International Journal of Bio-Inorganic Hybrid Nanomaterials

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: [email protected]

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)


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


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-


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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344


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



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


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


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).


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).


351

Figure 7: Average viscosity of grape juice treatments during storage. Values displayed with different letters are



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



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


352

Figure 9: General sensory acceptance of grape juice treatments during storage. Values displayed with different letters are



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,


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



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

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 355-364Moghimi A et al

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


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.


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.


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


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


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


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.


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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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


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



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.


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

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 365-371 Heydari A et al

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


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


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.

REFERENCES

1. Rowlings E.D., Annu. Rev. Microbiol., 4 (2005),

65.

2. Rodriguez Y., Ballester A., Blazquez M.L.,

Gonzalez F., Munoz J.A. Geomicrobiol. J., 20

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8. Mybodi S.M., Microbiology PhD Thesis,

Islamic Azad University Science and Research

Branch, (2008), 334.

9. Pacholewska M., Appl. Environ. Microbiol, 37

(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),

1.

12. Sand W., Rohde K., Sobotke B., Zenneck C.,

Appl. Environ. Microbiol, 58 (1) (1992), 85.

13. Rawlings D.E., Tributsch H., Hansford G.S.,

Microbiology, 145 (1999), 5.


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-


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



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.


2.1. 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).


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


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.

REFERENCES

1. Fan J.C., Bachner F.J., J. Electrochem. Soc., 122

(1975), 1719.

2. Witt J.H., J. Solid State Chem., 20 (1977), 143.

3. A.J. Wilson, 1992. The International Union of

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 373-378Farahmandjou M

377

Figure 4: X-ray diffraction pattern of SnO2 and ITO nanoparticles.

(a)

(b)

(c)

(d)

Crystallography, International Tables for

Crystallography, Kluwer Academic Press.

4. Marezio M., Acta Crystallogr, 20 (1996), 723.

5. Frank G., Kostlin H., Appl. Phys. A, 27 (1982),

197.

6. Subbarao E.C., Sutter, P.H., Hrizo J., J. Am.

Ceram. Soc., 48 (1965), 443.

7. SujathaLekshmy S., Maneeshya L.V., Thomas

P.V., and Joy K., Indian J. Phys., 87 (2013), 33.

8. Sarmah S., Kumar A., Indian J. Phys., 84

(2010), 1211.

9. Diana T., Devi K.N., Sarma H.N., Indian J.

Phys., 84 (2010), 687.

10. Wang S.L., Xia D.L., Glass & Enamel., 32

(2004), 51.

11. Niesen T.P., Guire M.R., J. Electroceramics, 6

(2001), 169.

12. Betz U., Kharrazi M., Marthy J., Escola M.F.,

Atamny F., Surf. Coat. Technol., 200 (2006),

5751.

13. Ogi T., Iskandar F., Itoh Y., Okuyama K., J.

Nanoparticle Res., 8 (3)(2006) 343.

14. Chang W., Lee S., Yang C., Lin T., Mater. Sci.

Engineering B, 153 (1) (2008), 57.

15. Zhang Y., Ago H., Liu J., J. Cryst. Growth, 264

(1) (2004), 363.

16. Soulantica K., Erades L., Sauvan M., Senocq,

F., Maisonnat A., Chaudret B., Adv. Funct.

Mater., 13 (7) (2003), 553.

17. Ki H.S., Byrne P.D., Facchetti A., Marks T.J., J.

Am. Chem. Soc., 130 (38) (2008), 12580.

18. Arfsten N.J., J. Non-Cryst. Sol., 63 (1984), 243.

19. Xu J.J., Shaikh A.S., Vest R.W., Thin Solid

Films, 161 (1988), 273.

20. Yamamoto O., Sasamoto T., J. Mater. Res., 7

(1992), 2488.

21. Bisht H., Eun H.T., Mehrtens, A., Aegerter

M.A., Thin Solid Films, 351 (1999), 109.

22. Toki M., Aizawa M., J. Sol-Gel Sci. Technol., 8

(1997), 717.

23. Yoshinaka A., Onozawa K., New Ceramics, 4

(1996), 24.

24. Matsushita T., Ceramics, 21 (1986), 236.

25. Quaas M., Eggs C., Wulff H., Thin Solid Films,

332 (1998), 227.


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


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



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).


3.1. 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)



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).


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-

hanan U., Chalcogenide Lett., 6 (2009), 197.

9. Wang J., Gao L., Inor. Chem. Com., 6 (2003),

877.


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


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


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



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.


2.1. 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


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


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


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.


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


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


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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395

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


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


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



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:


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


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;


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


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


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


405

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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International Journal of Bio-Inorganic Hybrid Nanomaterials