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The mechanical properties of thin nanocomposite
coatings can be evaluated by using the nanoinden-
tation test [1-3]. This test is usually quick and easy
to do. In the early twentieth century, this test is
developed by Brinell using spherical balls and
smooth ball bearings which were measured the
plastic properties of the materials [1, 4 and 5].
During the past two decades, this testing method
has been expanded at the nanometer range. In the
newly developed system, very small loads at
nano-Newton ranges and the small displacements
of 0.1 nm can be exactly determined. Today,
nanotechnology is considered as an important tool
for studying the mechanical properties of the small
parts of matter. In this test, an indenter tip with the
known specific geometry is penetrated into the
surface of coating or film with an applied specific
load or penetration depth in static or dynamic mode.
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344
A Brief Review of Nanoindentation Technique and its
Applications in Hybrid Nanocomposite Coatings
Amir Ershad Langroudi
Associate Professor, Color, Resin & Surface Coating Department (CRSC), Polymer Processing Faculty
(PPF), Iran Polymer and Petrochemical Institute (IPPI), 14965/115 Tehran, Iran
Received: 5 June 2013; Accepted: 12 August 2013
Nanoindentation techniques are widely used for the study of nanomechanical properties of thin
nanocomposite coatings. Theoretical concepts and practical use of nanoindentation method are
summarized with reporting the applications of these tests in characterization of some particular
thin nanocomposite hybrid coatings prepared by sol-gel process. The better mechanical
properties can be obtained in the investigated hybrid coatings in compare with the pristine
polymer coatings. It is demonstrated that the adding nano inorganic fillers can be influenced on
physical-mechanical properties of coatings as well their microstructures. However, the adhesion
of nanocomposite coatings is dependent on the chemical bond in the interface, microporous and
defects in the network. Coating can be delaminated on exposure to extreme UV and humidity
conditions. The mechanism of coating's failure as well microstructural changes can be studied by
nanoindentation technique in statistic or dynamic modes.
Keyword: Nanoindentation; Coatings; Hybrid; Nanocomposite; Mechanical Properties.
ABSTRACT
1. INTRODUCTION
International Journal of Bio-Inorganic Hybrid Nanomaterials
(*) Corresponding Author - e-mail: A.Ershad@ippi.ac.ir
The various nanomechanical properties can be
obtained based on the affected area such as elastic
modulus, elastic and plastic deformation, hardness,
wear and scratch resistance, etc [6-12].
In addition, nanoindentation can be used to
estimate the fracture toughness of thin films which
cannot be measured by other conventional
penetration tests [13-16]. With tangential force
sensors, nano-scratch and abrasion tests can also be
measured at ramping loads. Atomic force
microscopes (AFM) are ideal tools for monitoring
of nano-sized influence and provide the usefulness
of the information about the cracking and the
deformation of material as a result of nano-
indentation [17]. When the penetration force system
is used in joint of an atomic force microscope, the
in situ penetration image may be obtained
simultaneously [18]. Diamond is often used as a tip
of indenter because of its high hardness and elastic
modulus which minimize its share of influence on
the measured displacement [19, 20]. For measuring
properties such as hardness and elastic modulus at
the smallest possible scale, triangular pyramid
Berkorvich tip is preferred over Vickers or Knoop
indenter because three-sided pyramid tip is simply
stable than the two others four-sided tips on one of
the sharp point.
Continuous stiffness measurement (CSM) is a
recently significant development in nano-
indentation technique [1, 21-241. This technique is
ideal for mechanical studies of thin films, poly-
meric materials, multilayers which the micro-
structure and mechanical properties change with
indentation depth. In addition, this technique is less
sensitive to thermal deviation [1, 24-26] as carrying
out at frequencies greater than 40 Hz. In the CSM
test, the indentation load is applied by a small
sinusoidally varying motion of the indenter on the
material's surface and analyzed the response of the
material's surface by means of a frequency specific
amplifier data. The CSM technique allows the
measurement of mechanical properties at any point
along the loading curve and not just at the point of
unloading as in the conventional nanoindentation
test. The CSM technique gives opportunity for
measuring displacement and stress relaxation in
creep test, utilizing a sinusoidal shape load at high
frequencies allows doing fatigue tests at the
nanoscale in thin films and microbeams by
monitoring the change in contact stiffness because
the contact stiffness is sensitive to damage
formation. There are intensive studies and wide
research articles on the use of nanoindentation
techniques to characterize of the nanomechanical
properties of hybrid nanocomposite coatings
[1, 2, 8, 26 and 27]. The aim of this article is to
demonstrate a brief review on the theoretical
aspects and practical use of nanoindentation
methods with illustration the applications of these
techniques in characterization of some particular
thin nanocomposite hybrid coatings prepared by
sol-gel process. Figure 1 shows the standard
indentation instrument which includes three
essential elements: the first part of instrument is for
applying force, the second part of instrument is an
element through which the indent force is applied
on the surface sample and finally, the third part is
the sensors for measuring the indenter force and
displacement.
Figure 1: Different parts of a typical indentation
instrument [2].
The various shapes of indenter head used in nanoin-
dentation technique such as pyramidal, spherical,
cube corner or conical geometry according to
selected data. However, the most common shape of
indenter is a Berkovich pyramidal tip.
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344 Ershad Langroudi A
338
2. A typical nanoindentation curve
Figure 2 indicates a typical nanoindentation curve
including loading and unloading force as a function
of displacement of indenter head as well as a
schematic loading cycle force on the indenter in the
CSM technique. The displacement response of the
indenter at the excitation frequency and the phase
angle between the two are measured continuously
as a function of depth.
Figure 2: (a) A typical nanoindentation : load and unload-
ing -displacement curve and schematic loading cycle in
CSM technique (b) schematic deformation pattern of sub-
strate surface with an elastic-plastic behavior after inden-
tation test [1, 14].
Any inconsistency observed in the curve indicates
cracking, delamination or another failure in the
coating. In the coated substrate, it needs to pay
attention to the coating thickness in nano-
indentation assay. The penetration depth should not
exceed of the 10% of the coating thickness [8, 28].
Otherwise, the nanomechanical data is normally
influenced by the underlying substrates. The
coating is considered quantitatively with good
toughness if after the indentation test, no cracking
occurs, in it. However, this description needs the
measurement of crack length, which is extremely
difficult in thin films even under SEM observation
[6, 29].
Figure 3: Schematic presentation of cracking, delamina-
tion and spallation in failure of coating from substrate in
nanoindentation test [6, 30].
In addition, it depends on the type of the used
indenter head. Fracture can occur in three steps as
schematically represented in Figure 3. In the first
step, a ring form of crack surrounded around the
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344Ershad Langroudi A
339
(a)
(b)
1
2
3
indenter contact area, in the second step, high
lateral pressure induce delamination and buckling
of coating from substrate around the indented area
and in third step, the crack is seen as a second ring
and high bending stresses induce the spallation of
coating at the edges of the buckled area.
3. Hardness and Young's modulus of coating
Hardness (H) and Young's Modulus (E) of Coating
materials can be calculated from the load and
displacement curve as following equations:
(1)
A = C0hc2 (2)
(3)
Which Pmax is the maximum load and A is the
indented area, hc is the contact depth at maximum
load Pmax and C0 is a parameter depends on the
indenter tip. C0 is 24.56 for the Berkovich diamond
tip [1, 31]. S is stiffness that can be measured from
the slope of the unloading curve at Pmax and is the
passion ratio which is 0.75 for the Berkovich
diamond tip. Hardness analysis depends on the
calibration of indenter tip. Fused quartz silica with
known mechanical properties is usually used for
this purpose. The stiffness Smax can be measured
from the force-displacement indentation curve by
considering the fused silica has a constant elastic
modulus. The indented contact area A can be
calculated from following equation:
(4)
Where Er is the reduced elastic modulus
depends on the elastic modulus fused quartz silica
(Es) and it's of indenter tip (Ei), Er can be obtained
as following equation:
(5)
Where ν and E are the Poisson's ratio and elas-
tic modulus and index i and s are indicated for the
sample and the indenter, respectively. For diamond,
Ei = 1141 GPa and νi = 0.07 [1, 14].
The area function A(hc) calibration is needed to
obtain in practical nanoindentation testing. A can be
obtained by plotting of A versus hc and curve fitting
according to the following polynomial equation (6):
A=C0hc2+C1hc+C2hc
1/2+C3hc1/4+C4hc
1/6+C5 hc1/8
(6)
In this equation, C1 through C5 are constants. The
elastic modulus Es of the material can be
determined using Equations (5) and (7).
(7)
Where, Smax is the slope of unloading curve at
the Pmax which can be determined directly from
the unloading curve (i.e. at start of unloading in
Figure 2) and A is the contact area between tip and
the material at that point.
4. Dynamic mechanical behavior
The viscoelastic properties can be measured by
nanoindentation technique [2, 12, 23 and 32]. The
storage (E') and loss modulus (E″) of coating mate-
rials can be calculated under sinusoidal loading in
linear viscoelastic domain by Equation (8) and (9):
(8)
(9)
(10)
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344 Ershad Langroudi A
340
A
PH max=
S
PhH c
maxmax ε−=
2
max
4
=
rE
SA
π
i
i
S
S
r EEE
22 111 νν −+
−=
A
SEr
max
2
π=
ϕεσ
cos0
0=′E
ϕεσ
sin0
0=′′E
EiEE ′′+′=
Where σo is the stress, εo is the strain amplitude,
φ is the difference in phase of stress and strain. The
term of loss factor or tan φ, is also the ratio E'/E″represent the damping characteristic of a linear
viscoelastic material. In order to study the dynamic
nanoindentation of the material's surface, the
indenter head vibrates at a certain frequency, and
the resulting response is measured and subtracted
the contribution of instrument to determine the
unique response from the material.
5. Scratch test, wear resistance and coefficient of
friction
The scratch resistance of coating can be precisely
determined in the nanoscale as well as the
mechanism of deformation and delamination by
nanoindentation technique. In a typical scratch test,
a sharp indenter head is applied on the surface of
material at a constant or ramp-up load in the normal
direction as it moves simultaneously on the sample
surface in a lateral direction. By recording the
lateral force and normal displacement as a function
of time, Critical information such as the coefficient
of friction, cross profile topography, residual
deformation and pile- up of material during the
scratch can be measured as a function of scratch
distance. Scratch and wear resistance are
considered where scratch depth at a given load or
the load at which material fails catastrophically.
Scratch resistance is measured by in situ tangential
(friction) force and observed by light optical
microscopy (LOM) imaging of the scratches after
tests [1, 33-35]. By using a diamond head to scratch
a magnetic tape, nanoscratch data on magnetic
tapes and their individual layers can be
investigated.
In practical scratch experiment, an indenter head
with a tip radius of 1 mm conical diamond and an
included angle of 60° is drawn over the coating
surface. The load is ramped up until substantial
damage occurs. The coefficient of friction is
monitored during scratching. It needs to be
minimized for most sliding applications. In order to
minimize test duration, accelerated friction test are
commonly used by a ball-on-flat tribometer, for
example, a sapphire ball with a 3 mm diameter
under reciprocating motion. Normal and frictional
forces are measured with semiconductor strain
gages mounted on a crossed-I beam structure and
the data are digitized and collected on a personal
computer. Wear tracks of a tape can be monitored
by LOM imaging.
6. Nanoindentation test on thin nanocomposite
hybrid coatings
The nanocomposite hybrid coatings have been
widely used for good adherence to the substrate.
The varieties of organic resins and inorganic fillers
have been usually used in such coating composition
to obtain desiring formulation with good
mechanical properties. However, such coating
formulations are susceptible to consist of defect
sites such as pinholes and cavities that can be
influenced the coating properties and enable failure
of it. Davies et al. studied the epoxy adhesive joints
of different thicknesses between aluminum
substrates by nanoindentation test. Their results
indicate that the modulus value of the aluminum
substrate is about 70 GPa while it drops to 2 GPa
corresponding to the adhesive layer [36]. Shi et al.
studied the effect of inorganic filler in a commercial
epoxy resin. Their results indicated 1 wt% of SiO2
nanoparticles can be induced significant enhance-
ment in Young's modulus up to 10 times than that of
neat epoxy coating. However, the adding other
modified nanoparticle such as Zn, Fe2O3 and
halloysite clay in coatings did not show such
enhancement in the mechanical properties [37].
Woo et al. studied the mechanical properties of
nano clay modified epoxy based nanocomposites
after they were exposed to artificial weathering test.
They found the organoclay had little effects on the
variation of elastic modulus with UV exposure
time. However, an increasing in the modulus of
surface material was observed by nanoindentation
test after UV exposure, with less extent in the
nanocomposite in compare with the neat epoxy. It
may be attributed to embrittlement of top layer after
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344Ershad Langroudi A
341
UV light exposure [38]. Li et al. investigated the
mechanical properties of epoxy resin containing
various percentages of coiled carbon nanotubes
(CCNTs) and single-walled carbon nanotubes
(SWNTs) by the nanoindentation and tensile tests.
They found that the hardness and modulus of
nanocompsites depends on nanotube concentration
and dispersion [39]. In a separate study, the
physical and mechanical properties of a suspension
of nano-alumina in an epoxy acrylate resin were
investigated by nanoindentation and nanoscratch
tests. They found that the hardness of nano
composite films containing nano-alumina to be less
than that of the samples without any nano-
particles [40]. Lionti et al. synthesized hybrid silica
coatings based on 3-glycidoxypropyltriethoxysi-
lane (GPTES), tetraethylorthosilicate (TEOS) and
colloidal silica on polycarbonate (PC) by the sol-gel
method, in order to enhance scratch resistance of
substrate properties. Their results indicated that
scratch resistance can be improved by irrespective
of the alkoxysilanes/colloidal silica ratio or the sol
aging time [41]. Sun et al. studied the adhesion of
thin film interfaces by Cross-Sectional Nano-
indentation (CSN) technique. Figure 4 shows the
orientation of the three-sided Berkovich
diamond tip as well as its positioning with respect
to the interface in the CSN test which are critical
parameters for controlled delamination.
The optimum orientation of the indenter is
schematically shown in the figure, where one of the
sides of the triangular indentation mark is parallel
to the interface and the optimum distance tip to the
interface (d) is 1 to 5 [42]. A sudden Jump in Load-
displacement curve can be interpreted as delamina-
tion (see Figure 4c) [42]. Tiwari et al. have recent-
ly reported the basic fundamental principles as well
as the experimental analyzing in modern nano-
indentation techniques with a brief survey of
silicone based nanocomposite coatings [9]. Barth et
al. investigated thin Al2O3-nanoparticles coatings
on solid stainless-steel substrates. The influence of
particle size and width of the particle size
distribution on the mechanical properties was
studied by nanoindentation technique. Their results
indicated the maximum indentation force decreases
with decreasing particle size to a minimum, and
then it increases in very small sizes of the
nanoparticles. In addition, the micromechanical
properties and coating structure can be varied by a
change in the width of the particle size distribution
[43].
The mechanical behavior of nanocomposite
coatings containing silane modifed and unmodified
nanosilica fillers into a UV cured urethane acrylate
resin was recently investigated using nanoindenta-
tion, nano scratch and micro-hardness and dynamic
mechanical thermal analysis [44]. The results
indicated the surface modification of nanoparticles
can be induced stronger interfacial interaction with
the polymeric matrix and improved storage
modulus. In addition, based on nanoindentation and
microindentation measurements it proposed a
homogenous reinforced structure was formed in the
bulk and surface of hybrid coatings by the modified
nanosilica [44].
Figure 4: (a) A schematic presentation of cross-sectional
nanoindentation (CSN) technique in multi-layer thin films,
(b) Berkovich indenter with respect to thin film interface,
(c) a sudden jump in Load-displacement curve corre-
sponding to thin film delamination [42].
7. CONCLUSIONS
Nanoindentation and viscoelastic tests are very
effective techniques to investigate mechanical
properties of thin nanocomposite coatings.
However, there are many experimental nanoinden-
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344 Ershad Langroudi A
342
(a)
(b)
(c)
tation methods which provide quantitative
mechanical data such as elastic modulus, hardness,
scratch and wear resistance as well as viscoelastic
properties. The practical use of nanoindentation
technique was investigated in various organic-inor-
ganic hybrid nanocomposite coatings by sol-gel
process. It is demonstrated that the physical-
mechanical properties of these thin nanocomposite
coatings depend on the nature, particle size and dis-
tribution as well as the surface modification of the
inorganic nano fillers. They can be also influenced
on the microstructural properties of thin coatings.
In addition, the organic polymerization, cross
linking density of organic matrix can be affected the
materials stiffness. The adhesion of such nano-
composite coatings is dependent on the chemical
bonding by reactive functionality between coating
and substrate. However, the delamination of
coating layer can be produced on exposure to UV or
humidity conditions and artificial accelerating
weathering test. These microstructural changes can
be detected by various nanomechanical techniques
such as nanoindentation in statistic or dynamic
states.
