International Journal of Bio-Inorganic Hybrid Nanomaterials

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344

International Journal of Bio-Inorganic Hybrid Nanomaterials

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

ABSTRACT 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.

1. INTRODUCTION The mechanical properties of thin nanocomposite coatings can be evaluated by using the nanoindentation 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 (*) Corresponding Author - e-mail: A.Ershad@ippi.ac.ir

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.

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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 nanoindentation [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 nanoindentation technique [1, 21-241. This technique is ideal for mechanical studies of thin films, polymeric materials, multilayers which the microstructure 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

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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 nanoindentation 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.

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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.

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 nanoindentation 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].

1

2

(a) 3

Figure 3: Schematic presentation of cracking, delamination and spallation in failure of coating from substrate in (b)

nanoindentation test [6, 30].

Figure 2: (a) A typical nanoindentation : load and unloading -displacement curve and schematic loading cycle in CSM technique (b) schematic deformation pattern of substrate surface with an elastic-plastic behavior after indentation test [1, 14].

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

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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:

P H = max A A=

(1)

C0hc2

H c = hmax − ε

(2)

Pmax S

2

(4)

(5)

Where ν and E are the Poisson's ratio and elastic 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+C2hc1/2+C3hc1/4+C4hc1/6+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).

Er =

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:

340

1 1 −ν S2 1 −ν i2 = + Er ES Ei

(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:

π  S  A =    max   4   Er 

Ershad Langroudi A

π S max 2 A

(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 materials can be calculated under sinusoidal loading in linear viscoelastic domain by Equation (8) and (9):

E′ =

σ0 cos ϕ ε0

(8)

E ′′ =

σ0 sin ϕ ε0

(9)

E = E ′ + iE ′′

(10)

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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.

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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 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

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 enhancement 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

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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 nanoparticles [40]. Lionti et al. synthesized hybrid silica coatings based on 3-glycidoxypropyltriethoxysilane (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 Nanoindentation (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 Loaddisplacement curve can be interpreted as delamination (see Figure 4c) [42]. Tiwari et al. have recently reported the basic fundamental principles as well as the experimental analyzing in modern nanoindentation 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

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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 nanoindentation, 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].

(a) (b)

(c)

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 corresponding 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-

Ershad Langroudi A

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-inorganic hybrid nanocomposite coatings by sol-gel process. It is demonstrated that the physicalmechanical properties of these thin nanocomposite coatings depend on the nature, particle size and distribution 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 nanocomposite 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.

REFERENCES 1. Li X., Bhushan B., Mat. Charact, 48 (2002), 11.

2. Tiwari A., Adv. Chem. Eng. Sci., 2 (2012), 34.

3. Hu H., Onyebueke L., Abatan A., J. Min. Mat. Charact. Eng., 9 (2010), 275.

4. B. Bhushan, 1999. Handbook of micro/nanotribology. 2nd Ed. Boca Raton (FL): CRC Press.

5. Tabor D., Philos Mag A, 74 (1996), 1207.

6. Zhang S., Sun D., Fu Y., Du H., Surf. Coat.

Tech., 167 (2003), 113.

7. Mohseni M., Yahyaei H., Yari H., Ramezanzadeh B., Solid Mech. Appl., 203 (2014), 3.

8. Skarmoutsou A., Lolas G., Charitidis C.A. Chatzinikolaidou M., Vamvakaki M., Farsari M., J. Mech. Beh. Biomed. Mat., 25 (2013), 48.

9. Tiwari A., Agee P., Solid Mech. Appl., 203 (2014), 103.

10. Malzbender J., Den Toonder J.M.J.,

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344

Balkenende A.R., Materials. Sci. Eng. R: Reports, 36 (2002), 47.

11. Malzbender J., Den Toonder J.M.J., Thin Solid Films, 366 (2001), 139.

12. Mammeri F., Bourhis E.L., Rozes L., Sanchez C., J. Eur. Ceram. Soc., 26 (2006), 259.

13. Pharr G.M., Harding D.S., Oliver W.C., NATO ASI Series, 233 (1993), 449.

14. Oliver W.C., Pharr G.M., J. Mat. Res., 7 (1992), 1564.

15. Herbert E.G., Oliver W.C., Pharr G.M., J. Phys.

D: Applied Phys., 41 (2008), 074021.

16. Schiffmann K.I., Philos. Mag., 91 (2011), 1163.

17. G. Kaupp, 2006. Nanoscratching, in: Atomic Force Microscopy, Scanning Near field Optical Microscopy and Nanoscratching, Springer Berlin Heidelberg.

18. Bhushan B., Kulkarni A.V., Bonin W., Wyrobek J.T., Philos Mag A, 74 (1996), 1117.

19. Huang L.Y., Zhao J.W., Xu K.W., Lu J., Diamond Related Mat., 11 (2002), 1454.

20. B.A. Latella, M.V. Swain, M. Ignat, 2012.

Indentation and Fracture of Hybrid Sol-Gel Silica Films, Nanoindentation in Materials Science, Dr. Jiri Nemecek (Ed.).

21. Gotlib-Vainshtein K., Girshevitz O., Sukenik C.N., Barlam D., Kalfon-Cohen E., Cohen S.R., J. Phys. Chem. C, (2013), article ASAP. 22. Roussi E., Tsetsekou A., Skarmoutsou A., Charitidis C.A., Karantonis A., Surf. Coat.

Tech., 232 (2013), 131.

23. Ballarre J., Jimenez-Pique E., Anglada M., Pellice S.A., Cavalieri A.L., Surf. Coat. Tech., 203 (2009), 3325.

24. Xiong M., Zhou S., Wu L., Wang B., Yang L., Polymer, 45 (2004), 8127.

25. Hay J., Agee P., Herbert E., Exp. Tech., 34 (2010), 86.

26. Chen J., Beake B.D., Dong H., Bell G.A., Solid Mech. Appl., 203 (2014), 63.

27. Tan J.C., Cheetham A.K., Chem. Soc. Rev., 40 (2011), 1059.

28. Frutos E., Gonzalez-Carrasco J.L., Acta Mater, 61 (2013), 1886.

29. Li X., Bhushan B., Thin Solid Films, 330 (1999), 355.

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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 337-344

30. Bhushan B., Diamond Relat. Mater, 8 (1999), 1985.

31. Pharr G.M., Mat. Sci. Eng. A, 253 (1998), 151.

32. Zhou J., Komvopoulos K., J. Appl. Phys., 100 (2006), 114329.

33. Bhushan B., Gupta B.K., Azarian M.H., Wear, 181-183 (1995), 743.

34. Bhushan B., Li X., J. Mater. Res., 12 (1997), 54.

35. Babu B.S., Kumaraswamy A., Prasad B.A., Adv. Mat. Manufact. Chirac, 3 (2013), 169.

36. Davies P., Sohier L., Cognard J.Y., Bourmaud A., Choqueuse D., Rinnert E., Creachcadec R., Int. J. Adhes. Adhes, 29 (2009), 724.

