International Journal of Bio-Inorganic Hybrid Nanomaterials

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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 471-476

ISSN: 2251-8533

International Journal of Bio-Inorganic Hybrid Nanomaterials Improvement in Immobilization of Bread Yeasts by Sol-gel Method Combined with Functionalized Nanoporous Silica (LUS-1) Alireza Badiei1*, Golriz Dadashi2, Hossein Attar3, Nastaran Hayati-Roodbari4 1

Associate Professor, School of Chemistry, College of Science, University of Tehran, Tehran, Iran 2

M.Sc. Student, Department of Chemical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran

3

Associate Professor, Department of Chemical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran

4

Ph.D. Student, School of Chemistry, College of Science, University of Tehran, Tehran, Iran

Received: 27 August 2013; Accepted: 6 November 2013

ABSTRACT In this work, the effect of immobilization of bread yeast (Saccharomyces cerevisiae) by sol-gel technique combined with functionalized nanoporous silica was investigated in different weight ratios of silica containing materials (TMOS: LUS-1). The activities of immobilized yeast in days after immobilization were examined. The results showed immobilization maintain the yeast life for a longer time. The functionalization by C18 functional group improved the environmental conditions for yeast life. These results indicate that the immobilization technique in the gel matrix and porous solid is a good system to develop industrial fermentations. Keyword: Nanoporous silica, Sol-gel, Bread yeast, LUS-1, Fermentation, Immobilization, Mesopore.

1. INTRODUCTION Cells and Enzymes are immobilized by different methods including absorption, covalent linkage, entrapment, cross linking and microencapsulation [1]. Producing ethanol through consuming glucose is one of the PRVW VLJQLÂżFDQW DSSOLFDWLRQV RI \HDVW 'XH WR H[FOXGing yeasts through removing the ethanol, it is needed to stabilize the yeasts leading to decrease the costs of separation steps [2]. Immobilization of cells in a silica (*) Corresponding Author - e-mail: abadiei@khayam.ut.ac.ir.

0DWUL[ VKRZV GLIIHUHQW DGYDQWDJHV LQFOXGLQJ LQFUHDVHG metabolic activity, protection of environmental stresses DQG WR[LFLWLHV LQFUHDVHG SODVPLG VWDELOLW\ DQG DSSOLFDtion as a cellular storage systems in postponement of reactions [3]. Saccharomyces Cerevisiae (SC), a type of yeast bread, was utilized in this research. In order to immobilizing the yeast, entrapping technique by sol-gel method was used. Sol-gel method provides the

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SRVVLELOLW\ RI DSSO\LQJ SRURXV LQRUJDQLF PDWUL[HV KDYLQJ SOHQW\ DGYDQWDJHV UDWKHU WKDQ SRO\PHULF PDWUL[HV Utilizing the sol-gel process commonly is accompaQLHG ZLWK XVLQJ PHWDO DOFR[LGHV 7KH VWHSV RI 6RO JHO process includes solution formation, gelation, drying and agglomeration [4]. Pope and co-workers investigated the immobilization of SC into tetramethylortosilicate (TMOS) gel. One day after of immobilization, the yeast did not show any activity [5]. Fennouh et al immobilized the (VFKHULFKLD FROL EDFWHULD LQVLGH VLOLFD PDWUL[HV E\ XVH of entrapping method [6]. Nassif et al investigated the immobilization of Escherichia coli into TMOS and after two weeks the yeast activity were reduced and DIWHU IRXU ZHHNV LW ZDV GHDFWLYDWHG > @ 'HVLPRQ HW al compared the resistance of free and immobilized 6& LQ H[SRVXUH WR HWKDQRO > @ <X HW DO LPPRELOL]HG 0RUD[HOOD FHOO LQWR D JHO > @ The term “nanoporous materialsâ€? indicates the materials with pore diameters less than 100 nm [10]. LUS-1 is a type of silica with amorphous walls clasVLÂżHG LQ QDQRSRURXV PDWHULDOV 7KH V\QWKHVLV RI WKLV material was reported by Benneviot and Badiei in 2001 at Laval University [11]. Alvaro et al used nanoporous silica to immobilize the Lipas Enzyme [12]. Jang and et al in 2006 immobilized Tripsin Enzyme on SBA-15 (a type of mesoporous silica) with and without functionalized by thiol group [13]. As the immobilization into nanoporous silica leads to protection of yeast from unwanted environmental factors and in other side the functional group on surface of nanoporous material, help to remove the unsuitable materials such as ethanol. In this work, the effect of immobilization of bread yeast (Saccharomyces cerevisiae) by sol-gel technique combined with functionalized nanoporous silica was investigated in different weight ratios of silica containing materials (TMOS: LUS-1). The acWLYLW\ RI LPPRELOL]HG \HDVWV ZHUH H[DPLQHG WKURXJK the measurement of produced CO2 by consumption of glucose in days after immobilization and a sample able to maintain the activity of yeast after one month were determined. In comparison to the other methods which were used before including Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC), this method is more practical and 472

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convenient [15].

2. MATERIALS AND METHODS 2.1. Materials SiO2, cetyl-trimethylammonium bromide (CTMABr), +\GURFKORULF DFLG 6XOIXULF DFLG 7HWUDPHWK\ORUWRVLOLFDWH 7026 7ULFKORURRFWDGHF\OVLODQH (WKDQRO 7ROXHQH <HDVW ([WUDFW %LRFRQQHFWLRQ DQG ' *OXFRVH 0RQRK\GUDWH ZHUH SXUFKDVHG IURP 0HUFN &RPSDQ\ %UHDG <HDVW ZDV SXUFKDVHG IURP Fala Company. P-toluenesulfonicacid monohydrate (TSOH) obtained from Aldrich. 2.1.1. Characterizations

In order to characterize the functional groups on nanoSRURXV PDWHULDOV ,5 VSHFWURPHWHU PRGHO (TXLQR[ 55Bruker were applied. The morphology and shapes of synthesized materials were investigated by SEM GHYLFH PRGHO =HLVV '60 $ N: YROWDJH 3RUH diameter, surface area and adsorption-desorption isotherms were measured at 77 K using a BELSORPminiII porosimeter. BET (Brunauer-Emmett-Teller) HTXDWLRQ ZDV SHUIRUPHG WR FDOFXODWH VSHFL¿F VXUIDFH area and BJH (Barret, Joyner and Halenda). The low angle X-ray scattering spectrum was recorded E\ PRGHO ;œ3HUW 3UR 03' GLIIUDFWRPHWHU P$ N9 DW URRP WHPSHUDWXUH ZLWK &X .Ď UDGLDWLRQ within a range of 2θ of 0.6 - 10 degree. 2.2. Methods 2.2.1. Synthesis of LUS-1

LUS-1 was synthesized according to the literature [16], with a molar ratio of SiO2: 0.054 CTMABr: 762+ +2O. Prepared LUS-1 was washed with a solution of HCl (2 M) and ethanol, with a UDWLR RI IRU KRXUV DQG WKHQ ZDV ÂżOWHUHG RII XQGHU vacuum and dried in an oven overnight. 2.2.2. Functionalization of LUS-1

Acid washed LUS-1 was functionalized with Trichlorooctadecylsilane. 1 g LUS-1 with 30 mL dried ToluHQH DQG P/ 7ULFKORURRFWDGHF\OVLODQH GLOXWHG LQ P/ 7ROXHQH ZHUH UHĂ€X[HG IRU KUV DW ƒ& 7KHQ LW ÂżOWHUHG RII DQG ZDVKHG ZLWK 7ROXHQH > @

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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 471-476

Table 1: Texture properties of samples.

LUS-1

/86 &

/86 & 6F

as (m2/g)

236

Total pore Volume (cm3/g)

0.303

0.112

0.301

'p (nm)

2.4

2.3

2.7

2.2.3. Immobilization of bread yeast on functionalized LUS-1

Immobilization of yeast was performed through solgel method. 5 gel samples were prepared by adding different amounts of functionalized LUS-1 to 22.5 ÂľL RI +&O 0 DQG P/ 7026 LQWR LFH ZDWHU PL[WXUH DQG DGGLQJ J \HDVW H[WUDFW WR P/ ZDWHU 0L[HG PDWHULDOV ZHUH VWLUUHG IRU PLQXWHV DQG OHIW WR URRP WHPSHUDWXUH WR SURGXFH JHO > @ 'XULQJ VWLUring, the Si-O-R bonds were created. Hydrolysis process was catalyzed by HCl. The synthesized samples ZHUH NHSW LQ D UHIULJHUDWRU RQ ƒ& ,Q RUGHU WR LQYHVtigate pore size effect on immobilization of yeast different ratios of different silica materials were utilized. The silica amounts of TMOS were measured and then LUS-1 was added in weight ratios of 1:1, 0.75, 0.5, 0.375 and 0.3125.

Figure 1: Low angel X-Ray Diffraction pattern of the functionalized LUS-1.

was measure every 15 minutes. The reduced weight is equal to produced CO2 [15]. $OVR DERYH H[SHULPHQW ZHUH XWLOL]HG IRU IXQFWLRQDOized LUS-1 and Free yeast.

3. RESULTS AND DISCUSSION 2.2.4. Measurement of immobilized yeast activity

To investigate the remained yeast activity, the amount RI SURGXFHG FDUERQ GLR[LGH UHVXOWHG LQ JOXFRVH FRQsumption by yeasts were measured. The yeasts consume the Glucose through following reaction.

Figure 1 shows the functionalized LUS-1 X-ray diffraction pattern. Three well-known and characteristic ;5' SHDNV DW θ ƒ ƒ ƒ ZKLFK DUH GXH to diffraction peaks of (100), (110), and (200), are at-

Glucose (C6H12O6 ĺ (WKDQRO &2H52+ &22 $ PL[WXUH RI J *OXFRVH DQG J \HDVW H[WUDFW were dissolved in 75 mL deionized water and were DGGHG WR JHO 7ZR P/ (UOHQPH\HU ÀDVNV FRQWDLQing 50 mL H2SO4 0.1 M in one and Gel (feremantor) in another were joint through a tube. The lid of ÀDVNV ZHUH FORVHG DQG VHDOHG ZLWK SDUD¿OP WR LVRODWH the system. The produced CO2 in gel container were H[FOXGHG DQG OHDGHG WR +2SO4 container. Thus the weight in gel container reduced frequently. Its weight

Figure 2: IR Spectrophotometer: (a) LUS-1 (b) functionalized LUS-1.

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Badiei A et al

Figure 3: Producing CO2 from free and immobilized yeasts at days 1, 2, 7, 21 and 31.

WULEXWHG WR KH[DJRQDO 3 PP V\PPHWU\ IRU PHVRSRURXV VWUXFWXUHV > @ ,W FOHDUO\ FRQÂżUPHG WKDW IXQFWLRQDOL]DWLRQ KDYH QRW FKDQJHG WKH KH[DJRQDO VWUXFWXUH of LUS-1. LUS-1, functionalized LUS-1and gel samples (TMOS/LUS-1: 1:0.5) were investigated by nitrogen DGVRUSWLRQ GHVRUSWLRQ DQDO\VLV 'DWD IRU SRUH GLDPeters and surface area are provided on Table 1. Surface area and total pore volume in functionalized LUS-1 in comparison to LUS-1 illustrated a sig474

QLÂżFDQW GHFUHDVH FODULI\LQJ WKDW WKH IXQFWLRQDO JURXS are on surface of silica and in some parts pore blocking happened. In gel sample, surface area and total pore volume in comparison to functionalized one is increased indicating the functionalized LUS were placed inside gel structure and resulted data was attributed to pores of gel. The FTIR spectra (Figure 2) of LUS-1 based mateULDO H[KLELW ZHOO GHÂżQHG SHDNV GXH WR VLOLFD VXSSRUWV including a very strong band at 1110-1010 cm-1 rep-

Badiei A et al

resenting stretching vibration of Si-O-Si, a very broad band in the range of 3700-3200 cm-1 and a strong SHDN LQ WKH UDQJH RI DWWULEXWHG WR VXUIDFH K\GUR[\O JURXSV DQG 6L22 vibrations may be assigned WR WKH EDQGV DW DQG FP-1. Vibratios of H2O physisorbed onto the surface of silica appears at around 1645 cm-1 in spectra of all LUS-1 based material. Functional group, Cetyloctadecylsilane, on surface of LUS-1 was characterized by IR spectrophotometer )LJXUH 7ZR 6LJQLÂżFDQW EDQGV LQ ZDYHQXPEHUV RI FP-1 and 1462 cm-1 are related to stretching vibration of C-H bonds. Vital activity of immobilized yeasts and free yeasts were investigated at 0, 1, 2, 7, 21 days and one month after immobilization (Figure 3). According to different behaviour of gels illustrated in plots, Gels in comparisons to each other show different behaiviours. In all samples by spending time the activity is reduced. 7KH DFWLYLW\ RI IUHH \HDVWV ZDV PD[LPXP LQ ÂżUVW GD\V DQG LW GHFUHDVHG VLJQLÂżFDQWO\ DIWHU WLPH VSHQGLQJ In comparison to free yeasts, this reduction of activLW\ LQ LPPRELOL]HG RQHV ZDV OHVV 'HVFHQGLQJ UDWH RI cell life was followed by a smaller slope. Because of LQWHUQDO DQG H[WHUQDO SHQHWUDWLRQ OLPLWV WKH LPPRELlized yeasts show less reduction of activity. In order to achieve the yeasts, the substra should pass through mass transfer resistances such as boundary layer of ZDWHU DV DQ H[WHUQDO RQH SRURXV PDLQWDLQLQJ ODWWLFH (as an internal one). This fact reduces the amount of \HLOGV LQ ÂżUVW GD\ ZKLFK LV XVXDOO\ LQ LWV PD[LPXP level). Because of environmental effects and inappropriate conditions, the number of yeast was decreased. 7KH WUDSSHG FHOOV LQ 6RO JHO PDWUL[ VKRZ EHWWHU \LHOG Since Ethanol interefer with Fermentation ability

Figure 4: SEM image of the functionalized LUS-1.