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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 345-353
Evaluation of Coenzyme Q10 Addition and Storage
Temperature on Some Physicochemical and Organoleptic
Properties of Grape Juice
Zahra Goudarzi1*, Mahnaz Hashemiravan2, Sara Sohrabvandi3
1 M.Sc. Student, Department of Food Science and Technology, Varamin-Pishva Branch, Islamic Azad
University, Varamin, Iran
2 Assistant Professor, Department of Food Science and Technology, Varamin-Pishva Branch, Islamic Azad
University, Varamin, Iran
3 Assistant Professor, Department of Food Technology Research, National Nutrition and Food Technology
Research Institute, Faculty of Nutrition Sciences, Food Science Technology, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
Received: 15 June 2013 ; Accepted: 25 August 2013
Todays, parallel to growing in acceptance of functional products, various additives are used to
improve the characteristics of functional food products. The coenzyme Q10 is an essential
component for energy conversion and production of adenosine triphosphate (ATP) in the
membranes of all body cells and organelles, especially the inner mitochondrial membrane is
found. Coenzyme Q10 plays a vital role in cellular energy production. It also increases the body's
immune system via its antioxidant activity. The aim of this study was to evaluate the addition of
coenzyme Q10 on physicochemical properties of grape fruit juice. The variables were
concentrations of coenzyme Q10 (10 or 20 mg in 300 mL) and storage temperature (25°C and
4°C) and the parameters were pH, titrable acidity, brix, viscosity, turbidity and sensory evaluation
during three months of storage. By increasing time and temperature, pH was decreased and with
increasing concentration of coenzyme Q10, pH was increased. Time and temperature had direct
influence on acidity, and the concentration of coenzyme Q10 had the opposite effect on the
acidity. With increasing storage time and concentration of coenzyme Q10, Brix, viscosity and
turbidity levels were increased and with increasing time and concentration of coenzyme Q10, the
Brix, viscosity and turbidity were increased. The addition of coenzyme Q10 in grape juice showed
no negative effect on the physicochemical and sensory properties.
Keyword: Coenzyme Q10; Grape juice; Physicochemical properties; Sensory evaluation;
Storage temperature.
ABSTRACT
International Journal of Bio-Inorganic Hybrid Nanomaterials
(*) Corresponding Author - e-mail: goudarzi6302@yahoo.com
Coenzyme Q10 is a mediated electron transfer
between flavoproteins and cytochromes in
mitochondrial respiratory chain and has a cofactor
role in three mitochondrial enzymes. Coenzyme
Q10 in addition to energy transfer, as an
antioxidant, protects the oxidation of membrane
phospholipids and mitochondrial membrane protein
and low-density lipoprotein particles [1]. The
chemical name of Coenzyme Q10 is 2,3-
dimethoxy-5-methyl-6-polyisoprene parabenzo-
quinone. The letter 'Q' refers to quinone chemical
group and the digit '10' indicates the number of
isopernil chemical subunits [2]. The chemical
structure of coenzyme is shown in Figure 1.
Figure 1: The chemical structure of coenzyme Q10
Figure 2: Resources of coenzyme Q10
Table 1: Coenzyme Q10 levels in selected foods
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 345-353 Goudarzi Z et al
346
1. INTRODUCTION
O
O
H3CO
H3CO
CH3
H
CH36-10
Food Food
supplements
Synthesis
within the body
Resources of
coenzyme Q10
foodCoenzyme Q10
concentration [mg/kg]
Meat
- heart 113
- liver 39-50
- beef 16-40
- pork 13-45
- chicken 8-25
Fish
- sardine 5-64
- red flash 43-67
- white flash 11-16
- salmon 4-8
- tuna 5
Oils
- soybean 54-280
- olive 4-160
- grapeseed 64-73
- sunflower 4-15
Nuts
- peanuts 27
- walnuts 19
- sesame seeds 18-23
- pistachio nuts 20
- hazelnuts 17
- almond 5-14
Vegetables
- parsley 8-26
- broccoli 6-9
- cauliflower 2-7
- spinach up to 10
- rape 6-7
- Chinese cabbage 2-5
Fruit
- avocado 10
- blackcurrant 3
- strawberry 1
- orange 1-2
- grapefruit 1
- apple 1
CoQ10 levels in selected foods
Needed resources of coenzyme Q10 in the body can
be obtained in three ways, synthesis within the
body, food and food supplements, or a combination
of these factors (Figure 2) [2]. Due to the
complexity of the biosynthesis of this substance,
deficiency of coenzyme Q10 is possible [3]. Food
can usually provide in average 10 mg of needed
coenzyme Q10 in the body, while it have been
reported that the sufficient intake for a healthy body
is 30 mg per day [4]. Therefore, the obtained results
show the need to use coenzyme Q10 as a drug or
dietary supplement [5]. The results obtained about
stability of coenzyme Q10 in fortified dairy
products is consenting so that any changes in the
microbial, chemical and physical components of the
type has not seen yet [6-8]. Coenzyme Q10 levels in
some foods is shown in Table 1 [8].
Research in 2010 showed that use of fruits juice
such as grape fruit juice increased the absorption of
coenzyme Q10 in the human intestine [9]. Also, use
of coenzyme Q10 increased the vitamin content in
the liver and serum of rats [10]. According to the
survey results, fruit juice can be suitable to be
enriched with this invaluable coenzyme.
Biochemical and medical studies have shown that
grapes have phenolic content and antioxidant
properties and can be a good source of nutrition.
Grape juice has more than 2 times more
antioxidants than oranges, apples, grapefruit and
tomatoes [11]. The grape has antioxidant property
and actually has the capacity of free-radical
absorbance. This property is related to its phenolic
content [12]. Grapes help inhibit of heart disease,
neurological diseases, viral infections and
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 345-353Goudarzi Z et al
347
Figure 3: Flowchart of study
Alzheimer [13]. Grape juice inhibits platelet and
has anti-coagulation of blood property [14]. Grape
juice stimulates the production of nitric oxide
which is a vasodilator by platelets. This material
causes normal blood flow and actually reduces
blood pressure in people who are suffering blood
pressure [15]. In medical research studies have
reported potential benefits of grape juice on the
stage of the cancer start [16]. Grape juice is also
effective in the prevention and improvement of
atherosclerosis [17]. Anthocyanins in the grape
juice have significant antioxidant property and play
important biological role in mammals. They are
directly involved in the protection of DNA, and
indirectly can also reduce oxidative stress.
Anthocyanins enable detoxifying enzymes such as
Glutathione Reductase, Glutathione Geroxidase,
Glutathione S-Transferase and Oxidoreductase
quinone [18]. Anthocyanins may reduce body
weight and prevent fat accumulation and diabetes
which is caused by that [19]. The aim of this study
was to investigate the effects of adding coenzyme
Q10 into grape juice on its some physicochemical
properties and sensory attributes.
2. MATERIALS AND METHODS
2.1. Sample preparation
Coenzyme Q10 (Sensus, Netherlands) added into
300 mL grape juice (Takdaneh, Iran) at three levels:
0, 10 and 20 mg. The samples filled into sterile
bottles and were pasteurized at 90°C for 5 min.
Grape juice packs were kept in refrigerated temper-
ature at two temperatures (4 or 25 ± 2°C) for 3
months, per one-month intervals (Figure 3).
2.2. Physicochemical analysis and sensory
evaluation
Measurement of the pH were done with a pH meter
(Crison, Spain), Brix with a refractometer (Optech,
Germany), viscosity with a viscometers
(Brookfield, America), and turbidity with a
spectrophotometer (Cromtech, Taiwan ). Titrable
acidity was measured via titration method. Sensory
characteristics of the samples were examined using
a 5-point Hedonic test. The total sensory acceptance
was calculated and compared among treatments as
final sensory parameter.
Statistical analysis Experiments were performed
in triplicate and significant differences between
means were analyzed using two-way ANOVA test
from Minitab software. The design of experiment
was completely randomized design (full Factoriel).
Also, to clarify the relationship between the
characteristics of the Pearson correlation coefficient
was used.
3. RESULTS AND DISCUSSION
3.1. Effects Q10 addition on pH and titrable
acidity
Figures 4-9 shows the average pH, titrable acidity,
Brix, viscosity, turbidity and general sensory
acceptance of grape juice treatments during storage.
Concentration of coenzyme Q10 and dual effect of
temperature and time showed a significant effect on
pH of grape juice. With increasing temperature and
time, the pH was decreased. This may be due to the
growth of acid-producing bacteria in fruit juice.
Coenzyme Q10 concentrations also had a direct
effect on the pH of juice and the reason may be the
higher pH of Q10 and other accompanying
materials (pH = 7) [8, 21]. Q10 concentration had a
direct effect on pH (Figure 4). The results obtained
revealed that the highest pH was for treatments
A2B2C3 (containing 20 mg of Q10 in 300 mL of
juice stored 25°C for 1 month) and the lowest pH
was for treatment A2B4C1 (stored at 25°C for 3
months with no coenzyme Q10).
It was found that the factors of temperature, time
and concentration of coenzyme Q10 had significant
effect on the titrable acidity of the juice (Figure 5).
Storage time and temperature had a direct effect on
the titrable acidity of the juice, so that with
increasing temperature and time acidity increased
and with increasing concentrations of coenzyme
Q10, the acidity was decreased. The concentration
of coenzyme Q10 had reverse effect on titrable
acidity, since acidity has a reverse relation with pH
and according to the discussed reasons about pH
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348
changes, the numbers resulted about acidity seem to
be normal [20]. The highest titrable acidity was for
the treatments A1B4C1 and A2B4C1 (The both
stored for 3 months with no coenzyme Q10), and
the lowest was for the treatment A2B1C3 (At the
start of storage at 25°C and containing 20 mg of
coenzyme Q10 in 300 mL of juice).
3.2. Effect of adding Q10 on Brix and viscosity
It was determined that with increase of storing time
and concentration of coenzyme Q10, Brix levels
was increased due to increased dissolved solids.
Only time and concentrations of Q10 showed
significant effect while temperature had no effect.
When storage time and concentration of Q10
increased, Brix was increased. The maximum Brix
was for treatment A1B4C3 (containing 20 mg of
Q10 in 300 mL of juice stored 4°C for 3 months),
and the minimum Brix was for treatment A2B1C1
(At the start of storage at 25°C, with no coenzyme
Q10) (Figure 6). In parallel with increase in storage
time and concentration of Q10, juice viscosity was
increased (Figure 7). This could be due to the
interaction of juice particles with particles of Q10,
or creation of small lumps in grape juice over time.
Possible crystallization of sucrose and corn starch
with coenzyme Q10 could also mention as a reason
[21]. As the storage temperature increased,
viscosity of grape juice was reduced because lower
temperature (4°C compared to 25°C) resulted in a
more condensing matrix with an increased density
of the juice [21]. Also, at low temperature, the rate
of crystallization and creation of small particles of
crystals is increased. The maximum viscosity was
for treatment A1B4C3 (containing 20 mg of Q10 in
300 mL of juice stored 4°C for 3 months), and the
minimum viscosity was for treatment A2B1C1 (At
the start of storage at 25°C, with no coenzyme
Q10).
3.3. Effect of adding Q10 on turbidity
Results showed that storage time and concentration
of coenzyme Q10 had a direct effect on grape juice
turbidity. With increase of time and concentration
of coenzyme Q10, turbidity was increased
(Figure 8). The reason was associated with the
grape color of Q10. Results revealed that with
increase of temperature, turbidity of grape juice
was reduced and the reason could be associated
with the lower density of juice particles at higher
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349
Figure 4: Average pH of grape juice treatments during storage. Values displayed with different letters are significantly dif-
ferent. A = storage temperature (A1 = 4°C and A2 = 25°C); B = storage time (B1 = at the start of storage, zero,
B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0 mg/300
mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 345-353 Goudarzi Z et al
350
Figure 5: Average acidity of grape juice treatments during storage. Values displayed with different letters are
significantly different. A = storage temperature (A1 = 4°C and A2 = 25°C); B = storage time (B1 = at the start of storage,
zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0
mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).
Figure 6: Average Brix of grape juice treatments during storage. Values displayed with different letters are significantly
different. A = storage temperature (A1 = 4°C and A2 = 25°C); B = storage time (B1 = at the start of storage, zero,
B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0 mg/300
mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).
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351
Figure 7: Average viscosity of grape juice treatments during storage. Values displayed with different letters are
significantly different. A = storage temperature (A1 = 4°C and A2 = 25°C); B = storage time (B1 = at the start of storage,
zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0
mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).
Figure 8: Average turbidity of grape juice treatments during storage. Values displayed with different letters are
significantly different. A = storage temperature (A1 = 4°C and A2 = 25°C); B = storage time (B1 = at the start of storage,
zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0
mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).
temperatures [22]. The maximum turbidity was for
treatment A1B4C3 (containing 20 mg of Q10 in
300 mL of fruit juice stored at 4°C for 3 months),
while the minimum turbidity after the control was
for treatment A2B1C2 (containing 10 mg of Q10
per 300 mL of juice, at the start of storage at 25°C).
The Pearson correlation Table shows coefficients
between physicochemical characteristics of the
grape juice. As can be seen in the measured pH and
other characteristics had an inverse relationship
with each other while communicating with other
characters straight (Table 2).
3.4. Effect of adding Q10 on total sensory
acceptance
Most of treatments did not show significant
difference in total sensory acceptance (Figure 9).
The Transparency of juices kept at lower
temperature (4°C compared those stored at 25°C)
and samples with shorter storage time showed
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352
Figure 9: General sensory acceptance of grape juice treatments during storage. Values displayed with different letters are
significantly different. A = storage temperature (A1 = 4°C and A2 = 25°C); B = storage time (B1 = at the start of storage,
zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0
mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).
Table 2: Correlation between attributes in grape juice by pearson coefficient
attribute pH acidity brix viscosity turbidity
pH
acidity
brix
viscosity
turbidity
1
-0.751 **
-0.485 **
-0.451 **
-0.408 **
-0.751 **
1
0.690 **
0.617 **
0.426 **
-0.485 **
0.690 **
1
0.676 **
0.569 **
-0.451 **
0.617 **
0.676 **
1
0.404 **
-0.408 **
0.426 **
0.569 **
0.404 **
1
** = Difference between treatments is quite significant (P < 0/01).
higher score. Mentioned facts could be due to lower
unwanted interaction of coenzyme Q10 and other
ingredients in system. The older samples had signi
ficantly greater apparent turbidity. The changes in
sensory parameters during the storage, although
were significant, but fortunately, were not
considerable.
4. CONCLUSIONS
Addition of coenzyme Q10 into food products can
improve their functional characteristic due to its
healthful effects. On the other hand, grape juice is a
good vehicle for enrichment of Q10 because of its
remarkable antioxidant capacity, anti-microbial and
anti-fungal activity and having significant amounts
of vitamin C, tannins and estrogen. The results of
this study demonstrated that overall, addition of
coenzyme Q10 in grape juice showed no
considerable negative effects on the physico-
chemical and sensory properties.
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353
Trace amounts of metals are present in natural
biosphere. Presence of some of these metals in very
low concentrations and certain oxidation states are
necessary. Higher concentrations and other oxida-
tion states might be toxic and dangerous.
Unfortunately the difference between these two
levels is very small [1, 2]. Lead occurs in nature
mostly as PbS. It is used in batteries, tetraethyl lead,
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 355-364
Preconcentration of Pb(II) by Graphene Oxide with
Covalently Linked Porphyrin Adsorbed on Surfactant
Coated C18 before Determination by FAAS
Ali Moghimi1*, Majid Abdouss2, Golnoosh Ghooshchi3
1 Associate Professor, Department of Chemistry, Varamin (Pishva) Branch Islamic Azad University,
Varamin, Iran
2 Associate Professor, Department of Chemistry, Amir Kabir University of Technology, Tehran, Iran
3 M.Sc., General Physician, Varastegan Medical Educating Center, Mashhad, Iran
Received: 19 June 2013; Accepted: 1 September 2013
A simple, highly sensitive, accurate and selective method for determination of trace amounts of
Pb(II) in water samples is presented. A novel Graphene oxide with covalently linked porphyrin
solid-phase extraction adsorbent was synthesized by covalently linked porphyrin onto the
surfaces of graphite oxides. The stability of a chemically (GO-H2P) especially in concentrated
hydrochloric acid was studied which used as a recycling and pre-concentration reagent for
further uses of (GO-H2P). The method is based on (GO-H2P) of Pb(II) on surfactant coated C18,
modified with a porphyrin-treated graphite oxides (GO-H2P). The retained ions were then eluted
with 4 ml of 4 M nitric acid and determined by flame atomic absorption spectrometry (FAAS) at
283.3 nm for Pb. The influence of flow rates of sample and eluent solutions, pH, breakthrough
volume, effect of foreign ions on chelation and recovery were investigated. 1.5 g of surfactant
coated C18 adsorbs 40 mg of the Schiff's base which in turn can retain 15.2 ± 0.8 mg of each of
the two ions. The limit of detection (3σ) for Pb(II) was found to be 3.20 ng l-1. The enrichment fac-
tor for both ions is 100. The mentioned method was successfully applied on determination of lead
in different water samples. The ions were also speciated by means of three columns system.
Keyword: Determination of lead; Preconcentration; Graphene oxide with covalently linked
porphyrin (GO-H2P); C18; Solid-phase extraction; FAAS.
ABSTRACT
1. INTRODUCTION
International Journal of Bio-Inorganic Hybrid Nanomaterials
(*) Corresponding Author - e-mail: alimoghimi@iauvaramin.ac.ir
guns, solders and X-ray instruments [3]. Copper on
the other hand occurs as CuS, CuS2, CuFeS2,
CuSO4.5H2O and other forms. More than 75% of
copper production is used in electrical industries. It
is also used in pigments, metallic blends and
household. Hence determination of lead and copper
in industry and environment are both very
important. A preconcentration step is advisable in
trace analysis. Lead and copper have been so far
determined by various methods such as
spectrophotometry [5, 6], liquid-liquid extraction
[7-9], cloud point extraction [10, 11], and
electrochemical measurements [12]. Some of these
methods suffer from poor limit of detection and
harmful solvents are being used in some others. On
the other hand, effect of foreign ions on theanalyte
is not negligible in many instances. In such cases,
preconcentration of the analyte makes the
determination easier and the composition of the
sample less complicated. In recent years, solid
phase extraction (SPE) has offered attractive
possibilities in trace analysis. It has reduced the
solvent and time consumption drastically [13, 14].
In order to increase the preconcentration or
extraction power of SPE an organic or inorganic
ligand is used in conjunction with the sorbont.