37. Shi X., Nguyen T.A., Suo Z., Liu Y., Avci R., Surf. Coat. Tech., 204 (2009), 237.

38. Woo R.S.C., Zhu H., Leung C.K.Y., Kim J. K., Comp. Sci. Tech., 68 (2008), 2149.

39. Li X. F., Lau K.T., Yin Y.S., Comp. Sci. Tech., 68 (2008), 2876.

40. Kardar P., Ebrahimi M., Bastani S., Prog. Org. Coat., 62 (2008), 321.

41. Lionti K., B. Toury, Boissiere C., Benayoun S., Miele P., J. Sol-Gel Tech., 65 (2013), 52.

42. Sun B., Scherban T., Pantuso D., Sanchez J.M., Elizalde R., Martinez-Esnaola J.M., 2001.

Crosssectional nanoindentation: a novel technique to measure thin film interfacial adhesion oral reference: Oral reference: 10th ICF 100624 OR. 43. Barth N., Schilde C., Kwade A., Physics Procedia: Eur. Conf. Nano Films-ECNF2012, 40 (2013), 9.

44. Zamani Ketek Lahijania Y., Mohseni M., Bastani S., Tribol. Int., 69 (2014), 10.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

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

ABSTRACT 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 (25째C and 4째C) 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.

(*) Corresponding Author - e-mail: goudarzi6302@yahoo.com

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1. INTRODUCTION 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 2,3dimethoxy-5-methyl-6-polyisoprene parabenzoquinone. 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.

O H3CO

CoQ10 levels in selected foods food

Coenzyme 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

CH3

H3CO O

Table 1: Coenzyme Q10 levels in selected foods

CH3

- soybean

54-280

- olive

4-160

H

- grapeseed

64-73 4-15

6-10

- sunflower Nuts - peanuts

Figure 1: The chemical structure of coenzyme Q10

Synthesis within the body

27

- walnuts

19

- sesame seeds

18-23

- pistachio nuts

20

- hazelnuts

17

- almond

5-14

Vegetables

Resources of coenzyme Q10

Food

Food supplements

Figure 2: Resources of coenzyme Q10

346

- 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

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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

Figure 3: Flowchart of study

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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 temperature 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

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a 5-point Hedonic test. The total sensory acceptance was calculated and compared among treatments as final sensory parameter.

Statistical analysis Experiments were performed in triplicate and significant differences between means were analyzed using two-way ANOVA test from Minitab software. The design of experiment was completely randomized design (full Factoriel).

Also, to clarify the relationship between the characteristics of the Pearson correlation coefficient was used.

3. RESULTS AND DISCUSSION 3.1. Effects Q10 addition on pH and titrable acidity Figures 4-9 shows the average pH, titrable acidity, Brix, viscosity, turbidity and general sensory acceptance of grape juice treatments during storage.

Concentration of coenzyme Q10 and dual effect of temperature and time showed a significant effect on pH of grape juice. With increasing temperature and time, the pH was decreased. This may be due to the growth of acid-producing bacteria in fruit juice.

Coenzyme Q10 concentrations also had a direct effect on the pH of juice and the reason may be the higher pH of Q10 and other accompanying materials (pH = 7) [8, 21]. Q10 concentration had a direct effect on pH (Figure 4). The results obtained revealed that the highest pH was for treatments A2B2C3 (containing 20 mg of Q10 in 300 mL of juice stored 25°C for 1 month) and the lowest pH was for treatment A2B4C1 (stored at 25°C for 3 months with no coenzyme Q10).

It was found that the factors of temperature, time and concentration of coenzyme Q10 had significant effect on the titrable acidity of the juice (Figure 5).

Storage time and temperature had a direct effect on the titrable acidity of the juice, so that with increasing temperature and time acidity increased and with increasing concentrations of coenzyme Q10, the acidity was decreased. The concentration of coenzyme Q10 had reverse effect on titrable acidity, since acidity has a reverse relation with pH and according to the discussed reasons about pH

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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.

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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

Figure 4: Average pH of grape juice treatments during storage. Values displayed with different letters are significantly different. A = storage temperature (A1 = 4°C and A2 = 25°C); B = storage time (B1 = at the start of storage, zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0 mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).

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Figure 5: Average acidity of grape juice treatments during storage. Values displayed with different letters are significantly different. A = storage temperature (A1 = 4째C and A2 = 25째C); 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 = 4째C and A2 = 25째C); B = storage time (B1 = at the start of storage, zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0 mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).

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Figure 7: Average viscosity of grape juice treatments during storage. Values displayed with different letters are significantly different. A = storage temperature (A1 = 4째C and A2 = 25째C); B = storage time (B1 = at the start of storage, zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0 mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).

Figure 8: Average turbidity of grape juice treatments during storage. Values displayed with different letters are significantly different. A = storage temperature (A1 = 4째C and A2 = 25째C); B = storage time (B1 = at the start of storage, zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0 mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).

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Figure 9: General sensory acceptance of grape juice treatments during storage. Values displayed with different letters are significantly different. A = storage temperature (A1 = 4°C and A2 = 25°C); B = storage time (B1 = at the start of storage, zero, B2 = month 1, B3 = month 2, B4 = month 3); C = concentration of coenzyme Q10 in 300 mL of fruit juice (C1 = 0 mg/300 mL, C2 = 10 mg/300 mL, C3 = 20 mg/300 mL).

