and A1372/2900 (CI2 ), determine the crystallinity index which helps to reveal the orderly structure and crystallinity in cellulose samples. The crystallinity index increased from GNS to CNC-GNS (Table 2) which is in correlation with increase in crystallinity degree obtained from XRD analysis. The broad band from 3000 to 3600 cm−1 due to OH group gives considerable information related to hydrogen boding. The intra molecular hydrogen bonding of types O(2)H· · ·O(6) and O(3)H· · ·O(5) and intermolecular hydrogen bond of O(6)H· · ·O(3) play important role to determine the properties of cellulosic mate-

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Fig. 2. (A) FTIR spectra & (B) XRD pattern of (a) GNS (b) CPC-GNS and (c) CNC-GNS.

Table 2 Absorption bands & energies related to intra and inter molecular hydrogen bonds and crystallinity index for GNS, CPC-GNS and CNC-GNS. Sample

GNS CPC-GNS CNC-GNS

Intermolecular hydrogen bond of O(6)H· · ·O(3)

Intra molecular hydrogen bond of O(3)H· · ·O(5)

Intra molecular hydrogen bond of O(2)H· · ·O(6)

Crystallinity index

Absorption frequency (cm−1 )

Binding Energy (EH ) (kcal)

Absorption frequency (cm−1 )

Binding Energy (EH ) (kcal)

Absorption frequency (cm−1 )

Binding Energy (EH ) (kcal)

CI1

CI2

3231 3223 3216

6.1 6.23 6.34

3382 3367 3360

3.65 3.85 3.96

3525 3506 3487

1.24 1.65 1.86

1.52 1.67 3.4

0.34 0.36 0.74

rials. In order to describe the crystalline nature of these materials, energies of the concerned hydrogen bonds were calculated for GNS, CPC-GNS and CNC-GNS using the following relation (Ciolacu et al., 2010): EH = (1/k) ∗ [0 − /0 ] Where 0 = standard frequency for free OH groups (3600 cm−1 ),  = frequency of bonded OH groups and k = 1.68 × 10−2 /kcal. The increase in bond energy from GNS to CNC-GNS (Table 2) indicates increment in the structural regularity of ordered region of cellulose by increase in hydrogen bonding and consequently reveals the increase in crystallinity of samples as obtained from XRD studies and also explains the increase in the sharpness and intensity of OH peak in CNC compared to starting material. The hydrogen bonded OH stretching vibration between 3000 and 3600 cm−1 was resolved into three bands related to concerned intra and inter-molecular hydrogen bonds by deconvolution using Gaussian function (fig. 3). The related absorption bands are shown in Table 2. 3.3. Thermal analysis The thermal profile obtained for GNS, CPC-GNS and CNC-GNS through thermogravimetric analysis (TGA) measurement showed three step degradation in all cases (Fig. 4A). According to previous studies (Araki, Wada, Kuga, & Okano, 1998), the initial weight loss below 150 ◦ C is mainly related to removal of absorbed water, while the second step ranging from 200 ◦ C to 380 ◦ C basically corresponds to cellulosic chain degradation consisting of several steps such as depolymerisation, dehydration and decomposition of glycosidic units. The decomposition of carbonic residue into low molecular weight components above 380 ◦ C is responsible for third step of degradation. The corresponding data for samples in each degradation step are summarized in Table 3. As per the data obtained, the lower thermal stability of GNS than CPC-GNS can be ascribed to the presence of non-cellulosic components with lower degradation temperatures (Yang, Yan, Chen, Lee, & Zheng, 2007).