Some of the ligands used for determination of lead
and copper are: Amberlit XAD-2 with 3,4-dihidrox-
ybenzoic acid [15], silicagel modified with
3-aminopropyl triethoxysilane [16], Levatit with
di(2,4,4-trimethylpentyl)phosphinic acid [17],
silicagel functionalized with methyl thiosalicylate
[18], silicagel modified with zirconium phosphate
[19] and C18 diskes modified with a sulfur
containing Schiff's base [20, 28-32].
Comparing these examples with the presented
method, they have either a lower enrichment factor
or a higher limit of detection. On the other hand, the
C18 disks can be used only a few times, while the
proposed sorbent could be used more than 50 times
without loss of efficiency.
Surfactant coated alumina modified with
chelating agents has been used for extraction and
preconcentration of environmental matrixes and
metals [21, 22]. Here, the surfactant molecules have
been associated on the alumina surface forming an
admicell or hemimicell. Organic molecules attach
themselves on the hydrophobe part and low
concentration of metallic elements also on the
hydrophobe part, which includes the chelating
agent [22]. The Schiff's bases which are obtained
from salisylaldelyde are known as multidentate
ligands. These agents can form very stable
complexes with transition metal ions [23, 24].
The main goal of the present work is develop-
ment of a fast, sensitive and efficient way for
enrichment and extraction of trace amounts of
Pb(II) from aqueous media by means of a surfactant
coated C18 modified with, Graphene oxide with
covalently linked porphyrin (GO-H2P). Such a
determination has not been reported in the
literature. The structure of Graphene oxide with
covalently linked porphyrin (GO-H2P) (shown in
Scheme 1). Such a determination has not been
reported in the literature. The structure of Graphene
oxide with covalently linked porphyrin (GO-H2P)
is shown in Figure 1. The chelated ions were des-
orbed and determined by FAAS. The modified solid
phase could be used at least 50 times with
acceptable reproducibility without any change in
the composition of the sorbent, GO-H2P or SDS.
On the other hand, in terms of economy it is much
cheaper than those in the market, like C18 SPE
mini-column.
2. EXPERIMENTAL
2.1. Reagents and apparatus
Graphite oxide was prepared from purified natural
graphite (SP-1, Bay Carbon, Michigan, average
particle size 30 lm) by the Hummers [2]. Method
and dried for a week over phosphorus pentoxide in
a vacuum desiccators before use. 4-Isocyanato-
benzenesulfonyl azide was prepared from
4-carboxybenzenesulfonyl azide via a published
procedure [17]. All solutions were prepared with
doubly distilled deionized water from Merck
(Darmstadt, Germany). C18 powder for chromatog-
raphy with diameter of about 50 m obtained from
Katayama Chemicals from supelco. It was
conditioned before use by suspending in 4 M nitric
acid for 20 min, and then washed two times with
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2012), 355-364 Moghimi A et al
356
water. Sodium Dodecyl sulfate (SDS) obtained
from Merck (Darmstadt, Germany) and used
without any further purification.
2.2. Synthetic procedures
2.2.1. Preparation of GO-H2P
GO (15 mg) was stirred in 20 mL of oxalyl chloride
at 80°C for 24 h to activate the carboxylic units by
forming the corresponding acyl chlorides. Then, the
reaction mixture was evaporated to remove the
excess oxalyl chloride and the brownish remaining
solid (GO-COCl) was washed with anhydrous
tetrahydrofuran (THF). After centrifugation, the
resulting solid material was dried at room
temperature under vacuum. For the covalent
coupling between the free amino function of H2P
and the acyl chloride of GO, 15 mg of GO-COCl
was treated under anaerobic, dry conditions with 7
mg of H2P dissolved in 6 ml of dry THF at room
temperature for 72 h. The hybrid material, namely
GO-H2P, was obtained as brown-gray solid by
filtration of the reaction mixture through 0.2 mm
PTFE filter and the filtrate was sufficiently washed
with methylene chloride (4×20 mL) to remove
non-reacted free H2P and then with diethyl ether
(2×20 mL) before being dried under vacuum.
2.2.2. Column preparation
GO-H2P (40 mg) was packed into an SPE
mini-column (6.0 cm × 9 mm i.d., polypropylene).
A polypropylene frit was placed at each end of the
column to prevent loss of the adsorbent. Before use,
0.5 mol L-1 HNO3 and DDW were passed through
the column to clean it.
2.3. Apparatus
The pH measurements were conducted by an ATC
pH meter (EDT instruments, GP 353) calibrated
against two standard buffer solutions of pH 4.0 and
9.2. Infrared spectra of GO-H2P were carried out
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357
Scheme 1: A schematic illustration for the preparation of GO with covalently linked H2P. (i) H2SO4/HNO3 (2 : 1 v/v), (ii)
KClO3, 96 h, (iii) (COCl)2, 80°C, 24 h, (iv) 5-(4-aminophenyl)-10,15,20-triphenyl-21,23H-porphyrin, THF, r.t., 72 h.
from KBr pellet by a Perkin-Elmer 1430 ratio
recording spectrophotometer. Atomic absorption
analysis of all the metal ions except Zn(II) were
performed with a Perkin-Elmer 2380 flame atomic
absorption spectrometer. Zn(II) determinations
were performed by a Varian Spect AA-10. Raman
spectrophotometer analysis was performed with a
Perkin-Elmer.
2.3.1. Preparation of admicell column
To 40 mL of water containing 1.5 g of C18, 150 mg
of the above Schiff base-chitosan grafted
multiwalled carbon nanotubes was loaded after
washing acetone, 1 mol L-1 HNO3 solution and
water, respectively, solution was added. The pH of
the suspension was adjusted to 2.0 by addition of
4 M HNO3 and stirred by mechanical stirrer for 20
min. Then the top liquid was decanted (and
discarded) and the remained C18 was washed three
times with water, then with 5 mL of 4 M HNO3 and
again three times with water. The prepared sorbent
was transferred to a polypropylen tube (i.d 5 mm,
length 10 mm). Determination of Pb2+ contents in
working samples were carried out by a Varian
spectra A.200 model atomic absorption spectrome-
ter equipped with a high intensity hallow cathode
lamp(HI-HCl) according to the recommendations
of the manufacturers. These characteristics are
tabulated in (Table 1). A metrohm 691 pH meter
equipped with a combined glass calomel electrode
was used for pH measurements.
Table 1: The operational conditions of flame for determi-
nation of lead.
2.3.2. Procedure
The pH of a solution containing 100 ng of each
Pb(II) was adjusted to 2.0. This solution was passed
through the admicell column with a flow rate of 5
mL min-1. The column was washed with 10 mL of
water and the retained ions were desorbed with
1 mL of 4 M HNO3 with a flow rate of 2 mL
min-1. The desorption procedure was repeated
3 more times. All the acid solutions (4 mL all
together) were collected in a 10 mL volumetric
flask and diluted to the mark with water. The
concentrations of lead in the solution were
determined by FAAS at 283.3.
2.3.3. Determination of lead in water samples
Polyethylene bottles, soaked in 1 M HNO3
overnight, and washed two times with water were
used for sampling. The water sample was filtered
through a 0.45 m pores filter. The pH of a 1000
mL portion of each sample was adjusted to 2.0 (4 M
HNO3) and passed through the column under a flow
rate of 5 mL min-1. The column was washed with
water and the ions were desorbed and determined as
the above mentioned procedure.
2.3.4. Speciation of lead in water samples
This procedure is reported in several articles. The
method has been evaluated and optimized for
speciation and its application on complex mixtures
[26-29]. The chelating cation exchanger (Chelex-
100) and anion exchanger, Dowex 1X-8 resins were
washed with 1 M HCl, water, 1 M NaOH and water
respectively. 1.2 g of each resin was transfered to
separate polyethylene columns. Each column was
washed with 10 mL of 2 M HNO3 and then 30 mL
of water. The C18 bounded silica adsorber in a
separate column was conditioned with 5 mL of
methanol, then 5 mL of 2 M HNO3 and at the end
with 20 mL of water. 5 mL of methanol was added
on top of the adsorber, and passed through it until
the level of methanol reached just the surface of the
adsorber. Then water was added on it and
connected to the other two columns. A certain
volume of water sample was filtered through a 0.45
m filter and then passed through the three columns
system, Dowex 1X-8, RP-C18 silica adsorber and
Chelex-100 respectively. The columns were then
separated. The anion and cation exchanger columns
were washed with 10 mL of 2 M HNO3 and the C18
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358
Slit width 0.7 nm
Operation current of HI-HCL 10 mA
Resonance fine 283.3
Type of background correction Deuterium lamp
Type of flame Air/acetylene
Air flow 7.0 mL.min-1
Acetylene flow 1.7 mL.min-1
column with 10 mL of 1 M HCl. The flow rate of
eluents was 1 Ml min-1. The lead content of each
eluted solution were determined by FAAS.
3. RESULTS AND DISCUSSION
The treatment of Graphene oxide with covalently
linked porphyrin (GO-H2P) can lead to the deriva-
tization of both the edge carboxyl and surface
hydroxyl functional groups via formation of amides
[20] or carbamate esters [21], respectively.
3.1. Morphology
Initially, the GO-based hybrid material was studied
by AFM and TEM. Tapping mode AFM was
applied to identify the morphology of the GO-H2P
material (Figure 1a). Analysis of numerous AFM
images revealed the presence of graphene sheets
with heights ranging between 1.5-3.5 nm and
average lateral dimension of 150 nm. Considering
the height of a single GO sheet as 0.8-1.0 nm [20]
and the added contribution from the grafted
porphyrin moiety, the obtained images are
representative of single and/or bilayers of
exfoliated modified GO sheets. Moreover, TEM
images of GO-H2P were obtained and compared
with images of intact graphite, thus allowing the
observation of multiple-layered GO sheets with
various dimensions, most likely overlapped on the
peripheral edges (Figure 1b).
The formation of GO-H2P was followed by
ATR-IR spectroscopy. Initially, in the spectrum of
GO, the carbonyl vibration appears at 1716 cm-1,
while there are fingerprints at 3616 cm-1 and 3490
cm-1 due to the presence of hydroxyl species at the
basal plane of graphene. The covalent linkage of
H2P with the acyl chloride activated GO is evident
from the presence of a band at 1630 cm-1, which is
characteristic for the carbonyl groups of the amide
units [23] (see Figure S2, Electronic supplementary
information (ESI) available: Additional microscopy
and spectroscopy data). (See DOI: 10.1039
/c0jm00991a).
The amount of porphyrin attached onto the
graphene sheet was evaluated by thermogravimetric
analysis. As compared with the TGA results of pure
Figure 1: (a) Representative AFM image of GO-H2P and
profile analysis showing a height of 1.77 nm for the
enlarged region. Section analysis of other regions of the
image show height ranges of 1.5-3.5 nm. (b) TEM images
of the intact graphite (left panel) and GO-H2P hybrid
material (right panel).
The amount of porphyrin attached onto the
graphene sheet was evaluated by thermogravimetric
analysis. As compared with the TGA results of pure
graphite, which is thermally stable up to 900°C
under nitrogen, and GO which decomposes above
600°C, after having lost the oxygenated species at
240°C (i.e. 14.7% weight loss), the 6% weight loss
occurred in the temperature range 250-550°C for
the GO-H2P material, is attributed to the decompo-
sition of H2P (Figure 2). The GO-H2P material
forms a stable dispersion in DMF at a concentration
not exceeding 1 mg mL-1.
Figure 2: The TGA graphs of graphite (black), GO (blue)
and GO-H2P (red), obtained under an inert atmosphere.
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 355-364Moghimi A et al
359
(a)
(b)
Figure 3: The UV-Vis spectra of GO-H2P (black) and free
H2P (red), obtained in DMF.
The electronic absorption spectrum of GO-H2P in
DMF (Figure 3), shows (i) a broad signal mono-
tonically decreasing from the UV to the visible
region, which is attributed to GO and (ii) a
characteristic band at 420 nm (Soret-band)
corresponding to the covalently grafted H2P units
(the Q-bands at 516, 557, 589 and 648 nm were
flattened to the base line in the GO-H2P material).
Interestingly, the absorption of porphyrin in the
GO-H2P material is broadened, shortened and
bathochromically shifted (ca. 2 nm) as compared to
that of the free H2P, a result that corroborates not
only the linkage of porphyrin with the GO sheets
but also electronic interactions between the two
species (i.e. GO and H2P) in the ground state. These
results are in agreement with studies based on other
hybrid systems consisting of porphyrins covalently
grafted to carbon nanotubes and nanohorns [20].
3.2. Stability studies
The stability of the newly synthesized GO-H2P
phases was performed in different buffer solutions
(pH 1, 2, 3, 4, 5, 6 and 0.1 M sodium acetate) in
order to assess the possible leaching or hydrolysis
processes. Because the metal capacity values
determined in Section 3.2 revealed that the highest
one corresponds to Pb(II)s, this ion was used to
evaluate the stability measurements for the
GO-H2P phase [14]. The results of this study
proved that the GO-H2P is more resistant than the
chemically adsorbed analog especially in 1.0, 5.0
and 10.0 M hydrochloric acid with hydrolysis
percentage of 2.25, 6.10 and 10.50 for phase,
respectively. Thus, these stability studies indicated
the suitability of phase for application in various
acid solutions especially concentrated hydrochloric
acid and extension of the experimental range to
very strong acidic media which is not suitable for
other normal and selective chelating ion exchangers
based on a nano polymeric matrix [9]. Finally, the
GO-H2P phases were also found to be stable over a
range of 1 year during the course of this work. The
IGO is insoluble in water. Primary investigations
revealed that surfactant coated C18 could not retain
Pb(II) cations, but when modified with the GO-H2P
retains these cations selectively. It was then
decided to investigate the capability of the GO-H2P
as a ligand for simultaneous preconcentration and
determination of lead on admicell. The C18 surface
in acidic media (1<pH<6) attracts protons and
becomes positively charged. The hydrophyl part of
SDS (-SO3-) is attached strongly to these protons.
On the other hand, the GO-H2P is attached to
hydrophobe part of SDS and retains small
quantities of metallic cations [22].
Figure 4: Extraction percentage of Pb(II) against pH.
3.3. Effect of pH in does not occur
The effect of pH of the aqueous solution on the
extraction of 100 ng of each of the cations Pb(II)
was studied in the pH rang of 1-10. The pH of the
solution was adjusted by means of either 0.01 M
HNO3 or 0.01 M NaOH. The results indicate that
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360
complete chelation and recovery of Pb(II) occurs in
pH range of 2-4 and that of in 2-8 and are shown in
Figure 4. It is probable that at higher pH values, the
cations might be hydrolysed and complete
desorption occur. Hence, in order to prevent hydrol-
ysis of the cations and also keeping SDS on the C18,
pH= 2.0 was chosen for further studies.
3.4. Effect of flow rates of solutions
Effect of flow rate of the solutions of the cations on
chelation of them on the substrate was also studied.
It was indicated that flow rates of 1-5 mL min-1
would not affect the retention efficiency of the
substrate. Higher flow rates cause incomplete
chelation of the cations on the sorbent. The similar
range of flow rate for chelation of cations on
modified C18 with SDS and a GO-H2P has been
reported in literature [21, 22]. Flow rate of 1-2 mL
min-1 for desorption of the cations with 4 mL of
4 M HNO3 has been found suitable. Higher flow
rates need larger volume of acid. Hence, flow rates
of 5 mL min-1 and 2 mL min-1 were used for
sample solution and eluting solvent throughout
respectively.
3.5. Effect of the GO-H2P quantity
To study optimum quantity of the GO-H2P on
quantitative extraction of lead, 50 mL portions of
solutions containing 100 ng of each cation were
passed through different columns the sorbent of
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361
Diverse ion Amounts taken (mg) % Found % Recovery of
added to 50 mL Pb2+ ion
Na+ 92.2 1.19(2.9)a 98.6s(1.9)
K+ 92.2 1.38(2.1) 98.7(2.2)
Mg2+ 13.5 0.8(1.8) 96.9(2.7)
Ca2+ 23.3 1.29(2.0) 95.4(1.9)
Sr2+ 3.32 2.81(2.2) 98.2(2.1)
Ba2+ 2.26 3.16(2.4) 98.3(2.0)
Mn2+ 2.44 1.75(2.3) 98.5(1.8)
Co2+ 2.37 1.4(2.3) 98.1(2.2)
Ni2+ 2.25 2.0(2.14) 98.4(2.4)
Zn2+ 2.44 1.97(2.1) 98.7(2.2)
Cd2+ 2.63 1.9(2.0) 98.8(2.6)
Bi3+ 2.30 2.7(1.4) 98.4(2.7)
Cu2+ 2.56 2.81(2.3) 97.7(2.5)
Fe3+ 2.40 3.45(2.4) 97.6(2.8)
Cr3+ 1.30 2.92(2.2) 96.3(2.4)
UO2+ 2.89 1.3(2.2) 97.3(2.2)
NO3- 5.5 2.3 (2.3) 96.4(2.6)
CH3COO- 5.3 2.2(2.6) 95.5(2.2)
SO42- 5.0 2.9(3.0) 98.4(2.1)
CO32- 5.4 1.8(2.5) 96.3(2.5)
PO43- 2.6 2.1(2.0) 98.9(2.0)
Table 2: Effect of foreign ions on the recovery of 100 ng of Pb.
a: Values in parenthesis are CVs based on three individual replicate measurements.
which were modified with various amounts,
between 10-50 mg of the GO-H2P. The best result
was obtained on the sorbent which was modified
with 40 mg of the GO-H2P.
3.6. Figures of merit
The breakthrough volume is of prime importance
for solid phase extractions. Hence, the effect of
sample volume on the recovery of the cations was
studied. 100 ng of each cation was dissolved in 50,
100, 500 and 1000 mL of water. It was indicated
that in all the cases, chelation and desorption of the
cations were quantitative. It was then concluded
that the breakthrough volume could be even more
than 1000 mL. Because the sample volume was
1000 mL and the cations were eluted into 10 mL
solution, the enrichment factor for both cations is
100, which is easily achievable. The maximum
capacity of 1.5 g of the substrate was determined as
follow; 500 mL of a solution containing 50 mg of
each cation was passed through the column. The
chelated ions were eluted and determined by FAAS.
