International Journal of Polymer Analysis and

Characterization

ISSN: 1023-666X (Print) 1563-5341 (Online) Journal homepage: http://www.tandfonline.com/loi/gpac20

Isolation and characterization of cellulose

nanocrystals from Saharan aloe vera cactus fibers

Nagarajan K. J., A. N. Balaji & N. R. Ramanujam

To cite this article: Nagarajan K. J., A. N. Balaji & N. R. Ramanujam (2018): Isolation and
characterization of cellulose nanocrystals from Saharan aloe vera cactus fibers, International
Journal of Polymer Analysis and Characterization

To link to this article: https://doi.org/10.1080/1023666X.2018.1478366

Published online: 03 Aug 2018.

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INTERNATIONAL JOURNAL OF POLYMER ANALYSIS AND CHARACTERIZATION
https://doi.org/10.1080/1023666X.2018.1478366

Isolation and characterization of cellulose nanocrystals from

Saharan aloe vera cactus fibers
Nagarajan K. J.a, A. N. Balajia, and N. R. Ramanujamb
a
Department of Mechanical Engineering, K.L.N. College of Engineering, Pottapalayam, India; bDepartment of
Physics, K.L.N. College of Engineering, Pottapalayam, India

ABSTRACT ARTICLE HISTORY

Saharan aloe vera cactus leaves fibers (SACLFs) are a biodegradable and Received 12 April 2018
renewable agro textile fiber enriched with cellulose polysaccharides. The Accepted 16 May 2018
isolation of cellulose nanocrystals (CNCs) from SACLFs is an alternative
KEYWORDS
approach to reinforcement for bio-composites applications. In this study,
Cellulose microfibrils; CNCs;
we show that standard chemical methods for the extraction of cellulose FT-IR; XRD; TEM;
microfibrils and sulfuric acid hydrolysis (64 wt%) will result in usable CNCs. TGA analysis
The resulting CNCs were characterized by XRD, FTIR, TGA and TEM. Fourier
Transform Infrared Spectroscopy (FT-IR) confirms that the maximum
amount of noncellulosic materials is removed from SACLFs. High yield con-
tent (32%) of CNCs is achieved by sulfuric acid hydrolysis and sonication
process. It also enhances the crystalline size and crystallinity index of CNCs,
which are confirmed by XRD. TEM analysis of CNCs shows a rod-like struc-
ture with diameter (10–14 nm), length (200–255 nm) and high aspect ratio
of 18–20, which confers good reinforcing properties. These were tested in
biodegradable agar film, whose tensile strength is increased by 27% when
5 wt% of CNCs from SACLFs was incorporated into the matrix. The SACLFs
represent an interesting source of cellulosic reinforcing materials.

Introduction
In recent years, due to the deficiency of nonrenewable petrochemical resources and higher
demands of materials, numerous researchers have produced novel materials from renewable sour-
ces. Cellulose is the main component of all plants and is the most abundant biopolymer with nat-
urally occurring renewable polysaccharides on earth, it has also been made up of repeating link of
b-D-glucopyranose, and has been estimated that, globally, around 1012 tons are synthesized and
destroyed every year [1,2]. In recent developments, cellulose is highlighted as new high-tech mater-
ial based on the applications in nanotechnology. The productions of cellulosic fibers in nano
dimensions have enhanced the promising properties such as high mechanical strength, low ther-
mal expansion, high surface area to volume ratio, biocompatibility, low density, biodegradability,
decreased gas barrier properties, harmless and non-toxicity.[3] It finds application in the fields of
food packaging, papermaking, reinforcement in polymer composites, medical, energy and so on.[4]
The properties of nanocellulose are known to be strongly dependent, not only on the isolation
method but also on extraction sources, such as leaf, stem and roots, of the plants.[5] Compared to
microfibrils cellulose, the cellulose nanocrystals (CNCs) have a high aspect ratio and a large spe-
cific surface area. Due to this, the additive property, between the matrix and CNCs, can be
improved.[6] The Sulfuric acid hydrolysis is the most common chemical method to provide more

CONTACT A. N. Balaji balajime@yahoo.com Department of Mechanical Engineering, K.L.N. College of Engineering,

Pottapalayam, Tamil Nadu, India.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpac.
ß 2018 Taylor & Francis Group, LLC
2 N. K. J. ET AL.