Table 2: Correlation between attributes in grape juice by pearson coefficient attribute

pH

acidity

brix

viscosity

turbidity

pH

1

-0.751 **

-0.485 **

-0.451 **

-0.408 **

acidity

-0.751 **

1

0.690 **

0.617 **

0.426 **

brix

-0.485 **

0.690 **

1

0.676 **

0.569 **

viscosity

-0.451 **

0.617 **

0.676 **

1

0.404 **

turbidity

-0.408 **

0.426 **

0.569 **

0.404 **

1

** = Difference between treatments is quite significant (P < 0/01).

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

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with each other while communicating with other characters straight (Table 2).

3.4. Effect of adding Q10 on total sensory acceptance Most of treatments did not show significant difference in total sensory acceptance (Figure 9).

The Transparency of juices kept at lower temperature (4°C compared those stored at 25°C) and samples with shorter storage time showed

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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 physicochemical and sensory properties.

REFERENCES 1. Ernster L., Dallner G., Biochem Biophys Acta, 1271 (1) (1995), 195.

2. Crane F.L., J Am Coll Nutr, 20 (6) (2001), 591.

3. Quinzii C.M., Dimauro S., Hirano M., Neurochem Res., 32 (2007), 723.

4. Alhasso S., A review Hosp. Pharm., 36 (1) (2001), 51.

5. Kagan D., Madhavi D., J Int. Med., 11 (2010), 109.

6. Littarru G.P., Tiano L., An update Nutrition, 2 (2009), 1.

7. Coles L., Harris S., Adv Anti-Aging Med., 1 (1996), 205.

8. Pravst I., Prosek M., Wondra A.G., Acta Chim Slov., 56 (2009), 953.

9. Itagaki Sh., Ochiai A., Kobayashi M., Sugawara M., Hirano T., Iseki K., Food Chem., 120 (2010), 552.

10. Beketova N.A., Vrzhesinskaia O.A., Kosheleva O.V., Sharanova N.E., Soto S.K., Kulakova

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S.N., et al. Vopr Pitan., 795 (2010), 61.

11. Wang H., Cao G., Prior R.L., J. Agric. Food Chem., 44 (1996), 701.

12. Davalos A., Bartolome B., Gomez-Cordoves C., Food Chem., 93 (2005), 325.

13. Shankar S., Singh G., Srivastava R.K., Front Biosci, 12 (12) (2007), 4839.

14. Demrow H.S., Slane P.R., Folts J.D., Circ., 91 (1995), 1182.

15. Freedman J.E., Parker C., Li L., Perlman J.A., Frei B., Ivanov V., Deak L.R., Iafrati M.D., Folts J.D., Circ., 103 (2001), 2792.

16. Jung K.J., Wallig M.A., Singletary K.W., Cancer Lett, 233 (2) (2006), 279.

17. Vinson J.A., Teufel K., Wu. N., Atheroscl, 156 (1) (2001), 67.

18. Acquaviva R., Russo A., Galvano F., Galvano G., Barcellona M.L., Li Volti G., Cell Biol Toxicol., 19 (2003), 243.

19. Jayaprakasam B., Olson L.K., Schutzki R.E., Tai M.H., Nair M.G., J. Agric. Food Chem., 54 (2006), 243.

20. Sadras V.O., Petrie P.R., Moran M.A., Aust J Grape Wine Res., 19 (2013), 107.

21. Saravacos G.D., J. Food Sci., 35 (2) (1970), 122.

22. Meyer A.S., Zeuner B., Pinelo M., Food Bioprod Proc., 88 (2-3) 2010, 259.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

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

ABSTRACT 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 Âą 0.8 mg of each of the two ions. The limit of detection (3Ď&#x192;) for Pb(II) was found to be 3.20 ng l-1. The enrichment factor 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.

1. INTRODUCTION 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(*) Corresponding Author - e-mail: alimoghimi@iauvaramin.ac.ir

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,

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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-dihidroxybenzoic 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

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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 development 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 desorbed 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-Isocyanatobenzenesulfonyl 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 chromatography with diameter of about 50 Č?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

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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

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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

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.

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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 spectrometer 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 determination of lead.

Slit width Operation current of HI-HCL Resonance fine Type of background correction Type of flame

0.7 nm 10 mA 283.3 Deuterium lamp Air/acetylene

Air flow

7.0 mL.min-1

Acetylene flow

1.7 mL.min-1

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

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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 Č?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 (Chelex100) 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 Č?m filter and then passed through the three columns system, Dowex 1X-8, RP-C18 silica adsorber and Chelex-100 respectively. The columns were then separated. The anion and cation exchanger columns were washed with 10 mL of 2 M HNO3 and the C18

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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.

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(a)

3. RESULTS AND DISCUSSION The treatment of Graphene oxide with covalently linked porphyrin (GO-H2P) can lead to the derivatization 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

(b)

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 900째C under nitrogen, and GO which decomposes above 600째C, after having lost the oxygenated species at 240째C (i.e. 14.7% weight loss), the 6% weight loss occurred in the temperature range 250-550째C for the GO-H2P material, is attributed to the decomposition 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.

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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 monotonically 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

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and 10.0 M hydrochloric acid with hydrolysis percentage of 2.25, 6.10 and 10.50 for phase, respectively. Thus, these stability studies indicated the suitability of phase for application in various acid solutions especially concentrated hydrochloric acid and extension of the experimental range to very strong acidic media which is not suitable for other normal and selective chelating ion exchangers based on a nano polymeric matrix [9]. Finally, the GO-H2P phases were also found to be stable over a range of 1 year during the course of this work. The IGO is insoluble in water. Primary investigations revealed that surfactant coated C18 could not retain Pb(II) cations, but when modified with the GO-H2P retains these cations selectively. It was then decided to investigate the capability of the GO-H2P as a ligand for simultaneous preconcentration and determination of lead on admicell. The C18 surface in acidic media (1<pH<6) attracts protons and becomes positively charged. The hydrophyl part of SDS (-SO3-) is attached strongly to these protons.

On the other hand, the GO-H2P is attached to hydrophobe part of SDS and retains small quantities of metallic cations [22].

Figure 4: Extraction percentage of Pb(II) against pH.