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 471-476

growth rate of SC and compete to Glucose transfer which lead to slow and incomplete fermentation. As LUS-1 possess outstanding chemical, thermal and mechanical stability is able to act as a microprotective environment leading to avoid ethanols of prohibiting on yeast activity. According to the plots, the gel with TMOS: LUS-1 ratio of 1:0.5 provides the best conditions for yeast’s OLIH $OWKRXJK DW ¿UVW GD\V DOO \HDVWV VKRZ VDPH DFWLYLties, 21 days after immobilization the gel with 1:0.5 ratio maintains life and activity of yeasts for more time rather than the other ratios. So this ratio of TMSO and LUS-1 is selected as an optimum ratio. By comparing immobilized yeast’s activity on functionalized LUS-1 and LUS-1, the activity of yeasts on functionalized LUS-1 was more protected. The morphology of LUS-1 in Figure 5 shows its bush-like structure. By functionalizing LUS-1, the functional groups are placed into pores and LUS-1 scaffold. Since the diameters of yeasts are greater than pores, the yeasts were trapped into LUS-1 scaffold and immobilized. By consuming Glucose, CO2 and H2O are produced. CO2 LV H[FOXGHG WKURXJK SRUHV RI /86 and remained H2O improves the yeasts lifetime. Bond between functional groups and silica increase the hydrophobic, because the hydrophobic molecules are nonpolar and show trend to similar molecules. In other side, H2O molecules create hydrogen bonding and increase the moisture of gel. Nonpolar molecules, like CO2 DUH QRW DEOH WR FUHDWH ERQGV DQG H[FOXGH WKURXJK WKH SRUHV WKXV WKH WR[LFLW\ IRU \HDVWVœ OLIH DUH UHGXFHG

4. CONCLUSIONS The effect of immobilization of bread yeast (Saccharomyces cerevisiae) by sol-gel technique combined with functionalized nanoporous silica was investigated in different weight ratios of silica containing materials (TMOS: LUS-1). The activities of immobilized yeast LQ GD\V DIWHU LPPRELOL]DWLRQ ZHUH H[DPLQHG 7KH UHsults showed immobilization maintain the yeast life IRU D ORQJHU WLPH 7KH IXQFWLRQDOL]DWLRQ E\ & IXQFtional group improved the environmental conditions for yeast life. These results indicate that the immobili]DWLRQ WHFKQLTXH LQ WKH JHO PDWUL[ DQG SRURXV VROLG LV D 475

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good system to develop industrial fermentations. The HDV\ VHSDUDWLRQ RI WKH ÂżQDO SURGXFW DQG WKH ELRFDWDO\VW UHXWLOL]DWLRQ ZDV VLJQLÂżFDQW UHVXOWV

REFERENCES * %LFNHUVWDII HW DO Immobilization of enzymes and cells, Humana Press. & -HIIUH\ %ULQNHU * : 6FKHUHV The physics and chemistry of sol-gel processing, Academic Press Limited. :DQJ / :DQJ . 6DQWUD 6 HW DO Anal. Chem., 78 (3) 2006, 646. 6 :DWWRQ HW DO Progress in inorganic chemistry: coordination complexes in Sol-Gel silica materials -RKQ :LOH\ 6RQV 5. Pope E. et al., J. Sol-Gel Science & Tech., 4 225. )HQQRXK 6 *X\RQ 6 /LYDJH - DQG 5RX[ & J. Sol-Gel Science & Tech., 19 (2000), 647. 7. Nassif N. et al., Nat. Mater., 1 (2002), 42.

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'HVLPRQH 0 'HJURVVL - $TXLQR 0 'LD] / Biotechnol. Lett., 24 (2002), 1557. 6LQJ . 6 : HW DO Pure Appl. Chem., 57 603. ' <X - 9ROSRQL 6 &KKDEUD & - %ULQNHU $ Mulchandani, A.K. Singh, Biosens. Bioelectron, 20 (7) 2005, 1433. 11. Bonneviot M.M.L., Badiei A., Patent :2 01/55031 A1, 2001. 12. A. Mayoral, R. Arenal, V. Gascon, C.M. Alvarez, 5 0 %ODQFR , 'LD] Chem. Cat Chem., 5 (4) -DQJ 6 .LP ' 5RZ - & $KQ : J. Porous Mater, 13 < 0D / 4L - 0D < :X 2 /LX + &KHQJ Physicochem. Eng. Aspects, 229 (2003), 1. 9XOOR ' :DFKVPDQ 0 JFSE, 4 (2005). 16. Bonneviot L., Morin M., Badiei A., US Patent =KDR ' HW DO Chem. Mate., 12 (2) (2000), 275. <DPDGD 7 HW DO Adv. Mater., 15 (6) (2003), 511.

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ISSN: 2251-8533

International Journal of Bio-Inorganic Hybrid Nanomaterials Gradual Growth of Gold Nanoseeds on Silica for Silica@Gold Core-Shell Nanoparticles and Investigation of Optical Properties 0R]KJDQ .D]HP]DGHK 2WRXÂż1*, Nasser Shahtahmasebebi2, Ahmad Kompany2, Elaheh Kafshdargoharshadi3 1

M.Sc., Department of Physics, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran & Centre of Nanoresearch, Ferdowsi University of Mashhad, Mashhad, Iran

2

Professor, Department of Physics, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran & Centre of Nanoresearch, Ferdowsi University of Mashhad, Mashhad, Iran

3

Professor, Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran &Centre of Nanoresearch, Ferdowsi University of Mashhad, Mashhad, Iran

Received: 31 August 2013; Accepted: 3 November 2013

ABSTRACT Metal nanoshells consists of a dielectric core surrounded by a thin noble metal shell, possess unique optical properties that render nanoshells attractive for use in different technologies. This paper reports a facile method for growth of small gold nanoparticles on the functionalized surface of larger silica nanoparticles. Mono-dispersed silica particles and gold nanoparticles were prepared by the chemical reduction method. The size of the shell nanoseeds could be altered by repeating the stage of reducing HAuCl4 on Au/APTES/silica particles, and the time for which they react. The nanocore-shell particles prepared were studied using scanning electron microscopy (TEM), UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR) and PL spectrophotometer. The TEM images indicated that by growing gold nano-seeds over the silica cores a red shift in the maximum absorbance RI 89 9LVLEOH VSHFWURVFRS\ LV REVHUYHG )XUWKHUPRUH D UHPDUNDEOH LQWHQVL¿FDWLRQ KDSSHQV LQ WKH 3/ VSHFWUD RI silica@Au NPs in comparison with that of bare silica NPs. But, the existence of gold nanoseeds on the silica particles surfaces does not change the PL spectra peaks of these nanoparticles. Keyword: Core-shell; Silica; Gold; Nanoparticle; Surface functionalized; Initial growth.

1. INTRODUCTION Much recent research has focused on the fabrication of new types of nanoparticles, particularly those with optical and electrical properties that can be controlled with precision. There is increasing interest in the &RUUHVSRQGLQJ $XWKRU H PDLO RWRXÂż #JPDLO FRP

design and synthesis of topological structures composed of monocrystals of various size and shape. Such materials may have unusual optical properties as a UHVXOW RI LQFUHDVLQJ WRSRORJLFDO FRPSOH[LW\ &RUH

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VKHOO VWUXFWXU DUH RI FHQWUDO LQWHUHVW LQ WKLV FRQWH[W $V noble metals have received particular attention,because of the stability and the ease of preparation of Nanoparticles derived from, Hal and co-workers have recently reported a new hybrid nanoparticle system that consists of a dielectric core surrounded by a thin noble-metal shell [1-3]. These nanoparticles, termed “nanoshellsâ€?, possess unique optical properties, including a strong optical absorbance and a large third-order nonlinear optical susceptibility [4]. More importantly, the absorbance can be selectively tuned to any wavelength across the visible and infrared regions of the spectrum simply by adjusting the ratio of the dielectric core to the thickness of the metal overlayer. These features render nanoshells attractive for use in technologies rangingfromconducting polymer devices to biosensing and drug delivery [5-7]. At present, the most versatile nanoshell system is bed on the coating of silica nanoparticles with a thin OD\HU RI JROG :H FKRVH VLOLFD QDQRSDUWLFOHV DV WKH dielectric cores not only because methods for the functionalization of the surface of silica are well-known, but also because colloidal silica particles can be prepared with reproducibly spherical shapes and narrow size GLVWULEXWLRQV > @ To prepare these gold nanoshells,a silica QDQRSDUWLFOH FRUH LV ÂżUVW WUHDWHG ZLWK DQ DPLQH WHUminated surface silanizing agent (e.g., 3-aminoproS\OWULHWKR[\VLODQH $37(6 $V WKH JROG PHWDO KDV YHU\ OLWWOH DIÂżQLW\ IRU VLOLFD D VLODQH FRXSOLQJ DJHQW is used as the surface primer. The interaction between the amines and the negatively charged THPC gold nanoparticles might be electrostatic rather than coorGLQDWLYH LQ QDWXUH > @ 7KHUHIRUH WKH UHVXOWDQW WHUPLQDO amine groups act attachment points for small colloidal gold particles, which then serve nucleation sites for WKH FRDOHVFHQFH RI WKH WKLQ JROG RYHUOD\HU > @ Of all possible strategies [10], the reduction of FKORURDXULF DFLG ZLWK WHWUDNLV K\GUR[\O PHWK\O

phosphoniumchloride (THPC) affords relatively small gold particles (e.g., 2 nm) with a net negative LQWHUIDFLDO FKDUJH > @ :KLOH WKHVH VPDOO FROORLGDO particles can attach to APTMS-functionalized silica cores by coordinating to the lone pairs of the terminal amine groups, the attachment can be enhanced

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perhaps several fold by electrostatic effects, where in the negatively charged THPC gold nanoparticles are attracted to the amine groups, which are positively charged at the pH used for the attachment process. This strategy leads to silica nanoparticles in which 25% of the surface is covered by colloidal gold particles that can be used to nucleate the growth of the gold overlayer. In this work, we describe the preparation of gold nanoshellsby the chemical reduction method and characterize the nanoshells by using transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, ultraviolet visible (UV-Vis) spectroscopy and photo luminescence (PL) spectroscopy.

2. EXPERIMENTAL 2.1. Materials All reagents were purchased from the indicated supSOLHUV DQG XVHG ZLWKRXW IXUWKHU SXULÂżFDWLRQ WHWUDHWK\ORUWKRVLOLFDWH WHWUDNLV K\GUR[\PHWK\O SKRVSKRQLXP FKORULGH DPLQRSURS\OWULHWKR[\VLODQH VRGLXP K\GUR[LGH DPPRQLXP K\GUR[LGH DQG IRUPDOGHK\GH hydrogen tetrachloroaurate (III) (all from Merch Co.). Similarly, all solvents were received from the indicated suppliers: HPLC grade water, and absolute ethanol (Merch Co.). 2.2.Characterization methods 7R FROOHFW WKH 7(0 LPDJHV ZH XVHG D /(2 AB electron microscope operating at a bi voltage of 200 kV. Sample preparation involved deposition of the nanoparticles dispersed in water onto a 200 mesh copper grid. The grid was then set aside to allow for evaporation of any residual water before analysis. The FTIR data were collected using an AVA-TAR370-FTIR THERMONICOLET spectrometer using two separate procedures. The sample was impacted into a tablet shape and put onto a polished silicon wafer before analysis. UV-Vis spectra were collected XVLQJ D 89' /$%20(' 89 9LVLEOH VSHFtrometer over the range from 400 to 1100 nm. All samples were dispersed in water into a quartz cell for analysis.