stable aqueous suspensions of CNCs from natural raw fibers, having a diameter 4–25 nm and
length of 100–1000 nm.[7] This process needs lower acid content to attain high yield ratio at low
temperature for producing CNCs of similar dimensions with less time compared to other acid
hydrolysis process such as hydrochloric acid, or TEMP (2,2,6,6-tetramethylpiperidine-l-oxyl) oxi-
dation enzyme-assisted hydrolysis. The sulfuric acid which is concentrated enough is required to
break these strong crystalline contacts and to release cellulose in the form of free nanocrystal-
lites.[8,9] However, sulfuric acid concentration is the most important criteria to produce CNCs. It
produces highly amorphous low crystalline particles, when the acid concentration is more than
63 wt %. Most of the researchers have chosen the optimum level (57–60 wt%) of sulfuric acid con-
centration in order to produce CNCs at the range of temperature and time between 35–55
C and
40–60 min respectively.[10] Saharan aloe vera cactus plant is one of the renewable and sustainable
resources of cellulose fibers plant, which belongs to the Agavoideae family, abundantly available
in Morocco country, which is a part of the Sahara desert in the western tropical region of Africa.
In this country, people use these plant fibers for weaving clothes, tablemats, window curtains etc.
The world famous super fine Sabra silk sarees are manufactured in Morocco using these fibers.[11]
The characterizations of untreated and alkali treated Saharan aloe vera cactus leaves fibers
(SACLFs) are reported by Balaji and Nagarajan.[11] The result shows that the fiber has high tensile
strength due to the presence of a-cellulose enormously. Based on the literature studies, there are
little or no works been reported on utilizing SACLFs originated as a cellulose source for obtaining
CNCs. As per our knowledge, no works have been reported on the isolation and characterization
of CNCs from SACLFs based on the literature studies. The authors predicted that the SACLFs are
a source, of low-cost, which helps to increase the production rate of CNCs and also to meet a
part of the worldwide demand.
The aim of this study is to produce cellulose microfibrils and CNCs from SACLFs by using
chemical treatments such as acid-chlorite, alkaline and acid hydrolysis processes. The char-
acterization of the extracted microfibrils and isolated CNCs were studied by chemical analysis,
Fourier Transform Infrared Spectroscopy (FT-IR), X-Ray diffraction (XRD), thermo gravimetric
analysis (TGA) and transmission electron microscope (TEM).The reinforcing capacity of the
CNCs has been analyzed in agar films through their effect of tensile behavior.

Materials and methods

Materials
SACLFs were purchased from Chandra Prakash & Co., Jaipur, Rajasthan state, India. The Sulfuric
acid, acetic acid, sodium hydroxide pellets, sodium bisulfite, nitric acid, sodium chlorite, ethanol
and toluene (Sd-Fine Chemicals) chemicals were purchased from Sigma-Aldrich, Bangaluru,
Karnataka, India. Food grade agar, glycerol and distilled water were obtained from Srivas
Chemicals, Madurai, Tamilnadu state, India.

Extraction of cellulose microfibrils

The chopped SACLFs were dewaxed by refluxing with toluene-ethanol (2:1, v/v) at 70
C for
240 min in a Soxhlet apparatus. The dewaxed SACLFs were then delignified using 0.7 wt% sodium
chlorite at 100
C for 120 min in an acidic solution (pH 4–4.2 adjusted by 10 wt% acetic acid)
using liquor: SACLFs ratio as 50:1 (g/g). The pH value was measured using digital pH meter with
glass probe (S202, suntornics, India). The mixture was then allowed to cool down and the holo-
cellulose (a-cellulose and hemicellulose) were filtered using Whatman filter paper. The filtrate was
extensively washed using 2% sodium bisulfite and distilled water, then was dried at 110
C in an
oven for two hours. The holocellulose was treated with 17.5 (w/v) NaOH solution at room
 INTERNATIONAL JOURNAL OF POLYMER ANALYSIS AND CHARACTERIZATION 3

temperature (30
C) for 60 min. The treated fiber was washed with 10 wt% acetic acid, distilled
water and finally the cellulose was filtered using Whatman filter paper and was dried at 110
C in
an oven for 2 h. The crude cellulose was treated with volume concentrations of 80% acetic acid
and 70% nitric acid taken in the ratio of 10:1 and were stirred vigorously for 30 min at 120
C
using hot plate magnetic stirrer (VTMS-200, V-TECH, India). After being cooled, it was then
washed sequentially with 95 wt% ethanol and distilled water for several times until pH value was
in the range of 6.5–7.0. Finally, the purified cellulosic fibers were dried at 90
C in an oven for
5 h. The above procedure of extraction of cellulose microfibrils was adopted from Obi
Reddy et al.[12]