3.3. Effect of pH in does not occur The effect of pH of the aqueous solution on the extraction of 100 ng of each of the cations Pb(II) was studied in the pH rang of 1-10. The pH of the solution was adjusted by means of either 0.01 M HNO3 or 0.01 M NaOH. The results indicate that

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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 hydrolysis 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

Table 2: Effect of foreign ions on the recovery of 100 ng of Pb.

Diverse ion

Amounts taken (mg)

% Found

% Recovery of Pb2+ ion

added to 50 mL 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)

CH3

COO-

SO42-

5.3

2.2(2.6)

95.5(2.2)

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)

a: Values in parenthesis are CVs based on three individual replicate measurements.

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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

Moghimi A et al

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

Table 3: Recovery of Pb contents of water samples.

Diverse ion

Amounts taken (mg)

% Found

Pb2+ ion

added to 50 mL

Sample Distilled water

Pb

% Recovery of

-

-

-

0.050

0.043(2.40)a

96

0.100

0.094(2.60)

97

-

0.015(3.0)

-

0.050

0.068(2.42)

96

-

0.048(2.25)

-

0.100

0.155(2.30)

98

-

0.045(2.25)

-

0.100

0.143(2.40)

98

(100 mL)

Tap water (100 mL)

Pb

Snow water (50 mL)

Pb

Rain water (100 mL)

Pb

Synthetic sample 1 Na+, Pb

-

-

-

Ca2+, Fe3+, Co2+, Cr3+, Hg2+, 1 mg L-1 0.100 Synthetic sample 2 K+

Pb

-

0.104(2.40) -

98 -

Ba2+, Mn2+, Cd2+ , Ni2+, Zn2+, 1 mg L-1 of each cation 0.100

0.105(2.70)

a: Values in parenthesis are CVs based on three individual replicate measurements.

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Table 4: Results of speciation of Pb in different samples by three columns system.

Water sample (1000 mL)a

River water (50 mL)

Pb(µg)

Pb(µg)

Pb(µg)

Dowex 1X8

-

-

-

Silica C-18

-

-

-

Tap water (1000 mL)

Column

Chelex-100

0.012(4.0)b

0.104(2.9)

0.103(2.8)

a:

This was a solution containing 0.1 ȝg 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.

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 percentages 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 Department of Varamin branch Islamic Azad University for financial support.

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REFERENCES 1. Luoma S.N., Sci. Total Environ, 28 (1983), 1.

2. Hummers W., Offeman R., J. Am. Chem. Soc., 80 (1958), 1339.

3. R.E. Kirk, D.F. Othmer, 1979. Ecyclopedia of Chemical Technology, Vol. 14, 3rd Ed., John Wiley and Sons, New York.

4. L. Parmeggiani, 1983. Encyclopedia of Occupational Health and Safety, Vol. 1, 3rd, International Labor Organization, Geneva.

5. Choi Y.S., Choi H.S., Bull. Korean Chem. Soc., 24 (2003), 222.

6. Zaijan L., Yuling Y., Jian T., Jiaomai P., Talanta, 60 (2003), 123.

7. Kara D., Alkan M., Cakir U., Turk. J. Chem., 25 (2001), 293.

8. Sonawale S.B., Ghalsasi Y.V., Argekar A.P., Anal. Sci., 17 (2001), 285.

9. Diniz M.C.T., Filho O.F., Rohwedder J.J.R., Anal. Chim. Acta, 525 (2004), 281.

10.Manzoori J.L., Bavili-Tabrizi A., Microchem. J., 72 (2002), 1.

11. Chen J., Teo K.C., Anal. Chim. Acta, 450 (2001), 215.

12. Yuan S., Chen W., Hu S., Talanta, 64 (2004), 922.

13. Majors R.E., LC-GC., 4 (1989), 972.

14. Hagen D.F., Markell C.G., Schmitt G.A., Blevins D.D., Anal. Chim. Acta, 236 (1990), 157.

15. Lemos V.A., Baliza P.X., Yamaki R.T., Rocha M.E., Alves A.P.O., Talanta, 61 (2003), 675.

16. Tokman N., Akman S., Ozcan M., Talanta, 59(2003), 201.

17. Ibarra L., Jorda C., J Appl Polym Sci., 48 (3) (1993), 375.

18. Zougagh M., Torres A.G.D., Alonso E.V., Povon J.M.C., Talanta, 62 (2004), 503.

19. Matoso E., Kubota L.T., Cadore S., Talanta, 60 (2003), 1105.

20. Karousis N., Sandanayaka A.S.D., Hasobe T., Economopoulos S.P., Sarantopouloua E., Tagmatarchis N., J. Mater. Chem., 21 (2011), 109.

21. S.MB. March, 2001. J. March's advanced

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Moghimi A et al

organic chemistry: reactions, mechanisms, and structure, New York: John Wiley & Sons Inc. 22. Mermoux M., Chabre Y., Rousseau A., Carbon, 29 (3) (1991), 469.

23. Cataldo F., Fuller Nanotub Car N, 11 (1) (2003), 1.

24. H. Gunzler, H.U. Gremlich, 2002. IR spectroscopy, Winheim: Wiley-VSH. 25. H. Effery, 1991. Chemical Analysis, 5th ed., John Wiley & Sons, Inc., New York.

26. Abollino O., Aceto M., Sarzanini C., Mentasti E., Anal. Chim. Acta, 411 (2000), 233.

27. Bingye D., Meirong C., Guozhen F., Bing L., Xv D., Mingfei P., Shuo W., J. Hazard Mater, 219-220 (2012), 103.

28. Groschner M., Appriou P., Anal. Chim. Acta, 297 (1994), 369.

29. Lewis B.L., Landing W.M., Mar. Chem., 40 (1992), 105.

30. Stankovich S., Piner R.D., Nguyen S.T., Ruoff R.S., Carbon, 44 (2006), 3342.

31. Nayebi P., Moghimi A., Oriental J. Chem., 22 (3) (2006), 507.

32. Moghimi A., Oriental J. Chem., 22 (3) (2006), 527.

33. Dilovic I., Rubcic M., Vrdoljak V., Pavelic S.K., Kralj M., Piantanidab I., and Cindrica M., Bioorg Med Chem., 16 (2008), 518.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

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

ABSTRACT 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 30째C 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.