The maximum capacity of the sorbent for three
individual replecates was found to be 15.2 ± 0.8 µg
of each cation. The limits of detection (3σ) for the
catoins [30] were found to be 3.20 ngl-1 for lead
ions. Reproducibility of the method for extraction
and determination of 100 ng of each cation in a 50
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362
Diverse ion Amounts taken (mg) % Found % Recovery of
added to 50 mL Pb2+ ion
Sample Distilled water Pb - - -
(100 mL)
0.050 0.043(2.40)a 96
0.100 0.094(2.60) 97
Tap water (100 mL) Pb - 0.015(3.0) -
0.050 0.068(2.42) 96
Snow water (50 mL) Pb - 0.048(2.25) -
0.100 0.155(2.30) 98
Rain water (100 mL) Pb - 0.045(2.25) -
0.100 0.143(2.40) 98
Synthetic sample 1 Na+, Pb - - -
Ca2+, Fe3+, Co2+, Cr3+,
Hg2+, 1 mg L-1
0.100 0.104(2.40) 98
Synthetic sample 2 K+ Pb - - -
Ba2+, Mn2+, Cd2+ , Ni2+,
Zn2+, 1 mg L-1 of each
cation
0.100 0.105(2.70) 99
a: Values in parenthesis are CVs based on three individual replicate measurements.
Table 3: Recovery of Pb contents of water samples.
mL solution was examined. As the results of seven
individual replicate measurements indicated, they
were 2.85% and 2.98% for Pb(II).
3.7. Analysis of the water samples
Effect of foreign ions was also investigated on the
measurements of lead. Here a certain amount of
foreign ion was added to 50 mL of sample solution
containing 100 ng of each Pb(II) with a pH of 2.5.
The amounts of the foreign ions and the percent-
ages of the recovery of lead are listed in Table 2. As
it is seen, it is possible to determine lead without
being affected by the mentioned ions.
3.8. Analysis of the water samples
The prepared sorbent was used for analysis of real
samples. To do this, the amounts of lead were
determined in different water samples namely:
distilled water, tap water of Tehran (Tehran, taken
after 10 min operation of the tap), rain water
(Tehran, 25 January, 2013), Snow water (Tehran, 7
February, 2013), and two synthetic samples
containing different cations. The results are
tabulated in Table 3. As it is seen, the amounts of
lead added to the water samples are extracted and
determined quantitatively which indicates accuracy
and precision of the present method.
Separation and speciation of cations by three
columns system is possible to preconcentrate and at
the same time separate the neutral metal complexes
of GO-H2P, anionic complexes and free ions from
each other by this method [27]. Water samples were
passed through the three connected columns: anoin
exchanger, C18-silica adsorber and chelating cation
exchanger. Each species of lead is retained in one of
the columns; anionic complexes in the first column,
neutral complexes of GO-H2P in the second, and
the free ions in the third. The results of passing
certain volumes of different water samples through
the columns are listed in Table 4. According to the
results, it is indicated that lead present only as
cations. On the other hand the t-test comparing the
obtained mean values of the present work with
those published indicate no significant difference
between them. We have proposed a method for
determination and preconcentration of Pb in water
samples using surfactant coated C18 impregnated
with a Sciff's base. The proposed method offers
simple, highly sensitive, accurate and selective
method for determination of trace amounts of Pb(II)
in water samples.
ACKNOWLEDGMENTS
The authors wish to thank the Chemistry Depart-
ment of Varamin branch Islamic Azad University
for financial support.
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363
Tap water (1000 mL) Water sample (1000 mL)a River water (50 mL)
Column Pb(µg) Pb(µg) Pb(µg)
Dowex 1X8 - - -
Silica C-18 - - -
Chelex-100 0.012(4.0)b 0.104(2.9) 0.103(2.8)
Table 4: Results of speciation of Pb in different samples by three columns system.
a: This was a solution containing 0.1 g of each cation in 1000 mL of distilled water.
b: Values in parenthesis are CVs based on three replicate analysis. The samples are the same as those
mentioned in Table 4.
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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 355-364 Moghimi A et al
364
The term biomining have been coined to refer to the
use of microorganisms in mining processes. On the
other hand, biooxidation implies the bacterial
oxidation of reduced sulfur species accompanying
the metals. For many years bioleaching was thought
as a technology for the recovery of metals from
low-grade ores, flotation tailings or waste material
[1, 2]. Today bioleaching is being applied as the
main process in large scale operations in copper
mining and as an important pretreatment stage in
the processing of refractory gold ores [2]. The main
advantages of biooxidation of refractory gold ores
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 365-371
Facilitate of Gold Extracting From Mouteh Refractory Gold
Ore Using Indigenouse Bacteria
Seyed Mansour Meybodi1, Maryam Asghar Heydari2*, Ismaeel Ghorbanali nejad1,
Masoud Mobini2, Mohammad Salehi2
1 Assistant Professor, Department of Microbiology, Islamic Azad University, Tonekabon Branch,
Tonekabon, Iran
2 Master of Science, Microbiology Group, Islamic Azad University, Tonekabon Branch, Tonekabon, Iran
Received: 27 June 2013; Accepted: 30 August 2013
The term biomining have been coined to refer to the use of microorganisms in mining processes
as in the biooxidation of refractory gold minerals. The biooxidation of refractory gold ores
presents similar characteristics when compared with roasting and pressure oxidation. Almost
without exception, microbial extraction procedures are more environmentally friendly. The
isolated bacteria in this study, were included a variety of oxidizing acidophilic autotrophic iron and
sulfur oxidizing that named F.O.C.B and C.L.L.B. Biological oxidation with shaking flask method
were done in the presence of 1 gr of the ore milled of Mouteh with a particle diameter of 150
microns (100 mesh) in 9K medium without iron , at 30°C and shaking speed 180 rpm, during the
7 days, during this period ferrous ions assessment were performed by colorimetric method with
orthophenantrolin. The results showed that F.O.C.B. bacteria reduced the amount of ferrous ion
from 0.63 to 0.015 gr/L and C.L.L.B. bacteria from 0.64 to 0.04 gr/L. Also mineral pyrite was
removed after 7 days. This study aimed to Optimization of gold extracting from sulfide ore Mouteh
using indigenous bacteria.
Keyword: Bioleaching; Isolation; Mouteh; Refractory Gold; Chemolithotrop; Ferrous ion.
ABSTRACT
1. INTRODUCTION
International Journal of Bio-Inorganic Hybrid Nanomaterials
(*) Corresponding Author - e-mail: mheydari17m@gmail.com
as compared with pyrometallurgy lie in its relative
simplicity, low capital costs, low energy input, and
in its friendliness towards the environment [3, 4].
The primary biomining organisms have several
physiological features in common. hemolithoau-
totrophs are major organisms in biomining process
that are able to use ferrous iron or reduced
inorganic sulfur sources (or both) as electron
donors [2]. These organisms are acidophilic and
most will grow within the pH range 1.5-2.0. This
extreme acidophily applies even to those biomining
organisms that can oxidize only iron [4, 5].
Chemolithoautotrophic mesophilic bacteria of
genera acidithiobacillus and leptosprillum are the
most commonly found leaching organisms.
Acidithiobacillus is the gram-negative rod shape
bacteria with length 1-3 and Width 0.5.
Leptosprillum is gram-negative aerobic and spiral
shape bacteria that obtained energy requirements
from the oxidation of ferrous ions [4, 5]. This study
aimed to optimization of gold extracting from
sulfide ore Mouteh using native bacteria.
2. MATERIALS AND METHODS
The Chemolithoautotrophic mesophilic iron oxi-
dizing bacteria used in this study have been
isolated from the chahkhatoon and senjedeh mines
located in mouteh gold Mines complex, Isfahan,
Iran. Total of 10 samples collected from
Chahkhatoon and Senjedeh minerals and dumps in
mouteh gold mine. 10 gr of each sample inoculated
in in 250 mL Erlenmeyer flasks containing 90 mL
9K medium (3.0 g/L (NH4)2SO4, 0.1 g/L K2HPO4,
0.5 g/L MgSO4.7H2O, 0.1 g/L KCl, and 0.013 g/L
Ca(NO3)2.4H2O, 44.2 gr FeSO4.7H2O, 1 mL
H2SO4 10 N, 1 L D.W.) and DSMZ882 medium
(132 mgr (NH4)2SO4, 53 mgr MgCl2.6H2O, 27 mgr
KH2PO4, 147 mgr CaCl2.2H2O, 20 gr
FeSO4.7H2O, 50 mL H2SO4 (0.25 N), 950 mL
D.W). The pH value was adjusted with sulfuric acid
to 2 before the inoculation was processed [2, 4].
The presence of iron-oxidizing bacteria in liquid
iron medium (9K and DSMZ882) was indicated by
the formation of ferric iron and the medium
becoming brick red in color. Ferrous iron was
analyzed at 509 nm using visible spectroscopy. 1,
10 Orthophenanthroline was used as the
complexing agent. For enrichment and refreshing,
10 mL of brick red color flasks was inoculate in 90
mL of 9k fresh media [2, 7 and 8]. We used 9K agar
(4 g/L agar-agar ultrapure) and 2:2 solid media (4.5
g/L agar-agar ultrapure) for single colony isolation
and morphological studies [6]. For enrichment of
pure cultures, single colony of iron-oxidizing
bacteria, were picked from the plates by using a
sterile inoculating loop and inoculated into 25 mL
sterilized vials containing 10 mL liquid iron
medium, pH 2.0 and was vortexed to spread the
colony. All the cultures were incubated at 30°C
until the color of the medium changed to brick red
indicating ferrous iron (Fe2+) oxidation by iron-
oxidizing bacteria. Such ordinary purification
procedures were repeated several times, finally
pure cultures were obtained. Selected isolates were
subjected to light and scanning microscopy for
morphological characterization [7]. Finally
leaching experiments were performed in 250 mL
agitation flasks for 7 days, in which the initial 1%
pulp concentration of 150 µ ore particle size and
bacterial inoculation was 10% V/V. Control
samples were made by the addition of 10 mL of
inactive bacteria. All experiments were done and
carried out in rotatory shaker at 180 rpm, 30°C for
7 days. During the leaching, Redox potential and
pH were measured daily [6, 8]. Bacterial ferrous
iron oxidation rate was determined calculating the
amount of Fe2+ remaining in the solution by
spectrophotometer using 1, 10 orthophenanthroline
ferrous complex as an indicator. Sulfate concentra-
tion was indirectly determined by atomic absorp-
tion spectroscopy analysis of Ba after precipitation
of BaSO4 [6, 8]. The chemical composition and
particle size distribution of ore was determined
prior and after of bioleaching experiments
(Table 1).
3. RESULTS
After 3-5 days of incubation in 9K and DSMZ882
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366
media at 30°C and 150 rpm under shaking
condition, samples of Chahkhatoon spring became
reddish-brown due to bacterial oxidation of Fe2+ to
Fe3+. After the gram staining different biochemical
activities were analyzed. The compound micro-
scopic observations of isolated strains of bacteria
revealed that these strains were Gram-negative,
motile, very small (1-2 µm in length), rod shape and
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 365-371Heydari A et al
367
Figure1: a) DSMZ 882 medium right before and left after bacterial growth C.L.L.B. b) Microscopic images of
bacteria C.L.L.B.
(a) (b)
Figure 2: a) The iron-oxidizing bacteria colonies on 9K agar medium. b) The iron-oxidizing bacteria colonies
on 2:2 agar medium.
Table 1: Composition of Mouteh pyritic ore concentrate.
% SiO2 % Al2O3 % FeS2 % Na2O % K2O Gold
13.98 2.99 78 0.99 0.27 ppm
spiral shape bacteria, singles or pairs bacteria. The
most frequently observed colonies in 9K agar
medium were semi-spheroidal and smooth-
surfaced, with a white or yellow band outside and
around centre, and a margin with many short
projections. Ferrous oxidation was studied for all
bacteria isolates. These bacteria oxidized Fe2+ to
Fe3+ and reduced sulfur compounds produced
sulfuric acid which followed a drop in initial pH-
value of the medium. Two strains showed the
strongest ability to oxidize ferrous ion. Depending
on colony appearance, they were classified into 2
different types. These strains were rod-shaped and a
spiral shape bacteria was named F.O.C.B. (Ferrous
Oxidizing Chakhatoon Bacteria) and C.L.L.B.
(Chahkhatoon Leptospirillum like Bacteria)
respectively. These bacteria did not grow in culture
TSI and NA media. Growth was inhibited at neutral
and alkaline pH. Based on morphological and
biochemical characteristics of one isolate of
Leptospirillum-like bacteria (C.L.L.B.) were found
to be resembled to the genus Leptospirillum (Figure
1). Based on morphological and biochemical
characteristics of other isolate were found to be
resembled to those of the genus species
Acidithobacillus ferooxidans.
Oxidation of Ferrous Iron (Fe2+) by F.O.C.B.
and C.L.L.B. was conducted in shake flasks
containing iron liquid medium (9K Fe2+) contain-
ing pH-value of 1.8. It was observed that ferrous
iron (Fe2+) was completely oxidized to ferric iron
(Fe3+) by the isolated strain during 3-5 days of
incubation time at 30°C and 150 rpm. In chemical
control flasks, only a negligible amount of ferrous
iron was oxidized due to air-oxidation under the
same experimental condition. As shown in the chart
1, F.O.C.B. reduced ferrous ions from 0.64 to 0.004
mg/L, but in bioleaching by C.L.L.B. these changes
was from 0.63 to 0.015 mg/L. this results, indicates
high biooxidation potential of both types of
bacteria.
XRD analysis of the after leaching processes for
both types of bacteria showed pyrite remove from
ore (Figure 3).
4. DISCUSSION
Gold is usually obtained from ores by solubilization
with a cyanide solution and recovery of the metal
from the solution. In ores known as refractory,
small particles of gold covered by insoluble
sulfides. The main mineral composition of this ore
was pyrite and arsenopyrite, therefore, removal of
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 365-371 Heydari A et al
368
Chart 1: Right: Mouteh gold sulfide mineral ferrous ion concentration changes in 9k medium iron lacking with F.O.C.B
bacteria within 7 days compared with control. Left: Mouteh gold sulfide mineral ferrous ion concentration changes in DSMZ
882 medium iron lacking with C.L.L.B bacteria within 7 days compared with control.
these minerals does it feasible for extracting using
cyanide. Several alternative technologies are
available, such as pressure oxidation, chemical
oxidation, roasting and biooxidation, the latter
currently being the alternative of choice. In the
biooxidation process, bacteria partially oxidize the
sulfide coating the gold microparticles. Micro-
organisms belonging to the Thiobacillus and
Leptospirillum genera are commonly used,
although an increasing interest exists in
thermophilic archeons. Gold recovery from refrac-
tory minerals can increase from 15-30% to 85-95%
after biooxidation. Currently studies are being
carried on for the development of processes for the
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 365-371Heydari A et al
369
Figure 2: XRD analysis of gold sulfide ore of Mouteh, a before and b after 7 day's biooxidation by F.O.C.B bacteria.
Blue peaks in figure a, indicate the presence of pyrite that in the figure b, have been removed from ore.
bioleaching of gold concentrates [6]. In this study,
for the first time, the Mouteh gold mine indigenous
bacteria were used for bioleaching of Mouteh
sulfidic gold ore, whereas in the previous studies
(Shahverdi et al. 1378 and 1379 and Meybodi.
1378), were used from thermophilic adapted
bacteria isolated from hot springs [8, 10 and 11].
Results indicate high biooxidation potential of
indigenous bacteria. They tend to adapt to the local
ores in which they are found and may be better
suited for more efficient extraction from that
specific ore, Therefore In bioleaching process using
indigenous bacteria adaptation Stage has been
removed and will spend less cost and time [6].
Chemolithoautotroph bacteria are very sensitive
to organic matter including the small quantities of
sugar present as impurities in polysaccharide based
gelling agents such as agar or agarose. Attempts to
use highly purified agars have not been very
successful, probably because some of the sugar
molecules in the gelling agent are released owing to
acid-hydrolysis at low pH, and the released sugars
inhibit cell growth, a number of alternative gelling
agents have met with partial success, but most of
these are difficult to work. Because of inhibitory
effects of agar as an organic compound on growth
of bioleaching bacteria, we modified these media
using 4.5 and 4 g/L agar-agar ultrapure for 2:2 and
9K solid media, respectively [6].
In order to evaluate physiological and
biochemical characteristics of sulfur oxidizing
isolates, the sulfur and ferrous oxidizing abilities
were investigated F.O.C.B and C.L.L.B isolates
could oxidize all of initial ferrous within 3-7 days.
Based on this experience, one isolates of
Leptospirillum-like bacteria (C.L.L.B) were
isolated from Chahkhatoon mine in this study. Their
morphological and biochemical characteristics
were found to be resembled to those of the genus
Leptospirillum. Sand (1992) and Rolling (1999)
Studies indicate that Leptospirillum-like bacteria
are less sensitive to the inhibitory effect of ferric ion
and the inhibitory concentration of this ion is more
than ten times higher than amount that for
Acidithiobacillus ferrooxidans like bacteria. Also
the activity of these bacteria increases in mixed
cultures compared with single culture [12, 13].
Pachvlvska (2003) results determined, although
Acidithiobacillus ferrooxidans can be in relatively
high ferric to ferrous iron in comparison with
Leptosprillum ferrooxidans has higher growth, but
when ferric iron concentration is high,
Leptosprillum ferrooxidans will win the
competition [9].
The result of this study showed that division
time of C.L.L.B. bacteria is longer than F.O.C.B.
and is longer time to reach the logarithmic phase.
