Anal. Methods Environ. Chem. J. 5 (4) (2022) 5-19
Research Article, Issue 4
Analytical Methods in Environmental Chemi s try Journal
Journal home page: www.amecj.com/ir
AMECJ
ZnO nano s tructure synthesis for the photocatalytic
degradation of azo dye methyl orange from aqueous
solutions utilizing activated carbon
Ahmed Jaber Ibrahim a,*
a Scientic Research Center, Al-Ayen University, ThiQar 64011, Iraq
ABS TRACT
In this s tudy, zinc acetate (as a precursor) and activated carbon
carboxylic acid derivative were used to create the nano s tructure of
zinc oxide (ZnO) as a matrix. The carboxylic acid derivative was
produced by modifying the oxidized activated carbon with nitric
acid (AC-COOH). The modied activated carbon’s surface was then
impregnated with zinc to load it. By using BET, XRD, and SEM to
characterize the ZnO nano s tructure, it was discovered that it was
composed of nanoparticles with a surface area capacity of 17.78
m2 g-1 and a size range of 21–31 nm. The photocatalytic hydrolysis
of the dye methyl orange in an aqueous medium served as a te s t
case for the cataly s t’s performance. The primary variables were
considered, including pH, cataly s t dose, s tirring eect, and s tarting
dye concentration. Measurements of activity below UV light revealed
satisfactory outcomes for photocatalytic hydrolysis of the methyl
orange (MO). In addition, the eciency of the methyl orange (MO)
photolysis cataly s t prepared with unmodied activated carbon was
also evaluated. The outcomes proved that zinc oxide (ZnO), made
using a derivative carboxylic acid of activated carbon molecules by
a matrix, had more good photocatalytic action than zinc oxide (ZnO)
made by the real activated carbon matrix.
Keywords:
Degradation,
Zinc oxide,
Nano s tructure,
Methyl orange,
Photocatalytic
ARTICLE INFO:
Received 3 Sep 2022
Revised form 29 Oct 2022
Accepted 18 Nov 2022
Available online 29 Dec 2022
*Corresponding Author: Ahmed Jaber Ibrahim
Email: ahmed.jibrahim@alayen.edu.iq
https://doi.org/10.24200/amecj.v5.i04.200
1. Introduction
Reactive dye-containing euents from various
sectors frequently generate environmental issues [1].
The ecosy s tem of the receiving surface waterways
is severely harmed by this pollution [2]. Many
researchers’ eorts have focused on removing
pollutants and toxins from wa s tewater from dierent
sectors [3]. A variety of chemical and physical
procedures, such as membranes [4], adsorption
methods [5], and photolysis, have been employed
to remove dyes [6] presently. Several researchers
have recently used photolysis as one of the advanced
oxidation processes (AOPs) to get rid of dyes from
wa s tewater [7]. Without altering the sub s trate,
the photocatalytic reaction is catalyzed by light
and can proceed more quickly [8]. Under the right
circum s tances, semiconductors function as cataly s ts
due to the down breaking energy between the
capacitance and conduction bands [9]. The process
of photocatalysis requires two levels of dierent,
equal energy. The movement of the electrons caused
by the absorption of this energy leads to a hole (h+)
and a pair of electrons(e-). Both the oxidation of
the electron donor species and the reduction of the
electron acceptor species might include electrons
[10]. To degrade pollutants, many materials are
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6Anal. Methods Environ. Chem. J. 5 (4) (2022) 5-19
utilized as photocataly s ts, including TiO2, ZnO,
ZrO2, CdS, MoS2, and WO3 [11]. TiO2 is one of
these materials frequently used as a photocataly s t
and has seen the mo s t application to date. TiO2 has
benets like environmental safety, non-toxicities,
chemical con s tancy, and the capacity for re s toration
and reuse. However, TiO2 has drawbacks, including
a high price tag and a UV absorption band. The
importance of ZnO as a suitable TiO2 alternative in
photocatalysis has lately increased [12]. One riche s t
s tructures, zinc oxide, has a variety of advantages.
As a result, ZnO has several uses in various scientic
projects [13]. ZnO has been produced using a variety
of techniques, including the soft chemical method
[14], the sol-gel method [15], the vapor-phase
growth [16], the vapor-liquid-solid process [17],
electrophoretic deposition [18], thermal evaporation
[19], homogeneous precipitation [20], chemical
vapor deposition [21], chemical bath deposition
[22], etc. In the aforementioned inve s tigations, ZnO
nanoparticles were only occasionally generated
through the activated carbon layer and by an auxiliary
matrix approach, as recommended by Park et al. [23].
In this s tudy, the photocatalytic activity of the
generated ZnO was used to break down the azo dye
methyl orange. Additionally, ZnO was produced
using modied activated carbon (containing
carboxyl functional groups).
2. Materials and Methods
2.1. Reagents
All chemical sub s tances were obtained with a high
degree of purity, including cau s tic soda (NaOH,
CAS Number: 1310-73-2, Sigma), hydrochloric acid
(HCl, CAS Number: 7647-01-0, Sigma, Germany),
activated carbon (AC, CAS Number: 7440-44-0,
Sigma), zinc acetate dihydrate (Zn(CH3CO2)2.2H2O,
CAS Number: 5970-45-6, Sigma), nitric acid (HNO3,
CAS Number: 7697-37-2, Sigma, Germany), and azo
dye methyl orange (C14H14N3O3SNa, CAS Number:
547-58-0, Sigma). Methyl orange was dissolved in
100 mL of deionized water (DI), 0.010 g at a time,
to create a s tock solution (100 g mL-1). All working
solutions were made at the necessary concentration
using di s tilled water to dilute the s tock solution.
2.2. Equipment
An ultraviolet-visible spectrophotometer (model
2600) to record Rayleigh UV–Vis spectra. A
Metrohm pH meter (model 744) to adju s t the
working solution’s pH to the desired values. Field
emission-scanning electron microscope (FE-SEM)
(model SU5000) to know the characterization
of the sample’s surface and shape morphology.
X-ray diraction in s trument (XRD) (model B8
ADVANCE) to record patterns via BRUKER. The
transformer coupled plasma (TCP) (model VIS tA-
PRO) to measure the presence of zinc in the samples.
Spectrophotometer (IR-470 Shimadzu) to record the
Infrared (IR) spectrum of samples. At the analytical
chemi s try laboratory of the college of education
(Ibn Al-Haitham) at the University of Baghdad,
experiments in the photocatalytic bleaching and
degradation of dye MO were carried out at a
photoreactor framework prepared there for it.
2.3. Synthesizing Zinc Oxide nanoparticles by
modied activated carbon particles
2.3.1. Activated carbon surface modication
According to Chang et al. [24], adding carboxyl
functional groups to the surface of activated carbon
caused the carbon particles to become activated.
To eliminate metal ions and other impurities, a
hydrochloric solution (10% v/v) solution was
r s t used to clean the activated carbon powder
for 24 hours. Following that, 300 ml of a 32.5
% (v/v) HNO3 solution was s tirred with 10 g of
pure activated carbon added, and the mixture was
heated at 60 °C for ve hours. The heterogeneous
mixture had ltered and neutralized with DI water
(deionized water) by wash and then dried below
decreased pressure for eight hours at 80 °C. the
carboxylic derivative of activated carbon makes up
the nal product (AC-COOH).
2.3.2. Synthesizing nanoparticles of zinc oxide
As a precursor for manufacturing ZnO nanoparticles,
100 mL of zinc acetate solution was mixed with 2
g of carboxylate-activated carbon (AC-COOH) in
dierent concentrations for 12 hours. The solution
was ltered, dried for 18 hours at 80 °C, and then
7
Photocatalytic Degradation of Azo Dye Methyl Orange by ZnO Ahmed Jaber Ibrahim
calcined for 4 hours at 500 °C in an electric oven.
Dierent concentrations of zinc acetate dihydrate
were explored to create zinc nanoparticles by
examining the impact of the concentration of zinc
acetate precursor on the description of zinc oxide
(particle size, percentage values, photocatalytic
capabilities, etc.). To do this, ZnO nanoparticles were
created using solutions containing concentrations
of 0.09, 0.02, and 0.01 M(Molarity) zinc acetate
dehydrate, respectively. XRD spectra of three
samples were taken to verify the production of ZnO
nanoparticle forms. The XRD bandwidth pattern
and Scherrer’s Equation 1 [25, 26] were used to
measure the cry s tal size of three samples.
D = K (ƛ ⁄ ß cosø ) (Eq.1)
Where D represents the size of cry s talline particle
in units of a nanometer (nm), The coecient is K
(that equals 0.89), λ represents the wavelength of
the X-ray radiation in a unit of a nanometer (nm), β
means FWHM (full width at half maximum) is an
experimental value in radians (rad) and diraction
angle expressed in degrees had represented θ.
2.3.3. Synthesis of Zinc Oxide nanoparticles
utilizing unmodied activated carbon
2.0 g of activated carbon had put in 200 mL of
hydrochloric solution (10% v/v) to eliminate
impurities for twenty-four hours to s tudy the surface
modication phase of activated carbon particles in
the formation of Zinc Oxide(ZnO). After that, the
product was added to a concentration of 0.09 M
zinc acetate dihydrate solution for 12 hours, and the
resulting combination was then ltered. The nished
product was dried at 80 °C for 18 hours before being
calcined in an electric oven for 4 hours at 500 °C. To
determine the sample’s cry s tal size using Scherer’s
equation, XRD spectra were taken on a sample made
with unmodied activated carbon.
2.4. Procedure of methyl orange decomposition
in photocatalytic experiments
Initially, a 250 mL beaker was lled with 100 mL
of methyl orange(MO) solution with a concentration
of 10 mg L-1 and a pH of 6. The solution was then
supplemented with 20 mg of Zinc Oxide (ZnO)
photocataly s t. Methyl orange was adsorbed onto
Zinc Oxide(ZnO) nanoparticles after being combined
with the solution utilizing a magnetic s tirring device
in a dark environment for half hour (30 min).
Afterward conrming the balance of adsorption,
0.2 mL of the solution was placed into a te s t tube
and then centrifuged for ve minutes at 3000 rpm
to collect the photocataly s t deposition particles.
A spectrophotometer captured the solution’s
adsorption spectra in the 200–600 nm range.
After the absorption spectrum was recorded, the
combination was put into a photoreactor, and a UV
light was turned on. Twenty minutes after exposure
to UV light, ten samples were collected at intervals
of one, and their absorption spectra were recorded.
The control solution’s adsorption spectrum was
recorded similarly without including a photocataly s t.
2.5. Procedure of batch adsorption
To nd the be s t circum s tances for bleaching and
degrading MO (methyl orange) in the exi s tence of
Zinc Oxide (ZnO) photocataly s ts, batch experiments
were carried out. It was thoroughly explored how
relevant factors including methyl orange(MO)
concentration, pH, solution s tirring, photocataly s t
dose, and solution oxygen aected the outcomes.
One variable at a time optimization was utilized to
improve factors that aected the reaction. With this
procedure, s tudies were carried out in a batch setting
with 100 mL of dye solution (10 mg L-1) placed into
a beaker (250 ml). The magnetic s tirrer was used to
mix the suspensions for 30 minutes in the dark before
centrifuging them for 5 minutes at 3000 rpm. A UV-
Vis spectrophotometer was used to evaluate the clear
supernatant. Equation (2) was used to determine the
rate of bleaching and dye degradation [27].
R= (C0 - Ct ÷ C0) × 100 (Eq. 2)
Where R (percentage) represents the dye removal
eectiveness, C0 represents the dye’s s tarting
concentration (mg L-1), and Ct represents the dye’s
concentration at time t following adsorption (mg L-1).
8
3. Results and Discussion
3.1. S tudy of Nanoparticles’ characteris tics
3.1.1. X-Ray Diraction (XRD) examination
The XRD spectra of three produced zinc oxide
(ZnO) samples are shown in Figure 1 to illu s trate
that the nanoparticles formed appropriately.
Figures 1 show that the sample’s hexagonal zinc
oxide cry s tallization has been veried. Table 1
compares the XRD pattern characteri s tics for
three produced samples and the reference sample
[22]. It can be seen from the XRD spectrum in
Figure 1 and the data in Table 1 that as zinc acetate
dihydrate concentration increased, ZnO peak
density too increased, and spectral noise intensity
Table 1. Compares the s tandard sample with the XRD samples (a), (b), and (c),
as well as a sample made with unmodied carbon
Fig. 1. XRD spectra of synthetic ZnO nanoparticles at various concentrations
of zinc acetate dehydrate (a:0.01 M; b: 0.02 M; c: 0.09 M)
Anal. Methods Environ. Chem. J. 5 (4) (2022) 5-19
9
decreased. This data may result from increased
ZnO nano s tructure production with rising zinc
acetate concentration. This data may be the result
of increased ZnO nano s tructure production with
rising zinc acetate concentration. According to Liu
et al. [20], the intensity of peak 005 in XRD spectra
is associated with carbon impurities that became
less intense as zinc acetate dihydrate concentration
was raised. Figure 2 displays the XRD spectrum of
the carbon-free synthetic sample. The production
of hexagonal ZnO has been conrmed based on
Table 1 and the dierentiation of the XRD data of
the produced samples (unmodied activated carbon
and the s tandard sample). Three samples that were
created using both modied and unmodied carbon
are shown in Table 2 by their cry s tal sizes.
Table 2. Scherrer’s equation-derived e s timated particle size
Fig. 2. XRD spectra sample using unmodied carbon and 0.09 M
of zinc acetate dihydrate.
Photocatalytic Degradation of Azo Dye Methyl Orange by ZnO Ahmed Jaber Ibrahim
10
3.1.2. S tudy analysis using TCP, BET, and SEM
The ZnO percent (%) in samples was calculated
using TCP analysis. ZnO content was 9.94, 10.74,
and 31.81 % in samples a, b, and c, respectively.
according to TCP analysis. These ndings sugge s t
that raising the zinc acetate concentration leads
to an increase in the samples’ ZnO content. ZnO
percent was 19.8% for unmodied carbon in the
TCP measurement, demon s trating that ZnO % is
decreased in the absence of surface modication
of activated carbon. The specic surface area of
produced ZnO nanoparticles was measured using
BET analysis (only for sample c). The specic
surface area of this material is higher than the
specic surface area of traditional ZnO particles
(4.49 m2 g-1), according to the results of the BET
s tudy, which also revealed a total pore volume of 0.1
cm3g-1 and average pore width of 51.6 nm [28]. The
inclusion of activated carbon in the produced ZnO
accounts for this elevated amount. ZnO, activated
carbon, and AC-ZnO surface morphology and
textural characterization are signicant criteria that
could improve the eciency of the photocatalytic
activity [29]. SEM images of samples a, b, and c
as shown in Figure 3, were taken to evaluate the
morphology feature of produced ZnO nanoparticles.
It is evident that when the concentration of zinc
acetate rises, ZnO particles ll the pores of the
activated carbon, achieving uniform coverage on a
large portion of the activated carbons. Even though
the large holes in the activated carbon were lled
with ZnO particles, which prevent the porosity of
the carbon surface, sample C nevertheless displays
a porous nature with a sizable amount of surface
area and pore volume [30].
3.2. S tudy Ultraviolet-Visible (UV-Vis)
spectroscopic examination
Two adsorption bands at wavelengths of 464 and
272 nm can be seen in the methyl orange adsorption
spectra. While the breakdown of the azo link, which
results in bleaching, causes the absorption band
at 464 nm to drop, the methyl orange absorption
band decreases at 272 nm due to the phenyl rings
degrading and completing mineralization. As seen
in Figure 4, decolorization rates are very low, and
there is no total degradation or mineralization when
ZnO photocataly s t is not present. In contra s t to
what is depicted in Figure 5, complete bleaching
and degradation take place when ZnO nanoparticles
are present as a cataly s t. For additional research,
Figure 6 shows the absorption trend over time at
wavelengths of 464 nm and 272 nm under three
dierent circum s tances: Ultraviolet radiation, dark
medium, and ultraviolet radiation with the cataly s t
present.
The following gure demon s trates that dye methyl
orange completely bleaches and degrades in the
presence of a ZnO cataly s t within 200 minutes; s till,
these processes were nonexi s tent in the absence of
Fig. 3. SEM characterization for three samples (a, b, and c)
Anal. Methods Environ. Chem. J. 5 (4) (2022) 5-19
11
a photocataly s t. After swirling the dye and cataly s t
combination in the dark for 20 minutes, data analysis
revealed that no simple degradation occurred and
that the adsorption of dye methyl orange onto the
surface cataly s t was s table. As a result, dye and
cataly s t mixtures were swirled for 30 minutes in
complete darkness in each experiment to guarantee
that adsorption equilibrium was reached.
Fig. 4. Shows the reaction sy s tem’s adsorption spectrum without a ZnO photocataly s t,
with 10 mg L-1 of methyl orange as the cataly s t.
Fig. 5. Shows the reaction sy s tem’s adsorption spectrum at pH 6, 200 mg L-1
of ZnO photocataly s t, and 10 mg L-1 of methyl orange.
Photocatalytic Degradation of Azo Dye Methyl Orange by ZnO Ahmed Jaber Ibrahim
12
3.3. pH eect
The pH signicantly aects the adsorption
capacity of the adsorbent and removal eciency
by changing the adsorption chemi s try of the
adsorbent-adsorbate [31]. The appropriate contact
time was used to dissolve 20 mg of photocataly s t
into 100 mL of MO solution (10 mg L-1) for the
pH-related experiments. To change the pH of the
solution, 0.1 M HCl and 0.1 M NaOH solution
were utilized. Figure 7 shows how the pH of the
solution aects how quickly MO is bleached and
degraded by AC-ZnO. It is clear that pH six results
in the fa s te s t bleaching and dye degradation of
methyl orange (MO). Therefore, pH six was chosen
for additional research. Changes in the electro s tatic
attraction between the dye MO and the ZnO surface
can explain this removal process’s pH-dependent
behavior. In comparison to acidic circum s tances
(where the driving force is higher), the pollutant
adheres to the adsorbent particles more eectively
under optimal electro s tatic attraction [32]. ZnO’s
surface charge is positive at low pH 9 [33]. As a
result, anions are more likely to bind to ZnO in an
aqueous environment at a low pH of 9. However,
the pKa for methyl orange has been reported to
be 3.8 ±0.02 [34]. As a result, at pH values higher
than pKa, the concentration of methyl orange in its
anionic form is greater than in its cationic form. The
amount of methyl orange that could be adsorbed
onto ZnO increased as the solution pH was raised
to 6. The rate of bleaching and dye degradation was
shown to decrease at increasing pH values above 6,
because the hydroxyl radical’s oxidation potential
decreases with the rising pH of the solution [35].
Additionally, the anionic form of methyl orange
competes with OH ions in the solution due to the
greater OH content, which lowers the capacity of
methyl orange to bind to ZnO [36].
Fig. 6. The light absorption at wavelengths of 272 and 464 nm changes over time under the conditions
at pH 6, 200 mg L-1 of photocataly s t, and 10 mg L-1 of methyl orange.
Anal. Methods Environ. Chem. J. 5 (4) (2022) 5-19
13
3.4. Photocatalys t dosage eects
To inve s tigate the eects of photocataly s t dose on
the bleaching and degradation of MO, a solution
with a primary methyl orange concentration of
10 mg L-1 and a reaction duration of 180 min
was added to a range of photocataly s t doses from
100 to 500 mg L-1. Figure 8 depicts the ndings
of these analyses. The result demon s trated that
bleaching and degradation increased when the
cataly s t concentration was raised to 200 mg L-1.
When the cataly s t concentration was increased
to 400 mg L-1, bleaching and degradation did
not change noticeably, but when the cataly s t
concentration was increased to 500 mg L-1,
bleaching and degradation decreased. As cataly s t
concentration grew, more surface active sites
were available. As a result, there is an increase
in the generation of hydroxyl radicals, which
increases ZnO’s photocatalytic activity. UV
light cannot penetrate the cataly s t’s surface
when the milky solution is in excess. As a result,
these occurrences can reduce the generation of
hydroxyl radicals, reducing the eectiveness
of dye deterioration and solution discoloration
[33,37]. For subsequent research, a dose of 200
mg L-1 photocataly s ts was used.
Fig. 7. The pH eects of methyl orange under the conditions of 200 mg L-1 photocataly s ts, and 10 mg L-1
methyl orange on; (a) the rate of bleaching, (b)the rate of dye degradation
Photocatalytic Degradation of Azo Dye Methyl Orange by ZnO Ahmed Jaber Ibrahim
14
3.5. MO concentration eects
Several methyl orange (MO) concentrations
(5-20 mg L-1) at a reaction duration of 180 min
and a primary pH of 6 were s tudied to ascertain
the impact of primary MO concentration on the
method’s eectiveness. Figure 9 displays the
outcomes of these analyses. The ndings showed
that raising the initial dye concentration reduced
bleaching and degradation. This might be brought
on by a decline in the number of active surface
sites. As a result, the generation of hydroxyl
radicals declines, which may result in decreased
photocatalytic activity. Furthermore, as the dye
concentration increases, the di s tance of a photon
into a dye solution shortens. To conduct additional
research, MO at a concentration of 10 mg L-1 was
chosen because, at higher dye concentrations, the
dye molecules may absorb more sunlight than
the cataly s t, which could reduce the cataly s t’s
eectiveness [38, 39].
Fig. 8. Eect of cataly s t amount of methyl orange under the conditions of pH 6, 180 minutes,
and 10 mg L-1 of methyl orange on; (a) the rate of bleaching (b)the rate of dye degradation
Anal. Methods Environ. Chem. J. 5 (4) (2022) 5-19
15
3.6. Eects of s tirring the mixture
To find out how s tirring the solution affected
the bleaching and degradation of MO, specific
te s ts were conducted at reaction periods of
180 minutes, pH levels of 6, primary MO
concentrations of 10 mg L-1, and cataly s t doses
of 200 mg L-1. Figure 10 percent (%) the findings
of these analyses. The results show that swirling
the solution exacerbated the bleaching and
deterioration. Fir s t, agitation causes turbulence
in the solution, which promotes the solution’s
absorption of oxygen. In the synthesis of
hydroxyl radicals, soluble oxygen is crucial.
Second, s tirring the solution shortens the time
needed for equilibrium by accelerating MO
transfer and surface diffusion [40].
Fig. 9. Eect of primary concentration of MO under the conditions
of pH 6, and 200 mg L-1 photocataly s ts on; (a) the rate of bleaching (b)the rate of dye degradation
Photocatalytic Degradation of Azo Dye Methyl Orange by ZnO Ahmed Jaber Ibrahim
16
4. Conclusion
In the current work, methyl orange from aqueous
solutions was subjected to a dye degradation process
employing ZnO as a photocataly s t. The results
showed that the AC-ZnO sy s tem successfully
de s troyed the MO dye. When ZnO was present,
the rate of deterioration was high, but when ZnO
wasn’t there, the degradation rate decreased. The
be s t dye degradation conditions were found at a pH
of 6.0 with 200 mg L-1 of photocataly s t for 10 mg
L-1 of MO based on agitating the dye solution in an
air environment. The outcomes also demon s trated
that synthetic photocataly s ts in the actual world
had much high ecacy and that recovering and
reusing photocataly s ts hurt the degradation rate
and bleaching. The current s tudy oered a novel,
co s t-eective adsorbent with great promise for
treating wa s tewater contaminated with dyes.
5. Conicts of intere s t
There are no conicts to declare
6. Acknowledgements
This research is supported by the Physical
Chemi s try Lab., Chemi s t Department, College
of Education for pure science (ibn-al Haitham),
University of Baghdad.
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Photocatalytic Degradation of Azo Dye Methyl Orange by ZnO Ahmed Jaber Ibrahim
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
Research Article, Issue 4
Analytical Methods in Environmental Chemi s try Journal
Journal home page: www.amecj.com/ir
AMECJ
Review Article: Development of biodegradable lms using
nanocellulose for food packaging application
Asha Valsalana,*, and Paramasivan Sivaranjana a
a Department of Chemis try, School of Advanced Sciences, Kalasalingam Academy
of Research and Education Krishnankoil, Srivilliputhur, Tamil Nadu 626126
ABS TRACT
Due to the development of nanotechnology and changing cu s tomer
demands for food safety and hygiene, the food packaging indu s try is
growing signicantly. In today’s worldwide market, active packaging
oers a number of advantages over traditional wrapping because of
its capacity to absorb or release sub s tances to improve the shelf life
of food. Traditional food packaging materials are dicult to recycle
and are made from nonrenewable fossil fuels. The development of
biodegradable lms using Nano cellulose can be a good replacement
for synthetic pla s tic packaging materials and can be a good solution
for this problem. Other than that it has multiple advantages regarding
tensile and physical properties, also as reducing health hazards.
Tensile and physical characteri s tics are improved and water vapor
permeability is decreased with the addition of cellulose nanoparticles
to the biodegradable lms/biodegradable composite lms. The
production of biodegradable materials employing Nano cellulose has
been covered in this review s tudy in four dierent ways, including
extracts from agricultural wa s te, rice husk, various plant extracts, and
biopolymer composite material in food packaging. The reason for
using Nano cellulose-based biodegradable lms in food packaging
is also reviewed in this article. The key points for future research in
overcoming the problems related to Nano cellulose and biodegradable
lms are also predicted in the paper.