1. INTRODUCTION 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 (*) Corresponding Author - e-mail: mheydari17m@gmail.com

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

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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. hemolithoautotrophs are major organisms in biomining process that are able to use ferrous iron or reduced inorganic sulfur sources (or both) as electron donors [2]. These organisms are acidophilic and most will grow within the pH range 1.5-2.0. This extreme acidophily applies even to those biomining organisms that can oxidize only iron [4, 5].

Chemolithoautotrophic mesophilic bacteria of genera acidithiobacillus and leptosprillum are the most commonly found leaching organisms.

Acidithiobacillus is the gram-negative rod shape bacteria with length 1-3 and Width 0.5.

Leptosprillum is gram-negative aerobic and spiral shape bacteria that obtained energy requirements from the oxidation of ferrous ions [4, 5]. This study aimed to optimization of gold extracting from sulfide ore Mouteh using native bacteria.

2. MATERIALS AND METHODS The Chemolithoautotrophic mesophilic iron oxidizing 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

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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 ironoxidizing 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 concentration was indirectly determined by atomic absorption spectroscopy analysis of Ba after precipitation of BaSO4 [6, 8]. The chemical composition and particle size distribution of ore was determined prior and after of bioleaching experiments (Table 1).

3. RESULTS After 3-5 days of incubation in 9K and DSMZ882

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Heydari A et al

(a)

(b)

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.

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 13.98

% Al2O3 2.99

% FeS2 78

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

% Na2O 0.99

% K2O

Gold

0.27

ppm

activities were analyzed. The compound microscopic observations of isolated strains of bacteria revealed that these strains were Gram-negative, motile, very small (1-2 Âľm in length), rod shape and

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Heydari A et al

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.

spiral shape bacteria, singles or pairs bacteria. The most frequently observed colonies in 9K agar medium were semi-spheroidal and smoothsurfaced, 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 pHvalue 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.

368

and C.L.L.B. was conducted in shake flasks containing iron liquid medium (9K Fe2+) containing 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 30째C 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

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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.

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 refractory minerals can increase from 15-30% to 85-95% after biooxidation. Currently studies are being carried on for the development of processes for the

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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

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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 (2003), 131.

3. Mukhopadhyay B.P., Ghosh B., Bairagya H.R.,

Heydari A et al

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Afr. J. Biotechnol., 11 (8) (2012), 1991.

4. Salari H., Afzali D., Oliaie M.S., Afr. J. Microbiol. Res., 5 (23) (2011), 3919.

5. Lindstrom E.B., Wold S., Kettaneh-Wold N., Saaf S., Appl Microbiol Biotechnol., 38 (1993), 702.

6. Zilouei H., Shojaosadati S.A., Khalilzadeh R., Nasernejad B., Iran J. Biotech., 1 (2003), 162.

7. Khan S., Haq F., In. J. Biosci., 2 (2012), 85.

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.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

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

ABSTRACT 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 hydrothermal 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.

1. INTRODUCTION 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 (*) Corresponding Author - e-mail: farahmandjou@iauvaramin.ac.ir

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-

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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].

lattice anion

b site cation

anion vacancy

d site cation

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

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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, liquid-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 precipitation, hydrothermal (high temperature hydrolysis), sol-gel (colloidal chemistry), radiation chemical synthesis, and so forth [18, 24].

In this paper, the indium tin oxide nanoparticles are first fabricated by liquid-phase co-precipitation, hydrothermal and pechini sol-gel method and the nanoparticles' structure is then compared by these methods. The morphology of indium tin oxide nanoparticles is studied by scanning electron microscopy and X-ray diffraction.

2. MATERIALS AND METHODS 2.1. Liquid phase co-precipitation synthesis The synthesis of indium tin oxide nanoparticles was carried out by liquid phase co-precipitation as follows. A certain quality of indium chloride (InCl3·4H2O 99%, Aldrich) and tin chloride (SnCl4·5H2O 99%, Aldrich) was dissolved in pure de-ionized water or ethanol, keeping the ratio of In2O3: SnO2 = 9: 1. Certain concentrations (5%) of ammonia solutions were made by mixing certain amount of ammonia (NH3·H2O, 25%) with pure water. The prepared InCl3 solution (0.3 mol/L) was transferred into fixed three-neck flask, keeping in 60°C temperatures under electromagnetic agitation.

The ammonia solution was added to the flask, controlling the stirring speed and testing the pH value till the required pH value was added as dispersant. The precipitate precursor of indium tin oxide was aged a certain time and washed with deionized water and absolute alcohol for three times, respectively. After washing, the precipitates were dried at 110°C for 1 hour. The dried samples

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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

Figure 2: The SEM images ITO nanoparticles prepared by co-precipitation method.

(a)

(b)

Figure 3: SEM images of ITO prepared by (a) hydrothermal and (b) pechini method.

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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 diffraction. The microstructure of the indium tin oxide samples were characterized by a KYKY-Ammray 2800 type SEM with 200 kV acceleration voltages.

To determine the nanoparticles' structure, the X-ray diffraction (XRD) measurement of the samples were performed using a Seifert with Cu-Kα radiation (wavelength = 1.54 Å).

3. RESULTS AND DISCUSSION Figure 2 shows the scanning electron microscopy image of indium tin oxide nanoparticles prepared by liquid phase coprecipitation method in the presence of ammonia solution. The size ITO nanoparticle is about 46 nm after 600°C calcination. As you can see the particles are in good uniformity 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).

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From the width of X-ray diffraction broadening, the mean crystalline size has been calculated using Scherer's equation:

D=

Kλ β cos θ

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

Table 1: The data of lattice parameters for In2O3 and ITO nanoparticles.

Sample name*

Preparation Method

D(nm)

d222(Å)

I222

I400/I222

In2O3

Actual value

----

2.921

----

29.3

ITO

Pechini sol-gel

>100

2.929

14112

28.2

ITO

Hydrothermal

>100

2.923

8653

21.67

ITO

Co-precipitation

46

2.917

8747

29.07

*

376

D=crystallite size, d222 =atomic planar distance of 222

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(d)

(c)

(b)

(a)

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

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 hydrothermal 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 650째C. 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 indicate that the intensity ratio of I400/I222 is increased to 29.07 percent by co-precipitation method.