An ill-defined hub was observed in the DTG curve of GNS (Fig. 4A) at 290 ◦ C corresponding to hemicelluloses decomposition (Moran, Alvarez, Cyras, & Varzquez, 2008) along with main cellulosic chain degradation. Moreover, the presence of non-cellulosic contents could stimulate higher char formation in GNS compared to CPC-GNS. The higher thermal stability, absence of hub in the DTG curve and lower char residue for CPC-GNS than GNS, confirms the gradual removal of non-cellulosic components during chemical treatment. These findings were found to be consistent with FTIR and XRD studies. CNC-GNS showed considerable difference from CPC-GNS in the decomposition behaviour. The degradation was found to take place at lower temperature than that of CPC-GNS. The lower thermal stability is more likely due to the combined effect of (1) high surface area of nanocellulose crystals which lead to more exposure of surface to heat and (2) the incorporation of sulphate groups on CNC surface, which have catalytic effect leading to the reduction in activation energy of cellulose chain degradation (de Morais Teixeira et al., 2010). Moreover, the flame retardant behaviour of sulphate groups (Roman & Winter, 2004) and highly crystalline nature of CNCs may lead to induce higher char residue in CNC-GNS (George, Ramana, & Bawa, 2011). The DSC study showed an endotherm below 150 ◦ C in both CPC & CNC-GNS thermo grams (Fig. 4B) which is attributed to water loss while second endothermic transition, occurring in the region of 200 ◦ C–400 ◦ C, is indicative of fusion of crystallites showing the typical nature of cellulose decomposition in this range (Rosa, Rehman, De Miranda, Nachtigall, & Bica, 2012). The significant difference in thermal behaviour of CNC-GNS from CPC-GNS was also manifested from DSC studies. In case of CPCGNS a broad endotherm from 280 ◦ C to 330 ◦ C was observed while CNC-GNS showed a sharp and narrow endotherm, starts around 210 ◦ C with a peak at 265 ◦ C. An exothermic transition near 385 ◦ C and 340 ◦ C can be detected in CPC & CNC GNS respectively. The onset of this event was found to be overlapped with the end of endothermic region and can be related to the depolymerisation of cellulose chain in both cases. These observations were relevant to TGA analysis.

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Fig. 3. Deconvoluted FTIR in the range from 3000 cm−1 to 3800 cm−1 for (A) GNS (B) CPC-GNS and (C) CNC-GNS.

Fig. 4. (A) TGA curves of (a) GNS (b) CPC-GNS & (c) CNC-GNS and (B) DSC curves of (a) CPC-GNS & (b) CNC-GNS.

Table 3 Onset temperature (Tonset ), degradation temperature at max weight loss (Tmax ) and char yield for GNS, CPC-GNS & CNC-GNS evaluated from TG and DTG curves. Sample

GNS CPC-GNS CNC-GNS

Step I (Evaporation of water)

Step II (Degradation of Cellulose chain)

Step III (degradation of Carbonic residue)

Tonset (◦ C)

Tmax (◦ C)

Tonset (◦ C)

Tmax (◦ C)

Tonset (◦ C)

Tmax (◦ C)

30 30 30

66 53 49

238 253 211

322 330 262

351 382 337

471 516 435

The difference in the thermal behaviour of CNC showed the involvement of different decomposition gasification process than CPC-GNS which undergoes pyrolysis above 300 ◦ C by transglycosylation process as described by Mamleev, Bourbigot, and Yvon, 2007. Considering literature, as reported by (Lu & Hsieh, 2010; Wang, Ding, & Cheng, 2007), the sulphate groups present on CNCs, catalyse the direct solid to gas phase transitions in nanocellulose crystals resulting in subtle decomposition before primary cellulose pyrolysis. Also, the sulfation process remarkably changes the crystalline structure and particle size, lead-

Char yield (%) at 600(◦ C)

7.31 2.54 12.15

ing to earlier onset of fusion in CNC-GNS than CPC-GNS (Mandal & Chakrabarty, 2011).

3.4. Analysis of sulphate group on cellulose nanocrystals surfaces The presence of sulphate group on the surface of CNC-GNS was confirmed by FTIR and EDX. The small peak appeared at 1205 cm−1 in FTIR spectrum of CNC-GNS was consequence of S O vibration present in sulphate group (Fig. 2A) as reported earlier (Lu & Hsieh, 2010; Silverio, Flauzino Neto, Dantas, & Pasquini, 2013). The EDX spectrum contains peaks of Carbon, Oxygen and Sulphur corresponding to their bonding energies (Supplementary