Keywords:
Nano cellulose,
Biodegradable Films,
Food Packaging,
Extraction methods,
Te s t methods,
Tensile and physical characteri s tics
ARTICLE INFO:
Received 16 Aug 2022
Revised form 20 Oct 2022
Accepted 11 Nov 2022
Available online 30 Dec 2022
*Corresponding Author: Asha Valsalan
Email: id-bs.ashav@sbcemail.in
https://doi.org/10.24200/amecj.v5.i04.207
1. Introduction
The rapid population expansion, high s tandards
of living, and high rates of energy and goods
consumption all contribute to signicant levels of
wa s te generation that, if not properly disposed or
recycled, represent serious risks to the environment
[1]. Pla s tic wa s te is a non-biodegradable component
that can linger in the environment for hundreds
of years. Both people and animals should avoid
them because of how much land they consume.
Additionally, as pla s tics are petroleum-based
materials, the ongoing engineering of pla s tics,
which results in the depletion of petroleum, oers
additional issues [2]. Over the pa s t few decades,
petroleum-based materials have been widely
used in a variety of indu s tries, especially for food
wrapping because of their aordability, exciting
technological features, as well as mechanical and
physical capabilities. The bulk of pla s tics made
from fossil fuels is bad for both public health and
the environment [3]. In order to replace petroleum-
based goods in food packaging applications,
more renewable alternatives are being sought
after. A large amount of the numerous tones of
------------------------
21
inedible plant debris produced each year gets
landlled. Reusing lignocellulose biomass wa s tes
has received attention recently as a healthy and
practical sub s titute for the usage of fossil fuels.
Due to the enormous amount of agricultural wa s te
produced annually, this reuse serves two purposes:
Reducing landll overow and Reducing reliance
on fossil fuels, with all the attendant environmental
advantages [4]. They might also be referred to
as bio-wa s te. Sludge from wa s tewater treatment
plants, food manufacturing plant wa s te, and trade
trash are all examples of biodegradable wa s tes
[5]. Nowadays, biodegradable wa s tes are used
in an eective manner for the manufacturing of
various products, especially in the food packaging
indu s tries. The food packing sector is currently
looking for lightweight, biodegradable packaging
in an eort to utilize fewer resources, produce
less wa s te, save transportation co s ts, maintain the
freshness of food materials, and also to reduce
health hazards [6]. Pla s tic food packaging materials
are replaced by producing biodegradable lms
incorporated with Nano cellulose extracted from
various types of biodegradable wa s tes like agri-
wa s te, plant extracts, biodegradable polymers, etc.
Biodegradable lms are produced by adding some
additives with them during the manufacturing
process. Biodegradable lms are an alternative to
petroleum-based and pla s tic-based lms.
2. Experimental
2.1. Nano Cellulose
Using various extraction methods, native cellulose
is converted into the di s tinctive and natural
molecule known as Nano cellulose. The amazing
properties of Nano cellulose, such as its di s tinct
surface chemi s try, exceptional physiochemical
toughness, and abundance of hydrophilic groups
for alteration, are increasingly attracting attention.
In addition to being environmentally friendly,
it has signicant biological qualities such as
recyclability, bioactivity, and non-toxicity [7]. The
term “Nano cellulose” refers to a class of cellulosic
nanoparticles with at lea s t one dimension up to
100 nm. Cellulose nanobers (CNF), cellulose
nanocry s tals (CNC), and bacterial Nano cellulose
(BNC) are the three varieties of “Nano cellulose”
that may be identied by their diameters [8]. The
picture of Nano cellulose is depicted in Figure 1.
2.2. Basic Extraction method of Nano cellulose
Many techniques have been developed to extract
Nano cellulose from cellulose ber. The diverse
extraction methods led to a variety in the kinds and
quality of the Nano cellulose that was produced.
The three fundamental extraction techniques
are acid degradation, enzymatic hydrolysis, and
mechanical procedure. Acid hydrolysis is one of
the main techniques for eliminating Nano cellulose
Review of food packaging application by nanocellulose Asha Valsalan et al
Fig. 1. The picture of Nano cellulose [8]
22
from cellulosic products. Because cellulose chains
include equally arranged and un s tructured regions,
the organized regions survive acid degradation
while the disorganized regions break down quickly.
The acid mo s t frequently used for acid hydrolysis
is sulfuric acid. Enzymes are used in the biological
process known as enzymatic hydrolysis to degrade
or modify brous material. The biological treatment
with enzymes may typically be carried out under
mode s t conditions, although a lengthy procedure
is needed. To solve this problem, enzymatic
hydrolysis is always used in conjunction with other
methods. A mechanical process isolates cellulose
brils, resulting in Micro reinforcing materials
cellulose, by using a powerful shear force to split the
cellulose bres along their longitudinal axis. The
three mechanical processes that are mo s t frequently
used are ball milling, high-pressure homogeneity,
and ultrasonication [9]. The separation of Micro
cellulose from biomass including lignocellulose is
depicted in Figure 2.
2.3. Types of Nano cellulose
Below is an explanation of the three dierent types
of Nano cellulose: cellulose nanobers (CNF),
cellulose nanocry s tals (CNC), and bacterial Nano
cellulose (BNC).
2.3.1. Cellulose Nanobers (CNF)
Length, ela s tic, and intertwined nanoscale bers
known as “cellulose nanobers” (CNF) can be
recovered from lignocellulose-containing crops.
Due to their superior hardness, rigidity, lightweight,
environmental friendliness, and recyclability,
CNFs are being researched for usage in a variety of
applications, including electronics, packaging, and
nanocomposites [10]. CNF have cry s talline and
amorphous regions, and they resemble ropes. When
dried, CNF form a highly connected network as a
result of s trong intermolecular hydrogen bonding
[11]. The SEM picture of CNF is displayed in the
following Figure 3.
2.3.2. Cellulose Nanocrys tals (CNC)
The particles known as cellulose nanocry s tals
(CNC) are small, s ti, and rod-shaped. It is also
known as Cellulose Nano whiskers. They are
typically created through the process of s trong acid
hydrolysis, which separates the s ti cry s talline
sections from the amorphous phases of cellulose
s trands [11]. Researchers in both research and
indu s trial applications have shown a great deal of
intere s t in cellulose nanocry s tals (CNCs) because
of their intriguing s tructural features and di s tinctive
physicochemical properties, like amazing s tructural
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
Fig. 2. The illu s tration of Nano cellulose extraction from lignocellulose biomass [9]
23
rigidity, large surface region, numerous hydroxyl
groups for chemical treatment, lightweight, and
biodegradability. CNCs are a s trong candidate for
use in a variety of indu s tries. In addition, cellulose
nanocry s tal extraction and surface modication
continue to advance in response to producers’
growing demand for cellulose nanocry s tal-based
goods [12]. Figure 4, presents the image of CNC.
2.3.3. Bacterial Nano cellulose (BNC)
Bacterial Nano cellulose (BNC), a naturally
occurring biopolymer of enormous signicance
in many technical domains, has exceptional
physicochemical and biological features. Specic
species of bacteria generate bacterial Nano
cellulose (BNC), a promising natural biopolymer,
as an exopolysaccharide of D glucopyranose. BNC
Review of food packaging application by nanocellulose Asha Valsalan et al
Fig. 3. SEM of Cellulose Nanobers (CNF) [11]
Fig. 4. Cellulose Nanocry s tals (CNC) [12]
24
is 99 percent water but has excellent mechanical
properties. Due to its ability to s tore water and
its Nanos tructured form, which is similar to the
extracellular matrix protein collagen, BNC is
particularly suitable for cellular immobilization
and adhesion. Bacterial Nano cellulose is suited for
a variety of uses since it has a number of unique
characteri s tics and is a product that is generally
regarded as safe (GRAS) [13]. The picture of BNC
[14] is displayed in the Figure 5.
2.4. Reason for using Nano cellulose based
biodegradable lms in food packaging
The main objective of food packaging is to
preserve the production of agricultural products
through s torage and delivery. As a result, it’s
critical to grow the shelf life of food goods by
avoiding issues such as microbial deterioration
and chemical pollutants, carbon dioxide, water
vapor permeation, ammable sub s tances,
dampness, and light exposure as well as outside
physical inuences. The materials used for
packaging mu s t ensure physical safety and
e s tablish suitable physicochemical conditions to
ensure food quality [15]. Hence Nano cellulose
incorporated biodegradable lms thus produced
plays a vital role as a food packaging material by
overcoming all these defects due to their benecial
amount of physical, chemical, water solubility,
and water absorption properties. These properties
are discussed briey in the upcoming sections.
In this paper, a review based on development of
biodegradable lms using Nano cellulose from
various extracts and useful analyzing in food
packaging applications was presented. This review
paper’s s tructure is followed as: the experimental
section evaluates the exi s ting research on
biodegradable lms using Nano cellulose in four
dierent directions, the results section gives the
summary of this paper, another section comes out
with the key points to be researched in the future
and conclusion.
Fig. 5. Bacterial Nano cellulose (BNC: A-D) [14]
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
25
3. Results of literature review
This section reviews the development of
biodegradable lms using Nano cellulose from
– agricultural wa s te, rice Husk, various plant
extracts, and biopolymer composite material in
food packaging. Agricultural Wa s te: Agro-wa s tes
come from a variety of materials, including rice
husks, wheat s traw, palm oil bers, pineapple,
orange, and tomato pomace, grape pomace, lemon
peels, and sugarcane bagasse [16]. Agro-indu s trial
wa s te is a byproduct of agricultural-based
businesses that is frequently rich in lignocellulose
resources and bioactive compounds. The indu s tries
where these pollutants are frequently disposed of
in uncontrolled procedures have weak regulations
for their management. These actions have had a
negative impact on the ecology and the economy
as a whole. Due to this, extensive research has been
done to extract useful materials from these wa s tes
[17]. Rice husk is a lignocellulose biomass, that
comes under non-woody biomass sources. Non-
woody plants are ones that have frail s tems and are
susceptible to yearly regrowth to the ground. They
go by the name herbaceous plants as well [18].
Some of the Plant extracts used for Nano cellulose
production, that we have discussed in the upcoming
section are as follows: sugarcane bagasse, olive
tree pruning scraps, yam beam, sunower oil cake
(SOC), Natural essential oil from the clove bud,
buered with fermented black tea and cellulose
nanocry s tals ber. Biopolymer composites are
reinforced polymer materials in which the polymer
functions as a matrix resin that reaches the bundles
of reinforcement and forms bonds with it [19].
The upcoming sections deeply describe the related
works of the above-mentioned directions.
3.1. Biodegradable lms using Nano cellulose
from agricultural was te
Ilya et al examined the eects of dierent
sugar palm nano brillated cellulose (SPNFCs)
reinforced sugar palm s tarch (SPS) concentrations
on the morphological, s tructural, and physical
characteri s tics of the bio nanocomposite lm [20].
S tarch granules and laments from sugarcane plants
are regarded as agricultural wa s te. A suspended
sentence of sugar palm Nano brillated cellulose
(SPNFCs) with a mean duration of many μm in
diameter and diameters of 5.5 and 0.99 nm was
made from sugar palm bres using a high-pressure
homogenization technique. SPNFCs were then used
to s trengthen the sugarcane bagasse carbohydrate
sequence for the creation of bio nanocomposites
using a remedy technique. The miscibility of SPS
and SPNFCs was shown to be good using FESEM
analysis of the ca s ting solution. The FTIR analysis
proved that intramolecular hydrogen bonds exi s ted
between the SPS and SPNFCs and that they were
compatible. SPS/SPNFC bio nanocomposite
lms outperform control carbohydrate bio
nanocomposite lms in terms of physical and
mechanical properties. The segmental molecular
chains of the carbohydrate bio composite became
less mobile and exible as a result of the addition
of Nano-reinforcements, which decreased the
elongation at break. The ductility s trength
and modulus of the nanocomposite lms were
dramatically increased from 6.80 to 10.68 MPa and
59.07 to 121.26 MPa, respectively, by the increase
in SPNFC reinforcement from 0 to 1.0 wt. percent.
Adriana Nicoletta Frone et al used plum shells’
agricultural residues to Nano cellulose as a
biopolymer reinforcement [21]. Cellulose
nanocry s tals (CN) and cellulose nanobers (CF) are
the two types of Nano cellulose derived from
plum seed shells. For the r s t time, CN and CF of
cherry fruit skins were used as reinforcing agents
in a polylactic acid/poly(3-hydroxybutyrate) (PLA/
PHB) matrix using a solution-ca s ting technique.
A co s t-eective and successful s trategy to utilize
agricultural wa s te as a source of production for
elevated goods is to adopt this technique. Some of
the CF type’s limitations in terms of morphological
characterization and thermal performance include
that type CN cellulose nanocry s tals are more
similar in shape, have a smooth texture, and have a
larger image size. The melting temperature of CN
was somewhat less than that of CF due to the sulfate
groups added to the cellulose’s external side during
the hydrolysis process, which led to the dewatering
Review of food packaging application by nanocellulose Asha Valsalan et al
26
of the cellulose ber and a decrease in thermal
properties. Thermal and XRD te s ts showed that
adding CN improved the PLA/PHB bio-composite
lm’s thermo s tability and cry s talline nature.
According to report of Reshmy et al, jackfruit
(Artocarpus heterophyllus) skin was used as the
hydrolysis source for pure Nano cellulose [22].
Using liquid water evaporation, the thin lms were
created using BS as the lling, activator, and NC
as the sub s trate. Solvent ca s ting Nano cellulose
and various pla s ticizers were used to make various
thin lms. FT-IR and XRD were used to describe
thin lms, and FESEM was used to explore surface
changes. The advantages of this s trategy are as
follows: (i) To avoid chlorine bleaching solutions
for natural bers, the raw material was bleached
with a 4 percent soapnut solution. (i) A unique
lling named Boswellia serrata (BS) was used to
enhance the properties of NC thin lms for future
applications. The breakage of bonds between NC
and pla s ticizers caused commodities to decay
during food s torage due to the large price of WHC
for NC alone and NC/Gly/BS. This resulted in less
moi s ture absorption and swelling compared to
other thin lms.
Sheng Xu et al s tated that Artemisia selengensis
s talks were used as a source of hemicelluloses (ASH)
and cellulose nanocry s tals to create biodegradable
lms (ASCNC) [23]. Acid hydrolysis was used to
separate the ASCNC from the ASC. SEM, TEM
and FTIR methods are used for the te s t results,
and OT and WVP are also checked. The composite
membranes enhanced by ASCNC exhibited
increased durability and performed much better as a
water vapor shield when contra s ted to the reference
ASH/PVA lm. Additionally, compared with the
control screen, the ASCNC-enhanced ASH/PVA
composite material decreased light transmission
considerably. In the morphology of composite
lms, the ASH/PVA lm’s cross-section had many
voids, and the s tructure was loose. With ASCNC
loading reaching 9%, the composite lm’s tensile
s trength improved by 80.1 percent to 36.21 MPa,
while the water vapor transfer rate fell by 15.45
percent when 12 percent ASCNC was added.
Banana pseudo- s tems were proposed as a potential
source of environmentally friendly Nano cellulose-
based recyclable pla s tic as an agricultural wa s te
by R. H. Fitri Faradilla et al [24]. This s tudy
looked closely at the impact of nanoclay (NC) and
graphene oxide (GO) as nanollers and glycerol
as a lubricant on the mechanical, morphological,
chemical, thermal, and impact resi s tance of banana
pseudo- s tem Nano cellulose lms. TEM, SEM,
FEI NOVA 230, AFM, Bruker, X-ray diraction
(XRD), panalytical Xpert multipurpose X-ray
diraction, thermos gravimetric analysis, and
dierential scanning calorimetry, ATR-FTIR,
Bruker IFS 66/S, and Mocon-OX-TRAN are the
te s ting methods used to nd the results. Synergi s tic
eects were seen when nanoparticles and glycerin
were combined. Tensile modulus and exibility
were both risen, and the contact area of the motion
pictures was considerably higher than that of lms
containing only nanoparticles. The thermopla s tic
had a massive eect on the barrier properties of
the composites, while the glycerol concentration
was positively correlated with the water vapour
permeability. Oxygen permeability, however, was
reduced when glycerol content increased. Also, the
lms’ tensile s trength was found to be improved by
NC and GO, but not their ela s ticity. These results
s trongly imply that the characteri s tics of the banana
pseudo- s tem Nano cellulose lm may be altered
by modifying the nature and amount of additional
chemicals.
It was sugge s ted to separate bulgur bran into
cellulose and hemicellulose-rich components,
opening the way for exploiting this under-utilized
agro-indu s trial biomass by Didem Sutay Kocabas
et al [25]. Commercial cellulose nanocry s tal
(CNC) and cellulose nanober (CNF) were
added to the hemicellulose sub s trate to remove
bottlenecks. The characteri s tics of plain and Nano
cellulose-reinforced lms were compared using
the thermogravimetric analysis (TGA), dierential
scanning calorimetry (DSC), and Fourier transform
infrared spectroscopy (FTIR) techniques. A dense
architecture was discovered by SEM analysis
of lms reinforced with CNC and CNF. The
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
27
hemicellulose channel’s tensile properties were
signicantly improved by adding CNC and CNF as
lls. After adding Nano cellulose, the lms’ water
vapour permeability (WVP), light transmittance,
overall mismatching, and biocompatible all fell.
Additionally, the hemicellulose precipitate contains
lignin (6.70 percent), s tarch, potassium acetate,
and other impurities. The proposed full-quadratic
model was shown to have excellent accuracy
within the 95 percent condence interval (R2 =
0.9877). According to the ndings, lms with 10%
(w/w) CNC and 10% (w/w) CNF incorporation had
a 21.3 percent lower FWS when compared to neat
pictures.
Krishnavani Pavalaydon et al extracted Micro
cellulose from cassava peel and coco ber
using chemical processes such as mercerization,
bleaching, and acid hydrolysis [26]. Taguchi design
is the technique used in the process of Fourier-
Transform(FT). The te s t techniques employed in
the procedure include DLS, transmission electron
microscopy, and infrared spectroscopic. Bio-
nanocomposite lms were created using the solvent
ca s ting method using polyvinyl alcohol (PVA) as
the matrix. Excellent sources of Micro cellulosic
include sugarcane bagasse and coir, which can be
used to create bio-composites having good s trength
properties. Nanocellulose, which is made from
bagasse, r s t appeared as crooked and minute
circular particles. The highe s t tensile s trength (38.2
MPa) was achieved for CNCs derived from coir at
a CNC/PVA loading of 0.5 wt%, which is a 96.9
percent improvement in s trength properties over
the un s trengthened PVA sub s trate.
According to Vu Nang An et al, the goal of the s tudy
is to separate high-cry s tallinity Cellulose Nano
Cry s tals (CNCs) from Vietnamese agricultural
residues (Nypa Fruticans trunk, coconut husk ber,
and rice husk) [27]. Using a three- s tep process that
involved pre-treatment with formic/peroxyformic
acids, processing with hydrogen peroxide/sodium
hydroxide, and disintegration by hydrolysis, so,
CNCs were extracted from the aforementioned
natural origin. After every phase of behavior, the
thermophysical characteri s tics of the obtained
resources were examined using XRD, TGA, TEM,
and FT-IR s tudies. Nano cellulose bres were
found to have improved thermal s tability through
thermogravimetric analysis, making them suitable
for the creation of bio nanocomposites for a variety
of uses, including the production of functional
paper, exible assi s tance for the synthesis of metal/
oxide metallic nanoparticles, and cell wall lters.
The amorphous portions of the cellulose s tructure
have been utterly de s troyed by the corrosive ions,
remaining the cry s talline s tructure unharmed.
Because of this, CNCs are both shorter and have
a higher CrI than cellulose. The CNCs nanobers
have a high cry s talline index (almo s t 80%),
increased heat s tability, and indicating an extensive
variety of applications. Hui Li et al aimed to isolate
cellulose nanocry s tals (CNC) from pea hull and
te s t their capacity to s trengthen carboxymethyl
cellulose (CMC) lm [28]. To better utilize and get
rid of cheap and plentiful farmed pea husk trash,
the needle-like CNC was nally recovered from
the trash by alkalization, washing, and sulfuric acid
degradation. The solvent ca s ting process produced
CNC, which was then used as a reinforcing
component in the creation of compound products
which was depended on CMC. The te s ts included
scanning electron microscopy, ATR-FTIR and X-ray
diraction analysis, hand-held digital microscopy,
gravimetric technique, and DSC. The CMC/CNC
nanocomposites saw improvements in their thermal
properties, ultraviolet layer, mechanical properties,
and water vapor barrier. The lower the endothermic
peak, the larger the additional concentration of
CNC. This was due to a lower hygroscopic anity
caused by the addition of additional sulfate groups
to the CMC/CNC nanocomposite. In comparison
to pure CMC lm, the 5-weight percent CNC
reinforced composite lm had a 53.4 percent lower
water vapour absorption and a 50.8 percent better
durability.
According to research of Jayachandra S. Yaradoddi
et al, the objective is to transform carboxymethyl
cellulose (CMC), which is generated from
agricultural residues, into a workable, biodegradable
pla s tic that often includes a packaging material [29].
Review of food packaging application by nanocellulose Asha Valsalan et al
28
Mixtures were created using CMC (trash generated),
gelatin, agar, and varied levels of glycerol; 1.5
percent (sample A), 2 percent (sample B), and 2.5
percent (sample C) were added. CMC was recovered
from agricultural residues, primarily cane sugar
wa s te. Thermogravimetric analysis(TGA), Fourier
Transform Infrared (FTIR) spectroscopy, and
Dierential Scanning Calorimetry (DSC) were used
to describe the physiochemical parameters of each
created biodegradable pla s tic (samples A, B, and
C). Sample C, which was made with gelatin, CMC,
agar, and 2.0 percent glycerol, was discovered to be
the be s t combination and ideal for possible future
use in food applications because it had identied
the s trengths like the smalle s t water vapour
permeability and the greate s t recyclability rate
when compared to other samples. As commercially
available CMS is currently too expensive, farm
wa s te-derived carboxymethyl cellulose (CMC) is
used largely to reduce the co s t of lm development.
As a consequence, sample C’s (gelatin+CMC+agar)
lm functioned better than samples A and B related
to the addition of glycerol at a softener concentration
of 2.0 percent.
Table 1 shows the overall review of the techniques
such as Nano cellulose produced, the material,
tes ting method, advantages, limitations, and
performance parameters. Table 1 explained the
direction - Biodegradable lms using Nano
cellulose from agricultural wa s te.
Table 1. Review on Biodegradable lms using Nano cellulose from agricultural wa s te
Technique Nano cellulose
Produced
Material Te s ting
Method
Advantages Limitation PP Ref.
HPHM & SCM SPNFCs AW-SPF FESEM and
FTIR MP> CSBNF Reduced the
elongation at break
Aecting mobility
and ductility of
the biopolymer’s
segmental
TS=6.8 - 10.7 MPa
Modulus= 59.1 - 121.3
MPa
[20]
Solution-ca s ting
method
CF and CN Agri wa s te-
PSS
Thermal
and XRD
analyses
Co s t-eective and
well-organized
Compared to the
CF type, type CN’s
morphology is much
more regular, its
surface is ner, and
its aspect ratio is
higher.
The thermal s tability
was improved by
the calculation of
CN and cry s tallinity
of the PLA/PHB
biocomposite lm
[21]
Acid hydrolysis,
BS ller and NC Solvent ca s ting
Nano cellulose
(i)Agri wa s te
- Jackfruit
peel
(ii)Bleaching
Agent
- Soapnut
solution
FT-IR, XRD
and FESEM (i)Chlorine bleaching
solutions
(i)Boswellia serrata (BS)
improve the characteri s-
tics of NC thin lms
Because of the
tall value of WHC
for NC alone
and NC/Gly/BS,
commodities spoiled
during food s torage
due to breakdown of
bonds between NC
and pla s ticizers
When compared
to other thin lms,
this resulted in less
moi s ture absorption
and swelling
[22]
Acid hydrolysis Hemicelluloses
(ASH) and
cellulose
nanocry s tals
(ASCNC)
Agri wa s te
- Artemisia
selengensis
s traw
SEM, TEM
and FTIR
methods,
OT and
WVP is also
checked.
water vapour shield
eectiveness, and light
transmission reduction.
ASH/PVA lm’s
cross section had a
lot of voids and the
s tructure was loose
With ASCNC loading
reaching 9%, the
composite lm’s
tensile s trength
improved 80.1 percent
to 36.21 MPa
[23]
Modied Nano
cellulose lm Banana pseu-
do- s tem Nano
cellulose lms
Agri wa s te
- banana
pseudo- s tem
TEM, SEM,
FEI NOVA
230, AFM,
Bruker,
XRD, ATR-
FTIR,
(i) Enhanced tensile
power and exibility,
(ii) The greate materials’
contact angle
(iii) The lm resi s tance
aected by the pla s ti-
ciser.