Isolation of CNCs
Acid hydrolysis was carried out using 60 wt% H2SO4 solution and was stirred continuously
using mechanical stirrer at a speed of 1200 rpm for 45 min at 45
C. The time of hydrolysis
was fixed at 45 min, as it was considered as the optimum time for the study.[13] The ratio of
the cellulose microfibrils to liquor was 5:100 (wt %) (5-gram cellulose microfibrils: 100 g
H2SO4 solution). The hydrolysis reaction was quenched by adding the excess amount of cold
deionized water (12
C) to the reaction mixture. The quenched ivory white cloudy suspension
was centrifuged (5180R, Eppendorf, USA) at a speed of 8000 rpm for 20 min. The supernatant
was discarded, and the precipitate was washed with deionized water. Both the centrifugation
and washing processes are repeated until the suspension achieved the pH value of about
3.5–4.0 (about five cycles). The collected precipitate was then dialyzed with deionized water in
a dialysis bag until the constant pH value of about 5.0–6.5 was reached (about 10 days).
Subsequently, the suspension was kept in water ice bath at 18
C and sonicated using probe-
type ultrasonicator (ENUP 750W, Poland, 13mm probe diameter) for 30 min at 20 KHz (pulse
6s on 2s off) to break down the unfractionated cellulose into small nano-metric particles.
Thereafter the suspension was filtered with Whatman #541 qualitative filter paper to remove
the agglomerated and large particles. To obtain the CNCs, the suspension was finally freeze-
dried at 20
C for 24 h using freeze dryer (L-200, Lyovapour) and was stored in a refriger-
ator at 5
C and was taken for further analysis.

Characterization of SACLFs, cellulose microfibrils and CNCs

The chemical compositions of SACLFs and cellulose microfibrils regarding the a-cellulose, hemi-
cellulose lignin, and wax content were determined using standard test methods.[14–16] The mois-
ture content of the microfibrils and SACLFs was identified using Sartorius MA45
moisture analyzer.[11]
X-ray diffraction (XRD) pattern of the SACLFs, cellulose microfibrils and CNCs were analyzed
using a PAN alytical Xpert pro-MRD diffractometer (Amsterdam, Netherlands). The XRD spectra
were recorded using Cu-K a-radiation (wavelength of 0.154 nm) and a nickel monochromatic fil-
tering wave at a voltage of 40 KV and a current of 30 mA, respectively. All samples were scanned
over the range of diffraction angle 10–80
at the rate of 0.8
/min. The Crystallinity Index (CI) of
the fibers is calculated by using Equation (1)

I200  Iam
CI ¼  100; (1)
I200

where Iam represents the minimum intensity of the peak, which contributes to the amorphous
fraction and I200 represents the maximum intensity of the peak, which contributes to the
4 N. K. J. ET AL.

crystalline fraction. The crystalline size (CS) of the samples was calculated using Scherer’s formula
(2) for the plane (2 0 0)
CS ¼ Kk=bCOSðhÞ; (2)
where K is the Scherrer constant (0.84), k is the X-ray wavelength (0.154 nm), h is the Bragg angle
and b is the peak’s full-width at half-maximum.
The functional groups of SACLFs, cellulose microfibrils and CNCs were analyzed using iTR-
ATR Nicolet iS10 FTIR spectrometer. The samples, SACLFs which were in powder form and cel-
lulose microfibrils were mixed with potassium bromide (KBr) separately. The dried sample of
CNCs was grinded and pelletized using potassium bromide (KBr). The transmittance spectra of
the samples were recorded in the range of 4000–500 cm1 with the scan rate of 32 scans per
minute at a resolution of 4 cm1 at atmospheric conditions.
Thermal stability and degradation behavior of the samples were determined using thermogravi-
metric analyzer (Model STA 449 F3, NETZSCH, Germany). For this analysis, 5 mg of sample was
taken in a standard alumina crucible and the empty cup was used as a reference. It was then
heated from room temperature to 600
C at the rate of 10
C/min under nitrogen flow of
20 ml/min.
The yield of CNCs was expressed in terms of percentage, and it was the ratio of the mass of
the freeze-dried CNCs (Mf) to the mass of microfibrils fibers (Ma).
Mf
% Yield ¼  100: (3)
Ma
The morphology of CNCs was examined using FEITecnai model TF20 TEM. Before the ana-
lysis, a 10 ml dilute suspension of CNCs was poured over a plain Cu grid with ultra-thin carbon
film, dried under table lamp for 3 h, and then, stained with Ruthenium vapor for 3 min.
The stability of CNCs in suspension was determined using the equipment of HORIBA,
Zetasizer Nano ZS90. A 10-fold dilution of the CNCs in a total volume of 1 ml of distilled water
and was subjected to a particle size analyzer at room temperature. The measurement depends on
the electrophoretic mobility (mm/s) of the particles which was then converted to zeta potential by
means of an inbuilt software, which is based on the Helmholtz–Smoluchowski equation.
The structure of the cellulose microfibrils and the CNCs reinforced composite films are exam-
ined using CARL ZEISS model V18 Scanning Electron Microscope (SEM). The film samples were
cut into small pieces and were directly mounted on a specimen holder for analysis.