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 methods. Finally, the preferential growth and orientation of the indium tin oxide prepared by the hydrothermal and pechini sol-gel methods is the <400> orientation.

4. CONCLUSIONS

REFERENCES

In conclusion, indium tin oxide nanoparticles were successfully synthesized by liquid phase co-precipitation, hydrothermal and sol-gel methods. The results indicate that the size of ITO prepared by

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

ACKNOWLELDGMENTS The author is thankful for the financial support of Karaj material and energy research center for analysis and the discussions on the results.

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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.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

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

ABSTRACT 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.

1. INTRODUCTION In an analogous fashion to traditional bulk metallurgy, some properties of bimetallic nanoparticles can be modified by changing their compositions. 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 (*) Corresponding Author - e-mail: prmashayekhi@gmail.com

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 immiscible 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 600째C [1]. The interaction between the two metals plays an important role in the properties of bimetallic nanoparticles. These characteristics are

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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

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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

Figure 1: Schematic of samples preparation using Wet-chemical.

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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 D5000siemens 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).

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)

3. RESULTS AND DISCUSSION 3.1. SEM Characterization The SEM image of 10-50% Cu doped Au nanoparticles is shown in Figure 2. In addition, more uniform and homogeneous distribution of nanoparticles was obtained by doping Cu into the

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.

Table 1: Detailed experimental parameters and dopant amounts for preparation of copper doped with Au nanoparticles.

Concentration Concentration Concentration Doping of of of Morphology CuSO .5H O HAuCl.3H O percentage of ascorbic acid 4 2 2 Cu % (Mol L-1) ( Mol L-1) (Mol L-1)

Surfactant (the type and the Concentration)

Sediment color

Spherical

0.0087

0.05

10%

0.2

Anhydride maleic ( 0.001)

Black

Spherical

0.026

0.05

25%

0.2

Anhydride maleic ( 0.001)

Black

Spherical

0.078

0.05

50%

0.2

Anhydride maleic ( 0.001)

Black

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(a)

(b)

(c) Figure 2: SEM image of the Cu doped Au nanoparticles: (a) 10%, (b) 25% and (c) 50%.

Table 2: Size of Cu doped Au nanoparticles with various doping percent copper at temperature.

%Doping of Cu

Average size of particles for samples

10%

12.6

25%

9.82

50%

5.43

382

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).

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(c)

(b)

(a)

Figure 3: X-ray diffraction patterns of (a) 10% Cu, (b) 25% Cu and (c) 50% Cu.

(a)

(b)

(c)

Figure 4: Optical absorption spectrum of Cu doped Au nanoparticles (a) 10%, (b) 25% and (c) 50%.

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4. CONCLUSIONS Cu doped Au nanoparticles were synthesized using wet-chemical method. We used anhydride maleic as surfactant agent. The formations of the nanoparticles 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., Madhusudhanan U., Chalcogenide Lett., 6 (2009), 197.

9. Wang J., Gao L., Inor. Chem. Com., 6 (2003), 877.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

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

3

Assistant Professor, Department of Food Science and Technology, Islamic Azad University, Varamin-

Agricultural Engineering and Technology, University of Tehran, 31587-11167 Karaj, Iran Pishva Branch, Iran Received: 9 July 2013; Accepted: 16 September 2013

ABSTRACT 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 (25-50째C), time (20-40 min) and frequency (0-130 kHz). In this study thermal process (85째C, 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.

1. INTRODUCTION Carrot juice is one of the most high consuming vegetable juice [1] containing high amounts of A provitamin (such as beta carotene). Therefore, it is used for production of ATBC (alpha tocopherol (*) Corresponding Author - e-mail: s_dolat2003@yahoo.com

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

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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 reduction 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

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0.6-0.7 µ and can live on a broad range of substrates [18]. Quality characteristics of carrot juice (such as pH, total soluble solids, turbidity and antioxidant activity) are also investigated in this study. Moreover the browning index of carrot juice samples is studied by the way of Duncan's multiple rang test.

2. MATERIALS AND METHODS 2.1. Chemicals Analytical grade of Methanol (99.9%), hydroxide sodium, phenolphetalein, 2,2-Diphenyl-1-picrylhydrazyl (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 inoculation 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

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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 spectrophotometer (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.

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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 Re m =

[DPPH ] t [DPPH ] of

control sample

[DPPH] of control sample is the initial concentration 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

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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].

BI =

x=

[100(x − 0.31)] 0.17

(a + 1.75 L ) (5.645 L + a − 3.012b )

2.3. Experimental design and statistical analysis In this study Response Surface Methodology was used to evaluate the effect of ultrasound treatment

Dolatabadi S et. al

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

Table 1: Matrix of the face central composite design (FCCD) and experimental data obtained for the response variables.

Response variable a

Independent variables RUN

Frequency Temprature (°C) (kHz)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 aEscherichia

388

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

Time (min)

1E.C survival

2EC50

pH

3TSS

4Turb

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

coli1 (cfu/mL), Antioxidant activity2 (%), Total soluble solids3 (%), Turbidity 4(NTU).

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independent variables on each response with their coefficients, respectively.

5

5

i =1

i =1

4

Yk = βk 0 + ∑ β kiXi + ∑ β kiiXi 2 + ∑

(K = 1,2,3,...,5)

5

∑ βkijXiXj

i =1 j =i =1

(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

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.

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Figure 2-A: Effects of time, temprature and frequency of ultrasound on pH.

Figure 2-B: Surface plots of effects of time temperature and frequency of ultrasound on pH.

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.

390

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

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Figure 3: Effect of time and ultrasound on browning index

Figure 4: Effect of temperature and ultrasound on

at constant temperature.

browning index at constant time.

Table 2: ANOVA and regression coefficients of the second-order polynomial models for the response variables.