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2.3. Preparation of silica nanoparticles An aliquot (3.0 mL) of ammonia (30% NH3 NH4OH say) was added to 50.0 mL of absolute ethanol. The PL[WXUH ZDV VWLUUHG YLJRURXVO\ DQG D VXEVHTXHQW aliquot (1.5 mL, 6.7 mmol) of Si(OC2H5)4 (tetraethyl orthosilicate, TEOS) was added dropwise. Previous studies have shown that there is usually a concentrationdependent induction period required to form the SiO2 nucleus from the TEOS monomer. For the concentrations employed here, the induction SHULRG ZDV DSSUR[LPDWHO\ KRXU MXGJHG E\ WKH change of the solution from clear to opaque white. On the basis of previous work from our laboratories, the concentration of the resultant silica nanoparticles was 7Ă—1012 particles / mL. Analysis by TEM indicated that the silica nanoparticles were spherical in shape with 115 nm diameters. 2.4. Functionalization ofsilica nanoparticlesurfaces with APTES The silica nanoparticles were then surface functionalized by grafting them with 12 mM APTES in volume ratio of 3:7 under constant heating and vigorous VWLUULQJ DW ƒ& IRU K WR JLYH D WHUPLQDO DPLQH group on their surface. Under this condition, the $37(6 XVHG ZDV LQ PRODU H[FHVV WR DFKLHYH D FRPplete surface functionalization. The amine grafted silica particles were then cooled to room temperature and washed with at least 2 cycles of centrifugation and redispersion in absolute ethanol and distilled water at 10,000 rpm for 15 min each to remove residual reactants before resuspending them in 1 mL of water for every 0.3 g of silica used for surface functionalization with amine. 2.5. Preparation of colloidal gold nanoparticles To a 45 mL aliquot of HPLC grade water was added 0.5 mL of 1 M NaOH and 1 mL of THPC solution (prepared by adding 12 Âľ/ PPRO RI THPC in water to 1 mL of HPLC grade water). The UHDFWLRQ PL[WXUH ZDV VWLUUHG IRU PLQ ZLWK D VWURQJ YRUWH[ LQ WKH UHDFWLRQ Ă€DVN $IWHU WKH DOORWWHG WLPH P/ (27 mmol) of HAuCl4 1% in water was added quickly to the stirred solution, which was stirred further for 30 min. The color of the solution changed very quickly from colorless to dark reddish yellow (Figure 1),

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Figure 1: THPC gold nanoparticles solution

which we call “THPC gold nanoparticlesâ€?. Although the size of the THPC gold nanoparticles can be varied, our gold seeds were consistently 2-3 nm in diameter. The solution of THPC gold seeds was stored in the UHIULJHUDWRU IRU DW OHDVW GD\V EHIRUH IXUWKHU XVH 'ULHG samples of the gold nanoparticles were dark brown in color. The particles were near the detection limit of our TEM. 2.6. Attachment of colloidal gold nanoparticles to APTMS functionalized silica cores An aliquot of APTES-functionalized silica nanoparticles dispersed in ethanol (6.7 mL, 2.4Ă—1013 particles/ mL) was placed in a centrifuge tube along with an H[FHVV RI JROG QDQRSDUWLFOHV P/ RI JROG FROORLG solution, 3.5Ă—1014 particles/mL). The centrifuge tube was shaken gently for a couple of minutes and then DOORZHG WR VLW IRU K 7KH PL[WXUH ZDV WKHQ FHQWUL fuged at 2000 revolutions/min, and a red-colored pellet was observed to settle to the bottom of the tube. After drying, a red-colored pellet was left, which was redispersed and sonicated in HPLC grade water. The SXULÂżHG $X $37(6 VLOLFD QDQRSDUWLFOHV ZHUH WKHQ redispersed in 5 mL of HPLC grade water and used described in the following subsection (Figure 2 b). 2.7. Growth of gold nanoshells To grow the gold overlayer on the Au/APTES/siliFD QDQRSDUWLFOHV ZH ÂżUVW KDG WR SUHSDUH D VXLWDEOH solution containing a reducible gold salt. In a reacWLRQ Ă€DVN ZH GLVVROYHG PJ PPRO RI SRWDVsium carbonate (K2CO3) in 100 mL of HPLC grade water. After 10 min of stirring, 1.5 mL (20 mmol) of a

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solution of 1% HAuCl4 in water was added. The solution initially appeared transparent yellow and slowly became colorless over the course of 30 min. To a vigorously stirred 4 mL aliquot of the colorless solution, we injected 200 ÂľL of the solution containing the Au/APTES/silica nanoparticles. :H WKHQ DGGHG D ÂľL (0.36 mmol) aliquot of formaldehyde. Over the course of 2-4 min, the solution changed from colorless to blue, which is characteristic of nanoshell formation. The nanoshells were centrifuged and re-dispersed in HPLC grade water until use.

3. RESULTS AND DISCUSSION 3.1. Imaging by TEM $V GHVFULEHG DERYH ZH ÂżUVW SUHSDUHG PRQRGLVSHUVH spherical silica nanoparticleswith a size of about 120 nm, and then attached small colloidal particles of gold to APTES-functionalized silica nanoparticles cores and then used the attached gold particles to template the growth of a gold overlayer. NH3 is the most effective parameter in thespherical shape of silica nanoparticles [11]. By increasing theconcentration of TEOS and H2O, the size of the nanoparticles increases. It FRXOG EH GXH WR WKHLU LQĂ€XHQFH LQ LQFUHDVLQJ WKHUate of condensation and hydrolysis reactions [12]. Figure 2 shows TEM images of the different stages of our synthesis of gold nanoshells produced using this strategy. Figure 2(a) displays single silica

(a)

(b)

.D]HP]DGHK 2WRXÂż 0 HW DO

nanoparticles. Figure 2(b) shows the desirable arrangement of the small THPC gold nanoparticles with a narrow size of <3 on functionalized silica cores. This improves our success in properly functionalizing silica particles with a layer of bifunctional APTES molecules, and also in preparing THPC gold nanoparticles in narrow size of <3 nm and good aged. Figure 2(c) demonstrates growth of monodispersed gold seedsto a narrow size of <10 nm and thus, a homogeneous shell would be obtained by repeating the last process of reducing HAuCl4 on SiO2@Au nanoparticles as seen in Figure 2(a-c). Therefore, we also did this last part of coating for one time more on the nanoparticles of Figure 2(c), whichhas shown as the resultant shell growth in Figure 2(d). Thus as it is seen, the used method in this paper for the fabrication of SiO2@Au core/shell nanoparticles has some EHQHÂżWV VXFK DV IDFLOH URXWH WKH VDPH DQG VSKHULcal size for silica nanoparticles and also uniform attachment of colloidal gold nanoparticles to APTESfunctionalized silica cores. 3.2. XRD analysis In order to indicate identity of the particles, X-ray difIUDFWLRQ ;5' DQDO\VLV ZDV SHUIRUPHG 7KH ;5' pattern of the resultant nanoparticles (corresponding WR )LJXUH G

VKRZHG LQ )LJXUH H[KLELWHG FKDUDFWHULVWLF UHĂ€HFWLRQV RI IFF JROG -&3'6 1R 7KH GLIIUDFWLRQ IHDWXUHV DSSHDULQJ DW Č™ ƒ ƒ DQG ƒ ZKLFK UHVSHFWLYHO\ FRUUHVSRQGV WR the (111), (200) and (220) planes of the standard cubic phase of Au.

(c)

(d)

Figure 2: TEM images of different stages of preparing gold shell, (a) bare silica nanoparticles, (b) small colloidal THPC JROG QDQRSDUWLFOHV RQ $37(6 VLOLFD QDQRSDUWLFOHV F ÂżUVW VWDJH RI JURZWK RI JROG VHHGV RQ WKH FRUHV DQG G VHFRQG VWDJH of (the resultant) shell growth.

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ous SiO2 peaks, Si–OH bonding and peaks due to the residual organic group’s one. After coating these particles with gold, the intensity of Si–O–Si and 6L¹2+ SHDNV KDYH EHHQ UHGXFHG VLJQL¿FDQWO\ 7KLV indicates the presence of gold seeds on silica particles.

Figure 3: XRD spectra of the resultant silica-gold nanoparticles.

3.3. FTIR analysis The structure study, i.e.bonding of these core-shell particles was also done using Fourier transform infrared spectroscopy (FTIR). The results are shown in Figure 4. The Figure shows the FTIR spectrum of silica, functionalized silica and silica-gold core-shell particles. It indicates an intense characteristic absorption band between 3300 and 3500 cm-1 assigned to O–H stretching in H-bonded water. Also this band can be cross checked through the 1635 cm-1 band due to the scissor bending vibration of molecular water. For SiO2 and SiO2@Au particles peak 1105 cm-1 can be signed WR DV\PPHWULF YLEUDWLRQ RI 6L¹2 ERQG SHDN FP-1 FDQ EH DWWULEXWHG WR 6L¹2+ ERQG DQG SHDN FP-1 relates to symmetric vibration of Si–O. For functionalized silica particles peaks at 3215 cm-1 is signed to NH bond. 7KH DEVRUSWLRQ EDQGV EHWZHHQ DQG FP–1 have been described as a superimposition of vari-

3.4. UV-Vis analysis Optical absorption was recorded at various stages of addition of gold nanoparticles in every method using UV–Visible absorption spectroscopy. Optical absorpWLRQ RI SXUH JROG VROXWLRQ ZDV IRXQG DW QP VKRZQ in Figure 5(a). The singly attached Au NPs have a similar spectrum shape to that of free gold nanoparticles

(a)

(b) Figure 5: (a) UV-Vis spectra of attached THPC gold NPs/ APTES/silica has no noticeable plasmon peak shift in comparison with THPC gold pure NPs, (b) By growing gold nanoseeds on the silica cores, in the stage of gold nanoseed Figure 4: FTIR spectra of silica, silica@THPC attached Au

growing to obtain a complete gold nanoshell, a red shift in

and the resultant silica@Au particles prepared.

the maximum absorbance is observed.

,QW - %LR ,QRUJ +\EG 1DQRPDW 9RO 1R

DW DSSURSULDWH FRQFHQWUDWLRQ DQG OLNH WKDW LV ÀDW LQ the plasmon resonance region. In contrast, the absorption spectrum for gold nanoparticles attached in clusters to silica nanoparticles shows an enhanced absorption in the plasmon resonance region. This result is interpreted as a collective effect of the gold nanoparticles in the cluster which would indicate the presence of gold nanoparticle clusters on the silica nanoparticles and the effect of plasmon-plasmon interactions on the absorption of the group of gold nanoclusters on a VLOLFD FRUH KDV DSSHDUHG > @ :H KDYH VHHQ IURP the UV–Visible spectra of silica-gold core-shell particles that after two coatings (in SiO2@Au1 and SiO2@ Au2) the Plasmon peak demonstrated more spreading and red shift from 622 to 662 nm respectively (Figure 5(b)). So it reveals that as more gold chloride has reduced on the attached gold particles and the particles has begun to grow and merge, Their aspect ratio has increased and this has leaded to a red shift of the abVRUSWLRQ PD[LPXP 7KH PHFKDQLVP IRU WKH VSUHDGLQJ FRXOG EH UHODWHG WR WKH H[WLQFWLRQ FURVV VHFWLRQ DQG electron mean free path in the metal shell [15].

.D]HP]DGHK 2WRXÂż 0 HW DO

4. CONCLUSIONS Silica@gold core-shell particles were synthesized by reducing gold chloride on THPC attached silica nanoparticle cores for several stages. The morphology of these particles was also studied using TEM. TEM images demonstrated the growth of monodispersed gold seeds in narrow sizes up to 10 nm and making a whole shell by their linkage. Therefore, a uniform shell was obtained by repeating the last process of reducing HAuCl4 on these particles on the nanometer scale. UV–Vis absorption spectroscopy shows a red VKLIW IURP WR QP ,W LQGLFDWHV WKDW WKH 3ODVmon resonance peak position of gold depends upon the sizes of gold shell seeds. Therefore, by changing the sizes of gold seeds on core surfaces and thus by changing shell thicknesses; it is possible to design a material with desired optical properties.The SUHVHQFH RI JROG FRDWLQJ ZDV FRQ¿UPHG E\ )7,5 spectroscopy.

ACKNOWLEDGEMENTS 3.5. PL analysis Figure 6 shows the PL emission spectra under 540 QP H[FLWDWLRQ ZDYHOHQJWK RI WKH VLOLFD 13V DQG silica@Au NPs. The silica@Au NPs prepared by two methods in water display one strong emisVLRQ EDQG DW QP $V WKLV )LJXUH VKRZV WKH H[LVWHQFH RI JROG QDQRVHHGV RQ VLOLFD QDQRSDUWLFOHV does not shift the emission peak position.

Figure 6: PL spectra of silica and silia@Au in comparison with each other.