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materials Fig. S1). The result accounted for 0.65 atomic% sulphur as elemental impurity along with carbon and oxygen as main component of CNCs (Supplementary materials Table S2). This sulphur impurity in CNC-GNS is more likely originates from negatively charged sulphate groups introduce on CNC-GNS surface after acid hydrolysis as described earlier (Man et al., 2011). Being negatively charged, the sulphate groups present on CNC surfaces induce electrostatic repulsion and help to improve the separation of CNCs from each other, leading to homogeneous and stable aqueous suspension of CNCs (Beck-Candanedo, Roman, & Gray, 2005). 3.5. Zeta potential Zeta potential is an important parameter to explore the dispersion stability of CNCs in aqueous suspension which is explained by effective electrostatic repulsion between the charged species present on particles, restricting them to coagulate or flocculate. In this study, the average value of zeta potential obtained for CNC-GNS suspension in neutral water was −22.5 mV. A negative value of zeta potential insures the presence of negatively charged groups. This result shows that the CNC-GNS suspension has good stability as the value obtained is more than −15 mV which is minimum value to represent the onset of agglomeration (Zhou, Fu, Zheng, & Zhan, 2012). 3.6. Morphological analysis 3.6.1. Electron & Atomic Force Microscopic studies By FESEM it was possible to investigate the morphological changes that take place during different stages of purification by chemical treatments. According to the studies made by Rosa et al., 2010, FESEM micrographs (Fig. 5), confirm that the lignin, hemicelluloses and other non-cellulosic components act as cementing agent around the cellulose fibrils and help in the formation of compact structure in GNS (supplementary Fig.S3(a)). The removal of these components, at different stages of purification, results in defibrillation and individualisation of cellulose microfibers (Fig. 5a and b). Undoubtly, the diameter of CPC-GNS fibres reduced to large extent compared to others (Fig. 5b) which indicates that almost all components, responsible for binding in GNS were removed efficiently thus enabling the separation of cellulose fibres into individual form. Each CPC-GNS micro fibril in turn is considered as a bundle of elementary nano fibrils consisting of amorphous and crystalline regions. Upon acid hydrolysis the amorphous region get cleaved leaving the crystalline domains unaffected. This treatment results in defibrillation of fibres on nano scale level. These features are shown in micrograph of CNC-GNS having rod shaped morphology (Fig. 5c). The TEM & AFM analysis was made to precisely confirm the rod shaped structures of CNC-GNS as shown in Supplementary materials Fig. (S3(b) & S3(c)). The TEM micrograph of CNC-GNS shows that the nano-cellulose crystals are well separated with length varying from 67 to 172 nm and width from 5 to 18 nm. Based on 100 samples of nanocrystals, the average value of length and thickness was 111 nm and 9 nm respectively. The average aspect ratio was near 12 which is comparable to the values reported in literature for nanocellulose extracted from different sources such as bagasse pulp, kenaf fibers and rice husk (Bras et al., 2010; Johar, Ahmad, & Dufresne, 2012; Kargarzadeh et al., 2012). This indicates potential application of CNC-GNS as reinforcement in composite materials. The AFM topography in height mode shows aggregated CNCs revealing the presence of strong intermolecular hydrogen bonding between them.

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Table 4 SLS analysis parameters for Mw , Rg and A2 of CNC-GNS. Stock solutionsa

Mw (g/mol)

8S-0.5C 8S-0.25C 8S-0.125C 8S-0.0625C 6S-0.5C 6S-0.25C 6S-0.125C 6S-0.0625C 4S-0.5C 4S-0.25C 4S-0.125C 4S-0.0625C

(6.78 ± 0.72) × 10 (4.20 ± 0.37) × 105 (3.51 ± 0.41) × 105 (2.75 ± 0.35) × 105 (9.44 ± 0.54) × 105 (8.45 ± 0.22) × 105 (7.68 ± 0.66) × 105 (7.51 ± 0.53) × 105 (5.80 ± 3.60) × 106 (4.00 ± 2.20) × 106 (3.60 ± 2.00) × 106 (1.55 ± 0.23) × 106 5

Rg (nm)

A2 ×10−3 (cm3 mol/g2 )

77.9 ± 8.9 89.5 ± 8.1 68.1 ± 9.5 60.0 ± 11.0 106.8 ± 5.3 103.7 ± 6.2 93.0 ± 7.3 89.8 ± 6.2 148 ± 61 140 ± 53 130 ± 51 124 ± 16

(4.02 ± 0.36) (4.62 ± 0.68) (4.92 ± 0.73) (5.10 ± 1.70) (3.92 ± 0.44) (3.39 ± 0.42) (3.58 ± 0.40) (3.61 ± 0.39) (1.95 ± 0.79) (1.60 ± 1.20) (1.10 ± 1.80) (4.77 ± 0.62)

a The alphabets S & C stands for the salt (LiCl) and CNC respectively while the first and second numeral respectively indicates the concentrations of LiCl (present in solvent) and CNC used for the preparation of a particular solution.