(i)Oxygen permea-
bility, reduced when
glycerol content
increased
(ii)Film’s tensile
s trength was found
to be improved by
NC and GO, but not
their ela s ticity
By varying the type
and concentration
of added additives,
the properties of the
banana pseudo- s tem
Nano cellulose lm
might be modied.
[24]
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
29
3.2. Biodegradable lms using Nano cellulose
from rice Husk
Rice Husks were cleaned, chemically hydrolyzed,
and ultrasonically processed at a low temperature
by Pedro Nascimento et al to produce Nano
cellulose [30]. An agricultural sector byproduct
called rice husk can be utilized to make Nano
cellulose. SEM, TEM, XRD, FTIR, TGA, and DSC
are the te s ts undergone to nd the characterization
of Nano cellulose-reinforced s tarch-glycerol lms.
When added as reinforcement to the s tarch lms,
the Nano cellulose created webs of connected, tiny
bers (about 100 nm in diameter) that reduced
opacity, increased mechanical characteri s tics, and
were less permeable to water vapor. The inclusion
of 2.5 percent (w/w) of the nano s tructures to
s tarch-glycerol lms increased the mechanical
characteri s tics, water vapor permeability, and
opacity of s tarch lms (made by extrusion). After
the alkaline pre-treatment, the bre surface has
been less tiny as well as its actual con s truction
has been altered. This outcome showed that the
exterior non-cellulosic layer, which is made up
of hemicelluloses and lignin, had been partially
removed. The produced Nano cellulose displayed
lower lignin levels than 0.35 percent, greater
thermal s trength than the raw sub s tantial, and
higher cry s tallinity (up 70%). Sumira Rashid et al
used rice grains with short, medium, and long husks
to extract Nano cellulose [31]. During the s teps of
delignication and acid hydrolysis, the noncellulose
amorphous and noncry s talline cellulose fractions
Bulgur bran hemi-
cellulose
Cellulose nano-
cry s tal (CNC)
and Cellulose
nanober (CNF)
Agri wa s te -
Bulgur bran
TGA,
DSC,
FTIR
(i)Revealed a compact
s tructure
(ii)Increased the tensile
s trength
(i)Water vapor per-
meability (WVP),
light transmittance,
overall colour
dierence, and
biodegradability all
are decreased
(ii)Contains lignin
(6.70%), s tarch,
potassium acetate
(i) Have great accuracy
(R2 = 0.9877)
(ii)10% (w/w) CNC-
and 10% (w/w) CNF
incorporated lms had
a 21.3 percent decrease
in FWS
[25]
Bleaching,
Acid hydrolysis
and
Solvent ca s ting
process
Bio-nanocom-
posite lms
with polyvinyl
alcohol (PVA)
Agri wa s te
- Sugarcane
bagasse and
coir
FTIR,
SEM,
DLS
(i)Good bases of Nano
cellulose
(ii) employed to create
bio-composites with
good s trength properties.
Bagasse-derived
Nano cellulose
emerged as irregular
and tiny circular
particles
The ultimate compres-
sive s trength (38.2
MPa) for CNCs of
coir,
[26]
(i)Pre-
treatment
with HCOOH
(ii)treatment
with
H2O2/
NaOH
(iii)disintegration
Cellulose Nano
Cry s tals
(CNC)
Vietnamese
agricultural
wa s te (Nypa
Fruticans
trunk,
coconut husk
ber, and rice
husk)
XRD, TGA,
TEM, and
FT-IR anal-
yses
(i)Nano cellulose bers
have improved thermal
s tability
(ii)The cellulose
material’s un s tructured
regions have been
attacked and de s troyed
by the acidity ions
CNCs have a greater
CrI than cellulose
and have a shorter
length than cellulose
CNCs nanobers have
a high cry s talline index
(almo s t 80%) and in-
creased heat s tability
[27]
(i)Alkali treat-
ment, Bleaching,
For CNC
(ii)Solution ca s t-
ing For CMC
Cellulose nano-
cry s tals (CNC)
Agri wa s te
- Pea hull
wa s te
SEM,
ATR-FTIR
analysis,
Gravimetric
Method,
DSC
CMC/CNC hybrid
sheets’ UV barrier,
mechanical properties,
and heat resi s tance were
enhanced.
The presence of
more sulphate
groups in the CMC/
CNC composite
lm resulted in a de-
creased hygroscopic
anity
(i)50.8 percent im-
provement in tensile
s trength
(ii)53.4 percent de-
crease in water vapor
permeability
[28]
Bleaching Carboxymethyl
cellulose
(CMC)
Agri wa s te
- Sugar cane
bagasse
TGA,
FTIR,
DSC
Sample C:(i)be s t for-
mulation (ii) food pack-
aging (iii) lowe s t water
vapor permeability
Expensive The lm generat-
ed from sample C
outperformed the other
samples A and B
[29]
PP: Performance Parameters
TS: Tensile s trength
HPHM & SCM: High-pressure homogenization method and Solution-ca s ting method
SPNFCs: S tarch reinforced with sugar palm Nano brillated cellulose
AW-SPF: Agriculture wa s te- Sugar palm bers
MP: Mechanical properties
CSBNF: Control s tarch bio nanocomposite lms
CF: Cellulose nanobers CN: Cellulose nanocry s tals
PSS: Plum seed shells
Review of food packaging application by nanocellulose Asha Valsalan et al
30
were successfully removed because of increased
cry s tallinity and altered infrared diraction.
Nanocelluloses are more heat resi s tant than
cellulose. The results are obtained using the te s ts
methods: SEM, TEM, AFM, NT-MDT, SOLVER
NANO, ZP, ATR-FTIR, XRD, TGA, DSC, and HA.
Delignication in conjunction with bleaching led to
gradual depigmentation and turned the material’s
hue to white. The size, cry s tallinity, s trength, and
thermal s tability of long husks built at the nanoscale
were better than those of medium and short husks.
Cellulose nanomaterials are totally consi s tent with
fortifying biopolymers, according to J. F. Delgado et
al [32]. The eects of rice husk cellulose nanobers
(RHCNF) and bacterial nanocellulose (BNC) on
water vapour transport and mechanical behavior
were examined in yea s t biomass lms made from
dispersions (processed by greater homogenized
and subsequently thermal treatment) at pH 6 and
11. BNC was created using a culture of the NRRL
B-42 s train of Gluconacetobacter xylinus. The te s ts
used to analyze the results are NMR, XRD, AFM,
SEM, and WVP. Nano bers could be successfully
added to yea s t matrices and both increased tensile
s trengths, while BNC was more eective than
RHCNF at improving the mechanical properties
of yea s t lms. Despite having identical diameters
and co s ting more to produce than RHNCF, only
BNC improved Young’s modulus, ela s tic modulus,
modulus of rupture, and mechanical hardness of
the yea s t matrix simultaneously. Although they had
little eect at pH 6, both supplements had a 5-weight
percent reduction in water vapor permeability in
lms made at pH 11. The impact of Nano cellulose
on the lm properties of edible coatings was s tudied
by Jeya Jeevahan Jayaraj et al [33]. To produce Nano
cellulose from rice husk, a three- s tage biochemical
process involving alkaline solution, whitening, and
acid hydrolysis was applied. Using native potato
s tarch, glycerin, and varying amounts of Nano
cellulose, the edible coatings (potato s tarch lms)
were produced using the solution ca s ting method (0
percent, 5 percent, 10 percent & 15 percent). AS tM
E96, Digital colorimetric method, and AS tM D882
are the te s t analysis methods conducted to determine
the WVTR, Film color, and Powered s trength of the
come s tible lms. It was discovered that the addition
of Small amounts of cellulosic had created lms with
a lower Water Vapor Transmission Rate (WVTR),
more mechanical s trength, and greater transparency
compared to the control lms. Mechanical s trength
did not increase as the Nano cellulose content was
raised above 10%. With the rise in Nano cellulose
percentage from 5 % to 15 %, the WVTR of bio
nanocomposite edible lms reduced. S tarch
granules only have between 40 and 60 percent
visibility compared to Nano cellulose’s greater
than 95 percent visibility. According to A. Ganesh
Babu et al, bio-lms were produced utilizing
the solution ca s ting process employing liquid
polyvinyl alcohol (PVA) and variable amounts
(5-25wt percent) of rice husk our as reinforcing
ller [34]. FTIR, XRD, TGA, DSC, Tensile te s t,
Surface morphology inve s tigations, WVP, and
Antibacterial te s ts were used to examine the impact
of RHP on the PVA matrix. Some of the advantages
are (i) Bio-lms could tolerate temperatures of up
to 350 °C (ii) The s tronger interaction of polymer
chains is present with lower WVP levels (iii)
Exhibit s trong antibacterial activity (iv) Bio-lms
were clearly homogenous, free of fractures and
phase separation (v) Enhance the biolm’s thermo-
mechanical properties. The presence of hydrogen
bonding makes lms less exible. Tensile modulus
and tensile properties s teadily rise as RHP is infused
into the matrix, reaching their maximum values
at a concentration of 25 percent RHP in PVA and
23.32 MPa and 684 MPa, respectively. Himanshu
Gupta et al focused on using leftover lignocellulose
biomass (such as sugarcane bagasse and rice hulls)
to make carboxymethyl cellulose (CMC), which is
then transformed into a biodegradable lm [35].
The methods of Mercerization and Etherication
are used to create CMC from SCB Cellulose
and Rice Hulls. The characterization is done by
the FTIR, XRD, MC, and TS te s t methods. The
biopolymer lm made from sugarcane bagasse
CMC had the highe s t s trength and elongation
when compared to lms manufactured from
conventional CMC and CMC made from rice
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
31
hull. The bio-composite material formed from
mixed carbohydrate manufactured CMC solution
has exhibited superior mechanical properties in
comparison to the lm made from blended S tarch-
Commercial CMC solution (TS and Elongation).
Because rice husk contains a lot of sodium chloride
and sodium glycolate, (i) it has an adverse eect
on the material’s s trength properties and (ii)it has
lower DS and TS than the SCB. The degree of
CMC sub s titution and the DS of CMC both grow
as do the lm’s transparency, water levels, and
solubility. Table 2, describes an overall review of
the techniques such as Nano cellulose produced, the
material, te s ting method, advantages, limitations,
and performance parameters which was explained
under the biodegradable lms using Nano cellulose
from the rice husk..
3.3. Biodegradable lms using Nano cellulose
Table 2. Review on biodegradable lms using Nano cellulose from rice husk
Technique Nano
cellulose
Produced
Material Analysis Advantages Limitation PP Ref.
(i)NC - Bleaching,
Acid hydrolysis,
and Ultrasonic
(ii)SGF -Extrusion
(i)NC from
rice hulls
(ii)SGF
Rice hull SEM, TEM,
XRD, FTIR,
TGA, DSC
(i)Reduced opacity
(ii)Increased mechanical
characteri s tics
(iii)Less permeable to
water vapor
Less dense bre
layer with lessral
shape and the
exterior non-
cellulosic layer
removed
(i)Displayed lower
lignin levels than
0.35% (ii)Higher
thermal s tability than
the raw material (iii)
Higher cry s tallinity
(up 70%)
[30]
Delignication
and Acid
hydrolysis
Nano cellu-
lose
Rice grains
with short,
medium, and
long husks
SEM, TEM,
AFM,
NT-MDT,
SOLVER
NANO, ZP,
ATR-FTIR,
XRD, TGA,
DSC, HA
(i)Noncellulosic and
noncry s talline cellulose
removed
(ii)Increased cry s tallinity
(iii)Nanocelluloses are
more heat resi s tant than
cellulose and husks
Delignication
led to gradual
depigmentation
and turned the
material’s hue to
white
The size, cry s tallinity,
s trength, and thermal
s tability of long husks
were better than
medium and short
husks
[31]
(i)Yea s t biomass
lms - Dispersions
at pH 6 and 11
(ii)RHCNF and
BNC
Rice husk
cellulose
nanobers
(RHCNF)
and Bacterial
Nano cellu-
lose (BNC)
(i)For RHCNF
(ii)For BNC
(iii)For Yea s t
Biomass Films
NMR, XRD,
AFM, SEM,
WVP
(i) BNC was more
successful than RHCNF
in enhancing the
mechanical properties of
yea s t lms
(ii)BNC and RHCNF,
both boo s ted tensile
s trengths
(i)Only BNC
enhanced the
yea s t matrix’s
Young’s
modulus, tensile
s trength,
(ii) BNC is
more expensive
than producing
RHNCF
Water vapor
permeability was
reduced by 5 weight
percent in both
reinforcements in
lms created at pH 11.
[32]
(i)Nano cellulose
- Alkaline
treatment,
Bleaching, and
Acid hydrolysis
(ii)Potato s tarch
lms - Solution
ca s ting method
Nano cellu-
lose
(i)For NC –
Rice Husk
(ii)For Potato
S tarch Film -
glycerol with
varied NC (0
- 15 % )
AS tM E96,
Digital
colorimetry
method, AS tM
D882
(i)Lower Water Vapour
Transmission Rate
(WVTR)
(ii)Greater mechanical
s trength
(iii)Greater transparency
Mechanical
s trength did not
increase as the
Nano cellulose
content was
raised above 10%
(i)Rise in Nano
cellulose percentage
from 5 % to 15
% - WVTR of bio
nanocomposite edible
lms reduced. (ii)The
transparency of Nano
cellulose is greater
than 95%
[33]
Solution ca s ting
method
Polyvinyl
alcohol (PVA)
Rice Hull
powder as ller FTIR, XRD,
TGA, DSC,
Tensile te s t,
Surface
morphology
s tudies,
WVP and
Antibacterial
te s ting
(i)Tolerate up to 350 °C
(ii)High interaction
of polymer chains
(iii)Exhibit s trong
antibacterial activity
(iv)Bio-lms were
clearly homogenous,
electrical and thermal
qualities.
Lower exibility
of lms is caused
by the exi s tence
of hydrogen
bonds
Tensile s trength
and tensile modulus
- 23.32 MPa and
684 MPa, at a
concentration of 25
percent RHP in PVA.
[34]
Mercerization and
Etherication CMC
from SCB
Cellulose and
Rice Hulls
Rice husk and
Sugarcane
bagasse
FTIR, XRD,
MC, TS
(i)Had the highe s t tensile
s trength and elongation
(ii)Improved machine-
driven possessions (TS
and Elongation)
(i) negatively
aect the s trength
of the material
(ii) the DS and
TS of the rice
hull CMC were
lower than those
of the SCB
With an increase in
DS of CMC and as
the degree of CMC
sub s tituition grew
the lm’s opacity,
moi s ture content,
and solubility also
increases
[35]
NC: Nano cellulose
SGF: S tarch glycerol lms
Review of food packaging application by nanocellulose Asha Valsalan et al
32
from Various Plant Extracts
Using a more eective, economical enzymatic
hydrolysis pathway, R. Reshmy et al proposed a
s traightforward method for the extraction of Nano
cellulose from sugarcane bagasse [36]. It was
possible to extract NC bres from sugarcane
bagasse. NC was produced by alkaline treatment,
bleaching, and acid hydrolysis. The solvent
ca s ting method was used to create thin lms. FT-
IR, XRD, FESEM, DLS, and AS tM D 2216
methods were used to characterize the lms. Non-
edible su s tainable material usefulness, co s t
eciency, simple ease of processing, minimal
energy usage, non-hazardousness, and simple
degradation rate are advantages of this upgraded
technology. These thin lms might degrade well
in situations with soil, salt, acid, and alkaline
conditions. Glycerol, a pla s ticizer, is present in
NC, which lessens its tendency to inate. The
acid resi s tance is increasing due to the use of
glycerol as a pla s ticizer and the reduction in
weight loss from 50% to about 40% is a result of
the pla s ticizers included in NC. Nano cellulose
was sugge s ted to be added to polyvinyl alcohol by
Mónica Sánchez-Gutiérrez et al in order to
enhance the technical prowess of the composite
coating used for food packaging (PVA) [37]. PVA
lms reinforced with (L)CNFs derived from olive
tree trimming leftovers were manufactured using
the solvent ca s ting method. Micro cellulosic was
created from pulp that had been both dyed and
unbleached using a mechanical and TEMPO
preparation. The te s t methods are as follows:
Perkin Elmer UV-Vis Lambda 25
spectrophotometer, FTIR, TGA, SEM, XRD, and
AS tM E96/E96M-10. From six percent for the
pure PVA lm to 50 percent and 24 percent,
respectively. For unbleached and bleached Nano
cellulose, the UV barrier was increased in terms
of optical properties. Associated with pure PVA
lm, the antioxidant capacity of mechanical Nano
cellulose lms made without bleaching
signicantly increased (5.3%). The mechanical
Nano cellulose lms with a 5% unbleached
component demon s trated noticeably greater
tensile s trength as compared to pure PVA lm.
The 5 percent Nano cellulose lms were also
improved in terms of their thermal properties and
impermeability. They oered an oxygen shield
akin to aluminum layers and pla s tic lms while
reducing water vapour leakage by 38–59%.
Because they are more sensitive to environmental
conditions like humidity and temperature. (L)
CNF-reinforced lms obtained by mechanical
pretreatment (MU and MB) needed a lengthier
s tabilization period than (L)CNF-reinforced lms
obtained through TEMPO pretreatment (TU and
TB). This behavior is displayed by other materials,
such as EVOH, which are greatly aected by the
surrounding humidity. According to Mochamad
Asro et al, a Yam Bean (YB) s tarch sub s trate and
Micro Cellulosic Water Hyacinth Fiber (WHF)
reinforcing were employed to develop bio
nanocomposites utilizing the ca s ting process [38].
The secret to creating good bio nanocomposites
was adding Micro viscose as a solution to the YB
s tarch matrix, allowing it to gel, and then briey
sonicating it. The eect of Nano cellulose
suspension loading on the YB s tarch matrix was
examined using mechanical te s ting, Scanning
Electron Microscopy (SEM), X-Ray Diraction
(XRD), Thermogravimetric Analysis (TGA),
Fourier Transform Infrared (FTIR), and
wettability. After the addition of Nano cellulose,
tensile s trength (TS) and tensile modulus (TM)
greatly increased. With higher Micro
lignocellulosic content, heat resi s tance and water
resi s tance were also improved. Bio-
nanocomposites have a rougher fracture surface
than pure YB s tarch sheets. The greate s t amounts
of Nano cellulose (1 wt. percent) were used to
achieve the maximum values for TS (5.8 MPa)
and TM (403 MPa). With ju s t a little more than 1
weight percent of extra Nano cellulose, the bio-
nanocomposite’s cry s tallinity index (CrI)
increased by more than 200 percent. Zineb Kassab
et al proposed that Sunower oil cake (SOC) was
found to be a bio-sourced resource for the
manufacture of cellulose nanocry s tals (CNC)
after chemical processing and sulfuric acid
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
33
hydrolysis [39]. This s tudy also looked into the
newly created CNC’s polymer nanoreinforcing
capabilities. PVA-based nanocomposite lms
with CNC30 concentrations of 1, 3, 5, and 8
weight percent were produced using the solvent
ca s ting technique. The rheological properties of
CNC solutions at dierent percentages were
evaluated using sy s temic resi s tance s tiness
s tudies and cyclic dynamic experiments. When
the mechanical and transparency properties of
CNC-lled PVA nanocomposite lms were
examined at various CNC contents (1, 3, 5, and 8
wt%), clear nanocomposite products with high
hardness properties were produced. The resultant
CNC displayed remarkable saturated solution
s tabilization and gel-like properties at very low
CNC concentrations. Nanocomposite materials
with signicantly better tensile characteri s tics
were produced by incorporating CNC into a PVA
polymeric matrix. The addition of CNC causes
slight changes in the FTIR spectra of PVA
nanocomposites that are lled with CNC. It is
possible to see a tiny variation in the OH s tretching
vibration’s intensity. When 8-weight percent
CNC was added to PVA-based nanocomposite
lms, the tensile s trength and ela s tic modulus
improved by 107 and 78%, respectively. Swarup
Roy and Jong-Whan Rhim showed that an
extremely s table nanoscale Pickering
emulsion(PE) was made using natural clove bud
essential oil s tabilized with nanocellulose bre
[40]. The PE was used to produce gelatin and agar
functional lms. The gelatin/agar-based
bidirectional compound lm was made using the
solution ca s ting technique, and the cellulose
nanober-based PE was made by preparing a
cellulose nanober solution. AS tM D 882–88,
TGA, FESEM, FTIR, and Chroma meter are te s ts
taken to predict the characterization. The inclusion
of PE only slightly changed the mechanical
properties and vapor impermeability of the
gelatin/agar-based lm, with no discernible
impact on temperature. Without aecting the
lm’s transparency, the inclusion of PE also gave
it exceptional UV-barrier qualities. Additionally,
the composite lm had s trong antioxidant
properties. The power of the gelatin/agar lm was
signicantly impacted by the addition of PEC.
The lm has high transmittance to UV and visible
light, with corresponding T280 and T660 values
of 26.9%, 88.0%, and 1.4 %. The neat gelatin/
agar lm’s minimally changed WVP was 0.59 x
10-9 gm.m-2Pa-1s-1. The utilization of s tatic
intermittent fed-batch (SIFB) equipment and a
cheap medium, such as fermentation black tea,
according to Chhavi Sharma et al, this s tudy
proposes a technique with indu s trial signicance
for the creation of inexpensive and
environmentally friendly bacterial Nano cellulose
(BNC) lms [41]. Chitosan, a natural polymer,
successfully altered the BNC lm (BNC-chitosan
lm). SCOBY, black tea, and tomatoes were the
materials used. The lms were characterized
using FE-SEM (Field Emission Scanning Electron
Microscopy), ATR-FTIR (Attenuated Total
Reectance and Fourier Transform Infrared
Spectrometry), X-ray diraction (XRD), and
thermogravimetric analysis (TGA). Because of
their high tensile properties, cry s tallinity, air
resi s tance, and tomato shelf life evaluation, BNC-
chitosan coatings have a considerable potential to
be used for economical encapsulation, which is
unque s tionably wanted by the packaging sector.
The surface morphology of BNC changed after
chitosan treatment. BNC yield was higher in this
modied bioprocess (29.2 g L-1) than in the
s tandard s tatic approach (13.3 g L-1) with a BNC
yield of 29.2 g L-1. Segal method calculations
showed that the CrI of BNC formed via the SIFB
technique was 79.2 percent(%), which is nearly
identical to the CrI of BNC previously shaped
under the traditional xed technique (79.4 %).
The upcoming Table 3, describes an overall
review of the techniques such as, Nano cellulose
produced, the material, tes ting method,
advantages, limitations, and performance
parameters which was explained under the
biodegradable lms using Nano cellulose from
various plant extracts.
3.4. Biodegradable lms using Nano cellulose
Review of food packaging application by nanocellulose Asha Valsalan et al
34
Gelatin and carbohydrate sub s trates were
examined by S.M. Noorbakhsh-Soltani et al for
the integration of Nano-cellulose [42]. Chitosan
enhances the mechanical, anti-fungal, and
waterproong properties of materials. The response
surface approach is used in the design and analysis
of experiments. Acid hydrolysis is used to create
Nano cellulose, which is then wet-processed and
added to base matrices. Films are produced by
lm ca s ting techniques. The s trength properties,
Table 3. Review on biodegradable lms using Nano cellulose from various plant extracts
Technique Used Nano
cellulose
Produced
Mate-
rial
Analysis Advantages Limitation Performance
Parameters
Ref.
(i)NC - Alkaline
treatment,
Bleaching, and
Acid hydrolysis
(ii)Thin lms -
Solvent ca s ting
method
NC Fibres Sug-
arcane
bagasse
FT-IR,
XRD,
FESEM,
DLS,
AS tM D
2216
Non-edible renewable
feed s tock utility, co s t-
eectiveness, easy
processibility, less
energy consumption,
non-hazardous and easy
degradability
Glycerol, a
pla s ticizer, is
present in NC,
which lessens
its tendency to
inate
(i)Acid resi s tance
is increasing (ii)
The reduction in
weight loss from
50% to about
40% is a result of
the pla s ticizers
included in NC
[36]
(i)Nano cellulose
- Mechanical
and TEMPO
preparation
(ii) PVA lms
with (L)CNF
reinforcement -
Solvent ca s ting
PVA lms
reinforced
with (L)
CNFs
Olive
tree
pruning
scraps
Perkin
Elmer
UV/VIS
FTIR,
TGA,
SEM,
XRD,
AS tM E96/
E96M-10.