7KH DXWKRUV H[SUHVV WKHLU JUDWLWXGH WR )HUGRZVL 8QLYHUVLW\ RI 0DVKKDG IRU VXSSRUW RI WKLV SURMHFW

REFERENCES :DQJ 0 + +X - : /L < - <HXQJ ( 6 Nanotechnology, 21 2. Huang F., Baumberg J.J., Nano Lett., 10 (2010), +D\QHV & / 'X\QH 5 3 9 J. Phys. Chem. B, 105 3 & +LHPLQ] Principles of Colloid and Surface Chemistry 0DUFHO 'HNNHU 1HZ <RUN +DOH * ' -DFNVRQ - % 6KPDNRYD 2 ( /HH T.R., Hal N.J., Appl. Phys. Lett., 78 (2001), 1502. :HVW - / +DO 1 - Curr. Opin. Biotechnol, 11 (2000), 215. 6HUVKHQ 6 5 :HVWFRWW 6 / +DO 1 - :HVW - / J. Biomed. Mater. Res., 51 .DQGSDO ' .DOHOH 6 .XONDUQO 6 . Pramana J. Phys., 69 (2007), 277.

.D]HP]DGHK 2WRX¿ 0 HW DO

$ 8OPDQ An Introduction to Ultrathin Organic Films $FDGHPLF 1HZ <RUN 6RXQGHU\D 1 =KDQJ < Recent. Patents. Biomed. Eng., 1 6WREHU : )LQN $ J. Colloid Interface Sc., 26 9DQGHUNRR\ $ &KHQ < *RQ]DJD ) %URRN 0 $ ACS Appl. Mater. Interfaces, 3

,QW - %LR ,QRUJ +\EG 1DQRPDW 9RO 1R

.LP - + &KXQJ + : /HH 7 5 Chem. Mater, 18 (2006), 4115. /X + & 7VDL , 6 /LQ < + JPCS, 188 102. 15. Park J., Estrada A., Sharp K., Sang K., Schwartz - $ 6PLWK ' . &ROHPDQ & 3D\QH - ' .RUJHO % $ 'XQQ $ . Opt. Express, 16

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 485-490

ISSN: 2251-8533

International Journal of Bio-Inorganic Hybrid Nanomaterials Evaluation of Casein and Inulin Effects on Droplet Size and pH of Nano-emulsion, Morphology and Structure of Microcapsules of Fish Oil Mahnaz Hashemiravan1*, Maryam Saberi2, Nazanin Farhadyar3 1

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

2

M.Sc., Department of Food Science and Technology, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran

3

Assistant Professor, Department of Chemistry, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran 5HFHLYHG 6HSWHPEHU $FFHSWHG 1RYHPEHU

ABSTRACT 9DULRXV FRPSRXQGV KDYH EHHQ XVHG IRU PLFURHQFDSVXODWLQJ RI ÂżVK RLOVR IDU EXW LQ WKLVZRUN IRU WKH ÂżUVW WLPH LQXOLQ DQG FDVHLQ ZKLFK DUH ERWK NQRZQ IRU WKHLU IXQFWLRQDO SURSHUWLHV ZHUH XVHG DV WKH FRDWLQJ PDWHULDORI ÂżVK RLO 0LFURHQFDSVXODWLRQ RI ÂżVK RLO ZDV GRQH LQ VWHSV )LUVW WKH ZDOO ZLWK GLIIHUHQW FRQFHQWUDWLRQV RI LQXOLQ DQG FDVHLQ ZHUH PL[HG ZLWK GLVWLOOHG water. In the next step, an ultrasound generator was usedwith an intensity of 24 kHz for 120 seconds to prepare nanoHPXOVLRQV DQG ÂżQDOO\ WKH HPXOVLRQV ZHUH FRQYHUWHG LQWR SRZGHU LQ D ODE VFDOH VSUD\ GU\HU 7KH HPXOVLRQ S+ ZDV measured and emulsion droplet size was examined by a particle size analyzer. The microstructures of the powders were analyzed by scanning electron microscopy (SEM). The results showed that the type and concentration of the compounds used as the wallare effective on the properties of nano-emulsion. Comparing the two compounds and their concentrations GHPRQVWUDWHG WKDW FDVHLQKDV PRUH GHVLUDEOH SURSHUWLHV DV LI WKH ORZHVW VL]H RI QP FRUUHVSRQGV WR WKH WUHDWPHQW ZLWK FDVHLQ 0RUHRYHU D VLJQLÂżFDQW QHJDWLYH FRUUHODWLRQ ZDV REVHUYHG EHWZHHQ WKH VL]H DQG S+ RI WKH QDQR VL]H HPXOVLRQ RI ÂżVK RLO 7KH FRPSDULVRQ EHWZHHQ Z Z DQG Z Z ÂżVK RLO VKRZHG WKDW E\LQFUHDVLQJWKH UDWLR RI FRUH WR ZDOO IURP WR WKH VL]H RI QDQR HPXOVLRQ VLJQLÂżFDQWO\ UHGXFHG S 0LFURHQFDSVXODWHGSDUWLFOHV FRQWDLQLQJ higher concentrations of casein showed much less wrinkle and depression compared to the samples containing higher concentrations of inulin, because of the smaller size of the nano-emulsions containing higher concentrations of casein. ,Q WKLV VWXG\ WKH HPXOVLRQ GURSOHW VL]H ZDVDW WKH QDQR VFDOH DQG WKH LPDJHV VKRZHG WKH VLJQLÂżFDQFH RI ZDOO PDWHULDO properties and their concentrationsaffecting droplet size and morphology ofmicrocapsules.

Keyword: Casein; Inulin; Fish oil; Nano-emulsion; Scanning electron microscope.

1. INTRODUCTION Fish oil is inherently functional and possesses many components that are good for human health. There is DQ LQWHUHVW LQ LQFUHDVLQJ WKH DPRXQW RI ÂżVK RLO LQ WKH diet due to the many associated health of omega-3 (*) Corresponding Author - e-mail: m_hashemiravan@yahoo.com.

fatty acids [1]. Encapsulation has been used to mask unpleasant taste in food sciences as well as to protect DJDLQVW OLJKW DQG DLUERUQH R[LGDWLRQ > @ ,W LV D FRDWLQJ technology, small solid particle, liquid or gaseous as

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 485-490

core materials are packaged within wall materials to form microcapsules [3]. 6HYHUDOVKHOO RU PDWUL[ PDWHULDOV KDYH EHHQ WHVWHG IRU ÂżVK RLO HQFDSVXODWLRQ .RODQRZVNL HW DO XVHG PRGLÂżHG FHOOXORVH DV ZDOO PDWHULDO RWKHU ZDOO PDWHULals studied were Pectin, Sodium alginate and chitosan 'LD] 5RMDV HW DO VR\EHDQ SURWHLQLVRODWH &KR HW DO PDOWRGH[WULQFRPELQHG ZLWK PRGLÂżHG starch and whey protein were used as wall material by -DIDUL HW DO > @ ,Q WKLV VWXG\ ÂżVK RLO ZDV XVHG DV WKH FRUH PDWHULDO and Casein combined with Inulin were used as the wall material. A casein micelle is, in effect, a natural QDQR GHOLYHU\ V\VWHP > @ ,QXOLQ LV D 1RQ VWDUFKpolysaccharides consisting of a chain of fructose molecules. It is a polymer of β Äş OLQNHG ' IUXFWRVH units, constituting chains of different lengths each of them with a terminal glucose unit [10]. 7KH DLP RI WKLV VWXG\ ZDV WR DVVHVV WKH LQĂ€XHQFH RI two different type of wall material (Casein and Inulin) on microcapsule morphology and properties of nanoemulsion.

2. MATERIALS AND METHODS 2.1. Materials ,Q WKLV VWXG\ ÂżVK RLO +,'+$ 1 1X PHJD LQJUHGLents, Brisbane, Australia) was used as the core material (Ď NJ P3, Ρ PSD V DW ƒ& 5, 7KH wall material was Casein and Inulin. Casein (Casein soluble in alkali with bulk density 450 kg/m3 and soluELOLW\ J / ƒ&

ZDV SURYLGHG E\ 0HUFN &RPpany and Inulin was obtained from Sigma Chemical &R 6W /RXLVH 0R 86$ 7KH HPXOVLÂżHU XVHG ZDV 3RO\VRUEDWH 7ZHHQ WKDW ZDV VXSSOLHG E\ 0HUFN &RPSDQ\ $QDO\WLFDO JUDGH KH[DQH DQG SURSDQDO ZHUH SXUFKDVHG IURP 0HUFN &RPSDQ\ 'LVWLOOHG ZDWHU used for the preparation of all solution.

Hashemiravan M et al

LQJ DQG VWULQJ LQ D ERLOLQJ ZDWHU EDWK IRU K DW ƒ& 7KHQ OHIW LW IRU K $IWHUZDUG ZH PL[HG ÂżVK RLO J J FRQFHQWUDWLRQ DQG WZHHQ J J WKHQ added to the pre-emulsion. The solution was placed in DVRQLFDWRU PRGHO 6 PLVRQL[ IRU PLQ 2.2.2. Particle size determination

The mean particle size and the size distribution of the nano-emulsions were measured by dynamic light VFDWWHULQJ '/6 XVLQJ 30; & 3DUWLFOH0DWUL[ (German). The nano-emulsion was diluted 40-fold in deionized water before measurement. 2.2.3. pH determination

The pH Measurement bypHmeter (Swiss, Metrohm ZDV FDUULHG RXWLPPHGLDWHO\ DIWHUWKH XOWUDVRXQG 2.2.4. Spray-drying

The emulsions prepared were spray dried with a laboUDWRU\ VFDOH %XFKL VSUD\ GULHU 0LQL 6SUD\ GULHU % Switzerland) equipped with 0.7 mm diameter nozzle. Spray drying conditions were similar for all samples. 7KH DLU Ă€RZ UDWH RI IHHGLQJ DQG DVSLUDWLRQ ZHUH 600(l/h), 2(mL/min) and 100%, respectively. The inlet DQG RXWOHW DLU WHPSHUDWXUHV ZHUH PDLQWDLQHG DW ƒ& and 65 Âą ƒ& 7KH ÂżVK RLO HQFDSVXODWHG SRZGHUV were collected from the collecting chamber and VWRUHG LQ RSDTXH DLU WLJKW FRQWDLQHUV DW ƒ& XQWLO DQDOysis. 2.2.5. Scanning electron microscopy of encapsulated powders

Themorphology were evaluated with a scanning electron microscope (Model TESCAN//VEGA, England). The samples were placed on the SEM stubs using a two-sided adhesive tape. The specimens were subsequently coated with a thin layer of gold using a magnetron sputter coater (Model Emitech, England). 2.2.6. Statistical design

2.2. Methods 2.2.1. Preparation of emulsions

All emulsion produced in two stages. In pre-emulsion, Casein with various concentrations (5,10,15, 20 g/100g) and Inulin (5, 10, 15, 20 g/ 100g) concentration were solved in distilled water with heat

The independent variables were the ratio of core material to coating material (1:5; 2:5) and the concentration of wall material [Casein, Inulin]. Statistical designs are presented in Table 1.The dependent variables were the emulsion size, the pH of emulsion and the size of nano-emulsion.

Hashemiravan M et al

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 485-490

Table 1: 7KH IRUPXODWLRQV RI HQFDSVXODWHG ÂżVK RLO SRZGHUV

Formulation F1

Casein (%) 0

Inulin (%) 20

Fish oil (%) 4

7ZHHQ

1

'LVWLOOHG ZDWHU 75

F2 F3 F4 F5 F6 F7 F F F10

20 10 15 5 0 20 10 15 5

0 10 5 15 20 0 10 5 15

4 4 4 4

1 1 1 1 1 1 1 1 1

75 75 75 75 71 71 71 71 71

Table 2: pH nano-emulsion of casein at various concentration (5, 10, 15 and 20%wt) mixed with inulin 5, DQG ZW FRQWDLQLQJ DQG ZW ÂżVK RLO 9DOues in the same column shown with similar letters are QRW VLJQLÂżFDQWO\ GLIIHUHQW

Nano emulsion

S+ PHDQ “ 6'

F1 F2 F3 F4 F5 F6 F7 F F F10

“ b 5.756 ¹ 0.60a 5.773¹0.011a “ a “ a “ a 4.520¹0.070b 5.750¹0.070a “ a “ a

3. RESULTS AND DISCUSSION 3.1. pHofNano-emulsion According to the Table (2), the results show that thekindand concentration of combination of Casein and ,QXOLQ KDYH YHU\ VLJQLÂżFDQW LQĂ€XHQFH RQ S+ S EXW LQFUHDVLQJ WKH FRQFHQWUDWLRQ RIÂżVK RLOIURP WR VKRZV WKDW WKHUH ZDV QR VLJQLÂżFDQW FKDQJH (p<0.05) on the pH of nano-emulsions. 7KH S+ RI QDQR HPXOVLRQ RI ÂżVKRLOLV FKDQJHG E\ kind of wall material, when nano-emulsion was prepared at 20%wt inulin had the least pH of nano-emulsion on the other hand pH of Nano-emulsion had in-

Figure 1: (IIHFWV RI ÂżVK RLO DQG NLQG RI ZDOO PDWHULDO DQ GLWV concentration on the pH of nano-emulsion.