On the other hand, this bacterium tolerance of
power in high levels of ferrous ions is greater in
comparison with F.O.C.B. bacteria. As result in
long-term processes simultaneous use of these
bacteria will give better result. The results was
equalled with study Sand and Pachvlvska and
Rolling [9, 12 and 13].
5. CONCLUSIONS
XRF analysis of mouteh gold ore shows that high
value of iron (34.668%) and sulfur (13.686%),
created good conditions for the growth of iron and
sulfur oxidizing bacteria and it could be one of the
causes of high biooxidation potential of both types
of bacteria [8].
ACKNOWLEDGEMENTS
This study was conducted in Islamic Azad
University of Tonekabon Branch. Authors thereby
are acknowledgement from the officials and experts
called Branch.
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65.
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Branch, (2008), 334.
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(2003), 57.
10. Shahverdi A., Olia Zadeh M., Tabatabaei Yazdi
M., Seyyed Baqeri S.A., University College of
Engineering, 33 (2007), 97.
11. Shahverdi A.R., Yazdi M.T., Oliyazadeh M.,
Darebidi M.H., J. Sci. I. R. Iran, 12 (3) (2001),
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Appl. Environ. Microbiol, 58 (1) (1992), 85.
13. Rawlings D.E., Tributsch H., Hansford G.S.,
Microbiology, 145 (1999), 5.
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 365-371Heydari A et al
371
Sn-doped In2O3 is an n-type transparent conducting
oxide (TCO) with extensive commercial
applications, including flat-panel displays, solar
cells, and energy efficient windows [1]. Although
indium tin oxide (ITO) is a widely used TCO,
knowledge about its defect structure is limited. ITO
and In2O3 crystallize in the cubic bixbyite or Ia3
space group. The bixbyite structure is similar to the
fluorite structure, but one-fourth of the anions are
vacant, allowing for small shifts of the ions [2].
In2O3 has two nonequivalent six-fold coordinated
cation sites. Figure 1 shows the two cation sites,
which are referred to as equipoints "b" and "d" [3].
The b site cations have six equidistant oxygen anion
neighbors at 2.18 Å that lie approximately at the
corners of a cube with two anion structural
vacancies along one body diagonal [4]. The d site
cations are coordinated to six oxygen anions at
three different distances: 2.13, 2.19, and 2.23 Å.
These oxygen anions are near the corners of a
distorted cube, with two empty anions along one
face diagonal. Indium tin oxide exhibits higher con-
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 373-378
Synthesis and Morphology Study of Nano-Indium Tin
Oxide (ITO) Grains
Majid Farahmandjou
Assistant Professor, Department of Physics, Varamin Pishva Branch, Islamic Azad University,
Varamin, Iran
Electronic and Computer Department, Qazvin Branch, Islamic Azad University, Qazvin, Iran
Received: 1 July 2013; Accepted: 6 September 2013
In this paper, indium tin oxide (ITO) nanoparticles has been prepared by chemical methods under
given conditions with solution of indium chloride (InCl3·4H2O), tin chloride (SnCl4·5H2O) in
ammonia solution. The samples were characterized by X-ray Diffraction (XRD) and scanning
electron microscopy (SEM) analyses after heat treatments. The SEM results showed that, the
size of the ITO particles prepared by co-precipitation route decreased to 46 nm whereas the size
of the ITO prepared by hydrothermal and pechini sol-gel methods increased to 1 micron. The
XRD patterns revealed that, the size of crystallite ITO particles prepared by sol-gel and hydrother-
mal methods increased. Finally the intensity ratio of I400/I222 had a decrease of 21.67 percent for
ITO prepared by hydrothermal method.
Keyword: Liquid phase; Hydrothermal; ITO nanoparticles; Pechini sol-gel; Co-precipitation.
ABSTRACT
1. INTRODUCTION
International Journal of Bio-Inorganic Hybrid Nanomaterials
(*) Corresponding Author - e-mail: farahmandjou@iauvaramin.ac.ir
ductivities and carrier concentrations than pure
In2O3 because of the electron compensation of the
Sn species. An existing model of the defect
chemistry of Indium tin oxide has been inferred
from measured electrical properties of the material
[5], following the anion interstitial model for doped
In2O3 structures [2, 6].
Figure 1: Nonequivalent cation sites in ITO
The tradition deposition techniques of indium
tin oxide film are DC sputtering, RF sputtering, or
electron beam evaporation. It is the first step to
fabricate indium and tin alloy target or indium tin
oxide ceramic target. Afterwards the target is
sputtered to glass substrate by the controlled
electron beam. These techniques need costly
equipments, and the utilization rate of the target
materials is low [7-10]. Because indium is a rare
metal, it is necessary to explore a new route to
deposit indium tin oxide thin film with high-Indium
utilization rate. The synthesis nanoparticles of
metal oxide from aqueous solutions and deposition
thin films at low temperatures are an important way
for preparation of transparent conductive film [11].
Dip-coating or spray deposition of light transparent,
good conductive and low-membrane resistant
indium tin oxide film has been studied by the
researchers [12-14]. The fabrication of indium tin
oxide nanoparticle is important in emulsion
preparation for spray deposition or dip-coating ITO
film. The indium tin oxide thin film's quality is
related to the size and morphology of the
nanoparticles. With the development of nanometer
material research, several kinds of preparation
methods for nanosized ITO emerged. The current
methods for nanometer indium tin oxide
preparation mainly include solid-phase method, liq-
uid-phase method, and gas-phase method [15-17].
The liquid-phase method, with the advantages of
simple operation and controllable granularity, can
realize the atomic scale level of mixing. The doping
of components achieves easily, and the nanoscale
powder material has high-surface activity. The
liquid-phase methods include liquid phase precipi-
tation, hydrothermal (high temperature hydrolysis),
sol-gel (colloidal chemistry), radiation chemical
synthesis, and so forth [18, 24].
In this paper, the indium tin oxide nanoparticles
are first fabricated by liquid-phase co-precipitation,
hydrothermal and pechini sol-gel method and the
nanoparticles' structure is then compared by these
methods. The morphology of indium tin oxide
nanoparticles is studied by scanning electron
microscopy and X-ray diffraction.
2. MATERIALS AND METHODS
2.1. Liquid phase co-precipitation synthesis
The synthesis of indium tin oxide nanoparticles was
carried out by liquid phase co-precipitation as
follows. A certain quality of indium chloride
(InCl3·4H2O 99%, Aldrich) and tin chloride
(SnCl4·5H2O 99%, Aldrich) was dissolved in pure
de-ionized water or ethanol, keeping the ratio of
In2O3: SnO2 = 9: 1. Certain concentrations (5%) of
ammonia solutions were made by mixing certain
amount of ammonia (NH3·H2O, 25%) with pure
water. The prepared InCl3 solution (0.3 mol/L) was
transferred into fixed three-neck flask, keeping in
60°C temperatures under electromagnetic agitation.
The ammonia solution was added to the flask,
controlling the stirring speed and testing the pH
value till the required pH value was added as
dispersant. The precipitate precursor of indium tin
oxide was aged a certain time and washed with
deionized water and absolute alcohol for three
times, respectively. After washing, the precipitates
were dried at 110°C for 1 hour. The dried samples
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 373-378 Farahmandjou M
374
b site
cation
d site
cation
lattice
anion
anion
vacancy
were calcinated at 650°C for 1 hour to get the
indium tin oxide nanopowder.
2.2. Hydrothermal method
In this method, the acidity of indium (InCl3·4H2O)
and tin chloride (SnCl4·5H2O) were first controlled
by ammonia and then hexamethylenetetramine was
added to the solution as precipitant agent. The
reaction was transferred into fixed three-neck flask,
keeping in 120°C temperatures under electro-
magnetic agitation for 6 hours and then the solution
filtered and calcinated. The product was finally
annealed at 550°C for 2 hours.
2.3. Pechini Sol-gel method
In Pechini sol-gel method, ethylene glycol was first
added to the solution of a certain quality of indium
chloride (InCl3·4H2O 99%, Aldrich) and tin
chloride (SnCl4·5H2O 99%, Aldrich) in citric acid.
The solution was then dried at 80°C for 2 hours to
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 373-378Farahmandjou M
375
Figure 3: SEM images of ITO prepared by (a) hydrothermal and (b) pechini method.
(a) (b)
Figure 2: The SEM images ITO nanoparticles prepared by co-precipitation method.
remove the solvent. Finally, the ITO particles were
annealed at 600°C for 2 hours after purification.
The morphology and structure of the prepared
nanoparticles were characterized by means of
scanning electron microscopy and X-ray diffrac-
tion. The microstructure of the indium tin oxide
samples were characterized by a KYKY-Ammray
2800 type SEM with 200 kV acceleration voltages.
To determine the nanoparticles' structure, the X-ray
diffraction (XRD) measurement of the samples
were performed using a Seifert with Cu-Kα
radiation (wavelength = 1.54 Å).
3. RESULTS AND DISCUSSION
Figure 2 shows the scanning electron microscopy
image of indium tin oxide nanoparticles prepared
by liquid phase coprecipitation method in the
presence of ammonia solution. The size ITO
nanoparticle is about 46 nm after 600°C calcina-
tion. As you can see the particles are in good unifor-
mity in size.
Figure 3 shows the SEM images of indium tin
oxide particles prepared by hydrothermal and
sol-gel pechini methods. It is realized that the
particle size of ITO is more than 1 micron for both
of methods. But for the particles prepared by
hydrothermal method (Figure 3a) the uniformity
and crystallity is better than pechini method (Figure
3b).
From the width of X-ray diffraction broadening,
the mean crystalline size has been calculated using
Scherer's equation:
Where D is the diameter of the particle, K is a
geometric factor taken to be 0.9, λ is the X-ray
wavelength, θ is the diffraction angle and β is the
full width at half maximum of the diffraction main
peak at 2θ, is a function of the crystalline size.
In Table 1, the lattice parameters according to
XRD patterns are listed, including the size of
nanocrystals, D(nm), atomic planar distance d222
(Å), the intensity of diffraction peak, I222, and the
intensity ratio I400/I222. In 1998, Quaas and
co-workers reported that if tin oxide penetrates into
the indium oxide by 5%, the atomic planar distance
will decrease, and for penetration more than 5%,
the atomic planar distance will increase [25].
Comparing the atomic planar distance for the In2O3
sample d222 = 2.92 (Å), it is realized that the
penetration of Sn atoms into indium oxide is more
than 5% for indium tin oxide prepared by
co-precipitation, hydrothermal and sol-gel methods
with atomic planar distance d222 = 2.917 (Å),
d222 = 2.923 (Å) and d222 = 2.929 (Å) respectively.
By comparison of the I400/I222, it is found that the
ratio I400/I222 for ITO particles prepared by three
methods is less than 29.3%. The results show that
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 373-378 Farahmandjou M
376
θβ
λ
cos
KD =
Sample name*Preparation
MethodD(nm) d222(Å) I222 I400/I222
In2O3
ITO
ITO
ITO
Actual value
Pechini sol-gel
Hydrothermal
Co-precipitation
----
>100
>100
46
2.921
2.929
2.923
2.917
----
14112
8653
8747
29.3
28.2
21.67
29.07
Table 1: The data of lattice parameters for In2O3 and ITO nanoparticles.
* D=crystallite size, d222 =atomic planar distance of 222
the crystallite indium tin oxide particles have more
growth in <400> preferential orientation. In fact,
the ITO crystal growth is increased at the
preferential orientation with more atoms at higher
temperatures. Therefore, the penetration of Sn
atoms into the indium oxide prepared by hydro-
thermal and sol-gel approaches is more than indium
tin oxide prepared by the co-precipitation method.
Figures 4 shows the X-ray diffraction patterns
of SnO2 and indium tin oxide nanoparticles are
calcinated for 1 hour at 650°C. The large wide of
the picks for SnO2 pattern indicate that this
particles have the amorphous structure (Figure 4a),
while the ITO prepared by co-precipitation (Figure
4b), pechini sol-gel (Figure 4c) and hydrothermal
(Figure 4d) were intensively crystallized after
annealing and sharp picks indicate the body
centered cubic structure. The XRD results also indi-
cate that the intensity ratio of I400/I222 is increased
to 29.07 percent by co-precipitation method.
4. CONCLUSIONS
In conclusion, indium tin oxide nanoparticles were
successfully synthesized by liquid phase co-precip-
itation, hydrothermal and sol-gel methods. The
results indicate that the size of ITO prepared by
co-precipitation method is about 46 nm while the
size of indium tin oxide nanocrystals prepared by
hydrothermal and sol-gel methods is more than 100
nm, because of temperature. The X-ray diffraction
results indicated that the ITO particles are finely
crystallized body centered cubic structure. The
penetration of Sn atoms into indium oxide is more
than 5% for the indium tin oxide prepared by
co-precipitation, hydrothermal and sol-gel meth-
ods. Finally, the preferential growth and orientation
of the indium tin oxide prepared by the hydrother-
mal and pechini sol-gel methods is the <400>
orientation.
ACKNOWLELDGMENTS
The author is thankful for the financial support of
Karaj material and energy research center for
analysis and the discussions on the results.
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Figure 4: X-ray diffraction pattern of SnO2 and ITO nanoparticles.
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Crystallography, International Tables for
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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 373-378 Farahmandjou M
378
In an analogous fashion to traditional bulk
metallurgy, some properties of bimetallic nano-
particles can be modified by changing their compo-
sitions. However, the phenomena which one
expects here are not simply related to what happens
when the two corresponding metallic elements are
mixed to form a bulk alloy. That is, the metallurgy
for a certain bimetallic system at the bulk scale and
at the nano-scale may be somewhat different from
each other. In the bulk, Au can be mixed with Pt to
form a continuous solid solution at high
temperature (although these two species are immis-
cible at low temperatures) whereas bimetallic Au-Pt
nanoparticles of around 20 nm in size exhibit a
layer segregation between Au and Pt when annealed
at 600°C [1]. The interaction between the two
metals plays an important role in the properties of
bimetallic nanoparticles. These characteristics are
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 379-384
Structural and Optical Behavior of Cu Doped Au
Nanoparticles Synthesized by Wet-Chemical Method
Parivash Mashayekhi1*, Nazanin Farhadyar2
1 Ph.D. Students, Department of Chemistry, Science and Research Branch, Islamic Azad University,
Tehran, Iran
2 Assistant Professor, Department of Chemistry, Varamin-Pishva Branch, Islamic Azad University,
Varamin, Iran
Received: 7 July 2013; Accepted: 12 Sepember 2013
The nanoparticles of gold doped with various percentage of copper (Cu 10%, 25%, 75%) were
synthesized by wet-chemical method at room temperature. Copper (II) sulfate and gold (III)
chloride trihydride was taken as the metal precursor and ascorbic acid as a reducing agent and
anhydride maleic as surfactant. The reaction is performed with high-speed stirring at room
temperature under nitrogen atmosphere. X-ray diffraction (XRD), Scanning electron microscopy
(SEM) and DRS UV-Vis spectroscopy have been used for the characterization of the samples.
Moreover the X-ray diffraction results indicated that the synthesized Cu doped Au nanoparticles
had a pure single phase face-centered cubic structure and the average particle sizes were
between 5.43 - 12.6 nm. SEM images shows a spherical shape and dopant Cu influenced the
particles size of the powder.
Keyword: Anhydride maleic; Wet-chemical; Optical properties; Cu nanoparticle doped;
Surfactant.
ABSTRACT
1. INTRODUCTION
International Journal of Bio-Inorganic Hybrid Nanomaterials
(*) Corresponding Author - e-mail: prmashayekhi@gmail.com
quite sensitive to the medium in which the particles
are studied. This is because the elemental
arrangements of bimetallic nanoparticles depend
strongly on which method is used to produce them
[2], and the system of the two metals is generally
not in thermodynamic equilibrium. Moreover,
surface passivating ligands, which are normally
employed to prevent particle aggregation, may also
affect the relation between the metallic components
[2]. One of the most interesting kinds of element
arrangement for bimetallic nanoparticles is the
doping. The doping of transition metal ion such as
Mn, Cu, Co etc. opens up possibilities of forming
new class of material and new properties of the
material are expected [3]. Doping the impurities
into nanomaterials is an effect approach for tuning
the electronic, optical, mechanical and magnetic
properties of matrix nanomaterials [4-8]. The
growth rate of nanocrystals is strongly depending
upon doping concentration, capping agent
concentration and synthesis temperature. In order to
understand better these properties of doped
nanoparticles, the choice of sample preparation
method is therefore of greatest importance. The
preparation method should be the one that can
compel the doped ions into substitutional site and
have atomic scale homogeneous mixing with host
atoms without the formation of secondary phases,
nanoclusters etc. For the same, extensive research
efforts have been carried out worldwide to
synthesize nano-sized particles using various
methods [9] such as thermal decomposition,
chemical vapor deposition, sol gel, spray pyrolysis,
micro emulsions and wet-chemical. Among these
synthesis methods, wet-chemical method compared
with other traditional methods provides a simple
growth process for large scale production, and
which of course is an efficient and inexpensive way.
The distinctive feature of this process is that an
atomic scale homogeneous distribution of doped
ions the host matrix can be achieved.
2. EXPERIMENTAL
2.1. Material
Gold (III) chloride trihydrate (HAuCl4.3H2O,
99.9%) was obtained from sigma- Aldrich. Copper
(II) sulfate pentahydrate salt (CuSO4.5H2O, 98%),
ascorbic acid (C6H6O6, 99.7%), sodium hydroxide
NaOH (>98%), anhydride maleic (C4H2O3) were
obtained from Merck. All the chemical materials
were used without further purification. Deionized
water was purified for use during the synthesis.