Mechanical reinforcing capacity of the CNCs

Preparation of composite films
Agar and agar/CNCs suspension was prepared using different concentrations of CNCs based on
agar weight (0, 3, 5, 7 and 10 wt%). For this process, the predetermined amount of CNCs was dis-
persed in 200 ml distilled water and sonicated for 10 min at 20 kHz (pulse 6 s on 2 s off). Then,
1.2 g of glycerol was used as a plasticizer and was dissolved in the suspension and mixed continu-
ously for 30 min at room temperature (27
C). Finally, 4 g of agar was added slowly into the solu-
tion and mixed vigorously at 90
C using a hot plate stirrer for 30 min, until it completely
dissolved. Agar/CNCs composite films are prepared by using a solution casting method. The
mixed suspensions with different concentrations of CNCs were poured evenly onto a leveled
Teflon film-coated glass plate (30 cm  30 cm) and then dried for about 60 h at room temperature.
The dried films were peeled off from the casting plate for further studies. The thickness of the
samples was measured using a digital micrometer (Mitutoyo, Japan) with a sensitivity of
0.001 mm, at five random positions and this was measured for each and every film. The mean
value of the samples is considered for mechanical testing.
 INTERNATIONAL JOURNAL OF POLYMER ANALYSIS AND CHARACTERIZATION 5

Mechanical testing
The mechanical properties of composite films with the dimensions of (100 mm  25.4 mm) as per
the ASTM D 882-88 were studied using an Instron 5500R universal testing machine. The machine
was operated with an initial grip separation of 50 mm and 0.5 kN at a loading rate of 5 mm/min.
Five samples were tested for each combination of (agar/CNCs) films. The Young’s modulus (GPa)
of the composite films was calculated from the slope of the first linear region of the stress–strain
curve. The maximum tensile strength (MPa) of the composite films was calculated from
Equation (4)
Fmax
Ts ¼ ; (4)
Ao
where Fmax represents the maximum load and Ao is the original cross-sectional area of the film.
The elongation at break (%) of the composite films was calculated by using Equation (5),
Lf  L0
EB ¼  100; (5)
L0
where Lf denotes the extension at the rupture of the film and L0 is the initial gauge length of the
composite film.

Statistical analysis
It was used to determine the significance of the average values for mechanical properties and
water contact angle measurement. One-way analysis of variance (ANOVA) was conducted and
the significant difference (p < .05) between each mean property value is determined with
Duncan’s multiple range test.

Result and Discussion

Characterization of CNCs
Chemical composition analysis for cellulose microfibrils
The physicochemical and mechanical properties of cellulose microfibrils and SACLFs are summar-
ized in Table 1. The removal of wax, lignin and hemicellulose contents from the SACLFs was per-
formed by acidified chlorination, alkalization and acid hydrolysis treatments. In the acidified
chlorination process, a substantial breakdown of the lignocellulosic structure was created and it
resulted in depolymerization of lignin. From the alkalization treatment, the intermolecular binding
decreases and it leads to a high solubility of hemicellulose. The hemicellulose and lignin contents
are drastically reduced from 14.2% to 1.6% and 13.7% to 2.8%, respectively. It is observed that the
percentage of a-cellulose content is increased from 60.2% to 88.2% by the removal of non-
cellulosic and amorphous materials by chemical treatment. It may provide better mechanical
properties, due to the increase of solid cellulose, and it forms a microcrystalline structure with
high-order crystalline regions and low-order amorphous regions.[11] These above characteristics
have been proved in the following section of XRD analysis. After the chemical treatment process,