FUHDVHG VLJQLÂżFDQWO\ ZKHQ LQXOLQ PL[HG ZLWK FDVH LQ )LJXUH ,W KDSSHQHG EHFDXVH RI FDVH LQ ࣲV SURSHUWLHV that have relatively high electric charged and it is the result of the presence of phosphate groups bonded to serine [11]. 3.2. Size of nano-emulsion The results show that thekind and concentration ofcombination of Case in and Inulin have very signifLFDQW LQĂ€XHQFH RQ VL]H RI QDQR HPXOVLRQ S Figure (2) shows that the sample which contains 20% FDVHLQ KDV WKH PRVW S+ S+ ZKLOH LW KDV WKH OHDVW PHDQ VL]H RI HPXOVLRQ GURSOHWV QP ,W LV WKH UHVXOW RI FDVHLQ ࣲ V FKDUDFWHULVWLFV EHFDXVH RQ WKH

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 485-490

Hashemiravan M et al

Table 3: The mean size of nano-emulsion droplet of casein at various concentration (5, 10, 15 and 20%wt) mixed with inulin 5, 10, 15 and 20%wt, containing 4 DQG ZW ÂżVK RLO 9DOXHV LQ WKH VDPH FROXPQ VKRZQ ZLWK VLPLODU OHWWHUV DUH QRW VLJQLÂżFDQWO\ GLIIHUHQW

Nano emulsion

Size of nano emulsion PHDQ “ 6'

F1

“ a

F2

“ cd

F3

“ bc

F4

“ ab

F5

111.100Âą5.707bcd

F6

bcd

115.100Âą4.371

F7

“ d

F

“ cd

F

“ cd

F10

“ bcd

higher pH from it is Isoelectric point change between WR &OHDUO\ LW GHSHQGV RQ WKH NLQG RI WKH FDVH LQ ࣲ V )UDFWLRQV 6R ZKHQ FDVH LQ SUHVHQFH LQ HPXOVLRQ LQ the shared level of oil and water, it creates repulsion of electrostatic and also space prevent that avoids water closing each other [12]. 7KH VL]H RI WKHQDQR HPXOVLRQ UDQJ ZDV IURP WR QP DQG LV OLVWHG LQ 7DEOH ,W VKRZV RQ

Figure 3: Relationship between emulsion size (d43) and pH of the emulsion.

the Figure (3) that between the pH and the size of HPXOVLRQGURSOHW KDV QHJDWLYH VLJQL¿FDQW FRUUHODWLRQ (p<0.01). It means that the size of emulsion droplet was reduced by pH increasing. It mentioned on the Table (2) sample contain 20% inulinhad the least pH value (pH = 4.52) while it has themost size of emulVLRQ GURSOHW QP $FFRUGLQJ WR UHVHDUFKHU ࣲV study reducing of the pH from neutral to acidic caused increasing the size distribution of emulsion droplet [12]. The size distribution of emulsion depends onseverDOIDFWRUV OLNH WKH DPRXQWDQG W\SH RI HPXOVL¿HUV SKDVH DQG WKH PHWKRG RI SURGXFLQJ RI HPXOVL¿FDWLRQ DQGLWV pH [13]. So we investigate these items. Some agents

Figure 2: (IIHFWV RI ÂżVK RLO DQG NLQG RI ZDOO PDWHULDODQGLWV

Figure 4: Scanning electron microscope (SEM) of encapsu-

concentrationon the size of nano-emulsion.

ODWHG ÂżVK RLO SRZGHUV SUHSDUHG ZLWK FDVHLQ

Hashemiravan M et al

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 485-490

surfaces, but combined with low doses of inulin had no tangible impact, though the uniformity of microcapsule sizeswas affected. It showed the slower rate of KDUGHQLQJ RI WKH ZDOOV RI WKH VDPSOH PDWUL[ FRQWDLQLQJ FDVHLQ 7KH SLFWXUHV VKRZHG WKH VLJQLÂżFDQFH RI ZDOO material properties and their concentration saffecting the structure and morphology of microcapsules by the presence of cracks in the microcapsules containing greater amounts of inulin.

4. CONCLUSIONS

Figure 5: 6KRZHG WKH SUHVHQFH RI VRPH GHIHFWV VSHFLÂżcally the pores formed, which could explain the relatively KLJK YDOXHV RI VXUIDFH RLO IRXQG LQ WKH ÂżVK RLO HQFDSVXODWHG powder containing 20% Inulin.

OLNH WKH DPRXQW DQG NLQG RI HPXOVLÂżHU KHUH ZH XVHG 7ZHHQ DQG SKDVH NLQG RLO LQ ZDWHU DQG PHWKRG RI HPXOVLÂżFDWLRQ SURGXFWLRQ ZDV WKH VDPH IRU HYHU\ single samples. The only agents that changed was pH, it is because of kind of wall materials and different concentration so we use this factor in analyzing the result. According to results that we mentioned on the 7DEOH WKH OHDVW DPRXQW RI VL]H RI HPXOVLÂżFDWLRQ drops belong to sample that contain 20% case in and ÂżVK RLO ) VDPSOH KDG WKH OHDVW S+ DQG DOVR ) sample had the most pH so these results shows that ZKHQ S+ LV UHGXFHG WKH VL]H RI HPXOVLÂżFDWLRQ GURSV is increased. 3.3. Morphology and structure of microcapsules of ÂżVK RLO The SEM images (Figure 4) show that microcapsules containing 20% case in have much less shrinkage and GHSUHVVLRQ WKDQ WKRVH FRQWDLQLQJ LQXOLQ ([WHUQDO structure of the powder particles containing inulin was porous and has depression and some cracks shown in Figure 5. Adding Inulin to Casein had a profound impact on the structure and morphology of microencapsulated powders. Casein combined with high levels of inulin particles produces particles with rough

The study revealed that ultrasonic waves can be used WR FUXVK GURSOHWV DQG UHGXFH WKHLU VL]H WR WKH H[WHQW of nano and also energy of the waves can be used to produce food nano-emulsions and products in which the particle size as a parameter has an important role in the product quality. Results showed that the smallest particle size was related to the sample containing FDVHLQ DQG ÂżVK RLO DQG WKH JUHDWHVW SDUWLFOH size was related to the sample containing 20% inulin DQG ÂżVK RLO %\ FRPSDULQJ WKH VDPSOHV RI DQG ÂżVK RLO VWXGLHV VKRZHG WKDW E\ LQFUHDVLQJ WKH UDWLR RI FRUH ÂżVK RLO WR ZDOO FRPELQDWLRQ RI LQXOLQ DQG FDsein) from 1:5 to 2:5, the droplet size as the important characteristic of nano-emulsion decreased, thus the product quality improved. The lowest pH belonged to the treatment containing 20% inulin which was VLJQLÂżFDQWO\ S LQFUHDVHG E\ DGGLQJ RI FDVHLQ $ QHJDWLYH VLJQLÂżFDQW FRUUHODWLRQ ZDV REVHUYHG EHWZHHQ the pH and the emulsion droplet size as by increasing pH the emulsion droplet size was reduced.

REFERENCES 'DYLGRY SDUGR * 5RFFLD 3 6DOGDJR ' /HRQ E., Pedroza R., Am. J. Food Technol., 6 -DIDUL6 0 $VVDGSRRU ( %KDQGDUL % +H < Food Res. Int., 41 3. Loksuwan J., Food Hydrocolloid, 21 .RODQRZVNL : =LRONRZVNL 0 :HL%ERUGW - .XQ] % /DXIHQEHUJ * Eur. Food Res Technol., 222 (2006), 336.

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 485-490

'LD] 5RMDV ( , 3DFKHFR $JXLODU 5 /L]DUGL - $UJXHOOHV 0RUDOHV : 9DOGH] 0 $ 5LQDXGR0 *R\FRROHD ) 0 Food Hydrocolloid, 18 (2004), &KR < 6KLQ ' 6 3DUN - Korean J. Food Sci. Technol., 32 (2000), 132. 7. Klinkesorn U., Sophanodora P., Chinachoti P., 'HFKHU ( $ 0F&OHPHQWV ' - Food Hydrocolloid, 19 (2005), 1044. 6XPPHU $ 3 )RUPXJJLRQL 3 0DODFDUQH 0 Ann. Fac. Medic., 21 6HPR ( .HVVHOPDQ ( 'DQLQR ' /LYQH\ < ' Food Hydrocolloid, 21

Hashemiravan M et al

%DUFOD\ 7 *LQLF 0DUNRYLF 0 &RRSHU 3 Petrovsky N., J. Exc. Food Chem., 1 (2010), 27. < + +XL Dairy Science and Technology Handbook, VCH Publisher; Eurika, California, USA. 'LFKHQVRQ ( 5DGIRUG 6 - *ROGLQJ 0 Food Hydrocolloid, 16 (2003), 153. ,]TXLHUGR 3 (VTXHQD - 7DGURV 7 ) 'HGHUQ & *DUFLD 0 - $]HPDU 1 6RODQV & Langmuir, 1 (2002), 26.

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 491-494

ISSN: 2251-8533

International Journal of Bio-Inorganic Hybrid Nanomaterials pH and Properties of Synthesized Barium Hexa-Ferrite by Co-precipitation Method Shaghayagh Marzban1*, Saeid Abedini Khorrami2 1 2

M.Sc. Student, Department of Chemistry, Tehran North Branch, Islamic Azad University, Tehran, Iran

Associate Professor, Department of Chemistry, Tehran North Branch, Islamic Azad University, Tehran, Iran 5HFHLYHG 6HSWHPEHU $FFHSWHG 1RYHPEHU

ABSTRACT Synthesis of BaFe12O19 magnetic nano particles via precipitation in different pH conditions have been reported. The certain molar ratio of Fe/Ba = 12 selected and sodium hydroxide was used as a precipitant agent. X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and vibrating sample magnetometer (VSM) were used to consider the structural, morphological and magnetic properties of barium hexaferrite nano-particles, respectively. Results demonstrated that pH plays an important role in phase composition; so affected sample properties. The broad hysteresis loop shows that the barium hexaferrite powder was in good crystalline nature. Keyword: Barium Hexaferrite; Co-precipitaion; pH; Magnetic Properties; Nanoparticles; Hard Ferrites; XRD.

1. INTRODUCTION Barium ferrites are well known as a hard magnetic maWHULDO ZKLFK DUH EDVHG RQ LURQ R[LGHV 7KH\ DUH WHFKQRORJLFDOO\ QRWHZRUWK\ EHFDXVH RI WKHLU H[FHOOHQW SURSHUties such as chemical stability, corrosion resistivity and high coercive force. Because of these they could not be HDVLO\ UHSODFHG E\ DQ\ RWKHU PDJQHWV > @ +H[DJRQDO IHUULWHV DUH D ZLGH IDPLO\ RI IHUURPDJQHWLF R[LGHV 7KH FU\VWDO VWUXFWXUH RI WKH GLIIHUHQW NQRZQ W\SHV RI KH[DJRQDO IHUULWHV 0 : ; < = DQG 8 LV YHU\ FRPSOH[ and can be considered as a superposition of R and S (*) Corresponding Author - e-mail: sh_marzban@yahoo.com.

EORFNV DORQJ WKH KH[DJRQDO F D[LV 565 6 IRU 0 W\SH [3]. They have potential application in contrast agent in magnetic resonance imaging (MRI), recording media, radar absorbing material and as microwave absorber materials [4-6]. Some of the other applications are apSOLHG DV )ODPH UHWDUGDQW LQ SODVWLFV FRDWLQJV ÂżEHU DQG WH[WLOHV > @ &RQYHQWLRQDO SUHSDULQJ PHWKRGV RI 0 W\SH barium ferrite nanoparticles, such as ball milling rout, requires a high calcination temperature around 1200 ƒ& DQG XVHV D PLOG PL[WXUH RI EDULXP FDUERQDWH

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 491-494

VDOW DQG IHUULF R[LGH )XUWKHUPRUH WKHVH SURFHVVHV UHsult in entrance of impurities into the compositions, generation of lattice strains in the molecular structure and made irregularity in the particle shape. The high temperature insures the formation of barium ferrite; ODUJHU SDUWLFOHV DUH DOVR SURGXFHG LQ WKLV SURFHVV > @ 1RZDGD\V %D KH[D IHUULWHV DUH REWDLQHG E\ GLIIHUent chemical methods such as hydrothermal [10, 11], sol-gel auto-combustion [12,13], co-precipitation [1417]. Among these methods, co-precipitation is one of the simplest techniques. This method uses accessible, environment-friendly and cheap precursors such as FKORULGHV DQG QLWUDWHV VDOWV DQG VRGLXP K\GUR[LGH This process accrues at lower temperature conditions so, known as green synthesis methods. In the present work, BaFe12O powder has been prepared by coprecipitation method using metallic nitrates of barium and iron as precursors. Characterization of nano-particles showed the success process.

2. EXPERIMENTAL

Marzban Sh. et al

Âł3KLOLSV ;ÂśSHUW´ XVLQJ &X .ÄŽ UDGLDWLRQ DW N9 DQG 30 mA. A “Philips XL-30â€? scanning electron microscope was used to characterize the morphologies and microstructure of the samples.