2.2. Method
All glassware were cleaned with an aqua regia
solution (3:1, HCl: HNO3), and then rinsed. In this
work, at first time, we prepared four solutions
namely 0.05 M HAuCl4.3H2O (Solution A),
0.0087 M CuSO4.5H2O (Solution B), 0.026 M
CuSO4.5H2O (Solution C), 0.078 M CuSO4.5H2O
(Solution D). These were used inpreparing Cu
doped Au precursor solutions with different ratios
as shown in Table1. Combination of solution A and
B is labeled as concentration 1, solution A and C is
labeled as concentration 2, and solution A and D is
labeled as concentration 3. 0.001 M anhydride
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 379-384 Mashayekhi P et al
380
Figure 1: Schematic of samples preparation using Wet-chemical.
maleic polymer solution was used throughout the
synthesis. Then, with constant stirring and under N2
atmosphere mixture ascorbic acid (0.2 M) and
sodium hydroxide (0.2 M) added to the synthesis
solution. Color change occurred in the aqueous
phase to black. When the solution color did not
change, the reaction was ceased. After separation
from the mixed solution, the precipitation washed
3-4 times by de-ion water and the 2-3 times by
ethanol.
The powder of Cu doped with Au nanoparticles
was characterized by scanning electron microscopy
(SEM) and X-ray diffraction (XRD) and DRS
UV-Vis spectroscopy. X-ray powder diffraction
(XRD) analysis was performed on a D5000-
siemens with Cu Kα radiation (λ = 1.541Å) using a
30 KV operation voltage and 40 mA current.
Scanning electron microscopy (SEM) images were
obtained using a LEO 1430 VP microscopy. DRs
UV-Vis spectra of the synthesized materials were
recorded in the scan range 200-1000 nm, using a
UV-Visible spectrophotometer (S-4100, scinc
Korea).
3. RESULTS AND DISCUSSION
3.1. SEM Characterization
The SEM image of 10-50% Cu doped Au
nanoparticles is shown in Figure 2. In addition,
more uniform and homogeneous distribution of
nanoparticles was obtained by doping Cu into the
Au nanoparticles. All the nanoparticles exhibited
spherical morphology. Moreover the increasing
percent copper leads to the decreasing grain size.
3.2. XRD Diffraction analysis
The XRD patterns of the prepared samples were
recorded by an X-ray diffractometer are shown in
Figure 3. It is noteworthy that no secondary
diffraction peaks were detected in the XRD
patterns. All the diffraction peaks can be well
indexed to face-centered cubic (FCC) Au according
to the JCPDS card (NO.1-1172). Four pronounced
Au diffraction peaks (111), (200), (220) and (311)
appear at 2θ = 37.36°, 44.70°, 63.94° and 76.94°
respectively. The four most intense peaks of the
XRD pattern of sample show a slight shifting of the
center of the diffraction peaks toward a lower angle.
The shifting of the XRD lined suggests that Cu has
been successfully substituted in to Au host structure
at the Au site.
The crystalline size has been estimated from the
broadening of the first diffraction peak using
Debye-Scherrer formula:
D = 0.9λ /βcosθ (1)
Where D is crystallite size, θ is Bragg angle, λ is
wave length and β is Full-width at half maximum of
peak. The grain size of the samples was calculated
from Eq. (1) using (111) reflection in XRD pattern.
The average particle size of Cu: Au nanoparticles
have been obtained between 5.43 - 12.6 nm.
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 379-384Mashayekhi P et al
381
Morphology
Concentration
of
CuSO4.5H2O
( Mol L-1)
Concentration
of
HAuCl.3H2O
(Mol L-1)
Doping
percentage of
Cu %
Concentration
of
ascorbic acid
(Mol L-1)
Surfactant
(the type and the
Concentration)
Sediment color
Spherical
Spherical
Spherical
0.0087
0.026
0.078
0.05
0.05
0.05
10%
25%
50%
0.2
0.2
0.2
Anhydride maleic ( 0.001)
Anhydride maleic ( 0.001)
Anhydride maleic ( 0.001)
Black
Black
Black
Table 1: Detailed experimental parameters and dopant amounts for preparation of copper doped with Au nanoparticles.
Table 2: Size of Cu doped Au nanoparticles with various
doping percent copper at temperature.
3.3. DRS UV-Vis spectra
The DRS UV-Vis of Cu doped Au nanoparticles
prepared at various dopant percentages are shown
in Figure 4. It exhibits an intense peak centered at
375 nm and another peak with low intensity at 475
nm as shown in Figure 4. Optical absorption
measurements indicate blue shift in the absorption
band edge with increase dopant percentages. It is
clearly shown in Figure (4) the absorption edges
reveal a large shifting (30 nm) with increase dopant
percentage (Cu).
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 379-384 Mashayekhi P et al
382
Figure 2: SEM image of the Cu doped Au nanoparticles: (a) 10%, (b) 25% and (c) 50%.
%Doping of Cu Average size of particles for
samples
10%
25%
50%
12.6
9.82
5.43
(a) (b)
(c)
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 379-384Mashayekhi P et al
383
Figure 4: Optical absorption spectrum of Cu doped Au nanoparticles (a) 10%, (b) 25% and (c) 50%.
Figure 3: X-ray diffraction patterns of (a) 10% Cu, (b) 25% Cu and (c) 50% Cu.
(a)
(b)
(c)
(a)
(b)
(c)
4. CONCLUSIONS
Cu doped Au nanoparticles were synthesized using
wet-chemical method. We used anhydride maleic as
surfactant agent. The formations of the nano-
particles were confirmed by XRD peaks and result
shows that the samples have cubic phase. The effect
of doping percent of samples has been studied. In
addition, the Cu doping can control size of resulting
nanoparticles.
REFERENCE
1. Braidy N., Purdy G.R., and Botton G.A., Acta
Materialia, 56 (2008), 5972.
2. Ferrando R., Jellinek J., and Johnston R.L.,
Chem. Rev., 108 (2008), 845.
3. Dong L.S., FU X.F., Wang M.W., Liu C.H., J.
Lumin., 87-89 (2000), 538.
4. Cheng C., Xu G., Zhang H., Wang H., Cao J., Ji
H., Mater. Chem. Phys., 97 (2006), 448.
5. Quan Z., Wang Z., Yang P., Lin J., J. Fang,
Inorg. Chem., 46 (2007), 1354.
6. Park K., Yu H.J., Chung W.K., Kim B.J., Kim
S.H., J. Mater. Sci., 44 (2009), 4315.
7. Chandra B.P., Baghel R.N., Chandra V.K.,
Chalcogenide Lett., 7 (2010), 1.
8. Murugadoss G., Rajamannan B., Madhusud-
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9. Wang J., Gao L., Inor. Chem. Com., 6 (2003),
877.
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 379-384 Mashayekhi P et al
384
Carrot juice is one of the most high consuming veg-
etable juice [1] containing high amounts of
A provitamin (such as beta carotene). Therefore, it
is used for production of ATBC (alpha tocopherol
beta carotene) drinks [2, 3]. Carotenoids such as
beta carotene act as antioxidants in human immune
systems [4]. This product also contains B (B1, B2,
B6 and B12) vitamins and minerals [5]. 100 g of
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395
Investigation on Escherichia Coli Inactivation and Some
Quality Changes in Carrot Juice by Ultrasound Technique
Sima Dolatabadi1*, Zahra Emam-Djomeh2, Mahnaz Hashemi Ravan3
1 M.Sc. Student, Department of Food Science and Technology, Islamic Azad University, Varamin-Pishva
Branch, Iran
2 Professor, Transfer Phenomena Lab (TPL), Department of Food Science and Technology, Faculty of
Agricultural Engineering and Technology, University of Tehran, 31587-11167 Karaj, Iran
3 Assistant Professor, Department of Food Science and Technology, Islamic Azad University, Varamin-
Pishva Branch, Iran
Received: 9 July 2013; Accepted: 16 September 2013
In this study Response Surface Methodology was used to optimize process conditions and to
evaluate the effect of ultrasound on quality attributes (antioxidant activity, pH, total soluble solid,
turbidity) and the inactivation of Escherichia coli bacteria in carrot juice. Independent variables in
this study were temperature (25-50°C), time (20-40 min) and frequency (0-130 kHz). In this study
thermal process (85°C, 10 min) was chosen as control sample. The Browning index (BI) was
used to evaluate the color changes of carrot juice. Results showed that linear effect of frequency
(X3) and also interaction effect of frequency-time (X2-X3) were significant (p<0.05) in the
inactivation of E. coli. Moreover about antioxidant activity, it was shown that, linear and
quadratic effects of time were significant (p<0.05). The pH of samples was changed significantly
(p<0.05) under the effect of linear (X2) and quadratic effects of time and linear (X3) and
quadratic (X22 ) effects of frequency and also interaction effect of temperature-frequency (X1-X3).
None of parameters had significant (X32) effect on turbidity and total soluble solid (p>0.05).
Control sample showed higher value for browning index comparing other treatments.
Keyword: Ultrasound; Carrot juice; Antioxidant activity; E. Coli inactivation; Browning index;
Optimization.
ABSTRACT
1. INTRODUCTION
International Journal of Bio-Inorganic Hybrid Nanomaterials
(*) Corresponding Author - e-mail: s_dolat2003@yahoo.com
fresh carrot juice contains 0.08 g Ca, 0.53 g P and
0.001 g Fe. Carbohydrate, fat and proteins are
found at the amounts of 2.6, 0.10 and 0.9 g in 100 g
of carrot juice respectively. Regarding beta
carotene, this value is 1980 µg in100 g of fresh
carrot juice [6]. Concerning acidity, carrot juice is
considered as a low acid food due to its moderate
(pH = 6), due to its pH, a bacterial infection control
is required [7]. Heat treatment is a common
expensive way of microorganisms' inactivation in
fruit juices which reduces the number of most
resistant pathogens to 5 logs [8]. Furthermore, this
method has some undesirable effects on food
quality in terms of flavor. Thus tendency is to
propose a new method that can improve shelf-life
of the product while decrease these effects [9].
Membrane filtration, osmotic dehydration,
electrical pulse, irradiation, high pressure and
ultrasound are some non-thermal new methods
[10]. The intensity of micro-organism's inactivation
by ultrasound treatment depends on the type of
microorganism, environmental conditions and
process parameters. It has been reported that this
non thermal technique didn’t have damaging effects
on spherical cells as well as on spores [11]. Power
ultrasound (high intensity) combined with other
methods have been successfully applied for the
disinfection of various food products. Other
methods consist of heat treatment, chlorination, and
the use of hydrogen peroxide and etc. [12, 13]. In a
study the use of sonication (50W, 20 kHz) along the
concentration and storage at high pressure led to
decrease in salmonella count in orange juice [14].
In another study in 2011, it was found that
sonication can improve the quality of lemon juice
[15]. Sonication is an effective method for reduc-
tion in process time and enhancing output due to its
low energy consumption [16, 17].
In this study Response Surface Methodology
(RSM) was used to optimize ultrasound treatment
conditions including temperature, time and
frequency and base on some response variables to
evaluate the ability of ultrasound in Escherichia
coli destruction. in Escherichia coli is a gram
negative rod shaped non sporogenic bacterium with
a length of 2 µ, diameter of 0.5 µ and volume of
0.6-0.7 µ and can live on a broad range of
substrates [18]. Quality characteristics of carrot
juice (such as pH, total soluble solids, turbidity and
antioxidant activity) are also investigated in this
study. Moreover the browning index of carrot juice
samples is studied by the way of Duncan's multiple
rang test.
2. MATERIALS AND METHODS
2.1. Chemicals
Analytical grade of Methanol (99.9%), hydroxide
sodium, phenolphetalein, 2,2-Diphenyl-1-picrylhy-
drazyl (DPPH) were purchaced from Merck Co.
(Darmstadt, Germany). Culture mediums including
Tryptic Soy Agar (TSA), Tryptic Soy Broth (TSB)
were also bought from Merck Co. (Darmstadt,
Germany).
2.2. Methods
2.2.1. Carrot juice preparing
Carrot cultivar (Daucus carota L.) in the best
quality and value of 50 kg were obtained from local
market (IRAN, Boen Zahra region) and kept at
ambient temperature until juice extraction.
According to the mentioned method in [19] with a
little modification, whole of carrots were peeled
slightly and washed with potable water and cut into
smaller size and immediately converted into carrot
juice using juice extractor (Toshiba juicer Jc-17E,
Japan). Then prepared carrot juice was filtered by a
sterile 3-fold cotton cloth and homogenized using a
sterile tool like spoon and kept in PET bottles at
40C. This procedure was done in order to use the
same batch of carrot juice during all experiments.
2.2.2. Activation of Escherichia coli and inocula-
tion in carrot juice
The bacterium tested was prepared as lyophilized
ampoule from Iranian industrial collection of
bacteria and fungi. All contents of the ampoule
were transferred to 20 mL culture medium (Becton
Dikinson) and incubated at 35°C for 24 hours [20].
Then it was used for preparation of culture inside
the sterile micro tube. The inoculation of bacterium
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395 Dolatabadi S et. al
386
into carrot juice was performed according to
method described in [20, 21] with a little
modification. Before inoculation of bacterium to
carrot juice samples, in order to obtain the same
concentration of inoculated volume for all
experiments, light density of inoculated suspension
was measured at 600 nm by UV-Vis spectropho-
tometer (CECILE-2, UK) according to Mack Far
land standard, and all of the inoculated carrot juice
samples were exposed to ultrasound treatment after
10 min of inoculation.
2.2.3. Ultrasound treatment
Glass bottles involved inoculated carrot juice, were
put into an ultrasonic bath model (UP200S,
Hielscher Ultrasonic GmbH, Teltow, Germany).
Temperature was kept constant by re-circulating
coolant setting part (ethylene glycol: water, 50:50)
during the procedure. Frequency (0-130 kHz) was
also performed by adjusting the ultrasonic system
in time periods of (20-40 min). Each experiment
was done in triplicate.
2.2.4. Thermal treatment
A same sample was prepared as the control sample
and it was exposed to thermal pasteurization
treatment (85°C, 10 min) and after that it was
cooled until 20-25°C. This procedure was done in
triplicate.
2.2.5. Survival assay
Immediately after ultrasound process different
dilutions (6 dilutions) were prepared by ringer
solution under sterile conditions. Two plates of each
dilution were incubated on medium (TSA) surface
(Becton Dikinson) at 35°C for 48 hours. The
survival rate was expressed as cfu/mL [20].
2.2.6. Antioxidant activity
According to method mentioned in [22] with a
slight modification, 2,2-Diphenyl-1-picrylhydrazyl
methanolic solution (DPPH) was used to measure
antioxidant activity of treated carrot juice samples.
1 mL of different diluted of treated carrot juice
samples was mixed with 3 mL of DPPH solution in
methanol (25 mg/L) which was daily prepared.
After mixing (IKA, vortex Genius 3, Germany),
samples were kept in a dark place for about 30 min
without any movement. Then samples were
centrifuged for 10 min at 5000 rpm. Samples
absorbance was measured at 515 nm by UV-Vis
(CecilCE2502, Cecil Ins., England). Similarly to
methods described in [22, 23] antioxidant activity
of samples was presented in terms of EC50.
Following equation was obtained by standard curve
of DPPH methanolic solution, Y= 27.968X+3.8801
(r2= 0.992). Remained DPPH concentration in
samples (Y) was obtained by the way of putting the
amount of samples absorbance (X). Furthermore,
the control solution was prepared with similar
proportions to the major samples using methanol
until the remained DPPH percentage is also
calculated.
[DPPH] of control sample is the initial concentra-
tion of DPPH and [DPPH]t is DPPH concentration
in treated sample.
2.2.7. Turbidity
Treated samples were diluted with distilled water
(1:10 v/v). Turbidity of treated carrot juices was
measured using a turbid meter (Portable TURB 350
IR, TUV) and was presented as Nephelometric
Turbidity Units (NTU).
2.2.8. Total soluble solids
Total soluble solids were measured using a
refractionmeter (ART.53000C, TR di Turoni &
c.snc, Forli, Italy) and expressed as Brix at ambient
temperature (approx. 25°C) [24].
2.2.9. pH
pH was evaluated at ambient temperature (approx.
25°C) using a pH meter (IKA, RCT, and Basic
Germany) which was calibrated with buffer 7.0.
2.2.10. Browning index
Color of treated carrot juices was determined using
a Hunter-Lab Color Flex (A60-1010-615 model
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395Dolatabadi S et. al
387
[ ][ ] samplecontrolofDPPH
DPPHDPPH t
m =Re%
colorimeter, Hunter Lab, Reston, VA). Three color
parameters (L, a, b) were used to describe exact 3D
situation of color. So samples were poured into the
instrument cell and color parameters were read
three times. Presence of browning pigments in
samples was calculated by browning index (BI),
where L, a and b are correlated with (light/dark),
(red/green) and (yellow/blue) spectrums
respectively [25, 26].
2.3. Experimental design and statistical analysis
In this study Response Surface Methodology was
used to evaluate the effect of ultrasound treatment
independent variables including temperature X1
(25-50°C), time X2 (20-40 min) and frequency X3
(0-130 kHz) on some responses (pH, TSS,
turbidity, antioxidant activity, and inactivation of
Escherichia coli) in carrot juice samples.