Table 1: Chemical composition analysis of cellulose microfibrils and SACL fibers

Chemical properties
α-Cellulose Hemicellulose Lignin Moisture Wax Ash
Forms of fiber (Wt. %) (Wt. %) (Wt. %) (Wt. %) (Wt. %) (Wt. %) Reference
SACLFs 60.2 14.2 13.7 6.2 1.5 4.1 Balaji and Nagarajan[11]
Cellulose microfibrils 88.2 1.6 2.8 3.9 – 3.5 Present work
6 N. K. J. ET AL.

Figure 1. XRD patterns of SACLFs, micro fibrils SACLFs, CNCs.

the moisture content of the fibers decreases from 6.2% to 3.5%. As a result, the moisture resist-
ance property of the fibers increases and the hydrophilic hydroxyl groups are reduced.[11]

XRD analysis
The X-ray diffractograms of the CNCs, cellulose microfibrils and SACLFs are shown in Figure 1.
The diffractograms of the samples have exhibited a well-defined main peak at 2h of 22.5
and the
shoulder peak around 18
at 2h, which are the characteristic diffraction patterns of cellulose I and
amorphous region, respectively. The peak of CNCs (2h ¼ 22.5
), which is obtained by acid
hydrolysis, is narrow and sharp than its non-hydrolyzed counterpart; it indicates that the crystal-
linity index and the crystalline size of the CNCs have increased.[17] This observation is in parallel
with the findings of Abeer et al.,[18] Jeevan Prasad Reddy and Jong-Whan Rhim.[19] However, the
intensity peaks of the CNCs are significantly higher than that of the cellulose microfibrils and
SACLFs. The CI of the CNCs, cellulose microfibrils and SACLFs are calculated from Equation
(1), and they are 74.78%, 70.5% and 52.6%, respectively. The CS of CNCs, cellulose microfibrils
and SACLFs are found to be 6.95 nm 6.46 nm and 5.6 nm, respectively, they have been estimated
from Equation (2). It is concluded that the CI and CS are increased after the acid hydrolysis treat-
ment. The CI and CS are increased because the amorphous regions of cellulose are removed due
to the sensitive of acid and the realignment of monocrystals.[20] The calculated CI value of CNCs
(74.78%) is higher than that of soy hulls (73.5%) and pineapple leaves (54%).[21,22]

FT-IR analysis
FT-IR transmittance mode spectrograms of the CNCs, cellulose microfibrils and SACLFs are
shown in Figure 2. The intensity peaks at 896 cm1 indicate the purity of the cellulose and it
resulted from C–H vibrations, which are found in the cellulose microfibrils, and CNCs. The peak
at wave number 1400 cm1 refers the O–H bending vibration of cellulose [23]. The band at
3400 cm1 corresponds to C–H and O–H stretching of the cellulose present in the fibers. The
peaks at 896, 1400 and 3400 cm1 are increased significantly after the treatment of acidified chlor-
ination, alkalization and sulfuric acid hydrolysis.[24] The peaks observed in the spectra at
1050 cm1 are significantly increased after the chemical treatments, as a result of C–O–C pyranose
 INTERNATIONAL JOURNAL OF POLYMER ANALYSIS AND CHARACTERIZATION 7

Figure 2. FT-IR transmittance spectra of SACLFs, cellulose micro fibrils, CNCs.

ring stretching vibration. The peak is observed at 1518 cm1 because of the presence of aromatic
skeletal vibration of the functional group of lignin and is significantly reduced after the acidified
chlorination, alkalization and sulfuric acid hydrolysis treatment.[10] The band observed at
1718 cm1 corresponds to C¼O stretching of the acetyl and uronic ester groups of hemicellulose
or ester linkage of carboxylic groups of ferulic and p-coumaric acids of lignin.[24] This band is not
observed after the chemical treatments of the fibers. This disappearance of the band could have
been caused by the removal of hemicellulose and lignin from the cellulose microfibrils
and CNCs.[24,25]