3. RESULTS AND DISCUSSION 7KH SKDVH LGHQWLÂżFDWLRQ RI WKH QDQR SRZGHUV ZDV recorded by X-ray diffraction with Cu-KÎą radiation source in the range of the 2θ %UDJJ DQJOH ƒ The X-ray patterns of sample powders C1, C2 and C3 SUHSDUHG DW S+ DQG DUH VKRZQ LQ )LJXUH 7KH ZHOO PDWFKHG SLFNV ZLWK UHIHUHQFH -&3'6 12 74-1121 picks show that BaFe12O with miller plates (1 1 4) and (1 0 7), is dominant phase in all of samples. In Figure 1 the miller plates (1 1 0) and (1 0 4) refers to the small amount of Îą-Fe2O3 as sub phase in sample C1 V\QWKHVL]HG DW S+ $OVR WKH PDWFKHG SLFN ZLWK (2 1 2) demonstrates that BaFe2O4 is impurity phase at sample C2 (synthesized at pH 10). As shown in Figure 1. the absence of any sub-picks demonstrates that C3 (synthesized at pH 12) has a good BaFe12O

2.1. Materials Barium nitrate Ba(NO3)2 0HUFN IHUULF QLWUDWH nona hydrated Fe(NO3)3 +22 0HUFN DQG VRGLXP K\GUR[LGH XVHG DV VWDUWLQJ PDWHULDOV ZLWKRXW DQ\ SXULÂżFDWLRQ 'HLRQL]HG ZDWHU DSSOLHG LQ SUHSDULQJ DOO solutions. 2.2. Synthesis of BaFe12O19 Synthesis of nano-sized powder of magnetic barium KH[DIHUULWH ZDV FDUULHG RXW DV IROORZV $SSURSULDWH amounts of Fe(NO3)3 +2O, Ba(NO3)2 kept at a molar ratio of 12:1. The salt solution was added dropwise WR WKH EDVH VROXWLRQ XQWLO WKH S+ UHDFKHG DW DQG 12 for each system, respectively. The red precipitates ZHUH ZDVKHG DQG ÂżOWHUHG UHSHDWHGO\ ZLWK GLVWLOOHG ZDter. This process took about 6 hours. The sample was WKHQ GULHG LQ DQ RYHQ NHHSLQJ WHPSHUDWXUH DW ƒ& for 3 hours. After attaining the powder by mortar and SHVWOH FDOFXODWLRQ SURFHVV ZDV FDUULHG RXW DW ƒ& IRU KRXUV WR JHW WKH ÂżQDO SURGXFW 2.3. Characterization process ; UD\ GLIIUDFWLRQ ;5' SDWWHUQ ZDV PHDVXUHG E\ D

Figure 1: X-ray diffraction pattern of BaFe12O19 synthesis in different pH.

Marzban Sh. et al

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 491-494

Table 1: Crystallinity percent, particle size and phases of samples prepare in different pH.

Sample

Ba:Fe

pH

Phases

Particles size (nm)

Percent Crystallinity (%)

C1

1:10

Fe2O3, BaFe12O

C2

1:10

10

Fe2O3, BaFe2O4, BaFe12O

C3

1:10

12

BaFe12O

Table 2: Magnetic parameters of BaFe12O19 nanopowders prepared at pH= 12.

Sample

Ba:Fe

pH

Ms(emu g-1)

Mr(emu g-1)

Hc(Oe)

C3

1:10

12

single phase composition. The crystallite powders size was also measured by X-ray line broadening technique using the Scherer’s formula indicated in Equation (1): G

O E FRV T

(Eq. 1)

:KHUH ' LV WKH JUDLQ GLDPHWHU β is half-intensity width of the relevant diffraction, Îť is X-ray wavelength and θ the diffraction angle. The results revealed that the number of phases, particle size and percent cristallinity of BaFe12O KH[DJRQDO VWUXFWXUHV DUH LQĂ€XHQFHG E\ FKDQJLQJ S+ ;5' SDWWHUQ VKRZV WKDW KH[DJRQDO VWUXFWXUHV DW VHOHFWHG S+ DQG DUH WKH PDLQ VWUXFWXUH 1XPEHU RI SKDVHV

Figure 2: SEM imagining of BaFe12O19 nano-particles synthesized in pH= 12.

Figure 3: VSM Loop of BaFe12O19 nano-particles synthesized in pH= 12.

decrease with the pH rising. The effect of pH on the average size and percent crystallinity of nanoparticles is summarized in Table 1. As shown in Figure 2 the synthesized BaFe12O nanoparticles at pH= 12 has nonregular shape morphology at all. It was observed that individual grains are not distributed homogenously, but rather tend to agglomerate forming larger bundles. But anRWKHU LQWHUHVW LV VWDUWLQJ RI IRUPDWLRQ RI KH[DJRQDO shapes at high level pH. The magnetic properties were PHDVXUHG E\ XVLQJ 960 ZLWK DQ DSSOLHG ÂżHOG RI 7 at room temperature. Plot of magnetization (M) as a IXQFWLRQ RI DSSOLHG PDJQHWLF ÂżHOG + LV VKRZQ LQ )LJ

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 491-494

XUH 7KH VPRRWK K\VWHUHVLV ORRS FRQÂżUP ZLWK ; UD\ results demonstrated the formation of the pure barium KH[D IHUULWH 7KH PDJQHWLF SURSHUWLHV VXFK DV VDWXUDtion magnetization (Ms), remanent magnetization (Mr) and cervicitis (Hc) of sample C3, reported in Table 2. Even though from the SEM analysis particles are not uniformly distributed but the particle size and particle morphology are the main reasons for the low coercivity (Hc

4. CONCLUSIONS 6LQJOH SKDVH EDULXP KH[D IHUULWH SRZGHU ZDV V\QWKHsized successfully by co-precipitation technique. Results demonstrate that pH plays an important role in the phase formation process. As by pH value increasing, the main phase composition growing up and at last single phase obtained at pH= 12. Magnetic properties of sample C3 as a hard magnet, by single phase FRPSRVLWLRQ EDVHG RQ ;5' GDWD VHULHV LQGLFDWH DQG FRQÂżUP ZLWK ZKDW ZH H[SHFWHG IURP ZLWK OLWHUDWXUHV

REFERENCES 1RZRVLHVOVNL 5 %DELODV 5 'HUF] * 3DMDN / 6NRZURQVNL : JAMME, 27 2 Valenzuela R., Phys. Res. Inter. $UWLFOH ,' 3. Pullar R.C., Prog. Mater. Sci., 57 :DQJ < +XDQJ < :DQJ 4 +H 4 &KHQ / Appl. Surf. Sci., 259

Marzban Sh. et al

5. Ozah S., Bhattacharyya N.S., J. Magn. Mater., 342 /L 4 3DQJ - :DQJ % 7DR ' ;X ; 6XQ / Zhai J., Adv. Powder Technol., 24 7. Aksit A.C., Onar N., Ebeoglugil M.F., Birlik I., Celik E., Ozdemir E., J. appl. Polym. Sci., 113 6WDEOLQ + :RKOIUDWK ( 3 (GV )HUURPDJQHWLF Materials, North-Holland, Amsterdam, 3 ;X * 0D + =KRQJ 0 =KLX - <RX < +H =K J. Magn. Mater., 301 'URIHQLN 0 %DQ , 0DNRYHF ' =QLGDUVLF $ -DJOLFLF = +DQ]HO ' /LVMDN ' Mater. Chem. Phys., 127 (2011), 415. 11. Janasi S.R., Emura M., Landgraf F.J.G., Rodrigues ' J. Magn. Mater., 238 $GVFKLUL 7 +DNXWD < $UDL . Ind. Eng. Chem. Res., 39 13. Mali A., Ataie A., J. Al. Com., 399 (2005), 245. 14. (a) Packiaraj G., Nital P., Jotania R.B., J. Biomed. Bioeng., 1 E /LX < =KDQJ + /LX < :DQJ / /L < Adv. Mater. Res., 3052 (2011), &KHQ ' + &KHQ < < J. Colloid. Inter f. Sci., 235 16. Mallick K.K., Shepherd Ph., Green R.J., J. Eur. Ceram. Soc., 27 (2007), 2045. 17. Rashad M.M., Ibrahim I.A., J. Magn. Mater., 323

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 495-502

ISSN: 2251-8533

International Journal of Bio-Inorganic Hybrid Nanomaterials 3UHFRQFHQWUDWLRQ RI 0R ,, RQ 0LFUR &U\VWDOOLQH 0RGLÂżHG ZLWK Functionalized-Nano Graphene Ali Moghimi Associate Professor, Department of Chemistry, Varamin-Pishva Branch Islamic Azad University, Varamin, Iran

Received: 14 September 2013; Accepted: 21 November 2013

ABSTRACT A organic-solution-processable functionalized-graphene (SPFGraphene) material has been studied on preconcentration and determination of trace Mo(II) ions. In this process, the effects of pH solution, elution conditions on pre-concentration of trace Mo(II) were studied and the effect of interfering ions was also investigated. A selective method for the fast determination of trace amounts of Mo(II) ions in water samples has been developed. Method has been developed for preconcentration of Mo on organic-solution-processable functionalized-graphene 63)*UDSKHQH DGVRUEHQW LQ WKH S+ UDQJH SULRU WR LWV VSHFWURSKRWRPHWULF GHWHUPLQDWLRQ EDVHG RQ WKH oxidation of bromopyrogallol red at Îť QP 7KLV PHWKRG PDNHV LW SRVVLEOH WR TXDQWLWL]H 0R LQ WKH UDQJH RI 6.9Ă—10-9 WR ĂŽ 0 ZLWK D GHWHFWLRQ OLPLW 6 1 RI ĂŽ -9 M. This procedure has been successfully applied to determine the ultra-trace levels of Mo in the environmental samples, free from the interference of some diverse LRQV 7KH SUHFLVLRQ H[SUHVVHG DV UHODWLYH VWDQGDUG GHYLDWLRQ RI WKUHH PHDVXUHPHQWV LV EHWWHU WKDQ Keyword: Preconcentration; Micro crystalline; Nano graphene; Mo(II); SPE; FAAS; Organic-solution; Functionalized.

1. INTRODUCTION 7R[LFRORJLFDO VWXGLHV KDYH SURYHG WKDW WKH GHJUHH RI WR[LFLW\ RI DQ HOHPHQW GLUHFWO\ GHSHQGV RQ WKH VSHFLHV in which it is present. Mo(II) is a potentially carcinogenic agent [1]. Mo(II) at trace concentrations acts as ERWK D PLFURQXWULHQW DQG D WR[LFDQW LQ PDULQH DQG IUHVK water systems [2-5]. This element is needed by plants DW RQO\ YHU\ ORZ OHYHOV DQG LV WR[LF DW KLJKHU OHYHOV (*) Corresponding Author - e-mail: alimoghimi@iauvaramin.ac.ir.

At these levels, Mo(II) can bind to the cell membrane and hinder the transport process through the cell wall. Mo(II) at nearly 40 ng mL-1 is required for normal metabolism of many living organisms [6]. On the other hand, Mo(II) is an important element in many industries. Thus, the development of new methods for selective separation, concentration and determination of it in

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 495-502

sub-micro levels in different industrial, medicinal and environmental samples is of continuing interest. The determination of Mo(II) is usually carried out E\ ÀDPH DQG JUDSKLWH IXUQDFH DWRPLF DEVRUSWLRQ spectrometry (AAS) [7] as well as spectrometric PHWKRGV > @ 6ROLG SKDVH H[WUDFWLRQ 63( PHWKRGV DUH WKH EHVW alternatives for traditional classic methods due to selective removal of trace amounts of metal ions from WKHLU PDWULFHV 6ROLG SKDVH H[WUDFWLRQ GHWHUPLQDWLRQV FDQ EH FDUULHG RXW LQ GLIIHUHQW HI¿FLHQW ZD\V 2QH RI the most appropriative preformation features of SPE is achieved by using octadecyl silica membrane disks. 63( UHGXFH WKH XVH RI WR[LF VROYHQW GLVSRVDO FRVWV DQG H[WUDFWLRQ WLPH > @ The octadecyl silica membrane disks involves shorter sample processing time and decreased plugging due to the large cross-sectional area of the disk and small SUHVVXUH GURS ZKLFK DOORZV KLJKHU ÀRZ UDWHV UHGXFHG channeling resulting from the use of sorbent with smaller particle size and a greater mechanical stability of the sorbent bed [13]. ,Q RXU SUHYLRXV DWWHPSWV ZH PRGL¿HG 63( PHPbrane disks with suitable compounds for selective determination of chromium [14] and lead [11]. Meanwhile, other investigators have successfully utilized WKHVH VRUEHQWV IRU TXDQWLWDWLYH H[WUDFWLRQ DQG PRQLtoring trace amounts of lead [15], copper [16], silver > @ PHUFXU\ > @ FDGPLXP > @ SDOODGLXP > @ Ce [22] and UO2 [12]. The used ligand is new and fairly selective and will not interfere in the determination process of Mo(II). Absorption spectrophotometry method (after preconcentration) was applied for determination of Mo based RQ WKH R[LGDWLRQ RI EURPRS\URJDOORO UHG DW QP Various effective parameters have been evaluated, and the developed procedure has been successfully employed for the quantitation of ultra-trace amounts of Mo in water sample.