(i)UV barrier was raised
(ii)Antioxidant capacity
of mechanical Nano
cellulose lms is
increased
(iii)Had higher tensile
s trength
(iv)Thermal s tability (v)
Reduced water vapor
permeability by 38–59%
(vi) oxygen barrier
(L)CNF-
reinforced lms
required a longer
s tabilisation
period than (L)
CNF-reinforced
lms obtained
by TEMPO
pretreatment
(i)UV barrier
increased – 50%
& 24%
(ii)Antioxidant
capacity increased
– 5.3%
(iii)Reduced water
vapor permeability
- 38–59%
[37]
(i)NC- Gelation
and a brief Soni-
cation
(ii)Bionanocom-
posite Film -
Ca s ting method
(i)Nano
cellulose -
(WHF)
(ii)Film -
Yam Bean
(YB)
Yam
Beam
TT, SEM,
XRD, TGA,
FTIR, MA
(i)Tensile s trength (TS)
and Tensile modulus
(TM) greatly increased
(ii)Thermal s tability and
moi s ture resi s tance were
also raised
Bio-nanocom-
posites have a
rougher fracture
surface than pure
YB s tarch sheet
(i)TS- 5.8 MPa
and TM -403 MPa
(ii)CrI-increased
by more than 200
percent
[38]
(i)Nano cellulose
- Chemical
processing and
Sulfuric acid
hydrolysis
(ii)Film - Solvent
ca s ting method
(i)Cellulose
nanocry s tals
(CNC)
(ii)PVA-
based nano-
composite
lms
Sun-
ower
oil cake
(SOC)
observations
of con s tant
rheological
properties
and cyclic
dynamic
te s ts
(i)Has transparent
nanocomposite materials
with potent mechanical
properties
(ii)Exhibited exceptional
aqueous colloidal
s tability and gel-like
behavior
(iii)Has better tensile
characteri s tics
(i)Addition of
CNC causes
slight changes in
the FTIR of PVA
nanocomposites
with CNC.
(ii)A tiny
variation in the
OH s tretching
vibration’s
intensity
Ela s tic mechanical
and physical
properties improved
by 107 and 78%,
respectively.
[39]
(i)Nano cellulose
(ii) Film -
Solution ca s ting
method
(i)Nano
cellulose -
CNFPE
(ii)Film -
Gelatin/
Agar-based
BCF
CBN
s tabi-
lized
with
Nano
cellulose
ber
AS tM D
882–88,
TGA,
FESEM,
FTIR,
Chroma
meter
(i)Increased the
mechanical s trength
and decreased the vapor
barrier qualities of the
gelatin/agar-based lm
(ii)Good UV-barrier
qualities
(iii)Had s trong
antioxidant properties
The s trength
of the gelatin/
agar lm was
signicantly
impacted by the
addition of PEC
(i)High
transparency to UV
and visible light
- 26.9 1.3 %, 88.0
1.4 % T280 and
T660
(ii)Gelatin/agar
lm’s WVP - 0.59
10 9 g.m./ m2. Pa.s
[40]
S tatic intermittent
fed-batch (SIFB)
technology
(i)NC -
(BNC)
(ii)Film
- BNC-chi-
tosan lm
SCOBY,
black
tea and
toma-
toes
FESEM,
ATR-FTIR,
XRD, TGA
(i)Good mechanical
s trength
(ii)Cry s talline nature
(iii)Resi s tance to air
(iv)Shelf life evaluation
of tomatoes
The surface mor-
phology of BNC
changed after
chitosan treatment
(i)BNC yield was
higher - 29.2 gL-1
(ii)CrI of BNC
formed via SIFB
method - 79.2 %
[41]
WHF: Water Hyacinth Fiber
CNFPE: Cellulose nanober-based PE
CBNO: Clove bud natural essential oil
BCF: Binary composite lm
BNC: Bacterial Nano cellulose
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
35
s torage of food, clarity in visible and ultraviolet
light, and water contact angle are also conducted
on the Nano-composite lms. On the ideal lms,
DSC/TGA, SEM, TEM, XRD, and air permeability
te s ts are also carried out. The advantages are
include, (i) High ela s ticity, s trength properties,
extension to break, clarity, and possibly food s tu
s torage properties of both gelatin and s tarch bases
can be improved (ii) Decrease in UV transmittance
(iii) Gelatin lms oer greater transparency,
ela s tic modulus, and break length than s tarch
lms. According to the results of the s tress- s train
curves, which measure the mechanical s trength of
nanocomposite lms, some of the samples exhibit
thinning, others exhibit thickening, and some exhibit
a s traightforward linear response. According to the
results, increasing the amount of Nano cellulose to
10% raises the mechanical properties at the break
to 8121 MN m-2, while decreasing the ductility.
Furthermore, chitosan content can be increased
from 5% to 30% to enhance food s torage for up
to 15 days. Using the wire extension method,
Yasmim Montero et al sugge s ted making PBAT
active lms that were packed with nanocellulose
and infused with cinnamon essential oil [43].
The connections among NC-EO-PBAT were
inve s tigated, and the results demon s trated that the
direct closeness between the EO and the PBAT
matrix changed the conformations of the polymer
molecules. FT-Raman, FTIR, TGA, and WVP are
the te s ts undergone for the process. The modied
CNF lms displayed a controlled Fickian diusion,
a greater essential oil release, reduced water vapor
permeability, and eective ller dispersion. The
lm at 3085 cm1 lo s t its form and intensity after
3 wt% CNF was inserted. Fruits loaded in lms
with 0.5 weight percent modied-CNF have little
weight reduction, better quality maintenance, and
no fungal attack after 15 s torage periods. According
to the report of Syaq et al, liquid ca s ting was
used to generate biodegradable nanocomposite
lms using sugar palm s tarch (SPS), sugar palm
nanocry s talline cellulose (SPNCC), and cinnamon
essential oil (EO) [44]. By using an acid hydrolysis
technique, sugar palm Nano cry s talline celluloses
(SPNCC) was created. Solution-ca s t SPS/SPNCC
nanocomposite coatings with added cinnamon
essential oil were created. The SEM, AS tM D
644, AS tM 570, FTIR, disk diusion method
(DDM), and Agar disc method (ADM) are the te st
methods used to nd o ut the r esults. Mechanical
characteri s tics experiments on lms containing
cinnamon EO revealed improved tensile s trength
and tensile s tiness n umbers f rom 4 .8 t o 5.3
MPa and 122.49 to 130.52 MPa, respectively. In
addition, the density was lowered from 1.38 to 1.31
g cm-3 and the moi s ture content was reduced from
13.65 to 12.33 percent, respectively. The results
unambiguously demons trate that the introduction
of cinnamon essential oil caused a decrease in
the lms’ elongation at break, from 18.14 to 3.35
percent.
The eects o f d extran-coated s ilver nanoparticle
loading on the robotic, boundary, and antibacterial
activities of skinny movies produced from cellulose
nanobrils by solvent evaporation technique were
shown by Vesna Lazi’c et al [45]. They showed
an environmentally friendly and food-preservative
packing material. The lm is created using hybrid
materials based on CNF and Ag NPs covered with
dextran. The te s t methods employed to ascertain the
properties of the lm i nclude T EM, SEM, AS tM
D3985, and a Shimadzu AGS-X electromechanical
universal te s ting machine. The advantages of adding
dextran are as follows: (i) Acts as dispersing media
(ii) it is an additive that seals out moi s ture (iii) It
has insucient oxygen penetrability (iv) Keeps the
food safe from bacterial growth. These lms also
exhibit better Young’s moduli while maintaining
their exibility and tensile s trength. Both articial
subs trates like hydroxyapatite and magnetite, as
well as Ag NPs attached to macroporous polymer
sub s trates, demon s trate lesser antibacterial activity
again s t S. aureus than E. coli. The 99.9% suppression
of Escherichia coli after ve repetitive cycles of 24
h exposed to 0.9 percent NaCl aqueous solution
was demon s trated, supported by a su s tained release
of Ag+ ions (underneath the toxicants dangerous
criterion). Lower oxygen transmitting percentages
from 2.07 to 1.40-0.78 cm3 m-2 d-1, hydrophilicity
Review of food packaging application by nanocellulose Asha Valsalan et al
36
from 20.80 to 52.40 for MilliQ water, and from 350-
370 to 620-740 for 3 % acetic acid and 0.9 % NaCl
simulant solutions were obtained.
Nano-chitosan (NCH), Nano-cellulose (NCL), and
cellulose derivatives were employed by Narges
Jannatyha et al as biodegradable biopolymers [46].
Various amounts of nano chitosan or nanocellulose
were added using ca s ting procedures to the
carboxyl methyl cellulose (CMC) lm solution
(0.1, 0.5, and 1 percent). XRD, DSC, and DC
are the te s t methods conducted to predict the
results. Some of the advantages are: (i) When
the concentration of the nanocomposite rose, the
WVP of the polymer and nanoller decreased
(ii) By increasing concentration, the TS and
elongation at the break of a nanocomposite lm
were improved (iii) CMC/NCH provides more
benets than CMC/NCL biopolymer when
used as a biocompatible lm (iv) Particularly at
concentrations of 1%, physical characteri s tics like
water solubility(WS), moi s ture content(MC), and
moi s ture absorption(MA) were lowered by both
CMC/NCH and NCL and also causes the nanoller
in CMC lm to aggregate. The antibacterial
properties of CMC and CMC/NCL are absent. The
physical and thermal properties in CMC/NCH were
lower than CMC/NCL for the concentration p <
0.05. The melting points (Tg) of CMC, CMC/NCL
1 %, and CMC/NCH 1 % lms were, 206.31 °C,
221.97 °C, and 200.91 °C, respectively. Nanoller
utilized (1%) WS decreased to 18% and 33% for
CMC/NCL and CMC/NCH lms, respectively.
Sapuan et al reported that the mechanical, barrier
and thermal characteri s tics of nanocellulose-
reinforced polymer composites were improved
[47]. Enhancing the useful qualities of TPS, PLA,
and PBS for food packaging through the addition
of Nano cellulose is undoubtedly advantageous.
Thermopla s tic s tarch (TPS), polylactic acid
(PLA), and polybutylene succinate (PBS) were
selected as the alternatives because they are easily
accessible, biodegradable, and have high food
contact properties. FESEM and SEM are the te s ts
conducted. Reinforcing Nano cellulose has many
advantages such as, (i) Tensile s trength and ela s tic
modulus are improved by PLA biocomposites (ii)
Poor water barrier was improved by TPS/Nano
cellulose (iii) The mechanical and oxygen barrier
characteri s tics of PLA and PBS were enhanced.
In comparison to pure PBSA, CNN decreased
the tensile s trength and elongation at break. Their
usable characteri s tics did not necessarily increase
with increased Nano cellulose loading. If the
amount of Nano cellulose in the polymers was too
high, agglomeration took place. The hydrophilic
Nano cellulose and hydrophilic PLA are unsuitable
and result in weak matrix interaction, hence only
low Nano cellulose loadings between 0.5 and
2 weight percent are required for the optimum
results. On the other hand, the addition of 2%
PA increased the s trength of PBS/CNN by about
120%. (95:5). Table 4 describes an overall review
of the techniques such as, Nano cellulose
produced, the material, tes ting method, advantages,
limitations, and performance parameters which
was explained under the direction - Biodegradable
lms using Nano cellulose from polymer composite
material in the food packaging.
4. Discussion
Petroleum-based products have already been
employed in a variety of indu s tries, but packaged
foods have beneted mo s t from their minimal price
and s trong mechanical and physical properties. But
it is non-biodegradable and also produces numerous
health hazards. Pla s tic or petroleum-based food
containers need to be replaced in order to do so,
various types of research have been going on for
producing biodegradable lms using Nano cellulose
extracted from various biodegradable wa s tes such
as agri-wa s te, various plant extracts, non-woody
biomass, biopolymer composite materials, etc.
Nano cellulose is produced by various extraction
methods and the basic method is discussed in
the paper. The three primary methods utilized
to create Nano cellulose from diverse extracts
are acid degradation, enzymatic hydrolysis, and
electromechanical process. The common method
used to make biodegradable composite lms is
a solvent-ca s ting method. The types of Nano
Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
37
cellulose are CNF, CNC, and BNC. The XRD,
SEM, TEM, DSC, FTIR, and FE-SEM are some of
the te s t methods used to characterize biodegradable
composite lms. The tensile properties and
physical characteri s tics of biodegradable lms
and biodegradable composite lms are enhanced
overall by the use of nanocellulose. Water vapor
permeability, Moi s ture content, etc., are reduced
thus enabling the biodegradable lms more suitable
and ecient for food packaging.
Table 4. Review on biodegradable lms using Nano cellulose from polymer composite material
in Food Packaging Reduced
Technique Nano cellulose
Produced
Material Analysis Advantages Limitation Performance Param-
eters
Ref.
(i)Nano
cellulose - Acid
hydrolysis
(ii)Film -
lm ca s ting
technique
Nano-compos-
ite lms Gelatin,
S tarch,
Chitosan
air
permeability,
tensile
s trength, food
preservation,
transparency
in visible and
UV light
(i) High ela s ticity, ela s-
tic modulus, extension
to break, clarity can be
improved.
(ii) Decrease in UV
transmittance
(iii)In comparison to
s tarch lms, gelatin
lms have better trans-
parency
the s tress- s train
curves , thinning,
thickening, linear
line response
(i)Nano cellulose
content to 10%, tensile
s trength at break -
increase to 8121 MN/
m2, lowers the elonga-
tion at break
(ii)Increasing the
chitosan content from
5% to 30% - improve
food preservation for
up to 15 days
[42]
Wire extension
method
NC-EO-PBAT
Films PBAT,
Cellulose
nanobers,
Cinnamon
oil
FT-Raman,
FTIR, TGA,
WVP
(i)Fickian diusion,
(ii)Greater essential oil
release
(iii)Reduced water
vapor permeability
(iv)Eective ller
dispersion
After the addition
of 3 wt. % CNF, the
lm at 3085 cm1
lo s t its form and
intensity
The fruits sealed in
lms with 0.5 weight
percent modied-CNF
have very little losing
weight, better fresh
preservation, and no
fungal attack after 15
s torage periods.
[43]
(i)Nano
cellulose - Acid
hydrolysis
process
(ii)Film -
Solution ca s ting
method
(i)Nano cellu-
lose - (SPNCC)
(ii)Film - SPS/
SPNCC
Sugar palm
s tarch
(SPS)/
(SPNCC)
and
Cinnamon
essential oil
(EO)
SEM, AS tM
D 644, AS tM
570, FTIR,
DDM, ADM
(i) Enhanced tensile
s trength and tensile
modulus values
(ii)The moi s ture content
and density were
decreased
Addition of cinna-
mon EO reduced
the lms’ elongation
at break to drop
from 18.14 - 3.35 %
to 13.9 - 5.57 %
(i) Enhanced TS and
TM - 4.8 to 5.3 MPa
and 122.49 to 130.52
MPa
(ii) MC and
thickness decreased -
13.65 to 12.33 % and
1.38 to 1.31 g cm-3
[44]
Solvent ca s ting
method
Ag NPs with
dextran coating
and CNF-based
composite
sheets
Dextran,
coated
AgNPs and
Cellulose
nanobrils
TEM, SEM,
AS tM D3985,
a Shimadzu
AGS-X
electro-
mechanical
machine
1. Dextran
(i) Act as dispersing
(ii)Moi s ture-resi s tant
sealable additive (iii)
Reduced oxygen
permeability
2.Films also exhibit
better Young’s moduli
while maintaining their
exibility and tensile
s trength.
Ag NPs as well as
inorganic supports
like hydroxyapatite
and magnetite
both exhibit lower
antibacterial
ecacy again s t S.
aureus than E. coli
(i)Reduced OTR -
from 2.07 to 1.40-0.78
cm
3
(ii)Hydrophilicity
- from 20.8◦ to 52.4◦
for MilliQ water, from
35-37◦ to 62-74◦ for
3 % acetic acid, 0.9
% NaCl simulant
solutions yielding a
99.9 % inhibition of
E-Coli
[45]
Ca s ting
technique
CMC/NCH and
CMC/NCL
Nano-
chitosan
(NCH),
Nano-
cellulose
(NCL),
Cellulose
derivative
and (CMC)
XRD, DSC,
DC
(i)Concentration of the
nanocomposite rose, the
VWP decreased
(ii) Enhanced TS and
elongation at break
(iii)CMC/NCH provides
more benets than
CMC/NCL
(iv)At 1%, physical
characteri s tics lowered
by both CMC/NCH and
NCL
(i)The antibacterial
of CMC and CMC/
NCL are absent
(ii)The physical and
thermal properties
in CMC/NCH were
lower than CMC/
NCL for the con-
centration p < 0.05.
(i)Tg of CMC, CMC/
NCL 1 %, and CMC/
NCH 1 % lms -
206.31 °C, 221.97 °C,
and 200.91 °C.
(ii)Nanoller utilized
(1%) WS decreased
- 18% and 33% for
CMC/NCL and CMC/
NCH lms
[46]
Technique s tat-
ed as in citation
[47]
Nano cellulose
reinforced poly-
mer composites
TPS, PLA,
and PBS
FESEM and
SEM
(i) Tensile s trength
and ela s tic modulus
are improved by PLA
biocomposites (ii) Poor
water barrier were
improved by TPS/Nano
cellulose
(i)In comparison
to pure PBSA,
CNN decreased the
tensile s trength and
elongation at break
(i)For be s t outcomes
- between 0.5 %
and 2 weight % are
necessary
(ii)Addition of 2 % PA
enhanced the tensile
s trength of PBS/CNN
by around 120 %
(95:5)
[47]
SPNCC: Sugar palm nanocry s talline celluloses
Review of food packaging application by nanocellulose Asha Valsalan et al
38
5. Challenges and Future Research
There are more recent advancements have emerged
in the development of biodegradable lms using
Nano cellulose from various biodegradable extracts
but there are s till some parameters to be enhanced.
Some of the key points to be noted for future
research are as follows: (i) Due to the large variety
of bio-based sub s trates and essential oils oered, it
is dicult to make general recommendations for the
creation of proactive packaging products when using
them. Greater attention should be paid to sensory
evaluation, making additional, and the synergi s tic
eects of numerous essential oils in order to enhance
the active packaging on various food products [48]
(ii) BNC oers intriguing uses in food packaging,
but these uses aren’t being fully inve s tigated
because to the material’s high production co s ts
and dicult commercialization in the packaging
indu s try [49] (iii) Even with new approaches that
have enabled controlled delivery of antibacterial
agents in the appearance of NC feasible, utilizing
bio-polymers as natural resources with appropriate
membrane characteri s tics, s trength properties, and
satisfying the regulations for packaged foods is
s till a dicult problem to tackle [49]. (iv) More
research is necessary to determine the eectiveness
and durability of Nano cellulose-based packaging
technologies during the real food warehouse and
transportation process [50]. (v) Biopolymer-based
nanocomposites begin to replace conventional
synthetic pla s tic products in the near future only if:
I. It would be energy and co s t-eective to isolate
cellulose and turn it into nanoparticles;
II. Hydrophilic polymers and hydrophobic natural
bers (cellulose) would be able to coexi s t
without conict;
III. It is advisable to lessen the variability of the
extracted ber’s characteri s tics
IV. It is advisable to produce compatibilizers,
coupling agents, and adhesives from renewable
sources;
V. Nanocomposites’ biodegradability and life
cycle assessment should be thoroughly s tudied;
VI. It is necessary to create new processing
technologies [51].
6. Conclusion
In this review paper, we have analyzed Nano
cellulose, its types, and the basic extraction
methods. The reason for using Nano cellulose-
based biodegradable lms in food packaging is
also discussed. Also, we have explored the recent
research on the development of biodegradable
lms using Nano cellulose from agricultural wa s te,
rice husk, various plant extracts, and biopolymer
composite materials on food packaging. The
techniques used, other Nano cellulose produced,
various te s t methods adopted to dene the
characteri s tics of the lms, advantages,
limitations, and performance parameters are
discussed briey in the experimental section.
The result section summarizes the paper, and
other sections give the future research points
that should be considered. From this review, we
learned that developing biodegradable lms using
Nano cellulose has various valuable parameters
in food packaging. A notable replacement for
synthetic products has been highlighted for
nanocomposites, particularly those that contain
Nano cellulose as reinforcement.
7. Funding and Conict of Intere s t
The authors declare that no funds, grants, or other
support were received during the preparation of
this manuscript. I hereby declare that the disclosed
information is correct and that no other situation
of real, potential or apparent conict of intere s t is
known to me.
8. Acknowledgment
We would like to thank Sree Buddha College
of Engineering, Pattoor, Kerala, and Chemi s try
department of Kalasalingam Academy of Research
and Education, Krishnakovil, India.
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Anal. Methods Environ. Chem. J. 5 (4) (2022) 20-42
Anal. Methods Environ. Chem. J. 5 (4) (2022) 43-54
Research Article, Issue 4
Analytical Methods in Environmental Chemi s try Journal
Journal home page: www.amecj.com/ir
AMECJ
Preparation of chitosan lms pla s ticized by lauric
and maleic acids
Sara Hikmet Mutasher a
and Hadi Salman Al-Lami a,*
a Department of Chemis try, College of Science, University of Basrah, Basrah, Iraq
AB S TRACT
The energy crisis and environmental concerns have increased
intere s t in natural polymers, and the bio-sourced materials eld is
experiencing rapid growth. A useful alternative to conventional pla s tic
packaging manufactured from fossil fuels is packaging con s tructed
of biodegradable polymers. Consideration has been given to the
in s trumental methods for examining modications to the chemical
composition and characteri s tics of modied chitosan. The molecular
weight and the kind of pla s ticizer present in these materials are the
two primary variables inuencing their usability and performance.
This s tudy set out to physically blend chitosan with two dierent
acids, lauric and maleic, to enhance chitosan ca s t lms’ physical
and mechanical properties. Dierent pla s ticizer ratios appeared to
have little eect on the various properties of the chitosan ca s t lms.
Examining the obtained lms by FTIR implies that chitosan’s native
s tructure was unchanged. The lms prepared had more exibility and
better solubility than those made with un-pla s ticized chitosan. It was
evident from an analysis of the mechanical properties of the lms
that both acid pla s ticizers enhanced the mechanical properties of the
chitosan.
Keywords:
Chitosan,
Lauric acid,
Maleic acid,
Films pla s ticizers,
Mechanical properties,
Solubility
ARTICLE INFO:
Received 5 Aug 2022
Revised form 14 Oct 2022
Accepted 25 Nov 2022
Available online 29 Dec 2022
*Corresponding Author: Hadi Salman Al-Lami
Email: hadi.abbas@uobasrah.edu.iq
https://doi.org/10.24200/amecj.v5.i04.209
------------------------
1. Introduction
The intere s t in natural polymers has grown as a
result of the energy crisis and environmental
concerns, and the eld of bio-sourced materials
is currently expanding quickly. In this situation,
the indu s try mu s t look for new sources of organic,
environmentally friendly, and biodegradable
polymers to replace those derived from petroleum.
Polysaccharides in this area have enormous potential
for use in active and intelligent packaging, smart
textiles and biomedical devices, environmental
remediation, and other applications. Chitosan is a
special cationic polysaccharide with an excellent
anity for various surfaces and out s tanding
cosmetic properties, even when left unaltered. It
is a naturally occurring cationic copolymer [1].
This biopolymer, which is abundant in nature
(the second one after cellulose), holds much
promise for various uses. It is a highly renewable,
biodegradable, environmentally friendly, and
non-toxic polymer. Indeed, chitosan has been
successfully used as a scaold for biomedical
applications, water engineering, treatment, the
food indu s try, lms, coatings, and con s truction
elds. The functionalization of its chemical
s tructure often enhances its characteri s tics until
it obtains properties equivalent to synthetic
products [1]. The development of chitosan-
based materials has attracted much attention
44 Anal. Methods Environ. Chem. J. 5 (4) (2022) 43-54
due to their great qualities, such as, nontoxicity,
biodegradability, biocompatibility, antibacterial
properties, and biofunctional characteri s tics, not
only in the biomedical sector but also in the eld
of food contact materials. The new or improved
properties of chitosan would be obtained through
the chemical modication of its s tructure via
the blending or attaching of various chemicals.
Therefore, chitosan does not present problems of
handling and disposal that may be encountered
with some of its synthetic counterparts [2].
Chitosan is a promising material based on its
chemical modications as dye-removing agents
and metal ion adsorbents. Recently, the progress
on chemical modications of chitosan has been
quite rapid, and we are condent that a more
extensive range of applications of chitosan
derivatives could be expected shortly [3, 4]. In
the pla s tics sector, pla s ticizers have long been a
popular element [5]. Examples of its numerous
applications include packaging, consumer
goods, medicines, s tructures, and con s truction
[6]. However, the indu s try is moving away from
phthalate-based pla s ticizers and toward bio-based
pla s ticizers due to environmental and health
concerns [7]. Low co s t per volume, low volatility,
diusivity, low specic gravity, good miscibility,
and s trong intermolecular interactions between the
pla s ticizer and the polymer resin are all desired
properties of pla s ticizers. A well-pla s ticized
product should be exible at low temperatures,
have a low ela s tic modulus, a low glass transition
temperature, and have good tensile elongation
but low tensile s trength [8]. Physical blending
is a practical and important method for altering
chitosan to serve various applications. Chitosan-
based lms’ s tructural and physical characteri s tics
have been extensively researched for use in
biomedical and other applications [9,10]. When
polymer chains separate from one another,
pla s ticizers ll the intermolecular spaces between
them. This reduces chain retraction and increases
free volume, enabling polymer chains to move
more freely [11]. The chitosan and pla s ticizer
hydrogen bonding interaction [12,13] regulates
the mechanical and physical characteri s tics of
the lms. The physically blended pla s ticization
of extracted chitosan with lauric acid and maleic
was carried out in this work. It also focuses on
the possible changes in molecular s tructure and
mechanical and water solubility properties to see
if they can produce appropriate chitosan lms for
packaging, which is a potential application for
chitosan.