Independent variables and their ranges were
determined by the way of preliminary experiments
and all of experiments were done in triplicate. RSM
is a statistical program to optimize the experimental
conditions. This method get a pattern called central
composite rotatable design (CCRD) to appointment
of experiment terms and includes of full factorial
design , central and axial points [27, 28]. In this
study a table consists of 20 runs (Table 1) with 6
central points obtained by CCRD design (Minitab
Version 16 software). The use of RSM allows
presenting mathematic models for each of
responses as Eq. (1) which showed the significant
linear, quadratic and interaction effects of
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395 Dolatabadi S et. al
388
( )[ ]17.0
31.0100 −=
xBI
( )( )baL
Lax
012.3645.5
75.1
−+
+=
RUNFrequency
(kHz)
Temprature
(°C)
Time
(min)
1E.C
survival2EC50 pH 3TSS 4Turb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0
65
65
0
130
0
65
130
0
130
130
65
130
65
65
65
65
65
0
65
25
37.5
37.5
25
50
50
37.5
25
50
37.5
50
50
25
37.5
37.5
37.5
37.5
25
37.5
37.5
20
20
30
40
40
40
30
40
20
30
20
30
20
30
30
40
30
30
30
30
450000
350000
350000
400000
18200
40000
380000
100000
203000
350000
160000
32000
1350000
360000
210000
250000
350000
250000
400000
230000
0.68
0.48
0.64
0.72
0.65
0.73
0.61
0.59
0.6
0.74
0.61
0.75
0.54
0.62
0.64
0.64
0.66
0.67
0.67
0.65
6.82
6.92
6.92
6.89
6.85
6.81
6.88
6.43
6.9
6.51
6.73
6.79
6.4
6.86
6.89
6.89
6.9
6.59
6.6
6.87
7.5
8
7.5
7.5
7.5
7.5
7.5
7.5
7.5
8
7.5
7
7
8
7
8.5
7
9
9
7.5
3735.7
4084.5
3960.2
3833.1
4062.2
4209.0
4012.3
4137.2
4175.7
4646.5
4339.1
4967.8
4398.2
3927.9
3932.2
3914.5
4001.7
2760.4
4490.8
3952.8
Table 1: Matrix of the face central composite design (FCCD) and experimental data obtained for
the response variables.
Independent variables Response variable a
aEscherichia coli1 (cfu/mL), Antioxidant activity2 (%), Total soluble solids3 (%), Turbidity 4(NTU).
independent variables on each response with their
coefficients, respectively.
(1)
Where Y is the predicted response and with
different subscripts is explanatory of constant
regression coefficients and is correlated with
linear, quadratic and interaction terms of
independent variables respectively. Analysis of
variance table gotten by RSM presents the effect in
surface lower than 5% that are explanatory as
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395Dolatabadi S et. al
389
∑ ∑ ∑ ∑= = = ==
+++=5
1
5
1
4
1
5
1
20i i i ij
kijXiXjkiiXikiXikYk ββββ
( )5,...,3,2,1=K
Figure 1-A: Effects of temperature and frequency of ultrasound on E. coli inactivation.
Figure 1-B: Surface plots of effects of time temperature and frequency of ultrasound on E. coli inactivation.
significant effects (Table 2) [29]. RSM also shows
the interaction effects of independent variables on
each of responses using contour and 3D surface
plots [30]. Color assay results were not entered into
response surface. Duncan's new multiple range test
was used to explain the color changes.
3. RESULTS
3.1. Escherichia coli inactivation
According to results of analysis of variance
(ANOVA) shown at Table 2, among of linear effects
only frequency (X3) has significant linear effect on
inactivation of the bacterium and among of
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395 Dolatabadi S et. al
390
Figure 2-B: Surface plots of effects of time temperature and frequency of ultrasound on pH.
Figure 2-A: Effects of time, temprature and frequency of ultrasound on pH.
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395Dolatabadi S et. al
391
Figure 3: Effect of time and ultrasound on browning index
at constant temperature.
Figure 4: Effect of temperature and ultrasound on
browning index at constant time.
Table 2: ANOVA and regression coefficients of the second-order polynomial models for the response variables.
Turb4 (NTU) TSS3 (%) PH EC502 (%)EC
survival1(cfu/mL) DF Source
PV Coefficient PV Coefficient PV Coefficient PV Coefficient PV Coefficient
0.977 83.590 0.467 3.88750 <0.001 7.14125 0.669 0.134 0.147 1867640 9 Model
0.286 142.125 0.507 0.15573 0.138 0.04265 0.084 -0.025716 0.856 9765 1 X1
0.704 61.753 0.810 0.06966 0.029 -0.08419 0.005 0.060655 0.253 -79273 1 X2
0.286 720.865 0.657 -0.52500 0.007 -0.45500 0.313 -0.072000 0.019 73987 1 X3
0.357 -1.555 0.496 -0.00204 0.222 -0.00044 0.085 0.0003226 0.286 -752 1 X12
0.678 -1.075 0.883 -0.00068 0.020 0.00146 0.004 -0.000991 0.699 415 1 X22
0.096 461.609 0.695 0.18182 0.003 -0.20364 0.116 0.045909 0.290 116509 1 X32
0.947 -0.080 0.818 -0.0005 0.783 -0.00007 0.537 0.00008 0.069 995 1 X1X2
0.439 -9.5 0.818 0.005 0.013 0.00750 0.414 0.002 0.204 -6648 1 X1X3
0.583 -8.357 0.818 0.00625 0.507 0.00212 0.537 -0.001 0.037 -14735 1 X2X3
<0.001 - 0.027 - 0.001 - 0.012 - 0.012 - 5 Lack-of-fit
- 0.5047 - 0.1578 - 0.86 - 0.7618 - 0.7967 - R2
aEscherichia coli1 (cfu/mL), Antioxidant activity2 (%), Total soluble solids3 (%), Turbidity 4(NTU).
interactive effects between independent variables
only frequency time interaction (X2-X3) was
significant (P<0.05). Model for inactivation is
obtained as followings:
Inactivation of Escherichia coli= 1867640 +
739870X3 - 14735X2 X3
As it can be seen from Figure 1-A, the survival rate
depends on time and time had more effect on
survival rate than frequency. Figure 1-B shows the
survival rate decreased with time which is more
pronounced at higher frequencies.
3.2. Antioxidant activity
Results of ANOVA presented at Table 2 showed
that only linear effect of time (X3) and quadratic
effect of time (X2) on antioxidant activity were
significant (P<0.05). Contour and surface plots of
samples showed that antioxidant activity was
decreased with time and the highest antioxidant
activity was observed at times less than 25 min.
Model for antioxidant activity of samples is
obtained as followings:
Antioxidant activity= 0.134000 + 0.060655X2 -
0.000991
3.3. pH
Based on results of analysis of variance X3
(frequency's linear effect), X2 (time's linear effect),
X22 (time's quadratic effect) and X32 (frequency's
quadratic effect) had negative effects on pH
(P<0.05). Among interactive effects, only X1-X3
interaction (temperature - frequency) had a signifi-
cant effect on pH. Since pH had a limited variation
range, the only factor exerting the highest effects on
pH was frequency so that an increase in frequency
led to pH reduction (Figures 2-A and B).
pH= 7.14125 - 0.08419X2 - 0.45500X3 + 0.00146
X22 - 0.20364X3
2 + 0.00750X1X3
3.4. Total soluble solids and turbidity
Results of AVOVA presented at Table 2 showed that
none of linear, non- linear and interactive effects on
total soluble solids were significant (P> 0.05). This
was the same for turbidity.
Turbidity= 83.590 + 142.125X1 + 61.753X2 +
720.865X3 - 1.555X12 - 1.075X2
2 + 461.609X32 -
0.080X1X2 - 9.500X1X3 - 8.357X2X3
Total soluble solids= 3.88750 + 0.15573X1 +
0.06966X2 - 0.52500X3 - 0.00204X12 - 0 . 0 0 0 6 8
X22 + 0.18182X3
2 - 0.00050X1X2 + 0.005 X1X3
+ 0.00625X2X3
3.5. Browning index
Figures 3 and 4 show that the highest value for
browning index belonged to control sample. At a
constant temperature (Figure 3) browning index
was increased with time and the use of ultrasound
had no effect on this index. Furthermore, at a
constant time (Figure 4) browning index of control
sample was higher than that of ultrasound treated
samples. It can be concluded that browning index
was increased with temperature.
4. DISCUSSION
4.1. Escherichia coli inactivation
As Figures 1 A and B show survival rate was
decreased with time especially at higher
requencies. Another study in 2011 showed that
microbial load reduced by sonication depended on
time. Considering the effect of sonication on total
plate count (TPC), they found that reduction in
microbial load occurred only after 60 minutes and
microorganism cellular wall was destructed only
when sonication time was increased to longer
periods. They also attributed microorganism killing
during sonication process to the series of physical
and chemical mechanisms occurred during
cavitation [15]. Ahmad and Russell (1975) obtained
the same result during inactivation of Bacillus
cereus and Candida albicans spores by ultrasonic
bath technique. They found that applying of
ultrasound was useful for time periods upper than30
min [31]. Another group of researchers (2008)
stated that there were several targets for killing cells
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395 Dolatabadi S et. al
392
by ultrasound waves including cellular wall,
cytoplasmic membrane, DNA, intracellular
structure and external membrane [32]. In
accordance with this, another research (1989)
showed that ultrasound treatment could have
crucial effect on cytoplasmic membrane and that
destruction rate of microbial cells by ultrasound
depended on experimental conditions and
microorganism species. Based on this study
ultrasound by itself can't kill spores [33]. Oyane et
al., (2009) attributed microorganism death to the
formation of free radicals and hydrogen peroxide
[34].
4.2. Antioxidant activity
It should be noted that antioxidant activity in the
diet is related to presence of bioactive phenolic
compounds, ascorbic acid, tocopherol and
carotenoides in plants which increases body
resistance to oxidative stress [35]. The same result
was obtained on ascorbic acid content of melon
juice under thermo-sonication treatment; in
mentioned study when process time was increased
from 0 to 10 minutes, ascorbic acid content was
decreased and at extreme conditions (the highest
amplitude, frequency and time) ascorbic acid
percent was reduced to 50% significantly (P<0.05).
Furthermore significant decrease was observed on
phenolic compounds content of melon juice when
temperature increased up to 45°C at higher
frequencies and times [36]. Ascorbic acid
decomposition can attribute to the intensified
physical conditions occurred in bubbles during
cavitation [37, 38] and to simultaneous or separate
disintegration of these bubbles. In other words
because these bubble are full of vapor and soluble
gases such as O2 and N2 they bring about
consequent sonochemical reactions [39]. Ascorbic
acid decomposition in higher frequencies and times
has also been attributed to oxidation by free radicals
[40]. Another same result was observed by Zhou et
al., (2006) on destruction of Astaxanthin (one kind
of carotenoid pigments) under ultrasound
treatment. They stated that these changes are more
severe at higher times and powers of ultrasound
[41].
4.3. pH
The only factor influencing pH significantly was
frequency which was probably due to the partial
decomposition of some compounds as a result of
ultrasound which leads to the formation of H+ ions,
higher solubility and enhancement of acidity. In a
study (2010) done on the use of ultrasound for
grape puree, it was found that sonication treatment
increased total acidity by 13.6% compared to
control treatment (traditional enzymatic treatment).
Their result was attributed to better derivation of
acidic compounds by ultrasound [42]. It should be
noted that effect of ultrasound on pH, depends on
intensity of frequency, treatment time, temperature
and type of juice. Thus Tiwari et al., (2009a) found
any significant effect on pH in treated orange juice.
They attributed this observation to extents of
applying frequency, temperature and time during
sonication [43]. Another study was done in 2006 on
apple cider and showed insignificant effect on pH
[44]. Dizadji et al., (2012) studied on the effect of
ultrasound in kiwi juice and found no significant
effect on pH due to buffer effect of kiwi juice [45].
4.4. Browning index
As Figures 3 and 4 show, the highest amounts of
browning substances were produced in control
sample. At higher intensities of ultrasound and
temperature beta carotene decomposition rate was
decreased. The reason was that bubbles formed due
to the cavitation process inhibited emission of
ultrasound waves under these conditions as a result
of enhanced size. Also disintegration of these large
bubbles led to decrease in cavitation effects [46].
Therefore the factor causing the highest changes in
samples color was temperature. This can be
attributed to the formation of dark compounds at
higher temperatures. In order to study [47] they
showed a relationship between the formation of
insoluble brown compounds and mechanisms of
hydrolysis or decomposition of anthocyanin caused
by heat. Formation of browning compounds
requires sugar. Presence of bacterium in samples
was not ineffective in color changes. Therefore, it
can be concluded that at higher temperatures
higher number of bacterium was killed and
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395Dolatabadi S et. al
393
consequently the consumption of sugar matters was
decreased. This can lead to the presence of higher
amounts of sugar substances for involvement in
browning reactions.
5. OPTIMIZATION
In this study two responses; Escherichia coli
inactivation and antioxidant activity; were
optimized by RSM. The main purpose of our study
was achieving to the highest level of Escherichia
coli inactivation and the least level of antioxidant
activity destruction. These two responses vary
inversely. It means that each factor causes further
Escherichia coli inactivation, it can also cause a
decrease in antioxidant activity which is not
desirable. The best conditions to obtain maximum
E. coli inactivation and minimum antioxidant activ-
ity destruction were determined as temperature=
48.73°C, time= 47.28 min and frequency= 130 kHz
by RSM optimization. In this optimized condition,
the residual amount of Escherichia coli will be
(approx. 1.003 × 104 cfu/mL) and in other
pronunciation we will receive to 100% of
Escherichia coli inactivation goal.
6. CONCLUSIONS
Analysis of variance (ANOVA) showed that in
Escherichia coli inactivation the linear effect of
frequency and also interaction effect of frequency-
time were significant (p<0.05). According to the
ANOVA, it was seen that regarding antioxidant
activity, linear and quadratic effects of time were
significant (p<0.05). The pH of samples was
changed significantly (p<0.05) under the linear and
quadratic effects of time and frequency and also
interaction effect of temperature-frequency. No
significant effect of any variables was found on
turbidity and total soluble solid of all samples
(p>0.05). About Browning index of samples the
highest level was found in control sample.
ACKNOWLEDGMENTS
Authors wish to express their especial thanks to the
University of Tehran, Department of Food Science
and Technology because of provided facilities for
this study in Transfer Phenomena Lab (TPL) and
also to Islamic Azad University, Varamin-Pishva
Branch for their assistance.
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Decomposition of hydrogen peroxide to supply
oxygen for the atmosphere is more suitable method
than superoxide or cholorate piles. For the
decomposition of hydrogen peroxide the active
inorganic metal oxides of manganese, iron, cobalt
and lead are used. If these metal oxides are prepared
at nanoscales their performance will be
strengthened and can accelerate the catalytic
decomposition of hydrogen peroxide [1]. Catalytic
Substrates or beds such as zeolites also increase the
surface area of the nanoparticles and their uniform
size distribution that improves the catalytic activity
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406
Catalytic Decomposition of H2O2 on MnFe2O4
Nanocomposites Synthesized by Various Methods in the
Presence of Silicate and Zeolite Supports
MirHasan Hosseini1*, Meysam Sadeghi2, Mohammad Javad Taghizadeh3
1 M.Sc., Nano Center Research, Imam Hussein University, Tehran, Iran & Payame Noor University, Germi
Moghan, Ardebil, Iran
2 M.Sc., Nano Center Research, Imam Hussein University, Tehran, Iran
3 Ph.D. Students, Nano Center Research, Imam Hussein University, Tehran, Iran
Received: 12 July 2013; Accepted: 19 September 2013
In this research iron manganese oxide nanocomposites were prepared by co-precipitation,
sol-gel and mechanochemical methods by using iron (III) nitrate, iron (II) sulfate and manganese
(II) nitrate as starting materials. These nanocomposites were prepared in the presence of various
catalyst beds. The polyvinyl pyrrolidon (PVP) was used as a capping agent to control the
agglomeration of the nanoparticles. Nanocatalysts were identified by FT-IR, XRD,SEM and TEM.
The sizes of nanoparticles were determined by XRD data and Scherer equation. The prepared
nanocatalysts were tested for decomposition of hydrogen peroxide. The hydrogen peroxide
decomposition activity of samples was determined by evolved oxygen volumetry technique. Also
based on surface area analysis and BET data, using of sodium metasilicate bed led to the high
surface area and catalytic activity. Therefore Coprecipitation method in the presence of sodium
metasilicate introduce as preferred method. To optimize the catalytic activities of nanoparticles
factors such as concentration, cations ratio, pH and calcination temperature were investigated.
Keyword: Hydrogen peroxide; Decomposition Nanocatalysts; Co-precipitation method; Iron
manganese oxide; Catalyst Supports; Surface area analysis (BET).
ABSTRACT
1. INTRODUCTION
International Journal of Bio-Inorganic Hybrid Nanomaterials
(*) Corresponding Author - e-mail: hoseiny.inorganic@gmail.com
in the decomposition of hydrogen peroxide.
Ahmed et al [2] synthesized iron-manganese
oxides by combustion and sol-gel methods. In
combustion method, stoicheiometric amounts of
manganese acetate Mn(CH3CO2)2·4H2O, ferric
nitrate Fe(NO3)3·9H2O and urea were mixed in an
agate mortar for few minutes. Urea was added to
the mixture (as fuel) and mixed again thoroughly
then transferred to a quartz crucible and synthesized
at 500°C for 1/2 h. At this temperature, the mixture
was reacted leading to the combustion and the reac-
tion was complete in 3-5 min. A foamy and highly
porous precursor mass was obtained. The ferrite
powder was calcined at 900°C. In sol-gel method
the raw materials, Mn(CH3CO2)2·4H2O and
Fe(NO3)3·9H2O, were first dissolved in ethylene
glycol and de-ionized water under stirring until a
homogeneous mixture was observed, heated to
70°C for 12 h and dried at 80°C for 24 h. The
resulting gel was calcined at 600°C. The smallest
nanosize was obtained in combustion method
(41 nm).
Shin-Liang Kuo et al [3] synthesized MnFe2O4-
carbon black (CB) composite powders by a
co-precipitation method in alkaline aqueous
solutions. MnSO4 was dissolved along with FeCl3with a stoicheiometric ratio of 2:1 in 1M HCl
aqueous solution with bubbling N2. The solution
was then added into another solution that contained
1.5 M NaOH and suspended CB powder under
vigorous stirring. Black precipitate was formed
immediately upon mixing. The powder was
prepared by drying at 50°C. A subsequent
calcination process was carried out at different
temperatures for 2 h in N2 atmosphere.