Moghimi A

89Âą9LV VSHFWURSKRWRPHWHU :3$ &DPEULGJH 8. 'LRGH $UUD\ 0RGHO 6 ZDV DSSOLHG IRU UHFRUGing the absorption spectra. A spectrophotometer (Perkin-Elmer model 35) with 10 mm glass cuvette was XVHG WR PHDVXUH WKH DEVRUEDQFH DW D Âż[HG ZDYHOHQJWK Controlling the reaction temperature was done by a ZDWHU EDWK WKHUPRVWDW *DOOHQNDPS *ULIÂżQ %- 9 and a stopwatch was used for recording the reaction time. The synthesis of the TPP-NHCO-SPFGraphene is illustrated in Scheme 1. 2.2. Reagents 'RXEO\ GLVWLOOHG ZDWHU DQG DQDO\WLFDO UHDJHQW JUDGH chemicals were used throughout. 2.2.1. Synthesis of TPP-NHCO-SPFGraphene

7KH ÂżUVW RUJDQLF VROXWLRQ SURFHVVDEOH IXQFWLRQDOized-graphene (SPFGraphene) hybrid material with porphyrins was prepared. The synthesis of the porphyrin-Graphene nanohybrid, 5-4 (aminophenyl)-10, WULSKHQ\O SRUSK\ULQ 733 DQG JUDSKHPH R[LGH molecules covalently bonded together vi a an amide bond (TPP-NHCO-SPFGraphene, Scheme 1 and 2) was carried out using an amine-functionalized prophyrin (TPP-NH2 DQG *UDSKHQH R[LGH LQ 1 1 GLPHWK\OIRUPDPLGH '0) IROORZLQJ VWDQGDUG FKHPLVWU\ /DUJH VFDOH DQG ZDWHU VROXEOH *UDSKHQH R[LGH ZDV SUHSDUHG E\ WKH PRGLÂżHG +XPPHUV PHWKRG > 25]. Results of atomic force microscopy characterizaWLRQ KDYH FRQÂżUPHG WKDW WKLV JUDSKHPH PDWHULDO FDQ EH HDVLO\ GLVSHUVHG DW WKH VWDWH RI FRPSOHWH H[IROLDWLRQ which consists of almost entire single-layered Gra-

1) SOCl2, 24 h Grapheme oxide

TPP-NHCO-SPFGraphene 2) TPP-NH2, EtN , '0) ƒ& K

TPP-NH2 =

2. EXPERIMENTAL 2.1. Apparatus The glass column with 10 mm i.d. and 200 mm height was used to make preconcentration column. An

Scheme 1: Synthesis scheme of TPP-NHCO-SPF Graphene [26].

Moghimi A

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 495-502

Scheme 2: Schematic representation of part of the structure of the covalent TPP-NHCO-SPFGraphene [26].

phene sheets in H2O [24, 25]. TPP-NH2 and Graphene R[LGH PROHFXOHV DUH FRYDOHQWO\ ERQGHG WRJHWKHU E\ DQ amide bond. Much care has been taken to make sure all the unreacted TPP-NH2 has been removed using H[WHQVLYH VROYHQW ZDVKLQJ VRQLFDWLRQ DQG PHPEUDQH ÂżOWUDWLRQ 'HWDLOV DUH JLYHQ LQ WKH ([SHULPHQWDO SDUW 7KH DWWDFKPHQW RI RUJDQLF PROHFXOHV WR *UDSKHQH R[ide has made TPP-NHCO- SPFGraphene soluble in '0) DQG RWKHU SRODU VROYHQWV > @ 2.2.2. Standard Mo solution

A standard solution of Mo(II), 1.0×10-3 M was prepared by dissolving 0.1111 g Mo nitrate (Merck) in water containing a drop of concentrated HCl and GLOXWLQJ WR WKH PDUN LQ D P/ YROXPHWULF ÀDVN $OO working solutions of Mo(II) were prepared by serial dilution of the stock solution. 2.2.3. Standard bromopyrogallol red solution

An aqueous solution of (1.0×10-4 M) bromopyrogallol red (Merck) was prepared by dissolving of 0.0140 g bromopyrogallol red in water and diluting to the mark LQ D P/ YROXPHWULF ÀDVN 8QLYHUVDO EXIIHU VROXtions in the range from 2.0 to 10.0 were prepared with acetate, phosphate, and borate. Glycine/HCl buffer was used for pH 1.0. Stock solutions (5.0×10-3 M) of interfering ions were prepared by dissolving suitable

VDOWV LQ ZDWHU K\GURFKORULF DFLG RU VRGLXP K\GUR[LGH solutions. 2.3. General procedure The column was packed with 3.0 g adsorbent and was conditioned with 1.0-2.0 mL of pH 5.0. Then, 10.0 mL of Mo solution (5.0Ă—10-5 M) was passed through the column at 0.1 mL min-1. The analyte was eluted from the column by 1.0 mL of HCl, 1.0 M. A sample solution was prepared by pouring 0.5 mL of buffer soluWLRQ S+ LQ D YROXPHWULF Ă€DVN DQG P/ of 1.0Ă—10-4 M bromopyrogallol red was added. The PL[WXUH GLOXWHG WR FD P/ ZLWK ZDWHU WKHQ P/ of Mo(II) (5.0Ă—10-5 M) (eluted solution from column) was added and the solution diluted to the mark with ZDWHU 7KH UHDFWLRQ PL[WXUH DV DJLWDWHG DQG WKHQ DQ DSpropriate amount of the solution was transferred to the spectrophotometric cell and variation in absorbance ZDV UHFRUGHG IRU WKH ÂżUVW PLQ IURP LQLWLDWLRQ of the reaction at 517 nm. A calibration graph was plotted with absorbance change (∆A = A5 -A0.5) versus Mo concentration.

3. RESULTS AND DISCUSSION Organic-solution-processable functionalized-gra

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 495-502

Moghimi A

Figure 1: Structure of Bromopyrogallol red structure.

phene (SPFGraphene) with the following structure (Scheme 1) is a new chelating agent which can form VWDEOH FRPSOH[ ZLWK 0R ,, %\ LPPRELOL]LQJ WKLV WULdentate bisamide ligand on microcrystalline naphthalene, Mo(II) can be adsorbed. Then desorption of Mo is carried out by using a strong inorganic acid. The Mo(II) concentrations were determined spectrophotometrically after passing solution through the column. 7KHUHIRUH ÂżUVW WKH RSWLPXP FRQGLWLRQV IRU VSHFWURphotometric procedure should be studied. 3.1. Effect of variables on the determination of Mo Bromopyrogallol red with following structure Figure LV R[LGL]HG E\ 0R DQG WKH DEVRUEDQFH RI WKH VROXtion decreases with time, at Îť = 517 nm. The change in the signal is proportional to Mo concentration. Figure 3 shows the absorption spectra of bromopyr-ogallol UHG 0R V\VWHP DW GLIIHUHQW WLPHV ([SHULPHQW VSHWrophotometric determination) on eluted Mo solution

Figure 2: Effect of pH on the reaction rate. Conditions: bro-

Figure 3: Effect of bromopyrogallol red concentration on the reaction rate. Conditions: pH 1.0; Mo(II), 1.0Ă—10-5 M; temperature, 30°C; measuring time, 5.0 min from initiation of the reaction.

was done at different pH values (1.0-5.0). Figure 2 shows the effect of pH on the net absorbance (∆A). 7KH PD[LPXP QHW DEVRUEDQFH LV DW S+ ZKHUHDV higher pH values cause decreasing in the signal. At KLJKHU S+ YDOXHV R[LGDWLRQ SRWHQWLDO RI EURPRS\rogallol red increases, thus the reaction rate and ∆A decreased. Therefore the pH of 1.0 was selected for WKLV VWXG\ 7KH LQĂ€XHQFH RI EURPRS\URJDOORO UHG FRQcentration on the reaction rate was tested at pH 1.0 with 1.0Ă—10-5 0 0R ,, DW ƒ& )LJXUH ,W FDQ EH seen that the best concentration for bromopyrogallol

Figure 4: Effect of temperature on the rate of reaction. Con-

mopyrogallol red, 1.0Ă—10 M; Mo(II), 1.0Ă—10 M; tempera-

ditions: pH 1.0; Mo(II), 1.0Ă—10-5 M; bromopyrogallol red,

ture, 30°C; measuring time, 5.0 min from initiation of the

3.0Ă—10-5 M; measuring time, 5.0 min from initiation of the

reaction.

reaction.

-5

-5

Moghimi A

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 495-502

red is 3.0Ă—10-5 M. At higher values the aggregation of bromopyrogallol red causes the reaction rate to be deFUHDVHG (IIHFW RI WHPSHUDWXUH RQ WKH PD[LPXP VLJQDO (∆$ ZDV VWXGLHG IRU WKH UDQJH RI ƒ& XQGHU RStimum conditions otherwise as previously described. Figure 4 shows that with increasing temperature up to ƒ& ∆A signal or the rate of reaction increases. So WHPSHUDWXUH ZDV Âż[HG DW ƒ& $W KLJKHU WHPSHUDWXUH bromopyrogallol red can be decomposed. 3.2. Effect of variables on the preconcentration 7KH HIIHFW RI S+ RQ SUHFRQFHQWUDWLRQ RI 0R ,, ZDV H[amined in range of 1.0-10.0, and the results are shown in Figure 5. The results show that in the pH range of 5.0-10.0, the analyte was adsorbed on microcrystalline naphthalene quantitate and the recovery was more WKDQ )RU S+ ORZHU WKDQ WKH FRPSOH[ ZLOO QRW be formed on adsorbent (at acidic media, active sites of ligand will be protonated) and at high pH values, Mo will precipitate on the column (precipitating instead of adsorption will occur). In order to obtain the best conditions for determination after preconcentration and to prevent the precipitation of Mo (especially at high concentrations), the most acidic pH from this UDQJH RI EXIIHUV ZDV VHOHFWHG 7KH LQĂ€XHQFH of analyte retention time was investigated by passing 10.0 mL of Mo(II) (5.0Ă—10-5 M) solution in the pH 5.0 ZLWK GLIIHUHQW Ă€RZ UDWHV DQG SHUIRUPLQJ WKH H[SHULment with the passed solution. The results show that

Figure 5: Effect of pH on the preconcentration recovery. Conditions: Mo(II), 5.0×10-6 0 ÀRZ UDWH P/ PLQ-1; optimum conditions for determination of Mo(II).

Figure 6: Effect of HCl concentration for elution conditions: Mo(II), 5.0Ă—10-6 M; Ph 1.0; HCl, 1.0 mL; optimum conditions for determination of Mo(II).

LQ WKH KLJKHU ÀRZ UDWHV 0R FDQQRW EH DGVRUEHG RQ microcrystalline naphthalene quantitatively. The best ÀRZ UDWH ZDV VHOHFWHG WR EH P/ PLQ-1. As the Mo FRPSOH[ LV XQVWDEOH LQ KLJK DFLGLF VROXWLRQV K\GURchloric acid was selected to desorb the adsorbed analyte. Figure 6 shows that Mo(II) can be desorbed from the adsorbent by elution with 1.0 mL, HCl, 1.0 M. For investigating the ability of microcrystalline naphthalene to adsorb Mo(II) after sequential elusions, the preconcentration process was repeated for many times. It was indicated that the results were satisfactory, even by using one column for 10 times, without changing the packing. The different volumes of Mo solution, 1.0×10 M in the range of 10-1000 mL were passed through the column and the signal of each eluted solution was compared with calibration curve data which is achieved from determination method. The obtained signals of concentrated Mo solutions presented that a preconcentration factor of 100 can be achieved by this method. The effect of ionic strength on the sensitivity was studied. The sensitivity would be slightly changed with increasing the ionic strength RI WKH UHDFWLRQ PL[WXUH 3.3. Retention capacity of the adsorbent The retention capacity of organic-solution-processable functionalized-graphene (SPFGraphene) adsorbent was determined by a batch method. The 20 mL solution of Mo(II) 1.0×10-4 M in pH 5.0 was transferred into a separating funnel and 3 g adsorbent was

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 495-502

Moghimi A

added. The separating funnel was shaken vigorously IRU PLQ &RQFHQWUDWLRQ RI 0R LQ WKH ÂżOWUDWH ZDV GHtermined according to calibration curve data and then adsorbed amount of Mo was calculated. The retention capacity (mg adsorbed Mo/ g adsorbent) was obtained to be 0.1672 mg g-1 of adsorbent or 2.01 mg g-1 of ligand. 3.4. Calibration graph, reproducibility and detection limit A series of standard solutions of Mo(II) were treated XQGHU WKH DERYH PHQWLRQHG RSWLPL]HG H[SHULPHQWDO conditions. Mo concentration can be determined in the UDQJH RI ĂŽ to 2.7Ă—10-5 M with linear equation; ∆$ ĂŽ& DQG UHJUHVVLRQ FRHIÂżFLHQW of r2 ∆A is absorbance signal after preconcentration and C is molar concentration of MoĂ—106). 7KH H[SHULPHQWDO OLPLW RI GHWHFWLRQ LV ĂŽ M 6 1 7KH UHODWLYH VWDQGDUG GHYLDWLRQ 5 6 ' for 10 replicate measurements of 5.0Ă—10 , 1.0 Ă—10 , 1.0Ă—10-7, 3.0Ă—10-6 and 1.0Ă—10-5 M of Mo(II) ZHUH DQG UHVSHFWLYHO\ ,QĂ€XHQFH RI IRUHLJQ LRQV 7KH LQĂ€XHQFH RI FRQWDPLQDQW VSHFLHV SUHVHQWHG LQ various samples on the determination of 5.0Ă—10-6 M, Mo(II)was investigated. The tolerance limit was deÂżQHG DV WKH FRQFHQWUDWLRQ RI DGGHG LRQV FDXVLQJ D relative error less than 3% (Table 1). Some metal cations can be adsorbed on microcrystalline naphthalene at different pH values. This proposed adsorbent is not only able to remove anions of the Mo(II) solution but also can decrease the interference of some cations. Some of important ions that can be found in the real

Table 1: Interferences effect on the determination of 5.0 Ă— 10-6 M, Mo(II).