2. Experimental
2.1. Reagents and Materials
Chitosan was obtained by the deacetylation
process of chitin extracted from local shrimp shell
wa s te as described in the literature [14,15]. It had a
viscosity average molecular weight of 2.702x105 g
per mole as determined by the viscosity technique
and a deacetylation degree of 80%. The acetic acid
(CH3CO2H; pure ≥99%; CAS No.: 64-19-7), Lauric
acid (CH3(CH2)10COOH. CAS No.: 143-07-7) and
maleic acid (HO2CCH=CHCO2H, MDL Number:
MFCD00063177; PubChem ID: 24896549, CAS
No.: 99110-16-7) used as pla s ticizers and acetic
acid as a solvent were purchased from Sigma-
Aldrich Company and utilized without further
treatment. The tris-maleate buer and sodium
maleate buer make of maleic acid and can be
used for the pretreatment of poplar tracheid cell
walls for the spectroscopic analysis of lignin.
2.2. Ins truments
An FTIR-8101M Shimadzu spectrometer in the
4000–400 cm-1 range was used to inve s tigate
the chemical s tructures of the unpla s ticized
and chemically pla s ticized chitosan lms. The
mechanical properties (tensile s trength, Young’s
modulus, and %elongation at break) of the
unpla s ticized chitosan and their pla s ticizer blend
lms were measured in the tensile mode (speed 5
mm min-1) with a BTI-FR2.5TN.D14 (ZwickRoell,
Germany) mechanical te s ting machine.The
s tandard tes t method (AS tM D882-10) for tensile
properties of thin plas tic sheeting and lms was
used to determine the mechanical properties of the
pla s ticized and unpla s ticized chitosan lms in the
45
Modications Chitosan Films by Lauric and Maleic Acids Sara Hikmet Mutasher et al
form of s tripes of 20 x 2 mm. This te s t method
covers the determination of tensile properties of
pla s tics in the form of thin sheeting and lms (less
than 1.0 mm (0.04 in.) in thickness).
2.3. Preparation of the chitosan lms
The unpla s ticized chitosan lm was prepared by
the solvent evaporation method by dissolving 1.0
g of chitosan in 100 ml of 2% (v/v) acetic acid
solution under s tirring at ambient temperature for
24 h. Then, it was poured into a leveled Petri dish
of 50 mm in diameter. The lm was removed from
the dish, dried for 12 hours at 45°C, and then s tored
before determining its s tructural, physical, and
mechanical properties [16]. In the same procedure,
pla s ticized chitosan lms with dierent ratios of
various acid pla s ticizers were prepared, as shown
in Table 1.
2.4. Film solubility
The amount of dry matter in the lm that dissolves
in water is used to calculate the solubility of the
lm. The solubility of the lms was evaluated
using previously described techniques with some
changes [17]. In a nutshell, the lms were divided
into 2 cm × 2 cm squares and entirely dried before
being s tored. The lms were weighed repeatedly
until a s table weight that matched the fully dried
lms was attained; this weight was then used as
the initial dry weight. The lms were s tirred at 25
°C for 24 hours while submerged in a glass beaker
in 50 ml of deionized water. After being taken
out of the beakers, the lms were dried at 105 °C
until they attained a con s tant weight. This quantity
served as the nal dry weight. The solubility
percentage was calculated using equation 1 [18].
(Eq: 1)
Table 1. Pla s ticizers used and their ratio to Chitosan
46
3. Results and Discussion
3.1. Cas t lm formation and appearance
The lms were easy to peel from the ca s t Petra
dish and simple to handle and treat further. The
ca s t lms were transparent, uniform, thin, exible,
and manageable.
3.2. FTIR characterization of unplas ticized
Chitosan lms
Figure 1(black) displays the spectrum of the
unpla s ticized chitosan lm produced by ca s ting a
2% acetic acid solution after it was removed from
the Petri plate and before s torage. The di s tinctive
features of the chitosan spectrum in this s tudy are
analogous to those in other inve s tigations [19,20].
Pure Cs exhibit characteri s tic polymer base-
s tate peaks, including those at 1037 cm–1 from
the vibration of C-O groups, 1562 cm–1 from NH
bending, and 1654 cm–1 from C=O s tretching (amide
I) O=C-NHR. The s tretching vibration of free
hydroxyl and the asymmetrical and symmetrical
s tretching of the N-H bonds in the amino groups
are correlated with the peaks between 3610 and
3000 cm–1 that are present in all of the lms under
examination [21]. The bands at 2912 and 2843 cm–1
indicate the vibrations of the aliphatic C-H [22].
3.3. FTIR inves tigation of Cs-lauric acid blended
lms
Figures 1 illu s trate the usual lauric acid peak,
which occurs between 2928 and 2844 cm–1 [23].
The amide II band for the C-O s tretch of the acetyl
group is represented by the band at 1654 cm–1,
while the band represents the N-H s tretch by the
band at 1597 cm–1. The linked C-O s tretch of the
glucosamine residue is represented by the skeletal
vibration at 1072 cm–1, and the asymmetric C-H
bending of the CH2 group is indicated by the band
at 1377 cm–1 [24]. The bands at 2916 cm–1 and 2850
cm–1, respectively, reect the s tretching vibrations
of CH2 and CH3, whereas the band at 1753 cm–1
represents the s tretching vibrations of C=O [25,26].
The band between 1750 and 1700 cm–1 represents
the carbonyl C=O s tretching.
4000 3500 3000 2500 2000 1500 1000 500
T (%)
Wavenumber (cm
-1
)
CS
Cs-Lauric acid (1:1)
Cs-Lauric acid (1:3)
Cs-Lauric acid (2:1)
Fig. 1. FTIR spectra of Cs-lauric acid lms
Anal. Methods Environ. Chem. J. 5 (4) (2022) 43-54
47
3.4. FTIR examination of Cs-maleic acid
blended lms
The FTIR spectra of the physical blend of chitosan
with maleic acid lms are shown in Figure 2.
The spectra exhibited a peak in the 3500-2500
cm–1, which widened due to the OH groups of
both diacids combining chitosan with each diacid
individually. This sugge s ts that integrating the two
materials raised the proportion of hydroxyl groups
rather than changing the type of functional groups
in the backbone complex [27,28]. The outcome
demon s trated that maleic acid successfully
interacted with chitosan’s amine group.
Additionally, the diacid C=O band at 1701 cm–1
[29] is present. Similar to this, the contributions
of both diacid C-O bonds led to a wider chitosan
C-O absorption at 1180 cm–1. Creating an amide
link between maleic acid and the chitosan amine
group is responsible for the remaining spectrum
alterations.
At 1708 cm–1, the carbonyl C=O s tretching
absorption became visible. The literature claims
that pure diacid has two C=O peaks around 1700
cm–1 and 1750 cm–1, respectively, and s tands for free
and hydrogen-bonded carboxylic acid groups [30].
The peak at 1750 cm–1 vanished after the chitosan
reaction, and there were no additional peaks in
the 1735 cm–1 region, indicating that e s terication
did not occur. According to a literature review
on maleic acid amides, the cyclic amide analog
emerged around 1770 cm–1, but the acyclic amide
displayed classic C=O absorption near 1620 cm–1
[31]. The cyclic s tructure cannot exi s t since there
are no peaks in the 1770 cm–1 area of the chitosantric
acid spectra. Examining peaks in the 563–675 cm–1
range proves that chitosan’s native s tructure was
unchanged. According to Mima et al. [32], these
peaks are sharpe s t for 99 percent deacetylated
chitosan and gradually fade away as acetylation
increases (amide production), and this is the case
here because the extracted chitosan used had about
81% degree of deacetylation.
4000 3500 3000 2500 2000 1500 1000 500
T (%)
Wavenumber (cm
-1
)
Cs
Cs-maleic acid (1:1)
Cs-maleic acid (1:2)
Cs-maleic acid (2:1)
Fig. 2. FTIR spectra of Cs-maleic acid lms.
Modications Chitosan Films by Lauric and Maleic Acids Sara Hikmet Mutasher et al
48
3.5. Tensile properties of Chitosan lms
Especially for single-use packaging when
the material is s tretched during use owing
to continuous wear and tear, flexibility is an
important property of pla s tics. The mechanical
properties of synthetic biopla s tics mu s t be
precisely s tudied to define their range of uses.
Polymer films’ s trength and ela s ticity can be
determined through mechanical te s ting. The
amount to which the film subjected to the applied
pull force reacts is defined by tensile s trength
measurements. A pla s ticizer is a sub s tance that,
when added to polymer materials, increases
their ela s ticity. This pla s ticizer is necessary
to get around the s tiffness of films made with
chitosan. The inter-polymer bond between
chitosan polymer chains and pla s ticizers may
become brittle and break. The pla s ticized and
unpla s ticized chitosan films were te s ted in dry
s tates according to AS tM D882-2010 “S tandard
Te s t Methods for tensile properties of thin plas tic
sheeting and films,”
3.6. Tensile properties of plas ticized Cs: lauric
acid lms
The eect of the addition of lauric acid pla s ticizer
resulted in a decrease of the tensile s trength,
Young modulus, and % elongation at break with an
increasing amount of lauric acid, which was shown in
Figures 3, 4, and 5. This leads to the indication that a
level of interfacial adhesion may be lacking between
chitosan and lauric acid. Adorna et al [33] obtained
the same line of results. This happened because too
many lauric acid pla s ticizer molecules were in a
separate phase outside the phase of the pla s ticized
blend. This sugge s ts that there may not be enough
interfacial adhesion between chitosan and lauric
acid. The intermolecular force between the chains is
reduced because of the conditions. It is concluded that
the decrease in the measured mechanical properties
of chitosan pla s ticized with increasing lauric acid
pla s ticizer amount is in good accord with other reported
results [34,35]. Their results were explained due to the
pla s ticizer’s eect on the promotion of intermolecular
forces, thus lowering the high intramolecular forces
inside the pla s ticized polymer mix chains.
Fig. 3. The eect of Cs: Lauric acid ratios on the tensile s trength of pla s ticized chitosan.
Anal. Methods Environ. Chem. J. 5 (4) (2022) 43-54
49
3.7. Tensile properties of plas ticized Cs: maleic
acid lms
Figures 6, 7, and 8 showed the eect of dierent
ratios of chitosan: maleic acid pla s ticizer on
the mechanical properties of the chitosan-
based blended lms, i.e., tensile s trength,
Young modulus, and %elengotion at the break,
respectively. It can be observed that Cs: maleic
acid lms with a ratio of 2:1 showed higher
s tress at maximum load and Young’s modulus
than those by (1:2) cross-linked by maleic acid,
accompanied by an increase in the %elongation at
break as well. This is because there is an excess
of cross-linking bridges, resulting in lower chain
s tiness and higher extendibility.
This result was also in agreement with Reddy and
Yang [36] and Thessrimuang and Prachayawarakorn
[37]. They reported using an acid pla s ticizer caused
by excess cross-linking, which led to an increase in
tensile s trength, and this was the case here.
Fig. 4. The eect of Cs: Lauric acid ratios on the Young modulus of pla s ticized chitosan.
Fig. 5. The eect of Cs: Lauric acid ratios on the % elongation at break
of pla s ticized chitosan.
Modications Chitosan Films by Lauric and Maleic Acids Sara Hikmet Mutasher et al
50
Fig. 6. The eect of Cs: Maleic acid ratios on the tensile s trength of pla s ticized chitosan.
Fig. 7. The eect of Cs: Maleic acid ratios on the Young modulus of pla s ticized chitosan.
Anal. Methods Environ. Chem. J. 5 (4) (2022) 43-54
51
Fig. 8. The eect of Cs: Maleic acid ratios on the % elongation at break
of pla s ticized chitosan.
3.8. Solubility of plas ticized and un-plas ticized
Chitosan lms
As measured by the lm water solubility of chitosan
lms with various chitosan: pla s ticizers ratios
(w/w), the impact of various pla s ticizers on the
water barrier qualities of the materials was s tudied.
The solubility of lms in water was measured in
triplicate. According to the type of pla s ticizer and its
relative ratio, the lms’ water solubility increased,
as shown in Table 2. This might be explained by the
pla s ticizers’ higher hydrophilicity than chitosan. By
adju s ting the kind and proportion of each pla s ticizer
used in the production of those lms, the solubility
of chitosan/pla s ticizer lms and blends may be
controlled, opening up a wide range of indu s trial
applications. While it may be necessary for certain
materials to be insoluble in specic applications
to ensure the dependability and durability of
the implemented product, lm solubility may
occasionally be desired before consumption. It will
therefore rely on how each pla s ticizer is used when
preparing the sample [38,39].
It may be worth mentioning here that the chitosan
lms always turned rubbery when submerged
in water, but they never retained their s tructural
integrity because the soluble pla s ticized portion
of the lm interfered with the s tructure. However,
the type and concentration of the pla s ticizer can
be adju s ted to modify the solubility of the lm,
making it necessary for more potential applications.
Table 2. Solubility of some pla s ticized chitosan
Cs-Pla s ticizer Ratio SW 1 (%) SW 2 (%) SW 3 (%) SW 4 (%)
Cs 0:0 2.1 2.8 3.3 3.9
Cs-Lauric acid 1:3 10.25 12.8 16.2 26.5
Cs-Maleic acid 2.1 2:1 48.7 53 55
Modications Chitosan Films by Lauric and Maleic Acids Sara Hikmet Mutasher et al
52
The soluble pla s ticized part of the chitosan lms
interferes with the s tructure, which may be worth
highlighting here. Despite usually turning rubbery
when submerged in water, they never maintained
their s tructural integrity. However, the kind and
amount of the pla s ticizer can be altered to change
how soluble the lm is, necessitating it for more
applications.
4. Conclusions
The purpose of this research was to examine how
the types of mono and di-acid pla s ticizers aected
the molecular s tructure, solubility, and mechanical
characteri s tics of chitosan ca s t lms. To improve
some of the physical and mechanical qualities of the
resulting pla s ticized ca s t pla s ticized chitosan lms,
the characteri s tics of the original chitosan lm were
evaluated in conjunction with those of the other
lms. The in s trumental techniques used to s tudy
changes in the chemical s tructure and properties
of modied chitosan have been considered. Every
pla s ticizer te s ted showed enhanced mechanical
performance, and regardless of the kind or quantity
utilized, they all demon s trated the typical pla s ticizer
action of increasing elongation and decreasing
lm s tiness. Data demon s trating the possibility
of using modied chitosan in food packaging as
su s tainably as possible have been presented.
5. Acknowledgement
The authors wish to thank the Department of
Chemi s try, College of Science, University of
Basrah for supporting this work.
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Anal. Methods Environ. Chem. J. 5 (4) (2022) 43-54
Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65
Research Article, Issue 4
Analytical Methods in Environmental Chemi s try Journal
Journal home page: www.amecj.com/ir
AMECJ
In-vitro evaluation of photoprotection, cytotoxicity and
phototoxicity of aqueous extracts of Cuscuta campe s tris
and Rosa damascene by MTT method and UV spectroscopy
analysis
Payam Khazaeli a,b, Atefeh Ameri b, Mitra Mehrabani c, Morteza Barazvana,d, Marzieh Sajadi Bami a,
and Behzad Behnam c,a,e,*
a Pharmaceutics Research Center, Ins titute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
b Pharmaceutical sciences and cosmetic products research center, Kerman University of Medical Sciences, Kerman, Iran
c Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran
d S tudents Research Committee, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran
e Extremophile and Productive Microorganisms Research Center, Kerman University of Medical Sciences, Kerman, Iran
AB S TRACT
Applying sunscreen is essential for protecting the skin from UV’s
acute and chronic eects. Some of these products on the market
display side eects and are expensive. There is a great demand for
eective, cheap, safe, and herbal sunscreens with a wide range of sun
protection activities. This s tudy aimed to evaluate the photoprotection,
cytotoxicity, and phototoxicity of aqueous extracts of Cuscuta
campe s tris (CC-AE) and Rosa damascena (RD-AE). The maceration
method prepared the CC-AE and RD-AE from the aerial branch. In-
vitro photoprotection was evaluated by determining the sun protective
factor (SPF) of CC-AE and RD-AE by a UV-visible spectrophotometer.
The cytotoxicity and phototoxicity s tudies were assessed using the
MTT assay on 3T3 cells. In the nal, the PIF (Photo Inhibitor Factor)
was calculated. The SPF values of CC-AE and RD-AE were found
at 11.10±0.05 and 1.36±0.04, respectively, at the concentration of 0.2
mg mL-1. The half maximal eective concentration (EC50) of CC-AE
and RD-AE was obtained at 35.05±0.91 µg mL-1 and 40.7±0.87 µg
mL-1, respectively. The phototoxicity analysis showed that CC-AE
and RD-AE had low PIF values and were considered as the probable
phototoxic. Overall, regarding the considerable SPF and PIFs values
plus the anti-inammatory and antioxidant properties of these extracts,
they can be evaluated for further pharmaceutical formulations.
Keywords:
Cuscuta campe s tris,
UV-visible spectrophotometer,
Rosa damascene,
Sun protective factor,
Phototoxicity
ARTICLE INFO:
Received 29 Jul 2022
Revised form 12 Oct 2022
Accepted 15 Nov 2022
Available online 30 Dec 2022
*Corresponding Author: Behzad Behnam
Email: behnamb@kmu.ac.ir
https://doi.org/10.24200/amecj.v5.i04.202
------------------------
1. Introduction
Solar ultraviolet (UV) radiation such as UVA (320–
400 nm) and UVB (~295–320 nm) have acute and
chronic inuences on the skin; they might nally
cause cancers of the skin [1]. UVB radiation can
cause acute eects such as erythema and edema,
and chronic eects such as immunosuppression and
carcinogenesis [2, 3]. However, UVA radiation can
induce tanning by the oxidation of melanin, and
photoaging by the de s truction of dermal s tructures, as
well as leading to damage of the macromolecules, and
oxidative s tress by the production of reactive oxygen
species (ROS) [2, 3]. The main de s tructive factors of
UV radiation on the skin are free radicals including
superoxide anions, hydroxyl radicals, singlet oxygen,
56
hydrogen peroxide, ferric ion, nitric oxide, etc. [3].
Photoprotection which is caused with using of
sunscreen, prevents the acute and chronic eects of
UV radiation. UV protectors are classied as UV
lters and UV absorbers based on types of cosmetic
materials [2, 4]. UV lters are divided into two classes
according to their chemical s tructure and mechanism
of action: inorganic, such as titanium dioxide and
zinc oxide, are of low irritation potential and exhibit
photo s tability and wide-ranging absorption spectra
and organic such as UVA, UVB, and broadband
absorbers that absorb the radiations based on their
chemical s tructure. The lter’s ability of organic
lters is classied as a photo s table, photo-un s table,
and photoreactive lters [2, 4, 5]. The sunscreens’
formulations that protect the skin from harmful UV
rays could be introduced as physical and chemical
sunscreens by blocking, reecting, scattering and
absorbing the UV rays [2, 5]. The ecacy rate of
sunscreens is usually measured by the sun protection
factor (SPF) e s timation, which represents an
accepted global characteri s tic of protection from
erythema after exposure to simulated solar radiation
[6]. Generally, the components of sunscreens have
shown side eects such as disruption in the endocrine
sy s tem and changes in the hypothalamic-pituitary-
thyroid (HPT) axis. In addition, they could be caused
reproductive homeo s tasis during long-term use [4, 7].
However, some sunscreens may have environmental
toxicity eects and can have detrimental eects on
the ecosy s tem [2, 4]. Be s t sunscreens should have
several characteri s tics, including safe, non-toxic, and
photo- s table, and be able to protect the skin from
UVA and UVB rays [4]. The natural photoprotectants
can be included the obtained extracts of plants such
as aloe vera, pomegranate, rambutan, grape, tomato,
the green tea, and the oils obtained from soybean,
olive, coconut, almond, and jojoba as well as the
mycosporine-like amino acids (MAA), etc. [5,
8-10]. Several s tudies have described the use of
plant extracts with photoprotection properties. For
example, Rangel et al [2] assessed the photoprotective
capability of extracts from red macroalgae. Permana
et al [11] showed a potential absorption of UVA and
UVB radiation by the hydrogel-containing propolis
extract-loaded phytosome and indicated their high
SPF value of them [11]. Natural combinations have
shown the desirable SPF and anti-inammatory
and antioxidant properties [9, 12]. Rosa damascena
mill, commonly known as Gole Mohammadi in Iran
[13], showed several medicinal properties including
antiviral, antimicrobial, antioxidant, antitussive,
hypnotic, anti-diabetic, and sedative eects on the
respiratory sy s tem [14]. This plant contains dierent
chemical compounds such as tannins, polyphenols,
carotenoids, quercetin, eugenol, citronellol, geraniol,
liquiritin, etc. [14, 15]. Generally, the extracts of
rose petals have shown high antioxidant activity
that correlated to the total phenolic, and avonoid
contents of rose [16, 17]. The analgesic and anti-
inammatory eects of rose have also been reported
[18-20]. The hydroalcoholic extract of R. damascene
can signicantly reduce edema, which may be
mediated by the inhibition of acute inammation
[13]. Cuscuta campe s tris Yuncker with the common
name dodder has analgesic, antipyretic, anti-
inammatory, and anti-cancer properties [21, 22].
This holoparasitic plant has been applied to treat
a liver injury, cancer prevention, sciatica, scurvy,
and scrofula derma [22-24]. Based on reported
works, polyphenolic compounds such as quercetin,
sinapic acid, kaempferol, isorhamnetin hesperidin,
and eugenol were identied in extracts from C.
campe s tris [25, 26]. The ethyl acetate extract of the
plant has the s tronge s t antioxidant eect due to the
highe s t content of avonoid compounds kaempferol
and quercetin [22]. A review of the literature did not
expose any previous s tudies on the photoprotective,
cytotoxicity, and phototoxicity activities of the
aqueous extract of Cuscuta campe s tris (CC-AE) and
Rosa damascena (RD-AE) plants by MTT method
and UV spectroscopy analysis. Generally, the UV/
visible spectrophotometric method were applied
to analyze the UV radiation protection capability
for probable sunscreen applications [2, 6]. One of
the mo s t important issue in pharmaceutical circles
is to optimizing the wright method for analyzing
the active ingredients in bulk drug materials, their
impurities and decompositions sub s tances, and
also pharmaceutical formulations and biological
Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65
57
products. Spectrophotometry is the quantitative
measurement of the reection or transmission
properties of a material as a function of wavelength.
The use of UV-Vis spectrophotometry, especially in
the analysis of pharmaceutical forms, has increased
rapidly in recent years [2, 3, 26]. In vitro methods
for evaluating the sunscreen potentials of materials
are generally of two types. Methods that involve
measuring the absorption or transmission of UV
radiation through sunscreen product lms on quartz
plates or bio-membranes, and methods in which the
absorption characteri s tics of sunscreen agents are
determined based on spectrophotometric analysis of
dilute solutions [2, 3, 26].
In the present s tudy, the UV absorption of each
sample was obtained and the Mansur equation was
applied to nd the nal SPF. Afterwards the eects
of extracts were evaluated in vitro in 3T3 cells to
obtain their probable photo-toxic or photo-protective
behaviors.
2. Materials and Methods
2.1. Chemicals
Trypsin, phosphate-buered saline (PBS), and
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-
tetrazolium bromide (MTT) were supplied by
Sigma company (S t. Louis, MO, USA). Fetal bovine
serum (FBS), Dulbecco’s modied Eagle’s medium
(DMEM), and Penicillin-S treptomycin solution
(100X) were obtained from Borna Pouyesh Gene
Company (BPGene Co., Kerman, Iran).
2.2. Extracts preparation
The plants (C. Campe s tris and R. Damascena) were
collected from Mahan, Kerman, Iran (30.0630°
N, 57.2875° E). The plants were then identied
by Dr. Mitra Mehrabani and kept in the Faculty of
Pharmacy herbarium (Kerman University of Medical
Sciences, Kerman, Iran). The aerial branches of
plants were washed three times with deionized water
and dried at room temperature. The dried aerial
branches were ground with a mill to obtain a ne
powder. The extracts of plants were prepared using
the maceration method. For this purpose, 10 g of the
ne powder was combined with the deionized water
(100 mL) in a laboratory ask with a volume of 500
mL. The mixture was heated at 80 °C for 30 min and
ltrated through Buchner funnel linked with Watman
lter paper (No.1). Finally, the ltrate was freeze-
dried (freeze dryer FD-550 purchased from Tokyo
Rikakikai Co., Ltd, Japan) [27]. The dried aqueous
extract of C. campe s tris and R. damascena were
labeled as CC-AE and RD-AE, respectively, and the
SPF of compounds was determined by UV-visible
spectrophotometer.