Thermogravimetric analysis
The thermal stabilities of the CNCs, cellulose microfibrils and SACLFs are analyzed using TGA
analysis and the results are presented in Figure 3(a,b). The TGA and Derivative ThermoGram
(DTG) curves have exhibited the weight loss pattern and the thermal decomposition occurs at
maximum temperature of the samples. The weight loss during thermal degradation of the samples
of CNCs, cellulose microfibrils and SACLFs occurs in two stages. The minor weight losses are 4%
for SACLFs and cellulose microfibrils and 5% for CNCs, which is occurred in the temperature
range of 60–120
C due to the evaporation of the absorbed water from the fibers, during the first
stage. In the second stage, the major weight loss occurs, due to the thermal degradation of cellu-
losic materials in the temperature region of 230–400
C. The thermal degradation curve shows
that the pattern of CNCs is clearly different from the SACLFs and cellulose microfibrils, that is,
the onset temperature (220
C) of thermal destruction of the CNCs is lower than that of cellulose
microfibrils (252
C) and SACLFs(238
C). The higher onset temperature of cellulose was due to
the removal of less thermos table lignin and hemicellulose as shown in the FTIR results. From the
DTG curve, it is clearly observed that the degradation temperature of the CNCs is decreased. The
maximum degradation temperature of CNCs (318
C) is decreased in comparison with cellulose
microfibrils (357
C) and SACLFs (355
C). This happens because of more heat energy is absorbed
and their thermal destruction is induced, due to the increase in surface area of the CNCs and sul-
fate groups on the surface of CNCs compared to SACLFs and cellulose microfibrils.[26] The CNCs
peak at 318
C in the DTG curve, is higher than that of CNCs which is isolated from oil palm
(250
C), kelp (Laminaria japonica) (240
C).[6] From the DTG curve, it is clearly observed that
8 N. K. J. ET AL.

Figure 3. (a) TGA curve of SACLFs, micro fibrils SACLFs, CNCs. (b) DTG curve of SACLFs, micro fibrils SACLFs, CNCs.

Table 2. Thermogravimetric parameters for the thermal degradation process of CNCs, cellulose
microfibrils and SACLFs.
S. No. Forms of fiber Onset temperature (
C) Degradation temperature (
C)
1 SACLFs 238 355
2 Cellulose microfibrils 252 357
3 CNCs 220 318

the thermal stability and degradation temperature of the CNCs are decreased. It also shows that
the CNCs can be preferably used as reinforcements in biopolymers if the processing temperature
is less than 200
C. However, the char content is increased from 20% to 30% at 550
C, after the
conversion of CNCs. The thermogravimetric parameters for the thermal degradation process of
CNCs, cellulose microfibrils and SACLFs are shown in Table 2.

Yield of CNCs and zeta potential

The yield obtained after the isolation of CNCs is about 32% (of initial weight) which is in agree-
ment with the yield of mengkuang leaves (28%), sugar palm (29%) and sisal (30%) as reported by
Ilyas R.A et al., Garcia de Rodriguez, Thielemans, and Dufresne, Sheltami et al., respect-
ively.[10,27,25] The differences in the gain of yield depend on the source of sample, pretreatment
and hydrolysis condition.[10]
 INTERNATIONAL JOURNAL OF POLYMER ANALYSIS AND CHARACTERIZATION 9

The formation of a negative electrostatic layer covering the CNCs and the dispersed medium
of water is due to the presence of negative charge developed by grafting of sulfate groups to sul-
furic acid. If the zeta potential is in the range of 15 to 15 mV, then the agglomeration of nano-
cellulose will occur. The occurrence of agglomeration because the nanocellulose does not have
enough charge to repulse each other. If the zeta potential is less than 30 mV and greater than
þ30 mV, then the suspension of nanocellulose is a stable one.[28] In this study, the isolated CNCs
have a negative zeta potential of 45.2mV as shown in Figure 4(f). The higher negative value of
zeta potential attached to the CNCs may be due to sulfate groups during sulfuric acid hydrolysis,
which creates repulsive forces to each other. The performance of reinforcement in CNCs is also
associated with surface charge. The higher negative value of zeta potential of the CNCs suspen-
sions can resist aggregation leading to increased degree of dispersion.