3.6.2. Hot stage microscopy (HSM) The hot stage microscopic study was made on freeze-dried CNCGNS to reveal the phase changes occurring with temperature and supports the conclusions drawn from TGA and DSC studies. It can be seen that there was no phase change observed up to 175 ◦ C and at 215 ◦ C the change starts to take place which relate to the onset of CNC decomposition (Supplementary materials Fig.S3 (d–f)). The considerable change occurs from 270 ◦ C to 320 ◦ C which is relevant to TGA and DSC studies and corresponds to the fusion or decomposition of CNC in this range. Above 320 ◦ C, there were only carbonic residues (Fig. 5(d–e)). 3.7. Light scattering studies 3.7.1. Particle size analysis by dynamic light scattering (DLS) The mean hydrodynamic radius of CNC-GNS obtained was 82 nm (Supplementary materials Fig.S2) which is smaller than the actual length of the particles, measured by TEM. This can be explained according to earlier studies made by Boluk and Danumah (2014). 3.7.2. Molecular weight determination by SLS The stock solutions for CNC-GNS with concentrations 0.5, 0.25, 0.125 and 0.0625 mg/ml were prepared for SLS measurements, using LiCl/DMAc system containing 4%, 6% and 8% (w/v) LiCl. The procedure for solution preparation and their designations is briefly described in Supplementary materials and shown in Table S3. Zimm plots obtained from SLS measurement are shown in supplementary Fig. S4. The data collected from Zimm plots are summarized in Table 4. It was observed that Mw obtained for CNCs was in range of 105 g/mol which is on average 10 times higher than the value (Mw = 20581) obtained from viscosity data (supplementary Table 1s). Similar finding was reported by Röder et al., 2001.This fact concludes the presence of supra-molecular structures in stock solutions. The presence of these structures may be attributed to (1) Association of cellulose molecules as a result of increment in the crystallinity after acid hydrolysis. (2) Presence of aggregate particles in solution and (3) Presence of water traces which cannot be neglected to create molecular aggregation as described by Potthast et al. (2002), Sjoholm et al. (2000) & Terbojevich, Cosani, Conio, Ciferri, and Bianchi (1985). From Table 4, it can be seen that Mw and Rg values increase with decrease in salt concentration for stock solution with particular concentration. This was similar to the findings of Röder et al. (2001), where samples showed higher Mw and Rg with LiCl/DMAc containing 6 wt% compared to 9 wt% LiCl. As per Sjoholm et al. (2000) and Terbojevich et al. (1985), LiCl/DMAC solution with higher salt

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Fig. 5. FESEM micrographs of (a) delignified GNS (b) CPC-GNS (c) CNC-GNS and hot stage microscopic images (d–f) of CNC-GNS.

concentration was found to be good for solubilising the cellulose molecules by disaggregating the molecular structures responsible for higher Mw and Rg . With salt concentration below 6%, cellulosic samples found difficult to get dissolve in LiCl/DMAc which results into large amount of aggregates particles leading to higher Mw and Rg . Higher the polymer concentration higher will be the molecular weight, due to unbroken intermolecular hydrogen bonds of cellulose molecules leading to the formation of high molecular weight cellulose aggregates, as explained by Sjoholm et al. (2000) and Terbojevich et al. (1985). Samples with low polymer concentrations result in better solution state by dissolving the molecular aggregates and help in reducing both Mw and Rg values (Röder et al., 2001). These findings support the data obtained for CNC-GNS solutions (Table 4).The Mw and Rg both decreased with decrease in concentration of polymer stock solution prepared in LiCl/DMAc with particular salt concentration. The positive value of second virial coefficient indicates good interaction between solvent and polymer.

4. Conclusions CNCs were successfully isolated from groundnut shells after purification and acid hydrolysis treatment, to a total yield of 12%. The obtained CNC-GNS presented rod shaped morphology with average aspect ratio of 12 along with thermal stability (>200 ◦ C) and high crystallinity (74%). These findings indicate that CNCs extracted from groundnut shells are worthwhile to be used as organic filler with good reinforcement in composite materials. Molecular weight is an important parameter for polymeric materials along with their physicochemical properties. In that respect, SLS measurements were carried out to estimate Mw of CNCs. Solutions of CNCs in LiCl/DMAc showed higher value of Mw in the range of 105 g/mol which was an interesting feature, showing formation of supra-molecular structure in the solution and requires further more discussion.

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