2.3. Determination of UV absorption spectra by
UV-vis spectrophotometer
The characterizations of UV absorption spectra
were carried out by analyzing an aqueous extract of
C. campe s tris and R. damascena at concentrations
20000, 10000, 5000, 2500, and 1250 μg mL-1. The
UV spectra were recorded using a Synergy TM 2
multi-mode microplate reader (BioTek In s truments,
Inc., Winooski, VT, USA) from 200 to 900 nm.
2.4. Determination of photoprotection activity of
plants extracts
The procedure of Khazaeli and Mehrabani [28],
with some modications, was used to measure
the photoprotection activity of plant extracts. For
this purpose, the obtained aqueous extracts of C.
campe s tris and R. damascene were individually
scanned in the range from 337.5 nm to 292.5 nm
with interval ve nm using a double beam UV/Vis
spectrophotometer (Optizen 3220 UV). Then, in vitro
SPF was measured by the following equation I [29].
(Eq.I)
Where T(λ), E(λ), and ε(λ) represents the
transmittance of the sample at λ, the spectral
irradiance of terre s trial sunlight at λ, and the
erythemal action spectrum at λ, respectively. The
E(λ) × ε(λ) values are showed in Table 1, the T(λ)
was three times measured and the obtained means
were applied to e s timate the SPF value for each
extract. Afterward, the graph relationship of SPF
versus LnC was used to calculate SPF in 2.0 mg
mL-1 solution for each extraction.
Analysis of CC-AE and RD-AE by MTT and UV spectroscopy Payam Khazaeli et al
58
2.5. Cell culture
The mouse embryonic brobla s t cells (3T3)
(ATCC Number: IBRCC10100) were provided by
the Iranian Biological Resource Center (IBRC) in
Tehran, Iran. The cell line was cultured in DMEM
medium supplemented with 10% (v/v) FBS, 100 U
mL-1 penicillin, and 100 µg mL-1 s treptomycin and
incubated at 37 °C in a 5% CO2 incubator [30].
2.6. Cytotoxicity assay
Based on methods reported in the literature [31-
34], in the exponential growth s tage, the cells were
harve s ted and seeded into 96-well tissue culture
plates (approximately 104 cells per well). After 24 h,
the samples of serial concentrations of CC-AE and
RD-AE (at the nal concentration range of 3.9–125
µg mL-1) were separately poured into the desired
wells. After 24 h, the medium in each well was
switched with 20 µL of MTT solution (5 mg mL-1)
and plates were incubated at 37 °C for a further 3
h. For dissolving the formazan cry s tals, the culture
media were removed from the wells and 100 μL of
fresh DMSO was added to each well of the plate.
The optical density of nal solutions was then read at
570 nm using a Synergy TM 2 multi-mode microplate
reader (BioTek In s truments, Inc., Winooski, VT,
USA). Doxorubicin (12 µg mL-1) was applied as a
positive control. All experiments were repeated in
triplicate on dierent days and EC50 values were
determined and analyzed by non-linear regression
analysis (SPSS software, SPSS inc., Chicago) and
the data were reported as mean (m±SD).
2.7. Evaluation of phototoxicity and
determination of PIF factor
For the purpose of evaluation of phototoxicity in the
presence and absence of UVA radiation [35], cells
were prepared as described in the cytotoxicity assay
into two plates (A and B). Plate A was exposed to
UVA light (1.8 mW cm-2) for 60 min. After 60 min,
the medium was discarded and the fresh medium
was added. Plate B was used as a non-irradiated
control. Both plates were incubated for 24 h at 37
°C in a 5% CO2 incubator. Afterward, the medium
in each well was discarded and MTT solution (20
µL, 5 mg mL-1) was added. Plates were incubated at
37 °C for 3 h and following the culture, media were
removed from the wells and 100 μL of fresh DMSO
was added to each well to dissolve the formazan
cry s tals. The absorption was then measured at 570
Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65
Table 1. Normalized product function used in the calculation of SPF.
Wavelength (nm) E(λ) × ԑ(λ)
292.5 1.139
297.5 6.510
302.5 10.00
307.5 3.577
312.5 0.973
317.5 0.567
322.5 0.455
327.5 0.289
332.5 0.129
337.5 0.046
E(λ): the spectral irradiance of terre s trial sunlight at each wavelength.
ԑ(λ): the erythemal action spectrum at each wavelength.
59
nm, and the EC50 values were e s timated. The PIF
(Photo Inhibitor Factor) was determined based on
below equation II:
(Eq.II)
In compliance with the OECD TG 432 [36], the
below Table was considered for analyzing the PIF
values (Table 2).
2.8. Inves tigation of protective eects of
plant extracts agains t of phototoxic eects of
chlorpromazine
In assessing of the ability of plant extracts to
prevent of the phototoxic eects of chlorpromazine
(CPZ), two culture plates (A and B) were seeded
with about 104 cells per well. Then, the CC-AE and
RD-AE were prepared at a concentration of 31.25
µg mL-1. The concentrations of chlorpromazine
were also trained at the range of 0.1, 0.5, and
1 µg mL-1. After 24 h, the culture media on the
cells were evacuated and 100 µL of the prepared
concentration of extracts and 100 µL of the
concentrations of chlorpromazine were separately
added into the desired wells of the culture plates.
Subsequently, the plate A was exposed to UVA light
(1.8 mW cm-2) for 60 min. Over time, the cultural
media were removed and the fresh media were
added. The plate B was maintained in darkness (as
a non-irradiated control). The culture plates (A and
B) were incubated at 37 °C for 24 h in a 5% CO2
incubator. The subsequent s teps were performed as
in Section 2.8. All experiments were repeated three
times in dierent days. Then, the cell viabilities
(%) were determined, and data were s tated as mean
results (m±SD).
2.9. S tatis tical analysis
Experimental data are presented as the mean
(m±SD) with at lea s t three determinations for
independent experiments. All data were analyzed
by non-linear regression analysis (SPSS software,
SPSS inc., Chicago) and the p-valve (p< 0.05) was
considered to be s tati s tically signicant.
3. Results and Discussion
3.1. UV absorption spectra and critical
wavelength
The UV absorption spectra of CC-AE and RD-AE
are shown in Figure 1. The max absorbance of CC-
AE (at 2500 µg mL-1) and RD-AE (at 2500 µg mL-1)
Table 2. The categorization of phototoxicity s tages
based on PIF values.
PIF value Type of hazard
PIF < 2 Non phototoxic
PIF > 2 and < 5 Probable phototoxic
PIF > 5 Potential phototoxic
Fig. 1. The UV absorption spectra of aqueous extracts of Rosa damascena
and Cuscuta campe s tris at concentration 2500 μg mL-1
Analysis of CC-AE and RD-AE by MTT and UV spectroscopy Payam Khazaeli et al
60
were at 240 nm and 320 nm, respectively (Fig. 1).
3.2. In vitro SPF assessment by UV
Spectrophotometry analysis
The SPF is a quantitative capacity of the eciency
of a sunscreen product. To prevent sunburn and
other skin damage, a sunscreen product should
have a broad absorption of between 290 and 400
nm. Antioxidants from natural resources, especially
plants, might be oered as novel potentials for the
treatment and prevention of diseases caused by UV
rays. There are reports on the correlation between
antioxidant activity and SPF values [2, 37]. Based
on previous reports of the excellent antioxidant
activity of CC-AE and RD-AE plants [16, 17, 25],
the current s tudy inve s tigated the SPF values of
aqueous extracts of plants by UV spectrophotometry
applying Mansur mathematical equation [6]. In
Table 3, the SPF values measured using the UV
transmission spectra of CC-AE and RD-AE are
li s ted. As shown in Table 3, the SPF values obtained
at 2 mg mL-1 were 11.10±0.05 and 1.36±0.04 for
CC-AE and RD-AE, respectively. Ebrahimzadeh
et al [38] assessed the SPF values of extracts from
Sambucus ebulus, Zea maize, Feijoa sellowiana, and
Crataegus pentagyna and reached the highe s t value
(SPF = 24.47) using ultrasonic extract of Crataegus
pentagyna. They also reported that there is a good
correlation between SPF and phenolic contents.
Hashemi et al [37] reported the highe s t SPF values
(0.841 and 0.717) for Cucumis melo leaf ultrasonic
extract and Artemisia absinthium shoots methanolic
extract, respectively. Da Silva Fernandes et al [36]
obtained a low SPF (2.5±0.3) for an aqueous fraction
(AF) from Antarctic moss Sanionia uncinata;
however, the SPF values increased more than three
times in association with UV-lters with AF. The
highe s t value (25.8±0.3) was reported in AF plus
3-(4 methylbenzylidene)-camphor [36]. In another
s tudy, the sunscreen formulations prepared by using
the combination of organic UV lters (w/w %),
and Olea europaea leaf extract (OLE, w/w %) and
measured in vitro photoprotective ecacy using a
UV transmittance analyzer for the determination of
SPF values [7]. The SPF values 56±3, 42± 5, and
21±2 were obtained by formulations that contained
5%, 3%, and 1% OLE, respectively [7]. Therefore,
the association of UV lters with dierent plant
extracts can be increased the eciency of sunscreen
formulations [7, 36].
3.3. Phototoxicity Analysis
The toxicity eects of the CC-AE and RD-AE on
the 3T3 cell line were analyzed using the MTT-based
colorimetric te s t after 24 h; however, phototoxicity
was evaluated by comparing the dierence in toxicity
between the sample plate that was not exposed to UVA
light and the sample plate exposed to UV light. The
half-maximal eective concentration (EC50), without
UVA light, for 3T3 cell line treated with CC-AE,
RD-AE, and was measured to be 35.05±0.91 µg
mL-1, 40.7±0.87 µg mL-1, and 16.79±0.35 µg mL-1,
respectively (Table 4). According to analyses of
Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65
Table 3. Calculation of SPF of the aqueous extracts of plants
in dierent concentrations by UV–visible spectrophotometry
Plant Concentration of aqueous extract SPFa
Cuscuta campes tris 10 0.070±0.04
50 2.000±0.05
500 7.450±0.04
2000 11.10±0.05
Rosa damascena 10 0.027±0.04
50 0.110±0.05
500 1.072±0.05
2000 1.360±0.04
a Data represent means±SE (n=3).
61
the PIF, CC-AE (PIF=3.55) and RD-AE (PIF=2.35)
were exhibited as probable phototoxic in the te s ted
doses (Table 2). Chlorpromazine (PIF=35.59) was
a potential phototoxic hazard and results were
obtained for the cell viability with a dierence
approximately 35-fold in EC50 values, with and
without UV light (Table 4). Amaral et al [39]
presented that the IC50 values for Caryocar
brasiliense supercritical CO2 extract (CBSE) in
the phototoxicity assay considered 6.50% w/v
in dark conditions and 35.53% w/v in irradiated
conditions. According to the PIF value, the CBSE
not exhibited phototoxic potential (PIF=0.18).
Da Silva Fernandes et al [36] reported that the
AF presented non-phototoxic (PIF=1.089) and
the AF in mixtures with UV lters did not oer
any phototoxic potential (PIF < 2). Svobodová
et al [40] assessed the phototoxic potential of
silymarin, an identical extract of the seeds of
Silybum marianum, and its bioactive components.
The obtained results showed that silymarin and its
major component had no phototoxicity. Nathalie et
al [35] assessed the phototoxic of some essential
oils and showed that the PIF values of lemongrass
oil, orange oil, and CPZ were 2.34, 2.21, and 31.24,
respectively, as probably phototoxic hazard by 3T3/
MTT procedure [35]. Consequently, in the present
s tudy, C. campe s tris and R. damascene aqueous
extracts can be identied as probable phototoxic
ingredients; however, additional inve s tigations are
needed to evaluate the health risks associated with
them in vivo.
3.4. Analysis and Evaluation of protective eects
of plant extracts on prevention of phototoxic
eects of chlorpromazine
The eect of combinations of C. campe s tris aqueous
extract, and/or R. damascena aqueous extract and
chlorpromazine as s trong phototoxic sub s tance were
assessed using the MTT-assay on the 3T3 cell line. These
experiments were evaluated using the combination of a
concentration of CC-AE or RD-AE (31.25 µg mL-1) with
three concentrations of CPZ (0.1, 0.5 and, 1 µg mL-1) in
the presence and absence of UVA light. The obtained
results of cell viability (%) are shown in Table 5. After
24 h, the measured cell viabilities (%) for the 3T3
cell line treated with a combination of the CC-AE
and the dierent ranges of CPZ were 53.70±1.51%,
49.15±1.01%, and 43.67±1.2%, respectively, in the
absence of UVA light; however, the measured cell
viabilities (%) were 49.59±2.00%, 45.44±1.51%,
and 37.47±0.93%, for similar concentrations, in
the presence of UVA light (Table 5). The measured
cell viabilities (%) for the s tudied concentration
of RD-AE on the dierent concentrations of CPZ
were 51.29±1.13%, 46.43±1.64%, and 41.82±0.86,
respectively, in the absence of UVA light. Measured
cell viabilities were 43.36±1.02%, 35.53±1.33%, and
47.78±2.1%, respectively, in the presence of UVA
light (Table 5). Generally, in the fact of UVA light, the
measured cell viabilities of CPZ alone were lower than
the combination of CC-AE and CPZ. The measured
cell viabilities of CPZ alone at concentrations 0.5 µg
mL-1 and 1 µg mL-1 were higher than the combination
of RD-AE and CPZ (Table 5).
Table 4. Evaluation of the cytotoxicity and phototoxicity of the aqueous extracts of plants
and chlorpromazine in murine brobla s ts cell (3T3)
Sample UV radiationaEC50
bPIF
Cuscuta campes tris aqueous extract
(CC-AE)
- 35.05±0.91 3.55
+ 9.86±0.61
Rosa damascena aqueous extract
(RD-AE)
- 40.7±0.87 2.35
+ 17.31±0.22
Chlorpromazine
(CPZ)
- 16.79±0.35 35.59
+ 0.467±0.06
a – or + represents the te s ts performed with and without UV light.
b Data represent the mean±SD of three experiments in dierent days
Analysis of CC-AE and RD-AE by MTT and UV spectroscopy Payam Khazaeli et al
62
4. Conclusion
Ultraviolet rays cause numerous injuries to the
skin, so there is a vital need to protect it again s t
its harmful eects. Natural materials usually have
the ability to protect again s t the toxic eects of
ultraviolet rays. Based on favorable antioxidant and
anti-inammatory properties of Cuscuta campe s tris
(CC-AE) and Rosa damascena (RD-AE) plants,
the current s tudy inve s tigated the photoprotection,
cytotoxicity and phototoxicity activities of
aqueous extracts of CC-AE and RD-AE in mouse
brobla s t cells (3T3 cells) by MTT method and
UV spectroscopy analysis. In this research, the
SPF values of CC-AE and RD-AE were evaluated
by UV–visible spectrophotometry applying the
Mansur equation. At the concentration of 0.2 mg
mL-1, the SPF values of CC-AE and RD-AE were
11.10±0.05 and 1.36±0.04, respectively. The EC50
of CC-AE and RD-AE was 35.05±0.91 µg mL-1 and
40.7±0.87 µg mL-1, respectively. The PIF values for
CC-AE and RD-AE are in the range of probable
phototoxic materials (PIF > 2 and < 5), but as these
numbers are decient and near the range of non-
phototoxic, they could be hypothesized for future
anti-solar formulations. Moreover, in the presence
of UVA light, the measured cell viabilities of CPZ
alone were lower than the combination of CC-AE
and CPZ. Overall, the presented data in this report
showed that RD-AE, with SPF and PIF of 11 and
2.35 and various prominent biological eects,
could be regarded as an ecient natural product to
be considered in sunscreen formulations.
5. Acknowledgements
We thank the Pharmaceutics Research Center,
In s titute of Neuropharmacology, Kerman
University of Medical Sciences (Kerman, Iran) for
their support (Grant number: 95000346).
Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65
Table 5. Evaluation of protective eects of plant extracts on prevention of phototoxic eects
of chlorpromazine in murine brobla s ts (3T3).
Plant
CAE* Chlorpromazine
UV radiation a
Cell viability b
(%)
Cuscuta campes tris 31.25 1 - 53.70±1.51
31.25 0.5 - 49.15±1.01
31.25 0.1 - 43.67±1.20
31.25 1 + 49.59±2.00
31.25 0.5 + 45.44±1.51
31.25 0.1 + 37.47±0.93
Rosa damascena 31.25 1 - 51.29±1.13
31.25 0.5 - 46.43±1.64
31.25 0.1 - 41.82±0.86
31.25 1 + 43.36±1.02
31.25 0.5 + 35.53±1.33
31.25 0.1 + 47.78±2.10
Chlorpromazine - 1 - 55.22±1.09
- 0.5 - 53.60±2.11
- 0.1 - 53.60±1.21
- 1 + 47.66±1.21
- 0.5 + 38.04±0.98
- 0.1 + 32.86±0.88
* CAE: Concentration of aqueous extract
a – or + represents the te s ts performed with and without UVA light
63
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Anal. Methods Environ. Chem. J. 5 (4) (2022) 66-76
Research Article, Issue 4
Analytical Methods in Environmental Chemi s try Journal
Journal home page: www.amecj.com/ir
AMECJ
Evaluating the eect of ethanol foliar feeding on the essential oil,
phenolic content, and antioxidant activities of Ducrosia anethifolia
Aliyeh Sarabandia, Amirhossein Sahebkarb,c,d,e, Javad Asilif, Moharam Valizadehg, Khalilollah Taherih,
Jafar Valizadeh,*, and Maryam Akaberif,*
a Department of Phytochemis try, Faculty of Science, University of Sis tan and Baluchis tan, Zahedan, Iran.
bApplied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
c Biotechnology Research Center, Pharmaceutical Technology Ins titute, Mashhad University of Medical Sciences, Mashhad, Iran.
d School of Medicine, The University of Wes tern Aus tralia, Perth, Wes tern Aus tralia, Aus tralia.
e Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
f Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
g Faculty of Environmental Sciences and Sus tainable Agriculture, Sis tan and Baluchis tan University, Zahedan, Iran.
h Department of Biology, Faculty of Science, University of Sis tan and Baluchis tan, Zahedan, Iran
AB S TRACT
Ducrosia anethifolia (Apiaceae) is a medicinal aromatic plant
di s tributed in Iran and Afghani s tan. This research aims to inve s tigate
the composition of the plant essential oil, determine the total avonoid
and phenolic contents, and evaluate its antioxidant activities after
ethanol foliar feeding. For this purpose, 0, 10, 20, 40, and 80%
v/v of aqueous ethanol solutions were sprayed on dierent batches
of the plants. Then, the essential oils were obtained using water
di s tillation. Compounds were analyzed by Gas chromatography-
mass spectrometry technique(GC-MS) using a validated method.
The method was validated as per the ICH guidelines for linearity,
precision, accuracy, robu s tness, LOD, and LOQ. The total contents
of phenols and avonoids were measured using spectrophotometric
methods. The antioxidant activity was evaluated using DPPH and
FRAP assays. The results showed that n-decanal, cis-verbenyl
acetate and dodecanal were the major compounds in all treatments.
However, alcohol could cause signicant dierences in the essential
oils qualitatively and quantitatively. The results showed that 40%
ethanol could increase the number of phenolics and avonoids and
consequently the antioxidant activity. Thus, ethanol foliar feeding
can be used as an appropriate approach to increase the essential oil of
D. anethifolia as well as its phenolic and avonoid contents.
Keywords:
Ducrosia anethifolia,
GC-MS,
Spectrophotometry,
Foliar feeding,
Essential oil,
Antioxidant
ARTICLE INFO:
Received 27 Jul 2022
Revised form 11 Oct 2022
Accepted 23 Nov 2022
Available online 28 Dec 2022
*Corresponding Author: Maryam Akaberi and Jafar Valizade
Email: akaberim@mums.ac.ir and djafar.walisade@gmail.com
https://doi.org/10.24200/amecj.v5.i04.213
------------------------
1. Introduction
Ducrosia anethifolia (DC.) Boiss. (A s teraceae) is
an aromatic herbaceous and biennial plant with
a height of 10–30 cm. The s tems are glabrous
and branched mo s tly from the base. The leaves
are ovate-oblong, 2–6 cm long, and branched,
with a petiole length of 5–18 cm. The edges of
the petals are jagged and slightly shaggy and the
compound umbel inorescence has white owers.
In Iran, the plant is known as Moshgak, Mushk
Boo, Darvishan Ginger, Reshgak, Khorkhundai,
Gavarshkh, and mount Coriander [1]. The genus
Ducrosia has three species in Iran including D.
anethifolia, D. assadii Alava, and D. abellifolia
Boiss. Ducrosia anethifolia grows wildly in
mountainous and plain areas on sandy soils in
67
dierent regions of Iran such as Kerman, Khorasan,
Zanjan, Shushtar, Behbahan, Shiraz, Kazerun,
Borazjan, Noorabad, Farashband, Firoozabad,
Jahrom, Darab, and Si s tan Baluche s tan [2]. It also
grows natively in countries from the Mediterranean
range to W. Paki s tan such as Afghani s tan, Paki s tan,
Iraq, Syria, Lebanon, and some Arab countries
[3]. Ducrosia assadii is endemic to Iran and D.
abellifolia is native to Syria, W. Iran, and Arabian
Peninsula [4]. Essential oils are one of the mo s t
pharmacologically important con s tituents in plants
belonging to Ducrosia. Terpene compounds are
reported to be responsible for many medicinal
activities of the oil of this medicinal plant. The
volatile compounds of this aromatic plant are
used as a avoring additive in various beverages
and desserts. Besides, Moshgak is also used as
an edible vegetable [5, 6]. Several s tudies have
inve s tigated the essential oil composition of D.
anethifolia revealing that the long chain oxygenated
hydrocarbons such as decanal, dodecanal, and
decanol con s titute the major compounds in the
essential oil. In addition, monoterpenes including
α-pinene, α-thujene, linalool, cis-citronellyl
acetate, and oxygenated sesquiterpenes such
as chrysanthenyl acetate have been reported as
the main con s tituents of the essential oil [7]. In
Iranian traditional medicine, the aerial parts of D.
anethifolia have been used to relieve various pains
such as headaches, back pain, and colic pain. It
has been also used for the treatment of seizures,
insomnia, heartburn, cataract, inammation of the
inner wall of the nose, and colds. Pharmacological
s tudies show that the plants belonging to this genus
have muscle relaxant, CNS depressant, and anti-
depressant properties. In addition, the essential oils
obtained from D. anethifolia have antimicrobial
properties again s t gram-positive bacteria, yea s ts,
and some dermatophytes. The essential oil of D.
anethifolia is reported to have antifungal properties
by preventing the growth of parasitic fungi such as
Candida albicans on the skin [8-10]. It could also
improve kidney function and lower the lipid levels
of the blood. Alpha-pinene as one of the major
compounds in essential oil is probably responsible
for the anti-anxiety eect of the plant. Myrcene,
as another main component of the plant, has
several pharmacological activities including anti-
radical, inhibitory eects, anti-cancer, and anti-
tumor properties [11-13]. The use of methanol and
ethanol foliar feeding is one of the mo s t important
approaches for increasing plant growth and harve s t
yield. Research has shown that ethanol becomes
acetaldehyde after penetration into the plant tissue.
Acetaldehyde is transformed into acetate (acetic
acid) by the acetaldehyde dehydrogenase enzyme.
Acetic acid also converts to acetyl coenzyme A,
which eventually turns into carbon dioxide and
dioxide. Methanol, ethanol, and other alcohols
are non-toxic to plants and can simply penetrate
the membrane of plant cells. The absorption
rate directly depends on the density of alcohol.
Therefore, the application of methanol and ethanol
foliar feeding on the aerial parts of C3 plants (the
plants that only use the s tandard method of carbon
dioxide xation by the enzyme Rubisco) [14], in
which their light breathing is large can compensate
for part of the s tabilized carbon losses and in this
way, increase pure photosynthesis and dry matter
production per unit area. As a result, some s tudies
conducted in the eld of agronomic C3 plants have
shown that methanol could aect the performance
of these plants positively [15]. Thus, the aim of
this s tudy was to evaluate the eect of ethanol
foliar feeding on the essential oil composition and
yields of D. anethifolia. In addition, the antioxidant
activities of the plant extract were inve s tigated and
the total phenol and avonoid contents of the plant
were measured.
2. Material and Methods
2.1. Planting and harves ting
This s tudy was performed at the research farm of
the research center for medicinal and ornamental
plants of Si s tan and Baluche s tan University with a
latitude of 29°27’N and a longitude of 60°51’E at an
altitude of 1410 m from sea level. In this experiment,
seeds of the plant samples were planted in 7 rows
of binary, each row was about 80 centimeters
and the di s tance between every two rows was 20
Determination and evaluation of the aromatic essential oil in plant Aliyeh Sarabandi et al
68
centimeters. The plants were divided into ve
treatment groups including control (di s tilled water)
and four concentrations of ethanol solution (10, 20,
40, and 80%). The ethanol spraying was performed
before the owering s tage of the plants every three
days 6 times. The spraying process began in early
May and the plants were harve s ted in early June
(spring 2016). After harve s ting, the aerial parts of
the plants were dried in the shade and s tored until
use. After collecting, the plants were transferred
to the laboratory and dried in the shade. Then, the
plants were milled, powdered, and prepared for
essential oil.