Turb4 (NTU)

TSS3 (%)

EC502 (%)

PH

PV

Coefficient

PV

Coefficient

0.977

83.590

0.467

3.88750

0.286

142.125

0.507

0.15573

0.138

0.704

61.753

0.810

0.06966

0.286

720.865

0.657

0.357

-1.555

0.678

Coefficient

DF

Source

PV

Coefficient

PV

Coefficient

0.669

0.134

0.147

1867640

9

Model

0.084 -0.025716

0.856

9765

1

X1

0.029 -0.08419

0.005

0.060655

0.253

-79273

1

X2

-0.52500

0.007 -0.45500

0.313 -0.072000

0.019

73987

1

X3

0.496

-0.00204

0.222 -0.00044

0.085

-752

1

X1 2

-1.075

0.883

-0.00068

0.020

0.004 -0.000991

0.699

415

1

X2 2

0.096

461.609

0.695

0.18182

0.003 -0.20364

0.116

0.045909

0.290

116509

1

X3 2

0.947

-0.080

0.818

-0.0005

0.783 -0.00007

0.537

0.00008

0.069

995

1

X1 X2

0.439

-9.5

0.818

0.005

0.013

0.00750

0.414

0.002

0.204

-6648

1

X1 X3

0.583

-8.357

0.818

0.00625

0.507

0.00212

0.537

-0.001

0.037

-14735

1

X2 X3

<0.001

-

0.027

-

0.001

-

0.012

0.012

-

-

0.5047

-

0.1578

-

aEscherichia

PV

EC survival1(cfu/mL)

<0.001 7.14125 0.04265

0.00146

0.86

-

0.0003226 0.286

0.7618

-

0.7967

5 Lack-of-fit -

R2

coli1 (cfu/mL), Antioxidant activity2 (%), Total soluble solids3 (%), Turbidity 4(NTU).

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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:

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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 - 1.075X22 + 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 + 0.18182X32 - 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 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 significant 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 - 0.20364X32 + 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

392

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

Dolatabadi S et. al

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 45째C 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].

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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

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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 activity 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 Ă&#x2014; 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 frequencytime 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.

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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.

REFERENCES 1. Marx M., Stuparic, M., Schieber, A., & Carle, R., Food Chem., 83 (2003), 609.

2. Carle R., Fruit Process, 9 (1999), 342.

3. Marx M., Schieber A., & Carle R., Food Chemistry, 70 (2000), 403.

4. Riccioni G., Current Atherosclerosis Reports, 11 (2009), 434.

5. Gupta A.K., Kumar A., and Singh B., J. Food Eng., 79 (2007), 471.

6. C. Gopalan, S.B.V. Rama, S.C. Balasubramanian, 1989. Nutritive Value of Indian Foods- revised edition. National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India.

7. Park S.J., Lee J.I., & Park J., J. Food Sci., 67 (2002), 1827.

8. U.S. FDA, Fed. Reg., 65 (2000), 71056.

9. S.M. Alzamora, A. Lopez-Malo, & M.S. Tapia, Overview. In S. Alzamora, M.S. Tapia, & A. Lopez-Malo, (Eds.), 2000. Minimally processed fruits and vegetables. Fundamental aspects and applications, Maryland, USA: Aspen Publishers, Inc. 10. Cheng L.H., Soh C.Y., Liew S.C., J. Food Chem., 104 (2007), 1396.

11. Seymour I.J., Burfoot D., Smith R.L., Cox L.A., & Lockwood A., Int. J. Food Sci. and Technol., 37 (2002), 547.

12. Lillard H.S.Z., J. Food Prot., 8 (1993), 716.

13. McClements J.D.Z., Trends Food Sci. Tech., 6 (1995), 293.

14. Chaves-Olarte E., Vallant-Barka F., & Wong E., Food Research International.Article in Press, (2010). doi:10.1016/J.Foodres.07.037.

Dolatabadi S et. al

15. Bhat R., Kamaruddin N.S.B.C., Mintze L., & Karim A.A., UltraSon Sonochem., 18 (2011), 1295.

16. Zenker M., Hienz V., & Knorr D., J. Food Prot., 66 (9) (2003), 1642.

17. Lopez P., Vercet A., Sanchez A.C., & Burgos J., Food Rec. Technol., 207 (1998), 249.

18. Vogt R.L., Dippold L., Public Health Rep, 120 (2) (2005), 174.

19. Zhou L., Wang Y., Hu X., Wu J., Liao X., IInnov. Food Sci. Emerg., 70 (2009), 321.

20. Pathanibul P., Taylor T.M., Davidson P.M., Harte F., Int. J. Food Microbiol., 129 (2009), 316.

21. Loning J.M., Horst C., Hoffmann U., Ultrason Sonochem., 9 (2002), 169.

22. Goupy P., Hugues M., Boivin P., & Amiol J.M., J. Sci. Food Agric.,79 (1999), 1625.

23. Cam M., Hisil Y., & Durmaz G., Food Chem., 112 (2009), 721.

24. Wang H.Y., Hu X.S., Chen F., Wu J.H., Zhang Z.H., & Liao X.J., Eur Food Res Technol., 223 (2006), 282.

25. Askari G.R., Emam-Djomeh Z., Mousavi S.M., Drying Technol., 26 (2008), 1362.

26. Maskan M., J. Food Eng., 48 (2001), 177.

27. Box G.E.P., & Wilson K.B., J. Roy. Statist. Soc. Ser. B13, 5 (1951), 1.

28. R.H. Myers, & D.C. Montgomery, 2002.

Response Surface Methodology, New York, NY: John Wiley & Sons Inc. 29. R.H. Myers, and D.C. Montgomery, 1995.

Response Surface Methodology, Process and Product Optimization Using Designed Experiments, 2nd Ed., John Wiley and Sons, New York, NY. 30. Gharibzahedi S.M.T., Mousavi S.M., Hamedi M., Khodaiyan F., & Razavi S.H., Carbohyd Polym., 87 (2) (2012), 1611.

31. Ahmed F.I.K., & Russell C., J. Appl. Bacteriol., 39 (1) (1975), 31.

32. Cameron M., McMaster L.D., and Britz T.J., Ultrason Sonochem., 15 (2008), 960.

33. Garcia M.L., Burgos J., Sanz B., and Ordonez J.A., J. Appl. Bacteriol., 67 (1989), 619.

34.Oyane I., Takeda T., Oda Y., Sakata T., Furuta

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 2 (2013), 385-395

M., Okitsu K., Maeda Y., Nishimura R. Ultrason. Sonochem, 16 (2009), 532.