The decomposition of hydrogen peroxide by
manganese oxide at pH= 7 is represented by a
pseudo first order model [4]. The maximum value
of the observed first order rates constants (kobs) was
0.741 min-1 at 11.8 of [H2O2]/ [MnO2] when
[H2O2]/ [MnO2] were ranged from 58.8 to 3.92.
The direct relation of both the concentration of the
initial hydrogen peroxide and manganese oxide on
the decomposition rates allows the first order
kinetics to be modified:
(1)
Pretty lahirri et al [5] synthesized a set of ferrites
of different composition by coprecipitation method.
Ferrites have wide applications in transformer and
communication field. Nanoparticles of spinel.
Manganese ferrite (MnFe2O4) is a common spinel
ferrite material and has been widely used in
microwave, magnetic recording and catalyst
applications.They found that some ferrous spinels
act as catalysts for the decomposition of H2O2 and
their effectiveness is dependent on the composition
of the catalyst. The catalytic activity of the ferrous
spinels for hydrogen peroxide decomposition was
evaluated by rate of evolution of oxygen from the
liquid phase. The rate of evolution of gaseous O2
was monitored with a gasometric assembly.
Nasr-Allah M. Deraz [6, 7] studied the hydrogen
peroxide decomposition activity by oxygen
gasometry of the reaction kinetics at 20-40°C on the
pure and ZnO-doped cobaltic oxide catalysts. The
results revealed that the treatment of Co3O4 with
ZnO at 40-700°C brought about a significant
increase in the specific surface area of cobaltic
oxide.
In the present work, iron manganese oxide
nanocomposites synthesized using different
preparation methods to achieve the high surface
area and catalytic activity in decomposition of
hydrogen peroxide. For this purpose different
synthesis methods, catalytic supports and
parameters such as concentration, cations ratio, pH
and calcination temperatures were investigated to
optimize the catalytic activity for increasing the rate
of hydrogen peroxide decomposition.
2. EXPERIMENTAL PROCEDURES
2.1. Reagents and instruments
Mn(NO3).4H2O, Fe(NO3)3.4H2O and polyvinyl
pyrolidone (PVP) as a capping agent were
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406 Hosseini M et al
398
[ ] [ ] [ ]22222
2OHMnOk
dt
OHdMnO ⋅≡=−
[ ]22MnOkk MnOobs ≡=
purchased from Merck company. Ethylene glycol,
sodium metasilicate and Zeolite 13X prepared from
Fluka company. The IR and UV spectrums were
recorded by IR-Perkin Elmer and UV-Shimadzu
respectively. The nanocomposites were character-
ized by XRD and scanning electron microscopy
(SEM) analysis.
2.2. Preparation of MnFe2O4 nanocomposites
2.2.1. Coprecipitation method
An appropriate amount of Mn(NO3).4H2O and
Fe(NO3)3.4H2O were dissolved in water and
heated to 40°C. While the solution was being stirred
rapidly, 20 mL of NaOH 0.1 M was added to the
solution. After 30 minutes the reaction was halted;
filtering and washing steps at pH= 7 were carried
out. As a result the precursors of MnFe2O4 i.e.
Mn(OH)2 and Fe(OH)3 were produced which were
left for 24 h at 60°C±10°C to be dried. The dried
precursors were calcinated and annealed at 300°C
for 2 h a heating and cooling rate of 10°C/min to
obtain MnFe2O4.
The ionic equation of the reaction is as followed:
Mn2+ + 2Fe3+ + 8OH- → Mn(OH)2↓ + 2Fe(OH)3↓→ MnFe2O4 + 4H2O (2)
2.2.2. Sol-gel method
Mn(NO3).4H2O and Fe(NO3)3.4H2O were
dissolved in ethylene glycol as a gelling agent.
While stirring deionized water was added until a
homogeneous mixture was observed this was heat-
ed at 70°C for 12 h and dried at 80°C for 24 h. The
resulting gel was ground and reheated at 100°C for
24 h and slowly cooled. Final calcination was car-
ried out at 500°C for 2 h at a heating rate of
10°C/min which was followed by cooling step to
room temperature at the same rate.
2.2.3. Mechanochemical method
Mn(NO3).4H2O and Fe(NO3)3.4H2O were mixed
and ground to have an uniform powder. Addition of
some distilled water converted the powder to gel
form which was dried at 50°C for 4h that was
calcinated at 300°C for 2 h to obtain MnFe2O4.
To increase the nanoparticles active surfaces in
above method an optimized amount of sodium
metasilicate, foam form of sodium metasilicate gel
and zeolite as catalytic beds were added to metal
salts. To have a gel form of the mixture some
deionized water was added to the mixture. The
obtained mixture was dried at 50°C and calcinated
at 300°C for 2 h to build the desired phase.
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406Hosseini M et al
399
Figure 1: Proposed mechanism of PVP intractions with metal ions [8].
2.2.4. Coprecipitation method in the presence of
PVP
The procedure of this method is similar to the
Coprecipitation method. The difference is the
acting of PVP as a capping agent that controls the
size of nanoparticles and prevents from agglomera-
tion. The interactions between PVP and metal ions
are represented schematically in Figure 1, which
shows that the manganese (II) and iron (III) ions are
bound by strong ionic bonds between the metallic
ions and the amide group in a polymeric chain or
between the polymeric chains. This uniform
immobilization of metallic ions in the cavities of
the polymer chains favors the formation of a
uniformly-distributed, solid solution of the metallic
oxides in the calcination process.
2.3. Measurment of catalytic activity of the
nanocatalysts on decomposition of hydrogen
peroxide
The catalytic activity of the nanoparticles on
hydrogen peroxide decomposition was evaluated by
rate of evaluation of oxygen from the liquid phase.
A measured amount of catalyst (0.1 g) was injected
into a thermostated reaction vessel containing 10
mL of 5% H2O2 (pH= 6.64) for each specimen.
H2O2 was standardized immediately prior to use by
standard KMnO4 solution. The peroxide decom-
position is represented by:
H2O (aq) → H2O (l) + 1/2 O2 (g) (3)
H2O2 undergoes an exothermic reaction to form
O2 and H2O. The rate of evolution of gaseous O2
was monitored with a gasometric assembly. The
time dependent volume, Vt of the evolved oxygen
was monitored at 0.5 min intervals in all cases.
The catalytic activity was calculated by Eq. 4:
a= k/(t.m) (4)
where a is the activity, k is a constant, t is a reaction
time and m is mass of catalyst (In this experiment it
is 0.1 g).
3. RESULT AND DISCUSSION
3.1. IR investigation
The IR transmission spectra were measured for
sample calcined at 300°C. Two bands with wave
numbers 470 cm-1 and 562 cm-1 are attributed to
Fe-O and Mn-O on octahedral and tetrahedral sites
with spinel structure (MnFe2O4) respectively
(Figure 2).
Figure 2: IR spectrum of MnFe2O4
Figure 3: XRD of MnFe2O4 (a: pure b: in the presence
of sodium metasilicate).
3.2. XRD investigation
The peaks that are present and are labeled by codes
220, 311, 400, 511, 440 belong to MnFe2O4 with
spinel structure (Figure 3). Basedon data obtained
from JCPDS-JCDD (joint committee for powder
diffraction international center for diffraction data)
our sample exhibits a cubic structure (space group:
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406 Hosseini M et al
400
(b)
(a)
Fd3m, JCPDS: 10-319) and no extra high exists
along the peak that implies the sample doesn't
contain any impurity. Using Scherer equation the
average size of nanoparticles was determined to be
45 nm. The XRD of the sample that was prepared
by coprecipitation method and PVP capping agent
demonstrates a decrease of 15 nm in particle size
that may be related to capping agent.
3.3. Investigating the UV spectrum
Considering the low solubility of transitional
elements in organic solvents and water, for
analyzing the UV spectrum the sample should be
left in suspended and disperse situation in
ultrasonic bath. Also, surface active and surfactant
agents like PVP can be used for this purpose. In
electromagnetic region of the spectrum, the
molecules experience the electronic transition.
Charge transition band in maximum wavelength
absorb (347 nm) and observation of broad peaks
before and after the incident indicates the formation
of ferrite compound [9] (Figure 4).
Figure 4: UV spectrum for MnFe2O4
3.4. SEM analysis
Analyzing the morphology aspect of the nano-
particles by studying the SEM (micrographs)
indicates that the synthesized nanoparticles are
quasi-spherical and the size is less than 100 nm.
That means the synthesized catalysts have nano
dimension. The information obtained from XRD
also confirms the above findings. The results
obtained from the calculation of average size of
nanoparticles by the aid of SEM images and
analytical Clemex image software is as followed:
The size of samples prepared by coprecipitation
method and synthesized in presence of a capping
agent are 50 nm and 40 nm respectively (Figures 5
and 6).
Figure 5: SEM of MnFe2O4 (coprecipitation method).
Figure 6: SEM of MnFe2O4 in the presence of PVP.
It should be mentioned that the deter mination of
nanoparticles size by aid of SEM is related to
morphology of the particles that means the reported
size of the nanoparticles verified by XRD and SEM
techniques are related to uniform distribution of
particle size. At this section of the article SEM
images of nanoparticles that are formed in the
presence of catalytic beds are investigated. SEM
images demonstrated the fact that the morphology
of majority of nanoparticle beds is quasi-spherical.
Another determination by SEM images is related to
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406Hosseini M et al
401
the size particles i.e. the formed particles exhibit
nano dimension and to be exact the size is under
100 nm. The size of particles formed on silica and
porous beds are smaller than those formed on zeo-
lite bed (Figures 7, 8 and 9).
Figure 7: SEM of nanocomposite with sodium
metasilicate support.
Figure 8: SEM of nanocomposite with porous beds.
Figure 9: SEM of nanocomposite with zeolite support.
Also, dispersion and distribution of nanoparticles
size in silica and porous beds are more than the
other beds. Hence it can be concluded that the
above reasoning are effective in incrementation of
catalytic activities of silica and porous beds. In
order to have a sharper remark the analysis of
samples surface area should be considered.
3.5. The role of catalyst support on surface area
and particle size
Presence of catalytic support of sodium metasilicate
illustrates various advantages associated with
nanoparticles such as simple work up procedure,
short reaction times, reduced particle size, high
total surface area (m2), high specific surface area
(m2/g), high product yield and easy recovery and
reusability of the catalyst. The use of sodium meta
silicate reduces the size of nanoparticles from 43
nm to 12 nm; increases the catalytic activity;
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406 Hosseini M et al
402
SampleWeight
(gram)Adsorb gas
Relative (p/p0)
Pressure
Total Surface
area (m2)
Specific
surface area
(m2/g)
in the
absence of
support
0.107 Nitrogen 0.15 0.5407 5.0531
in the
presence of
support
0.105 Nitrogen 0.5 11.2864 107.4899
Table 1: surface area analysis (BET)-iron manganese oxide nanocaomposites in the
presence of sodium meta silicate catalyst support.
increases the distribution of nanoparticles on the
support (the morphology of system improves).The
data that confirm the above claims are listed in
Table 1 and shown in Figures 10 and 11.
Furthermore presence of catalytic support incre-
ments the amount of evolved oxygen
(Figure 12).
Figure 10: SEM of iron-manganese oxide nanocompos-
ites in the absence of sodium meta silicate catalyst
support.
Figure 11: SEM of iron-manganese oxide nanocompos-
ites in the presence of sodium meta silicate catalyst
support.
It should be mentioned that three other catalyst beds
i.e. meso porous, molecular sieves and zeolit 13X
were also investigated, but none of them had the
effectiveness of sodium metasilicate on decompo
sition of H2O2 and as a result the amount of evolved
oxygen was in excess. Among the catalyst beds the
zeolit 13X bed had the less effect (Figure 12).
Figure 12: Catalytic activity of iron-manganese oxide
nanocomposites in the presence of catalyst beds on
hydrogen peroxide decomposition.
TEM images obtained for the ferrite nanoparticles
and sodium metasilicate ferrite nanocomposites are
shown in Figure 13a and b respectively. The
MnFe2O4 sample consists of nanoparticles of
approximately 40 nm which aggregate to form large
clusters. The size of these nanoparticles as deduced
by TEM inspection is in agreement with that
calculated by XRD analysis. The TEM image
obtained for the MnFe2O4/sodium metasilicate
composite (Figure 13b) shows that the MnFe2O4
are dispersed along the silicat, which exhibits a
large porosity.
3.6. Investigating the different variables providing
optimized conditions to accelerate H2O2 decompo-
sition
3.6.1. Effect of calcination temperature
In order to determine the optimized calcinations
temperature, different calcinations temperatures
were implemented on hydroxide precipitation
produced via co-precipitation method. Catalytic
activities was at maximum when the temperature of
calcination reached 300°C. At this temperature
decomposition of hydrogen peroxide occurred at
lower temperature which evolves more oxygen. In
higher calcination temperature the nanoparticles
stick together, so their sizes increments and the
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406Hosseini M et al
403
surface area for catalytic activities are reduced. At
lower calcination temperature oxidize phases is not
formed (Figure 14).
Figure 14: Effect of calcination temperature on catalytic
activity of hydrogen peroxide decomposition (samples
obtained from co-precipitation method).
3.6.2. Optimization of pH in co-precipitation
method
Decomposition of hydrogen peroxide is a variable
of pH. It was mentioned that the optimized pH for
the most catalytic activity of the samples is on the
basic region of 9-10 the samples in acidic region
(low pH) has the least catalytic activity.
Figure 15: Effect of pH on catalytic activity of hydrogen
peroxide decomposition (samples obtained from co-
precipitation method).
3.6.3. Effects of catalyst bed amounts and different
types of starting materials for supporting process
It was observed that for having the maximum
catalytic activity the amount of metal salts should
be four times of catalytic bed (Figure 16). In fact
the amount of catalytic bed should be exact because
if it is more the catalyst active sites are covered and
surface and catalytic activities are reduced;
Furthermore if it is less than the optimized amount
the surface activity is not altered by it. It was
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406 Hosseini M et al
404
Figure 13: TEM image of the iron-manganese oxide nanoparticles (a: pure b: in the presence of sodium
metasilicate).
(a) (b)
observed that if FeSO4 is used as a reactant instead
of Fe(NO3)3 the surface activity is reduced
dramatically; the reason could be related to the
poisoning nature of sulfate. In co-precipitation
method, catalytic activities are not poisoned
because of the washing and filtering processes. It
was confirmed that suitable starting material is
utilization of metal salts instead of metal
hydroxides and metal oxides, the reason that in
metal nitrates the ions are more freer to move about,
therefore the ion-exchange was carried-out better
(Figure 17).
Figure 16: Optimization of support weight in 2g catalyst.
3.7. Feasibility of reusing nanocatalysts
In order to examine the reusability of nanocatalysts
in decomposition of hydrogen peroxide, a few of
synthesized samples were chosen coincidentally,
and catalytic decomposition reaction was carried
out on them. At the end of reaction the samples was
collected, dried and weighted, then they were
reused for the decomposition of hydrogen peroxide,
Figure 17: Form of starting materials for supporting
process.
as the last step of the experiment the difference
between the catalytic activity and the weight of
nanocatalysts were considered. The slight
difference in initial and final weights of nano-
catalysts indicates that both the primary nano-
catalysts and synthesized samples have the
catalytic activity in hydrogen peroxide decom-
position. The results are summarized in Table 2.
4. CONCLUSIONS
For carrying out the hydrogen peroxide decompo-
sition reaction in short time (rapid reaction),
MnFe2O4 nanocomposites were used. In order to
reach the maximum speed in hydrogen peroxide
decomposition reaction, and as a result collecting
the maximum possible amount of oxygen in short
time, nanocatalysts with the highest catalytic
activity should have been used. Hence, major
Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 397-406Hosseini M et al
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Sample Initial weight Reaction Time Secondary weight Reaction Time
Coprecipitationmethod
Sol-Gel
Silica Support
(preferred method)
Zeolit 13X
0.1(g)
0.1(g)
0.1(g)
0.1(g)
30(s)
150(s)
12(s)
120(s)
0.098(g)
0.099(g)
0.091(g)
0.088(g)
33(s)
155(s)
13(s)
123(s)
Table 2: Feasibility of reusing nanocatalysts.
variables that were so effective in catalyst synthesis
should be considered, and optimized. Conditions of
each variable that had a major effect in incrementa-
tion of catalytic activity were investigated that
caused the augementation of nanoparticles active
surface area and their uniform distribution on
catalytic supports that are followed:
1. The best method for synthesis of nanocatalysts
was Coprecipitation method in the presence of
silicate that provided the maximum catalytic
activity for decomposition of H2O2. The suitable
pH for nanoparticles precipitation was around 9.
The optimized calcinations temperature was about
300°C. In lower temperature suitable oxidized
phase was not formed and in higher temperature the
nanoparticles were sticked together and as a result
the size of nanoparticles was incremented.
2. For controlling the nanoparticle size PVP
capping agent was applied by using co-precipitation
method. PVP agent also caused a 15 nanometer
reduction in nanoparticles size.
3. To accelerate hydrogen peroxide decomposition
sodium metasilicate was used as a catalytic support.
In presence of sodium metasilicate shorter time
needed to decompose a certain amount of H2O2.
Decomposition of H2O2 was implemented at the
least time when the amount of catalyst was four
times of the support because large amount of
support cover the active sites of catalysts.
4. Investigating the reusability of nanocatalysts
indicated that second use of them was accompanied
by a slight poisonous that was negligible; therefor
the nanocatalysts could be used for a few times. It
was also observed that the difference in weight of
consumed nanocatalysts is so small that demon-
strated the nanocatalysts participated in the
reactions as catalysts and not reactants.
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