2.2. Extraction Procedure
The dried aerial parts of the plants for each treatment
were powdered separately with an electric mill.
Then, the powdered samples were subjected to
extraction. The essential oil of the samples was
obtained by water di s tillation. For this purpose,
50 g of each dried plant sample was subjected to
hydrodi s tillation for 3 h using a Clevenger-type
apparatus (1000 ml water). The essential oils were
collected in separate glass vials and were dehydrated
with aid of sodium sulfate and magnesium sulfate
salts. In order to obtain the extracts of the samples,
the maceration method was used. About 5.0 g of
the powdered samples were weighed accurately
and added to separate Erlenmeyer asks containing
50 ml methanol (covered with aluminum sheets)
and placed on a magnetic s tirrer for 24 hours. Then,
the extracts were ltered with Watman lter paper
and the solvent was removed by a rotary evaporator
to obtain dry extracts (Schema 1).
2.3. GC-MS Analysis
In the next s tep, the essential oils were subjected
to the GC-MS analysis to separate the chemical
con s tituents and identify them according to their mass
characteri s tics and retention times. The analysis of the
essential oils was carried out using an Agilent sy s tem
equipped with an HP-5S column (30 m × 250 µm,
lm thickness 0.25 µm) interfaced with a quadrupole
mass detector (MS 5977A). Oven temperature 50-
250°C (3°C per minute), injector temperature 250°C,
injection volume: 0.1 μL, split injection with a split
ratio of 1:50 helium as the carrier gas with ow rate
1 mL min-1, ion source: 70 eV, ionization current:
150 μA, and scan range: 35-465. The method was
validated as per the ICH guidelines for linearity,
precision, accuracy, robu s tness, LOD, and LOQ
according to our previous s tudy [16]. Identication
of the chemical con s tituents of the essential oil was
carried out using AMDIS software (www.amdis.net)
and identied by its retention indices with reference
to the n-alkanes series (C6-C20), comparison of their
retention time, mass spectra, and computer matching
with the Wiley 7 nL and NIS t library database.
2.4. Determination of total phenolic content
Total phenol content was determined by the Folin-
Ciocalteu reagent. A dilute solution of the extracts
(0.05:1 g mL-1) or gallic acid ( s tandard phenolic
compound) was mixed with the Folin-Ciocalteu
reagent (2.5 ml, 1:10 diluted with di s tilled water)
and aqueous Na2CO3 (2 ml, 5%). The mixture was
allowed to s tand for 30 min and the phenolic contents
were determined by colorimetry at 765 nm. The total
phenolic content was determined as mg of gallic
Schema 1. Procedure for determination of avonoid, phenolic contents in Ducrosia anethifolia
Anal. Methods Environ. Chem. J. 5 (4) (2022) 66-76
69
acid equivalent using an equation obtained from the
s tandard gallic acid calibration curve [17].
2.5. Determination of total avonoid content
The avonoid contents of the extracts were measured
by aluminum chloride coloration using quercetin as
s tandard [18]. To extract avonoids, 0.1 g of each extract
was solved in 10 mL ethanol 80%. Then, 100 μL of the
solution was added to a te s t tube, and 100 μL of 10%
AlCl3, 100 μL of 1 M sodium acetate, 1.5 mL of ethanol
96%, and 3.2 mL of di s tilled water were added and
vortexed for 1 minute. The control treatment included
3.4 mL of di s tilled water, 100 μL of 1 M sodium
acetate, and 1.5 mL ethanol 96%. After 30 minutes,
adsorption was read at 415 nm.
2.6. DPPH free radical scavenging activity
This spectrophotometric method was used to evaluate
the antioxidant activity of the extracts. DPPH is
a reagent that measures free radical scavenging
activity [19]. Zero, 0.01, 0.02, 0.03, 0.04, 0.05 mL of
concentration 2000 mg L-1 of the extracts and positive
control (ascorbic acid) were added to 1.0 mL of 0.1
mM solution of DPPH (Sigma, S t Louis) in methanol.
The reaction mixture was shaken and then incubated
for 30 min at room temperature. The remaining
amount of DPPH was determined at 517 nm again s t
a blank using a spectrophotometer (Milton Roy
Company Spectronic 2OD). All te s ts were carried out
ve times.
2.7. Ferric-reducing antioxidant power (FRAP)
assay
The antioxidant capacity of the plant extracts was
done by Iron reduction (FRAP assay) according to
Sadeghi et al [17]. For this purpose, 300 mM acetate
buer (pH 3.610 ) mM TPTZ solution in 40 mM HCl,
and 20 mM FeCl3-6H2O solution were mixed for the
preparation of s tock. FRAP reagent was prepared
right away before analysis by mixing 25 mL acetate
buer, 2.5 mL TPTZ solution, and 2.5 mL FeCl3-
6H2O solution. Plant extracts (1000 μg mL-1) were
prepared. 200 μg mL-1 of the extracts was mixed with
1.8 mL of the FRAP reagent and was incubated at
37 ºC for 30 min in the dark condition before being
used. Then, readings of the colored products (ferrous
tripyridyltriazine complex) were determined at 595
nm again s t a di s tilled water blank. FeSO4-7H2O (100-
1000 μM) was used for calibration. Ascorbic acid was
used as a positive control. Results are expressed as
mM Fe2+ per mg sample [17].
3. Results
3.1. Chemical composition of the essential oils
Table 1 shows the major components of the essential
oil in the dierent treatments and control along
with the percentage of each compound. The results
revealed that ethanol spraying had a signicant eect
on the amount and yield of the essential oils. The yield
of the essential oil was enhanced by increasing the
amount of alcohol from 10% to 40% treatment with a
decline in 80% alcohol-treated samples. The chemical
composition of the essential oil of the control and that
of the 10% treatment were to some extent similar.
Intere s tingly, while in the two treatments 20% and
40%, the chemical composition is rather the same, their
chemical composition is dierent from the blank and
10% treatment. Although the chemical composition
of the 80% treatment was similar to 20% and 40%,
there were some dierences. For in s tance, compound
2-isopropyl-5-methyl-3-cyclohexene-1-one was only
identied in the 80% treatment. Decanal, cis-verbenyl
acetate, and dodecanal were major components in all
samples with variations in dierent treatments.
Table 2 shows that monoterpenes and other compounds
including alkanes are dominated in the essential oil
samples. The number of oxygenated monoterpenes
is increased in 20%, 40%, and 80% treatments while
hydrocarbon monoterpenes are decreased in these
samples compared to the control. Hydrocarbon
monoterpenes are absent in the 20% sample.
The number of oxygenated sesquiterpenes is almo s t
the same in all treatments with a small increase of
20% and 40%. The highe s t number of hydrocarbon
sesquiterpenes were observed for 20% sample.
3.2. Determination of total avonoid content
The highe s t and the lowe s t amount of total phenol
per 1g of the plant powder were observed for 40%
Determination and evaluation of the aromatic essential oil in plant Aliyeh Sarabandi et al
70
Table 1. The major compounds identied in the essential oil of the dierent treatments.
aRt bRI Compound Control 10% 20% 40% 80%
4.936 939 α-Pinene 5.272 4.678 - - 0.564
5.52 975 Sabinene 0.936 0.876 - - -
5.589 979 β-Pinene 0.251 0.223 - - -
5.737 990 β-Myrcene 1.428 1.341 - - -
6.287 1024 ρ-Cymene 4.217 3.593 - - 0.330
6.355 1029 d-Limonene 5.178 4.575 - - 0.348
7.271 1088 Terpinolene 0.343 0.329 - 0.523 -
7.46 1100 Nonanal 0.849 1.018 0.425 - 1.057
8.135 1137 cis-Verbenol 0.809 0.682 0.779 - 0.927
8.204 1140 Citronellal 0.801 0.902 - - -
8.364 1150 trans-Verbenol 0.247 0.210 0.962 0.607 0.253
8.432 1169 1-Nonanol 0.732 0.783 - - 0.710
8.713 1179 ρ-Cymen-8-ol 0.458 0.419 - - 0.620
8.776 1185 Cryptone 0.606 0.696 - - -
8.965 1200 Decanal 20.044 20.681 9.098 10.596 13.571
9.274 1250 3,7-Dimethyl-2-octen-
1-ol
1.709 1.479 - - 0.353
9.754 1274 cIMC Hexane - - - - 10.191
9.817 1282 cis-Verbenyl acetate 20.598 18.151 36.274 42.415 21.766
9.88 1286 5-Undecanol 1.816 1.560 1.038 0.778 1.311
10.155 1290 Lavandulyl acetate 0.915 0.828 1.562 1.315 0.623
10.367 1298 trans-Pinocarvyl
acetate
- - 0.420 1.093 -
10.395 1306 Undecanal 1.008 1.183 - - 1.131
10.498 1352 Citronellyl acetate 0.659 0.634 2.058 2.255 1.019
11.408 1381 Geranyl acetate 1.387 1.467 0.958 1.243 0.331
11.677 1408 Z-Caryophyllene 0.451 0.575 0.490 - 0.243
11.757 1410 Dodecanal 10.768 11.953 6.777 6.431 11.053
12.072 1420 β-Caryophyllene 1.666 1.731 1.359 1.308 0.351
12.501 1436 γ-Elemene 3.458 3.246 22.994 1.055 2.038
13.25 1510 dCPCP - - - 18.913 16.705
14.029 1578 Spathulenol 1.898 2.096 2.287 1.577 1.417
14.12 1583 Caryophyllene oxide 1.568 1.709 1.304 0.796 1.161
14.252 1612 Tetradecanal 0.342 0.452 0.467 0.425 0.814
14.515 1620 Unknown 2.536 2.689 1.287 1.165 0.604
14.652 1632 γ-Eudesmol 0.264 0.266 2.008 3.832 1.287
14.738 1677 Z-Nerolidyl acetate 0.778 0.833 0.724 0.462 0.225
14.887 1680 β-Eudesmol 0.506 0.576 1.168 - 1.723
14.939 1685 n-Tetradecanol 1.092 1.334 0.990 0.665 1.582
Total 73.176 90.761 95.429 97.454 94.287
aRetention time; bKovats Index
cMC Hexane: 2-Isopropyl-5-methyl-3-cyclohexen-1-one
dCPCP: 2-Cyclopentylidene cyclopentanone
Anal. Methods Environ. Chem. J. 5 (4) (2022) 66-76
71
and 80% ethanol samples, respectively (Fig. 1). The
highe s t and the lowe s t amount of total phenol per 1.0
g of the plant extract were observed for 40% and 10%
ethanol samples, respectively (Fig. 2).
3.3. Determination of total avonoid content
As shown in Figure 3, the 40% ethanol treatment
showed the highe s t amount of avonoid compared
to the re s t of the treatments.
Table 2. The amount of dierent volatile compounds in dierent treatments.
Compounds Control 10% 20% 40% 80%
Oxygenated Monoterpenes 28.189 25.468 43.013 59.524 36.083
Hydrocarbon Monoterpenes 17.625 16.453 - 0.523 1.272
Oxygenated Sesquiterpenes 5.014 5.480 7.491 6.667 5.813
Hydrocarbon Sesquiterpenes 5.575 5.552 24.843 2.363 4.670
Other Compounds 25.883 38.964 18.795 37.808 47.934
Fig. 1. The total phenol content in the plant powder of dierent treatments.
Fig. 2. The total phenol content of the extracts from dierent treatments.
Determination and evaluation of the aromatic essential oil in plant Aliyeh Sarabandi et al
72
3.4. DPPH free radical scavenging activity
The results of DPPH radical-scavenging activity
assay are shown in Figure 4. Considering the large
variation of IC50, the lowe s t antioxidant activity
was observed for 80% treatment compared to the
control. Treatments with 10%, 20%, and 40%
showed almo s t the same antioxidant activity, all
higher than that of the control.
3.5. FRAP assay
All analyzed extracts demon s trated signicant
antioxidant capacities with FRAP te s t. The 40%
treatment showed 53.00 mMFe2+/mg sample
with the highe s t antioxidant activity compared to
reducing power of ascorbic acid (69.00 mMFe2+/
mg sample) (Fig. 5).
Totally, the results from the determination of phenolic
and avonoid contents of the samples as well as
DPPH and FRAP assays revealed that the amount of
these compounds and the antioxidant capacity of the
plant samples were inuenced by the use of ethanol
spraying which was shown in Table 3. By increasing
the concentration of alcohol from 10% to 40%, the
phenol and avonoid contents and consequently the
antioxidant activity reached their maximum rate.
Fig. 3. The total avonoid content of the extracts from dierent treatments.
Fig. 4. DPPH free radical scavenging activities observed
for dierent treatments compared to ascorbic acid as positive control
Anal. Methods Environ. Chem. J. 5 (4) (2022) 66-76
73
4. Discussion
Foliar feeding is a technique of feeding plants by
applying liquid fertilizer directly to the leaves. Due
to the increased rate of absorption through the aerial
parts of plants, it is an excellent method to deliver
food and elements required for plants much fa s ter
[19]. S tudies show that using alcohols with dierent
concentrations would exert dierent eects on
dierent plant species. The mo s t important role
for methanol operating in C3 plants is to prevent
light respiration, probably due to increased CO2
concentration in leaves. If the concentration of CO2
increases in leaves, ribulose 1,5-bisphosphate will
react with CO2 in s tead of O2, and the carboxylation
function will occur. Therefore, the alcohol-induced
biomass increase of the C3 plants might cause the
plant to use methanol as a direct source of carbon
for serine biosynthesis and reduce carbon wa s te
through light respiration [15]. There are several
reports inve s tigating the eect of alcohol on the
function of dierent plants. For in s tance, Zbiec and
Podsiad (2003) inve s tigated the eect of alcohol
spraying and reported the increasing quantitative
and qualitative yield of this technique on plants such
as geranium, wheat, turnip, and sugar beet [21].
Iqbal Makhdum et al. s tudied the eect of methanol
spraying on cotton plants and observed that 30%
methanol treatment has been able to increase plant
function compared with the control treatment [22].
In another research, performed by Safarzadeh
Vishkaei who s tudied the eect of methanol on
peanuts, 30% methanol treatment could increase
the height of plant and grain function. Methanol
and ethanol (30%) spraying could increase plant
growth and the essential oil amount of peppermint
[23]. The application of alcohol foliar feeding
Fig. 5. The antioxidant capacity of dierent treatments compared
to ascorbic acid as a positive control
Table 3. Comparing the phenolic and avonoid contents as well as antioxidant activities of the samples.
Plant samples IC50
Fe2+ mM/
mg sample
mg total phenol/
1 gr powder
mg total phenol/
1 gr extract
mg quercetin/
1 gr extract
Control 2.87 0.69 5.33 20.44 21.7
10% 66.9 0.43 5.97 19.04 27.94
20% 30.22 0.45 6.71 20.58 30.62
40% 28.55 0.47 9.88 22.12 33.3
80% 91.73 0.44 4.43 18.46 26.54
Determination and evaluation of the aromatic essential oil in plant Aliyeh Sarabandi et al
74
would induce increasing the production of cytokinin
and plant growth [24]. In addition, foliar feeding
of alcohols in plants might induce increasing the
plant metabolites including essential oils. S tudies
show that plants exposed to environmental s tresses
might increase the production of their specialized
metabolites to confront the s timulant leading to
more metabolite synthesis.
The current s tudy showed that alcohol foliar feeding
had eects on the amount of essential oil of the
medicinal plant D. anethifolia and its composition.
Our results showed that monoterpenes and alkanes
such as n-decanal were the major components in
the essential oil of D. anethifolia. In mo s t of the
s tudies inve s tigating the essential oil composition
of this medicinal plant, n-decanal was reported as
the main con s tituent [25,26]. For example, Salari et
al. have reported n-decanal (22.29%) as the mo s t
abundant con s tituent of D. anethifolia essential oil
(Kerman), followed by alkanes decanol (22.18%)
and dodecanol (11/79%) [27]. There are also some
s tudies in which decanal has not been reported
as the major compound [3,28,29]. The results of
Arbabi et al. showed that cis-chrysanthenyl acetate
with an average amount of 44.77% was the main
compound of the essential oil of D. anethifolia
from Si s tan & Baluchi s tan [2].
Besides, our results revealed that this technique
had benecial eects on the amount of phenolic
and avonoid contents and consequently on the
antioxidant activity of the plant. In total, the be s t
eciency was observed for 40% ethanol treatment.
This can be due to the nutritional role of ethanol
as a carbon source, s timulus, and active sub s tance
in metabolic reactions. In addition, ethanol plays
an essential role in the biosynthetic pathway for
the production of terpenoids. The opposite eect
observed in the higher percentages of alcohol might
be due to the plant poisoning leading to the decreased
level of specialized metabolites and antioxidant
capacity. The results of this experiment showed
that the use of the hydroalcohol spraying could
increase essential oil production, so it is sugge s ted
to use of hydroalcoholic foliar feeding to increase
D. anethifolia metabolites in future research.
5. Conclusion
Taken together, the results of this experiment
showed that ethanol spraying could increase the
production of essential oil as well as phenolic and
avonoid compounds in D. anethifolia. Since the
plant is an important medicinal plant, this might
increase its ecacy. Thus, hydroalcoholic foliar
feeding can be a valuable s trategy to increase the
specialized metabolites of D. anethifolia in future
research.
6. Acknowledgements
The authors would like to thank the University of
Si s tan and Baluche s tan for their help and support
(Grant No. 2376081).
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Anal. Methods Environ. Chem. J. 5 (4) (2022) 66-76
Anal. Methods Environ. Chem. J. 5 (4) (2022) 77-86
Research Article, Issue 4
Analytical Methods in Environmental Chemi s try Journal
Journal home page: www.amecj.com/ir
AMECJ
Determination of tetrauoroborate in wa s tewaters by ion
chromatography after ion pair liquid-liquid dispersive
microextraction
Roman Grigorievich Sirotkina,*, Elena Valerievna Elipashevaa, Alexander Vladimirovich Knyazeva,
and Viktoriya Alekseevna Bobrovaa
a Lobachevsky S tate University of Nizhny Novgorod - National Research University (UNN), Nizhny Novgorod, Russian Federation
AB S TRACT
The ion chromatographic method was developed to determine
tetrauoroborate ion (BF4
-) in dierent types of water using ion
pair liquid-liquid dispersive microextraction. Tetrauoroborate was
extracted into an organic phase (1,2-dichloroethane) as an ion pair with
a tetrabutylammonium cation (TBA+). The mo s t complete formation
of [(ТBА+)(BF4
-)] was observed at ion-pair reagent concentration of
at lea s t 5 mmol L-1 (C(BF4
-) ≤ 1mg L-1). Ultrasonic irradiation was
used to disperse the extractant. The achieved concentration factor (K)
was 29±3, and the degree of extraction (R) was 50±5%. The limit of
detection of tetrauoroborate using the microextraction technique was
7×10-3 mg L-1. The method applies to the analysis of dierent water
origins. The presence of the main contained anions does not interfere
with the microextraction and chromatographic determination of
tetrauoroborate. The maximum molar ratio of BF4
- to diverse ions is
1:104 for uoride, chloride, bromide, nitrate ions, and 1:102 for sulfate
and perchlorate ions.
Keywords:
Tetrauoroborate,
Microextraction,
Ion chromatography,
Ion pair,
Wa s tewaters
ARTICLE INFO:
Received 6 Aug 2022
Revised form 19 Oct 2022
Accepted 25 Nov 2022
Available online 29 Dec 2022
*Corresponding Author: Roman G. Sirotkin
Email: roman_sirotkin94@mail.ru
https://doi.org/10.24200/amecj.v5.i04.214
1. Introduction
Today, the concept of “green chemi s try” remains
one of the main trends in analytical chemi s try [1-4].
Decreased volumes of toxic reagents, automation,
and miniaturization of analytical methods are the
critical points of this concept [3]. One of the leading
trends in green chemi s try is using ionic liquids
containing tetrauoroborate ions [5, 6]. The control
of its concentration in various objects is necessary
at the s tages of the synthesis and use of ionic liquids.
Indu s trial wa s tewater can also contain various
amounts of tetrauoroborate. It’s known that BF4
-
use in the composition of non-aqueous electrolytes
for chemical reactions and electrochemical
processes [7], as well as in electroplating to
improve the quality of the electroplated layer [8].
Another alternative application area for BF4
- is
agriculture. It is used there as a “green fungicide”
[9] and an herbicide [10]. The wide use of BF4
-
means a sub s tantial dierence in the composition
of the analyzed samples. This necessitates the
development of a universal method for analyte
determination.
Spectrophotometry [11, 12, 13] and ion
chromatography [14, 15] are the mo s t common
methods for determining tetrauoroborate in
aqueous solutions. The works [11, 12] are based on
the conversion of all boron forms in the sample to the
BF4
-, reaction with an organic dye, and photometric
determination of the product. However, the reaction
s tability depends on the temperature, the sample
------------------------
78 Anal. Methods Environ. Chem. J. 5 (4) (2022) 77-86
pH, and the dye concentration. Also, the high
consumption of toxic reagents is the disadvantage
of this work. In addition, these methods make it
possible to determine the concentration of total
boron in the sample. The ion pair preconcentration
of BF4
- and spectrophotometric determination are
described in [13]. But large amounts of sample (20
ml) and toxic extractant (10 ml) were used in this
work.
The determination of BF4
- in water samples by
ion chromatography is often not sensitive enough
(0.2 mg L-1 - 1.4 mg L-1) [14,15]. In this work, a
procedure for ion pair liquid-liquid microextraction
of BF4
- is proposed to increase the determination’s
selectivity and lower the detection limit. This
method involves forming a neutral ion pair between
the anion of the analyte and the cation of the ion-
pair reagent due to electro s tatic interaction [16].
It is known that cations of quaternary ammonium
bases (QABs) can form ion pairs with inorganic
anions from aqueous solutions [17]. The solubility
of such compounds in organic solvents is higher
than in water [18]. In addition to the lower detection
limits, microextraction also makes it possible to
reduce the load on the chromatographic sy s tem at
high concentrations of diverse ions. It is important
for the analysis of various samples, including
indu s trial wa s tewater.
2. Materials and methods
2.1. Microextraction technique
A specially designed centrifuge tube (4.5 mL
volume) was used for microextraction. 3.0 ml of
analyzed water was placed in a tube, and a solution
of the ion-pair reagent was added. Then the tube
was placed in an ultrasonic bath, and 500 μL of
the extractant was added. The resulting emulsion
was separated by centrifugation. Further, the
replacement of the organic matrix with an aqueous
one was held. To do this, the extract (400 μL) was
transferred in a Teon vial, and the extractant was
evaporated by using an infrared lamp. Then 50
µL of eluent was added to the dry residue. This
solution was injected into the chromatograph for
the determination of tetrauoroborate ion (BF4
-) in
water samples (Schema 1).
2.2. Chromatographic conditions
All experiments were carried out on an LC-20A ion
chromatograph (Shimadzu, Japan), which consi s ted
of a Model LP-20ADsp liquid delivery pump and
a conductivity detector Model CDD-10Avp. The
separating column was OKA (50x4 mm i.d., Vagos,
Tallinn, E s tonia), and the suppressor column was
KU-2x8 (100x4 mm i.d., Ekos-1, Moscow, Russia).
Both columns and the conductivity detection cell
Schema 1. Microextraction technique for determination of tetrauoroborate ions (BF4
-) in water
and wa s tewater samples based on ion chromatography
79
Determination of tetrauoroborate by ion chromatography Roman Grigorievich Sirotkin et al
were placed inside the CTO-20AC column oven
for temperature control (32 ºС). The eluent was
obtained by a mixture of 1.0 mmol L-1 Na2CO3 with
4.0 mmol L-1 NaHCO3, and the ow rate was set
at 3 ml min-1. This eluent has the optimum eluting
power for the selective and fa s t determination of
tetrauoroborate. The volume of the injection loop
was 50 μL.
2.3. Reagents
Ammonium tetrauoroborate was used to prepare a
s tandard solution supplied by JSC Vekton, Russia.
The solution was kept in a polyethylene container
for no more than a month. Sodium carbonate and
sodium bicarbonate were used to prepare an eluent
solution (JSC Vekton, Russia). All solutions were
prepared using deionized water. 1,2-dichloroethane,
chloroform, dichloromethane, and carbon
tetrachloride were used as extractants (JSC Vekton,
Russia). All reagents were analytical grade or
better. Ion-pair reagents were tetrabutylammonium
hydroxide TBAOH (40% aqueous solution, Sigma-
Aldrich, Switzerland, CAS Number: 2052-49-5)
and tetraethylammonium hydroxide TEAOH (25%
aqueous solution, Acros Organics, India).
2.4. Other Equipment
The extractant was dispersed using a PSB-Gals
1335-05 ultrasonic bath with a radiation generator
power of 50 W and a frequency of 35 kHz. SM-
6M centrifuge with a rotor speed of 1200 rpm was
used for separating the organic and aqueous phases.