35. Dimitrios B., Trends Food Sci. Tech., 17 (2006), 505.

36. Rawson A., Tiwari B.k., Patras A., Brunton N., Brennan C., Cullen P.J., Odonnell C., Food Res Int., 99 (2011), 1168.

37. Tiwari B.K., Muthukumarappan K., Odonnell C.P., & Cullen, P.J., Food Sci. Technol., 41 (2008a), 1876.

38. Tiwari B.K., Muthukumarappan K., Odonnell C.P., & Cullen P.J., J. Agr. Food Chem., 56 (2008b), 2423.

39. Korn M., Prim P.M., & de Sousa C.S., Microchem J., 72 (2002), 273.

40. Portenlanger G., and Heusinger H., Carbohyd Res., 232 (1992), 291.

41. Zho L., Zhao G., Chen F., Wang Z., Wu J., and Hu X., Coll. Food Sci. Nutr. Eng., 54 (21) (2006), 8346.

42. Lieu L.N., Le V.v. M., Ultrason Sonochem., 17 (2010), 273.

43. Tiwari B.K., Odonnell C.P., Muthukumara Ppan K., Cullen P.J., Food Bioprocess Tech., 2 (2009), 109.

44. Ugarte R.E., Feng H., Martin S.E., Cadwallader K.R., and Robinson S.J., J. Food Sci.,71 (2006), E102.

45. Dizadji N., Entezar P., Afsar A., WASET, 61 (2012).

46. Sun Y., Ma G., Ye X., Kakuda Y., & Meng R., Ultrason Sonochem., 17 (2010), 654.

47. K.L. Simpson, 1985. Chemical changes in food during processing. In R.T. & J.W. Finley (Eds.), Chemical Changes in Natural Food Pigments: Van Nostrand Reinhold, New York, USA.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

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 3

M.Sc., Nano Center Research, Imam Hussein University, Tehran, Iran

Ph.D. Students, Nano Center Research, Imam Hussein University, Tehran, Iran Received: 12 July 2013; Accepted: 19 September 2013

ABSTRACT 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).

1. INTRODUCTION 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 (*) Corresponding Author - e-mail: hoseiny.inorganic@gmail.com

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

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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 reaction 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 MnFe2O4carbon black (CB) composite powders by a co-precipitation method in alkaline aqueous solutions. MnSO4 was dissolved along with FeCl3 with 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:

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−

d [H 2O2 ] = k MnO2 [≡ MnO2 ]⋅ [H 2O2 ] dt

kobs = k MnO2 [≡ MnO2 ]

(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

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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 characterized 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 heated 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 carried 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.

Figure 1: Proposed mechanism of PVP intractions with metal ions [8].

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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 agglomeration. 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 decomposition is represented by:

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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

(a)

(b)

H2O (aq) â&#x2020;&#x2019; 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).

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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:

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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).

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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. Figure 4: UV spectrum for MnFe2O4

3.4. SEM analysis Analyzing the morphology aspect of the nanoparticles 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

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

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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 zeolite bed (Figures 7, 8 and 9).

Figure 9: SEM of nanocomposite with zeolite support.

Figure 7: SEM of nanocomposite with sodium metasilicate support.

Figure 8: SEM of nanocomposite with porous beds.

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;

Table 1: surface area analysis (BET)-iron manganese oxide nanocaomposites in the presence of sodium meta silicate catalyst support.

Sample in the absence of support in the presence of support

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Relative (p/p0) Total Surface area (m2) Pressure

Specific surface area (m2/g)

Weight (gram)

Adsorb gas

0.107

Nitrogen

0.15

0.5407

5.0531

0.105

Nitrogen

0.5

11.2864

107.4899

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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 increments the amount of evolved oxygen (Figure 12).

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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.

Figure 10: SEM of iron-manganese oxide nanocomposites in the absence of sodium meta silicate catalyst support.

Figure 11: SEM of iron-manganese oxide nanocomposites 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

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 decomposition 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 300째C. 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

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(a)

(b)

Figure 13: TEM image of the iron-manganese oxide nanoparticles (a: pure b: in the presence of sodium metasilicate).

surface area for catalytic activities are reduced. At lower calcination temperature oxidize phases is not formed (Figure 14).

Figure 15: Effect of pH on catalytic activity of hydrogen peroxide decomposition (samples obtained from coprecipitation method).

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.

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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

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Table 2: Feasibility of reusing nanocatalysts.

Sample

Initial weight

Reaction Time

Secondary weight

Reaction Time

Coprecipitation method

0.1(g)

30(s)

0.098(g)

33(s)

Sol-Gel

0.1(g)

150(s)

0.099(g)

155(s)

0.1(g)

12(s)

0.091(g)

13(s)

0.1(g)

120(s)

0.088(g)

123(s)

Silica Support (preferred method) Zeolit 13X

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 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 nanocatalysts indicates that both the primary nanocatalysts and synthesized samples have the catalytic activity in hydrogen peroxide decomposition. The results are summarized in Table 2.

Figure 16: Optimization of support weight in 2g catalyst.

4. CONCLUSIONS

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,

For carrying out the hydrogen peroxide decomposition 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

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variables that were so effective in catalyst synthesis should be considered, and optimized. Conditions of each variable that had a major effect in incrementation 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 300째C. 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 demonstrated the nanocatalysts participated in the reactions as catalysts and not reactants.

REFERENCES 1. Rathsberger K.D., Composition for Catalytic Decomposition of Hydrogen, US Patent 3,884,836.

2. Ahmed M.A., Okasha N., El-Dek S.I., Nanotechnology, 19 (2008), 065603.

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3. Kuo S.L., Wu N.L., J. Power Sources, 162 (2006), 1437.

4. Do S.H., Batchelor B., Lee H.K., Kong S.H., Chemosphere, 75 (2009), 8.

5. Lahirri P., Sengupta S.K., Can. J. Chem., 69 (33) (1991), 33.

6. Deraz N.M., Mater. Lett., 57 (2002), 914.

7. El-Shobaky G.A., Ghozza A.M., Deraz N.M., Mater. Lett., 58 (2004), 699.

8. Goodarz Naseri M., Binsaion E., Abbastabar Ahangar H., J. Magn Magn Mater., 323 (2011), 1745.

9. Toma L.G., Ion R.M., J. Optoelectron ADV M, 12 (10) (2010), 2113.