Morphology of cellulose microfibrils and CNCs

After the acidified chlorination, alkalization and acid hydrolysis (acetic acid / nitric acid) treat-
ment (Figure 4(d)) SACLFs fiber bundles were separated into micro-sized individual ribbon-
shaped fibers with rough surface due to the removal of noncellulosic and cementing materials. It
is apparent from the SEM image that the range of width of the microfibers was about 20–25 mm,
which was lower than that of the diameter of SACLFs (91.15 mm)[11]. The TEM image analysis of
the CNCs is shown in Figure 4(e). It clearly signifies the evidence of isolation of cellulose nanofi-
brils from SACLFs. It can also be observed that the dispersions contain more separated fibrils
(CNCs) with a rod-like structure and some agglomerated CNCs. The ultrasonic treatment present
after the sulfuric acid hydrolysis, centrifugation and dialysis process has probably promoted the
cleavage of the glycosidic bonds in cellulose chains and the length of distribution becomes uni-
form.[23,29] The geometrical characteristics such as diameter and length of 80 samples of CNCs
are measured using the Image-J software and its ranges are shown in Figure 5(b,c). The diameter,
length and aspect ratio are shown in the range of 10–14, 200–255 and 18–20 nm for most of the
samples. Comparisons of the CNCs from SACLFs with that of other CNCs from classical natural
fibers/shells are presented in Table 3 and SACLFs, cellulose microfibrils and isolated CNCs sus-
pension are shown in Figure 4(a–c).

Table 3. Comparisons of the CNCs from SACLFs with that of other CNCs from classical natural fibers/shells.
Sulfuric acid
concentration Temperature Time Diameter
S.No. CNCs (%wt) (
C) (min) (nm) Length (nm) Reference
1 Sharan aloe vera 60 45 45 10–14 200–255 Present study
cactus fiber
2 Rice husk 64 50 40 15–20 Nurain Johar et al.[30]
100–150
3 Cassava peelings 50 60 60 2.4 ± 0.8 176 ± 5
Anna Leticia Moron Pereira
4 Bagasse 50 60 60 2.3 ± 1.1 Leite et al.[31]
258 ± 36
5 Kapok fibre 60 45 60 4.2 ± 3.0 79.4 ± 12.8
Mohamad Azuwa
Mohamed et al.[32]
6 Mulberry pulp 47 60 180 20–40 – Jeevan Prasad Reddy et al.[19]
7 Roslelle 50 45 60 6.84–13.06 280.9 Kian et al.[33]
8 Cotton linter 60 45 90 15–50 210–480 Morais et al.[26]
9 Kelp waste 51 30 70 20–45 100–500 Zhenhua Liu et al.[6]
10 Kenaf bast fibers 65 50 60 3 100–500 Zaini et al.[34]
11 Oil plam empty 64 45 15–90 1–3.5 100000–20- Farh Fahma et al.[35]
fruit bunch 0000
12 Coffee husk 64 50 40 20 ± 04 310 ± 160 Soffa Collazo-Bigliardi et al.[36]
13 Sago seed sheels 64 45 45 50 — Naduparambath et al.[28]
14 Sugar palm fibers 60 45 45 9 ± 1.96 nm, 130 ± 30 Ilyas et al.[10]
(arenga pinnata)
15 Ushar seed 64 50 75 14–24 140–260 Oun et al.[37]
16 Pistachio shells 64 50 90 12 ± 1 187 ± 2 Josh marett et al.[38]
10 N. K. J. ET AL.

a b c

Figure 4. (a) SACLFs (b) cellulose micro fibrils (c) CNCS (d) SEM image of cellulose micro fibrils (e) TEM image of CNCs (f).
zeta potential of CNCs.

Mechanical properties
The Mechanical properties and water contact angles of agar, agar/CNCs composite films are sum-
marized in Table 4. The mechanical properties such as strength, flexibility and stiffness of the cel-
lulose-based agar composite films are determined by measuring the tensile strength, elongation at
break and Young’s modulus. With the addition of CNCs, the thickness of composite films is
increased significantly (p < .05) compared to that of the control agar film.
The tensile strength of the composite film is increased from 33.5 ± 1.5 MPa to 42.6 ± 1.1 MPa
since the CNCs (5 wt%) is added to control agar film. It shows that the interaction between the
CNCs and agar matrix is increased.[17] The dispersed CNCs particles in composite agar films are
shown in SEM images of Figure 5 (b,c). By the further addition of CNCs (7% wt) and (10% wt),
the effect of reinforcement is reduced, due to the aggregation of the CNCs as observed in the
 INTERNATIONAL JOURNAL OF POLYMER ANALYSIS AND CHARACTERIZATION 11

a b

c d

Figure 5. SEM images of the distribution of CNCs and surface of (a) control agar film, (b) agar/3 wt% of CNCs, (c) agar/5 wt% of
CNCs, (d) agar/7 wt% of CNCs (e) agar/10 wt% of CNCs.