During replacing the matrix, the extractant was
evaporated using a 300 W mirror infrared lamp.
The Anion 4100 conductometer was used to control
of purity of deionized water. Laboratory scales
AUX 320 Shimadzu, micro doses HTL 20-200 µL,
and Dragon LAB TopPette Pipette 2-20 µL were
also used in work.
3. Results and discussions
3.1. S tability of tetrauoroborate ion (BF4
-)
Hydrolysis of BF4
- ions occurs in aqueous solutions
at pH>1. Also, the rate of this process depends
on the temperature [19]. About 0.7% of BF4
-
decomposes in alkaline solutions within a day at
20°C [20]. So, we have s tudied the s tability of a
s tandard solution containing 1 mg L-1 of BF4
-. The
results (Fig. 1) showed that the s tandard solution is
s table for a month at a temperature of 3 ± 1ºС in a
polyethylene container.
Fig. 1. The s tability of BF4
- s tandard solution concentration.
Conditions: T = 3±1ºС, polyethylene container
80
3.2. Eect of ultrasonic irradiation time
The maximum concentration factor of BF4
- is
reached at 3 min of ultrasonic irradiation and does
not change further (Fig. 2). This dependence is valid
for all chosen extractants and ion-pair reagents.
3.3. Eect of dierent extractants and ion pair
reagents
Using tetrabutylammonium hydroxide as an ion-pair
reagent made it possible to achieve better microextraction
parameters (Table 1). Obviously, this is explained by the
longer alkyl chain of tetrabutylammonium compared
to tetraethylammonium cation. So, the extraction of
ion pairs from the aqueous phase increases [21].
Parameters of microextraction depend on the
polarity of the extractant solvent. Both the
permittivity of solvent ɛr and its dipole moment
μ are important complementary characteri s tics,
so the polarity of the extractant was e s timated
using the electro s tatic coefficient EF [22],
defined as the product of ɛr and μ. Thus, the
simultaneous influence of both parameters
is taken into account. The dependence of the
concentrating factor of tetrafluoroborate on the
electro s tatic coefficient of the used extractant is
shown in Figure 3. The higher the EF value of
the solvent, the higher the efficiency of [(TBA+)
(BF4
-)] extraction.
3.4. Eect of ion pair reagent concentration
The minimum concentration of QAB was 5 mmol
L-1 for analyte concentrations range of 0.01–1
Table 1. The concentrating factor (K) and the degree of BF4
- extraction (R) (n=3, P=0.95).
QAB )% ,К )R
chloroform 1,2-dichloroethane carbon tetrachloride dichloromethane
Tetraethyl-ammonium
hydroxide
0.1 ± 1.3
(0.2 ± 2.2)
0.3 ± 3.1
(0.5 ± 5.3)
0.1 ± 0.8
(0.1 ± 1.4)
0.2 ± 2.4
(0.3 ± 4.1)
Tetrabutyl-ammonium
hydroxide
2 ± 19
(3 ± 27)
3 ± 29
(5 ± 50)
0.2 ± 1.8
(0.3 ± 3.0)
3 ± 26
(4 ± 44)
Fig. 2. Eect of ultrasonic irradiation time. Extractant: 1,2-dichloroethane;
ion-pair reagent: TBAOH, concentration 5 mM (n=3, P=0.95)
Anal. Methods Environ. Chem. J. 5 (4) (2022) 77-86
81
mg L-1 (Fig. 4). Abundance of ion pair reagents
in the sample does not make the concentration
results worse. It is necessary to shift the chemical
equilibria to the formation of ion pairs.
Excess reagent was contained in the extract
[23]. So, tetrabutylammonium hydroxide and
tetraethylammonium hydroxide were chosen as
ion-pair reagents. The fact was that hydroxide ions
were neutralized by hydrogen ions in a suppression
column and entered the detector as water molecules.
It helped to decrease the background signal.
Fig. 3. Eect of extractant EF. Ion-pair reagent TBAOH, concentration 5 mM.
1 – carbon tetrachloride, 2 – chloroform, 3 – dichloromethane,
4 – 1,2-dichloroethane. (n=3, P=0.95) EF, 10-30 Cm-1
Fig. 4. Eect of ion pair reagent concentration. Extractant: 1,2-dichloroethane.
Ion-pair reagent: TBAOH, concentration 5 mM. С(BF4
-) = 1 mg L-1 (n=3, P=0.95)
C(TBA+), mmol L-1
Determination of tetrauoroborate by ion chromatography Roman Grigorievich Sirotkin et al
82
3.5. Detection limits
The limit of chromatographic detection Cmin
was dened as 3.3σ (σ: the s tandard deviation
of measurements of blank samples) [24]. The
detection limit using microextraction Cmin, ex was
calculated by dividing Cmin/K (Table 2).
3.6. Synthetic was tewaters analysis and eect of
diverse anions
The microextraction eciency and chromatographic
determination are also aected by the sample
composition. Many anions can compete with
tetrauoroborate in forming ion pairs with QABs.
Synthetic wa s tewater samples were prepared and
analyzed to assess the interfering eect of diverse
anions. The results are shown in Table 3.
Sulfate and perchlorate ions can have the greate s t
interfering eect. These anions also form s table
ion pairs with tetrabutylammonium cation. When
its concentration is over than shown in Table 3, the
co-eluting occurs with BF4
-. The peak geometry
is violated, and the calculation of its area is
dicult. However, under the chosen conditions,
the separation of all components is not dicult.
It is illu s trated by the synthetic wa s tewater
chromatograms (Fig. 5a, 5b).
Table 2. Summary of method parameters (Extractant : 1,2-dichloroethane. Ion-pair reagent:TBAOH,
concentration 5 mM; n=3, P=0.95)
Parameter name Value
Linear
Regression Equation
S = 4011C±180, where
S – value of BF4
- peak area,
mV×min, C – BF4
- concentration, mg L-1
Linearity Range of the calibration curve, mg L-1 100 - 0.5
% ,R 50±5
K 29±3
Сmin, mg L-1 10-1×2
Сmin, ex, mg L-1 10-3×7
Table 3. Eect of diverse anions
(Extractant − 1,2-dichloroethane. Ion-pair reagent − TBAOH, concentration 5 mM; n=3, P=0.95)
Anal. Methods Environ. Chem. J. 5 (4) (2022) 77-86
83
3.7. Example of was te waters analysis and
recoveries of BF4
-
Using the proposed method, 3 samples of
wa s tewater were analyzed to determine BF4
-.
Samples 1 and 2 were taken from the drain of the
university electrochemical laboratory, and sample
3 was wa s tewater from the electroplating indu s try.
All samples were ltered twice: r s tly, with an ash-
free paper lter with a pore size of 5-8 µm (Melior
XXI LLC, Russia), then with a syringe lter with a
pore size of 0.22 µm (Hawach Scientic Co., Ltd,
China). BF4
- concentration was calculated using the
calibration curve method. The results (Table 4) show
that the proposed method is suitable for analyzing
various origins samples. Recoveries of BF4
- are
shown in Table 5.
Fig. 5a. Chromatograms of synthetic wa s tewater samples with direct analysis of samples,
1 – F-, 2 – Cl-, 3 – Br-, 4 – NO3
-, 5 – SO4
2-, 6 – BF4
-, 7 – ClO4
-
Fig. 5b. Chromatograms of synthetic wa s tewater samples after microextraction.
1 – F-, 2 – Cl-, 3 – Br-, 4 – NO3
-, 5 – SO4
2-, 6 – BF4
-, 7 – ClO4
-
Determination of tetrauoroborate by ion chromatography Roman Grigorievich Sirotkin et al
84
4. Conclusion
A simple ion chromatographic method was developed
to determine BF4
- using ion-pair liquid-liquid dispersive
microextraction. The parameters of microextraction
(concentration factor and degree of extraction) were
calculated using ion-pair reagents with dierent
hydrophobicity and extractants of various polarities.
The higher the ion-pair reagent hydrophobicity and
the electro s tatic coecient of the extractant, the higher
the extraction eciency. The be s t combination is
using 1,2-dichloroethane and tetrabutylammonium
hydroxide. The maximum molar ratios of analyte
to diverse anions were e s tablished using synthetic
wa s tewater. Analysis of real samples of indu s trial
wa s tewater showed that other contained components
don’t interfere with the determination of BF4
-. The
proposed method can generally be applied to analyze
water samples of various origins.
5. Acknowledgment
The authors are sincerely grateful to the laboratory
attendants of the chemi s try department of the
Lobachevsky S tate University of Nizhny Novgorod
for their eorts.
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Table 5. Recoveries of BF4
- (n=3, P=0.95)
Original, mg L-1 Added, mg L-1 Found, mg L-1 Recovery, %
10-1×(4.0±0.4)
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Anal. Methods Environ. Chem. J. 5 (4) (2022) 77-86
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Component
Concentration, mg L-1
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*Fe 10-1·(2.0±0.4)10-1×(1.5±0.3) 0.4±1.9
*Ni 10-2×1> 10-2×1> 10-2×(2.0±0.4)
*Cu 10-2×1> 10-2×1> 10-2×(8.0±1.5)
*Cr 10-2×1> 10-2×1> 10-2×(3.0±0.8)
-F1.1±0.1 10-1×(9.3±0.9)11±2
-Cl 25±3 32±4 68±7
SO4
2- 5.0±0.6 8.1±0.9 36±5
BF4
-10-2×(7.2±1.4)10-2×(2.2±0.4)10-1×(4.0±0.4)
* determined by atomic absorption analysis
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Anal. Methods Environ. Chem. J. 5 (4) (2022) 77-86
Anal. Methods Environ. Chem. J. 5 (4) (2022) 87-95
Research Article, Issue 4
Analytical Methods in Environmental Chemi s try Journal
Journal home page: www.amecj.com/ir
AMECJ
Photocatalytic degradation of methyl orange using cerium
doped zinc oxide nanoparticles supported bentonite clay
Safoora Javana, Mohammad Reza Rezaei Kahkhab*, Fahimeh Moghaddama,
Mohsen Faghihi-Zarandic, and Anahita Hejazid
a Departement of Environmental Health Engineering, Neyshabour University of Medical Sciences, Neyshabbur, Iran
b Department of Environmental Health Engineering, Zabol University of Medical Sciences, Zabol, Iran
c Foreign Language Department, Shahid Bahonar University of Kerman, Kerman, Iran
d Engineering Department of Occupational Health & Safety at Work, Kerman University of Medical Sciences, Kerman, Iran
ABS TRACT
Methyl orange (MO) is a common anionic azo dye that is a serious
harmful pollutant to the environmental aquatic sys tems, so it mus t
be treated before it can be discharged. Photocatalys ts are usually
semiconducting solid oxides that create an electron-hole pair by
absorbing photons. These electron holes can react with molecules on the
surface of the particles. Photocatalys ts are used in water purication,
self-cleaning glasses, the decomposition of organic molecules, etc.
Photocatalys ts are environmental cleaning materials that remove
pollution from surfaces and can des troy organic compounds when
exposed to sunlight or uorescence. The photocatalytic process
follows the following principles. Bentonite mineral is a natural
adsorbent material that has good adsorption capacity. In this work,
zinc oxide nanoparticles doped with cerium were prepared by the sol-
gel method (SGM) and deposited on bentonite clay to degrade methyl
orange (MO) dye. Important parameters that aected degradation
eciency such as contact time, amount of nanocatalys t, and initial dye
concentration were inves tigated and optimized. Results showed that
100% degradation eciency was obtained at 60 mg of nanocatalys t
and 50 mg L-1 of methyl orange in 120 minutes. The Kinetics of the
degradation process was consis tent with pseudo-second-order and
the adsorption isotherm of MO dye on nanocatalys t was tted with
the Langmuir isotherm model. The reusability of the synthesized
nanocatalys t showed that the nanocatalys t was applied successfully
seven times without a signicant change in degradation eciency.
Keywords:
Photocatalys t,
Degradation,
Clay,
Bentonite,
Methyl Orange,
Dye
ARTICLE INFO:
Received 3 Aug 2022
Revised form 20 Oct 2022
Accepted 17 Nov 2022
Available online 30 Dec 2022
*Corresponding Author: Mohammad Reza Rezaei Kahkha
Email: m.r.rezaei.k@gmail.com
https://doi.org/10.24200/amecj.v5.i04.216
1. Introduction
Photocatalys ts are one of the essential elements
for advanced oxidation processes (AOPs) [1, 2].
Zinc oxide (ZnO) is often the rs t choice due to
its cheapness, non-toxicity, chemical s tability,
and high photocatalytic activity. Photocatalys ts
absorb light radiation (ultraviolet or visible) by
the catalys t, and electrons are transferred from the
semiconductor’s valence band to the conduction
band. This transition creates a hole in the valence
band, and an electron is produced in the conduction
band (Schema 1).
The hole in the reaction with water molecules
produces active hydroxyl radicals[3, 4].
Moreover, the produced electron is transferred
to the dissolved oxygen and forms a superoxide
------------------------
88
radical. These radicals can remove pollutants in
aqueous media. Since the produced electron and
holes are uns table and can recombine and return
to their original s tate, doping elements were used
with ZnO to prevent these phenomena[5]. Cerium
is one of the elements of the lanthanide family
whose redox couple Ce (III)/Ce (IV) causes the
production of CeO2 and Ce2O3 oxides. Ce (IV)
traps the electron created in the conduction band,
which has los t its s table electron conguration,
tends to donate its electron and become s table,
which is possible by electron migration to oxygen
absorbed on the surface and the formation of
superoxide radicals. Therefore, the electron of
the conduction band enters a new cycle, which
reduces the possibility of its access to the hole[6,
7]. Despite the advantages of nanocatalys ts, their
use in water purication processes is limited due
to the small size of the particles. Problems such
as the separation of suspended particles, non-
recycling and secondary pollution are among the
limitations of this method. One proposed method
to solve this problem is xing nanocatalys ts
on suitable subs trates[8]. Many researchers
developed dierent types of subs trates such as
silica gel[9], activated carbon [10], s tainless s teel
[11], glass bers [12] and wood foam [13], has
been used. Recently, ZnO nanoparticles were
immobilized on cellulose paper [14], which was
used to treat textile was tewater in a photoreactor.
Natural clays such as bentonite, montmorillonite,
and perlite are used as catalys t subs trates due
to their high porosity, chemical inertness, non-
degradability, and high mechanical and thermal
resis tance compared to the other mentioned
subs trates[15]. In this work, ZnO-Ce nanoparticles
were rs t synthesized by the sol-gel method.
Then, nanoparticles were xed on bentonite. The
performance of the synthesized nanocatalys t as a
catalys t was inves tigated in the removal of methyl
orange in a batch photoreactor.
2. Material and methods
2.1. Reagents and ins trumental
All reagents and chemicals are analytical grade
and used as received. Zinc acetate dihydrate
Zn(CH3COO)2·2H2O; CAS Number: 5970-45-6),
cerium (CAS Number: 7440-45-1), nitrate (SRM
from NIS t: NaNO₃ in H₂O 1000 mgL-1 NO₃,
Sigma), hydrochloric acid (CAS Number: 7647-01-
0), sodium hydroxide (CAS Number: 1310-73-2),
absolute ethanol (CAS Number: 64-17-5) and MO
Anal. Methods Environ. Chem. J. 5 (4) (2022) 87-95
Schema 1. Photocatalytic process
89
dye (Content 85 %; CAS Number: 547-58-0; EC
Number: 208-925-3; Sigma) were obtained from
Sigma and Merck (Germany). Bentonite (CAS
Number: 1302-78-9) was purchased from Sigma
(Sigma, USA). The pH of the solutions was adjus ted
using a Metrohm (Metrohm, Switzerland) pH
meter. The color concentration was measured using
a double beam-Unico 4802 spectrophotometer at
its maximum wavelength (584 nm). FTIR spectrum
was recorded by Bruker Tensor 27 device.
2.2. Synthesis of ZnO-Ce nanoparticles
The sol-gel method was used to prepare ZnO-Ce
nanoparticles. Firs t, 8.5 mL of zinc acetate was
added to 40 mL of absolute ethanol and the solution
was placed in an ultrasonic bath for 30 minutes
(Solution A). Then 0.12 g of cerium nitrate was
dissolved in 20 mL of absolute ethanol and 3 mL
of deionized water and 2 mL of hydrochloric acid
were added to it. Then, the solution was placed
in an ultrasonic bath for 10 minutes (solution B).
Solution B was added drop by drop to solution
A while s tirring to form a gel. To evaporate the
ethanol, the gel was placed in an oven with a
temperature of 80°C for 12 hours and then calcined
for 3 hours in an oven at a temperature of 550°C.
2.3. Synthesize bentonite nanocatalys t
The immersion method was used to synthesize
bentonite nanocatalys ts coated with ZnO-Ce
nanoparticles. For this purpose, 0.1 g of ZnO-Ce
nanoparticles was added to one liter of ethanol and
water with a ratio of 3:1. For homogenization, the
slurry solution was placed in an ultrasonic bath (35
kHz, 40 W) for 30 minutes. Next, bentonite was
immersed in the solution for one minute. Then
the bentonite was rs t dried at room temperature
and then dried in an oven at 80°C for 2 hours. To
increase the adhesion of nanoparticles to the surface
of bentonite, the granular bentonite was heated in
an oven at a temperature of 550°C for 2 hours.
2.4. Removal procedure
Photocatalytic removal of MO by ZnO-Ce
nanoparticles was s tudied using a batch reactor,
equipped with a UVC lamp at room temperature
(25 °C). To enhance removal eciency, the reactor
was covered with aluminum sheets. The appropriate
dose of ZnO nanoparticle was mixed with dierent
amounts of MO dye. The solution was s tirred at
300 rpm for 30 minutes while the UV lamp at 3800
W irradiated the solution. After the experiment,
30 ml of the sample was taken and in order to
separate the zinc oxide nanoparticles, the sample
was centrifuged at 5000 rpm and ltered. Remind
concentration was measured by spectrophotometer
at 530 nm (Schematic 2) shows the diagram of
degradation of MO).
Photocatalytic degradation of MO by ZnO/Ce/bentonite clay Safoora Javan et al
Schema 2. Schematic diagram of degradation of MO
90
The removal percentage of MO dye (%removal)
was calculated as Equation 1.
(Eq.1)
Parameters aecting the removal of MO dye,
including the amount of the nanocomposite (10-
100 mg), initial concentration of MO dye ( 25
–150 mg L-1 ), and contact time (30-210 min), were
inves tigated.
3. Results and discussion
3.1. Characterization of nanocatalys t
3.1.1. XRD pattern of ZnO/Ce/ bentonite
Schematic 3 showed an XRD pattern of the
synthesized nanocomposite. It can be seen in
schema 3 that by doping cerium as an impurity, the
peaks related to the rutile phase are removed and
only the anatase phase is observed. In other words,
the presence of cerium as an impurity greatly
improves the growth of anatase phase crys tals and
prevents the transfer of the anatase phase to rutile.
3.1.2. SEM image of synthesized ZnO/Ce/
bentonite
Schematic 4 showed an SEM image of a synthesized
nanocatalys t. It can be concluded that the presence
of cerium in the ZnO s tructure reduces the size of
nanoparticles. Considering the s trong dependence
of the properties of nanoparticles on their size, we
can expect signicant changes in the properties of
ZnO/Ce/ bentonite nanoparticles.
Anal. Methods Environ. Chem. J. 5 (4) (2022) 87-95
Schema 3. XRD pattern of ZnO/Ce adsorbent
91
3.2. The optimized parameters for photocatalytic
removal of MO
3.2.1.Eect of amount of nanocatalys t
The amount of ZnO /Ce / bentonite nanocatalys t
impacts the adsorption of the MO dye. The removal
eciency and the adsorption capacity were
inves tigated. For this purpose, experiments were
conducted using an adsorbent dosage in the range
of 10 to 120 mg. As depicted in Figure 1, the uptake
of the MO dye was signicantly increased, up to
60 mg. Furthermore, by increasing the amount of
nanocatalys t, the removal eciency was increased.
3.2.2. Eect of initial concentration of MO dye
on removal eciency
The eect of the initial concentration of dye
on removal percentage by ZnO/Ce/Bentonite
nanocatalys t was inves tigated in the range of 20
to 150 mg L-1. The result is shown in Figure 2. In
the early s tages of adsorption, the results showed
a signicant increase. The maximum percent
removal was achieved at 50 mg L-1 of MO dye.
After this point, the saturation of active sites on the
nanocatalys t has occurred, resulting decrease in the
adsorbent’s ability to the sorbent.
Photocatalytic degradation of MO by ZnO/Ce/bentonite clay Safoora Javan et al
Schema 4. SEM image of synthesized adsorbent
Fig. 1. Eect of ZnO/Ce/Bentonite amount on degradation eciency
92
3.2.3. Eect of time on degradation eciency
The eect of contact time on the degradation eciency
of MO dye by ZnO/ Ce/ Bentonite nanocatalys t was
inves tigated. The results are depicted in Figure 3.
The removal percentages of MO dye increased
signicantly in the early s tages. After a while, the
percentage degradation will rise slightly until an
equilibrium is reached. The results showed that the
bes t dye removal percentage was obtained at 120
minutes. Hence, this time was selected for subsequent
experiments.
3.2.4. Kinetic s tudy
Adsorption kinetic s tudies of MO dye onto ZnO/
Ce/ Bentonite nanocatalys t were inves tigated
using pseudo-rs t-order and pseudo-second-order
kinetics. The results are shown in Figure 4 and at
summarized in Table 1. The kinetic model that bes t
ts the adsorption of MO dye on the nanocatalys t was
determined by R2 values. Considering the reported
R2 values, the adsorption of MO dye on ZnO/ Ce/
Bentonite nanocatalys t was followed by a pseudo-
second-order kinetics model.
Anal. Methods Environ. Chem. J. 5 (4) (2022) 87-95
Fig. 3. Eect of time on degradation eciency
Fig. 2. Eect of dye concentration on degradation eciency
93
Photocatalytic degradation of MO by ZnO/Ce/bentonite clay Safoora Javan et al
Fig. 4. Kinetic s tudies of the adsorption of MO dye on nanocatalys t.
a) Psuedo Firs t order b) Psuedo second order
Table 1. Kinetics parameters for MO dye
Firs t order kinetics Second order kinetics
MO dye R2K1 (min-1) R2K1 (g.mg-1min-1)
0.8344 -6.51X10-3 0.9992 1.88X10-2
3.2.5. Adsorption isotherms
For the evaluation of adsorption isotherms,
Langmuir and Freundlich’s isotherms were used
to illus trate the mechanism. The Langmuir and
Freundlich isotherms for MO dye on nanocatalys t
were depicted in Figure 5 and Table 2. It was
found that the adsorption of Mo dye on ZnO/Ce/
Bentonite nanocatalys t followed from Langmuir
isotherm.
3.2.6. Reusability of Nanocatalys t
Evaluating the reusability of ZnO/Ce/Bentonite
nanocatalys t on the degradation of MO dye
photocatalytic experiments in optimal conditions was
repeated several times. Afterward, the nanocatalys t
was washed, dried, and reused for the next run.
Results showed that degradation eciency was
decreased from 100 to 98.1 after 7 repeated
experiments that conrmed the reusability of
the nanocatalys t. Also, for the evaluation of the
94
sorption capacity of the nanocatalys t, a s tandard
solution containing 100 mgL−1 of MO was applied.
The initial and nal amounts of MO dye were
determined by spectrophotometer after adsorption
on ZnO/Ce/ Bentonite. The maximum adsorption
capacity was dened as the total amount of
adsorbed MO per gram of the nano catalys t. The
obtained capacity was found to be 115 mg g−1.
4. Conclusion
The degradation eciency of methyl orange using
ZnO/Ce/Bentonite nanocatalys t as photocatalys t and
adsorbent was inves tigated. The optimal conditions
for the degradation eciency of the dye were found at
a nanocatalys t dosage of 60 mg , a contact time of 120
min yellow. At optimum conditions, 100% of methyl
orange was removed by synthesized nanocomposite.
Also, the nanocatalys t was reused after 7 repeated
cycles, and adsorption capacity was obtained115 mg
g-1. The isotherm data of MO dye were tted with
the Langmuir model, while the kinetic data were
modeled by the pseudo-second-order, revealing that
the nature of the kinetic adsorption is chemical. The
present s tudy showed that the ZnO/ Ce/ Bentonite
nanocatalys t is an eective adsorbent for the
degradation of MO dye from aqueous solutions.
Fig. 5. a) Langmuir isotherm of MO dye onto nanocatalys t,
b) Freundlich isotherm of MO dye onto nanocatalys t
Table 2. Isotherms parameter of MO dye
Langmuir Freundlich
MO dye RLKLKFN
Results 0.9874 0.3989 0.03345 0.9513 11.9674 2.5713
Anal. Methods Environ. Chem. J. 5 (4) (2022) 87-95
95
5. Acknowledgements
Authors at this moment thank from health laboratory
of Zabol University for their cooperation to perform
experiments.
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Photocatalytic degradation of MO by ZnO/Ce/bentonite clay Safoora Javan et al