Table 4. Mechanical properties and contact angle of agar and agar/CNCs films.
S.No Films Thickness (mm) Tensile strength (MPa) Elongation at break (%) Young’s modules (GPa)
1. Agar/0% (wt%) CNCs 52.2 ± 1.2d 33.5 ± 1.5d 18.8 ± 1.0c 0.67 ± 0.01d
2. Agar/3%(wt%) CNCs 54.1 ± 0.6c 37.8 ± 0.8b 19.5 ± 1.6b 0.75 ± 0.05d,c
3. Agar/5% (wt%) CNCs 54.8 ± 0.2c 42.6 ± 1.1a 20.4 ± 1.2a 0.89 ± 0.04a
4. Agar/7% (wt%) CNCs 56.4 ± 1.5b 35.2 ± 0.5c 20.1 ± 0.5b,a 0.85 ± 0.07b,a
5 Agar/10% (wt%) CNCs 58.7 ± 0.8a 29.2 ± 1.2e 18.5 ± 1.4d 0.78 ± 0.07c
Note: An Each value is the mean of three replicates with the standard deviation. Any two means in the same column followed
by the same letter are not significantly (p > .05) different from Duncan’s multiple range test.
12 N. K. J. ET AL.

SEM analysis of Figure 5(d,e). It may also reduce the interaction between the CNCs and polymer
matrix because of the nonuniform stress distribution in the film and the strength of the composite
film is reduced accordingly.[17] The similar mechanical performances have been reported due to
the addition of CNCs to agar and other biopolymers such as starch and chitosan.[17,19,39]
The Young’s modulus (GPa) of the composite film measures the stiffness of a film, which is
increased linearly with the concentration of CNCs. The stiffness is significantly (p < .05) increased
to 34%in accordance with the control agar film when 5%wt of CNCs is added to the control agar
film, because, the nanofiller has high elastic modulus.[19]
The elongation at break (%) is a measure of flexibility or ductility of agar film. This is similar
to the behavior of tensile strength and it varies accordingly with the addition of concentration of
CNCs to control agar film. The elongation of break increases up to 5 wt% of CNCs blended with
agar film and afterwards, it decreases. The decrease in flexibility of the composite film at a high
concentration of the filler may be due to the restricted mobility of polymer strand caused by the
increased stiffness of the film.[19]

Conclusions
In the presented work, CNCs are isolated from SACLFs by -chlorite, alkaline and sulfuric acid
hydrolysis, and sonication process. The chemical, physical and thermal properties of SACLFs, cel-
lulose microfibrils and isolated CNCs are extensively investigated. After the extraction of cellulose
microfibrils, a maximum amount of hemicellulose, lignin and wax contents are removed from the
SACLFs and the a-cellulose contents are enhanced from 60.2% to 88.2%. The hemicellulose and
lignin contents are removed due to the effect of -chlorite, alkaline and acid hydrolysis processes
and are confirmed by FT-IR analysis. TGA analysis confirms that the thermal stability is
decreased from 238
C (SACLFs) to 218
C, due to the increase in surface area of the CNCs and
sulfate groups on the surface of CNCs compared to SACLFs. The isolated CNCs has a negative
zeta potential of 45.2mV and yield (32%) content with minimum aggregation is attained from
the sulfuric acid hydrolysis and sonication process. The TEM analysis of CNCs shows the rod-like
structure with a diameter of (10–14 nm), length (200–255 nm) and aspect ratio 18–20. It is con-
cluded that these CNCs are an alternative to reinforcement in bio-based composite if the process-
ing temperature is less than 220
C. The properties of cellulose fractions from SACLFs make them
very adequate as reinforcing materials in biopolymer composites, especially nanosized reinforce-
ment (CNCs from SACLFs) which increased the tensile strength and elastic modulus of agar film
(5 wt% CNCs) by 27% and 34%, respectively. The SEM analysis has confirmed that 5 wt% of
CNCs is well dispersed in agar film and the surface is smooth in nature. The results confirm that
these CNCs represent an interesting source of cellulose reinforcing materials, whose use in differ-
ent application such as packaging materials and reinforcement in polymer composites could boost
the value of these fibers.

Acknowledgment
The authors are grateful for the support rendered by the Management of K.L.N. College of Engineering.

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