Tolerance limit :ion : Mo(II) )

Species

100

NH4 , Na , K , H3BO3, Hg , Ba , Cd , Co , Ca , Pb , Sn , Sr , Tl

Al , Mg , Cr , Cu

25

Fe , Fe

Zn

1

Sb , Ag

samples with Mo(II) such as Sn , Hg , Cd , Co , Ca , Pb and Tl do not have any interference on the determination of Mo(II). Al and Fe can be troublesome in the determination procedure but with preconcentration their interference decreases. The reported method is selective and simple and it KDV H[FHOOHQW FDSDFLW\ IDFWRU $PRQJ RWKHU PHQWLRQHG methods in Table 2 stripping voltammetry has a good VHQVLWLYLW\ EXW LW QHHGV H[SHQVLYH DSSDUDWXV DQG QHHGV WKH RSHUDWRU WR EH VNLOOIXO &RQYHQWLRQDO VROYHQW H[tractions are not sensitive enough and they consume a ODUJH DPRXQW RI VROYHQW $OVR PRVW RI H[WUDFWLQJ VROYHQWV DUH WR[LF DQG YRODWLOH On the basis of the results obtained from the Mo(II) standards, the recommended preconcentration method has been successfully applied prior to spectrophtomeric determination of low values of Mo in the tap water

Table 2: Comparison of some methods for preconcentration and determination of thallium with proposed method.

500

Reference

/'5 QJ P/Ă­ )

'/ QJ P/Ă­ )

Method

[21]

Âą

1

6ROLGÂąOLTXLG H[WUDFWLRQ

[22] [20]

0.1–100 5–250

1

Potentiometric stripping

[23] [24] –

5–20 ¹ 2.1–2000

4 20 0.3

/LTXLGÂąOLTXLG H[WUDFWLRQ Microcrystalline naphthalene Proposed method

Moghimi A

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 495-502

Table 3: Spectrophotometric of Mo(II) in the real samples after preconcentration.

Recovery %

ICP

Found (Ă—10Ă­ M)

Added (Ă—10Ă­ M)

–

“ “ “

– “ “ “

– 1.0 5.0

–

10.41 ¹ 0.07 – “ “ “ “

“ – “ “ “ 10.47 ¹ 0.076

10.0 – 1.0 5.0 10.0

Sample

'ULQNLQJ ZDWHU

River water

Table 4: Analytical characteristics of organic-solution-processable functionalized-graphene (SPFGraphene) for determination of Mo(II).

Parameter

Analytical feature

Linear range ( M ) r2 Limit of detection (ng LĂ­ Äą Q

5HSHDWDELOLW\ 5 6 ' a, %) (n = 10) Enrichment factorb Enhancement factor Sample volume (mL)

10Ă­ to 2.7Ă—10Ă­ Ă— 10Ă­ MĂ— 3.0 122 100 10.00

([WUDFWLRQ WLPH PLQ

(a)

<4

Mo(II) concentration was 20 nM for which R.S.D. was obtained;

(b)

Enhancement factor is the slope ratio of

calibration graph after and before extraction.

(Tehran, taken after 10 min operation of the tap), rain water (Tehran, 22 January, 2013) samples. The analysis was performed by using the standard addition technique. The results are summarized in Table 3. Good recoveries in all samples were obtained. This method was reliable through comparing with each other [2426]. Table 4 summarizes the analytical characteristics of the optimized method, including linear range, limit of detection, reproducibility, and enhancement factor. The calibration graph was linear in the range of ĂŽ to 2.7Ă—10-5 M of Mo. The limit of detection, GHÂżQHG DV &/ 6% P ZKHUH &/ 6% DQG P DUH the limit of detection, standard deviation of the blank and slope of the calibration graph, respectively), was

ĂŽ 0 7KH SUHFLVLRQ H[SUHVVHG DV UHODWLYH VWDQdard deviation of three measurements is better than 3.0%. The enhancement factor was obtained from the slope ratio of calibration graph after and before H[WUDFWLRQ ZKLFK ZDV DERXW

4. CONCLUSIONS 6ROLG OLTXLG H[WUDFWLRQ ZLWK PLFURFU\VWDOOLQH QDSKWKDlene is an effective separation and preconcentration technique for trace elements. The method has the adYDQWDJHV RI EHLQJ VLPSOH LQH[SHQVLYH DQG VHOHFWLYH this proposed preconcentration method has a high en501

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 495-502

richment factor (100) which develops possibility of determining concentration levels as low as sub micro amounts of Mo with eliminating the interference of some diverse ions. The selected determination procedure (after preconcentration) is convenient, sensitive and fairly selective.

ACHNOWLEDGMENTS The authors wish to thank the chemistry department of Varamin branch Islamic Azad University for ÂżQDQFLDO VXSSRUW

REFERENCES 1. Izatt R.M., Bradshaw J.S., Nielsen S.A., Lamb - ' &KULVWHQVHQ - - 6HQ ' Chem. Rev., 85 2. Izatt R.M., Pawlak K., Bradshaw J.S., Bruening R.L., Chem. Rev., 91 3. Izatt R.M., Pawlak K., Bradshaw J.S., Bruening R.L., Chem. Rev., 95 %ODNH $ - 'HPDUWLQ ) 'HYLDOORQRYD ) $ *DUDX A., Isaia F., Lippolis V., Schroder M., Verani G., J. Chem. Soc., Dalton Trans $UFD 0 %ODNH $ - &DVDE - 'HPDUWLQ ) 'HYLOlanova F.A., Garau A., Isaia F., Lippolis V., Kivekas R., Muns V., Schroder M., Verani G., J. Chem. Soc., Dalton Trans 6. Gomes M.M., Hidalgo Garcia M.M., Palacio Corvillo M.A., Analyst, 120 %RXGUHDX 6 3 &RRSHU : 7 Anal. Chem., 61 0DKPRXG 0 ( 6ROLPDQ ( 0 Talanta, 44 0DKPRXG 0 ( Talanta, 44

502

Moghimi A

10. Mahmoud M.E., Talanta, 45 11. Mahmoud M., Anal. Chim. Acta, 398 0 ( 0DKPRXG In: Proceeding of the 25th FACSS Conference, Austin, TX, USA, and 11–15 October. 7RQJ $ $NDPD < 7DQDND 6 Anal. Chim. Acta, 230 14. Moghimi A., Ghiasi R., Abedin A.R., Ghammamy S., Afr. J. Pure Appl. Chem., 3 15. Moghimi A., Chin. J. Chem., 25 (10) (2007), 640. 0RJKLPL $ 7HKUDQL 0 6 :DTLI +XVDLQ 6 Mater. Sci. Res. India, 3 (1a) (2006), 27. 17. Moghimi A., Abdouss M., Afr. J. Pure Appl. Chem., 6 :DQJ + =KDQJ + 6 &KHQJ - . Talanta, 48 =DUJDUDQ 0 6KRXVKWDUL $ 0 $EGRXVV 0 J. Appl. Polym. Sci, 110 20. Tabarzadi M., Abdouss M., Hasani S.A., Shoushtary A.M., Matwiss U., Werkstofftech, 41 (4) (2010), 221. 6KLQ ' + .R < * &KRL 8 6 .LP : 1 Ind. Eng. Res., 43 (2004), 2060. 22. Nayebi P., Moghimi A., Orient. J. Chem., 22 (3) (2006), 507. +DVKHPL 2 5 0DUJDU 5D]L . 5DRX¿ ) 0RJKLmi A., Aghabozorg H., Ganjali M. R., Microchem. J., 69 (2001), 1. 24. Ganjali M.R., Pourjavid M.R., Hajiagha Babaei L., Niasari M.S., Quim Nova, 27 (2004), 213. 25. Moghimi A., Shabanzadeh M., JCHR, 2 (2) (2012), 7. 26. Tang C., Tracz A., Kruk M., Zhang R., Smilgies ' 0 0DW\MDV]HZVNL . .RZDOHZVNL 7 J. Am. Chem. Soc., 127

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 503-510

ISSN: 2251-8533

International Journal of Bio-Inorganic Hybrid Nanomaterials Three-Component Procedure for the Synthesis Chiral Spirooxindolopyrrolizidines via Catalytic Highly Enantioselective 1,3-Dipolar Cycloaddition of Azomethineylides and 3-(2-Alkenoyl)-1,3-Oxazolidin-2-ones Mohammad Javad Taghizadeh1*, Khosrow Jadidi2 1

Ph.D.Student, Department of Chemistry, University of Imam Hossein, Tehran, Iran 2

Ph.D., Department of Chemistry, University of Shahid Beheshti, Tehran, Iran 5HFHLYHG 6HSWHPEHU $FFHSWHG 'HFHPEHU

ABSTRACT The catalytic highly regio- diastereo-, and enantioselective synthesis of a small library of spiropyrrolizidineoxindoles YLD D IRXU FRPSRQHQW GLSRODU F\FORDGGLWLRQ UHDFWLRQ RI D]RPHWKLQH\OLGHV GHULYHG IURP LVDWLQ ZLWK HOHFWURQ GHÂżFLHQW GLSRODURSKLOH ZDV GHVFULEHG 7KH SURFHVV RFFXUV DW URRP WHPSHUDWXUH LQ DTXHRXV HWKDQRO DV D JUHHQ VROYHQW DQG LQ WKH SUHVHQFH RI D ELGHQGDWHELV LPLQH Âą&X ,, WULĂ€DWH FRPSOH[ DV FDWDO\VW 7KH UHDFWLRQ PHFKDQLVP LV GLVFXVVHG RQ WKH EDVLV RI WKH DVVLJQPHQW RI WKH DEVROXWH FRQÂżJXUDWLRQ RI WKH F\FORDGGXFWV Keyword: &KLUDO VSLUR R[LQGRORS\UUROL]LGLQHV $V\PPHWULF GLSRODU &KLUDO DX[LOLDULHV $]RPHWKLQH\OLGH Three-component reaction; Proline; Sarcosine.

1. INTRODUCTION Catalytic asymmetric multicomponent reaction (CAM&5 LV RQH RI WKH PRVW HIÂżFLHQW SURFHVVHV LQ WHUPV RI chirality economy and environmental benignity. In addition, this strategy has manifested as a powerful tool IRU WKH UDSLG LQWURGXFWLRQ DQG H[SDQVLRQ RI PROHFXODU diversity [1]. It is therefore desirable to utilize and develop this method for the synthesis of important hetHURF\FOHV VXFK DV FKLUDO VSLURR[LQGROS\UUROL]LGLQHV DQG (*) Corresponding Author - e-mail: Mohammadjavadtaghizadeh31@yahoo.com.

VSLURR[LQGROSUROLQHV OLNH KRUVÂżOLQH > @ HODFRPLQH > @ U\FKQRSK\OLQH H[KLELW VLJQLÂżFDQW ELRORJLFDO DFWLYLWLHV [4] (Figure 1). Asymmetric multicomponent 1,3 dipolar cycloaddition of azomethineylides with alkenes can be a great interest and useful strategies for stereoselective synthesis and develop of these class of molecules and compounds having similar structure [5]. :H UHFHQWO\ UHSRUWHG WKH HQDQWLRPHULFDOO\ SXUH QRYHO

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Taghizadeh MJ et al

Me

N

Et NH

N

MeO

OMe

O

O

N H

N H

HO

(-) horsfiline

O HO

N H

CO2Me

richnophiline

( ) elacomine

Figure 1: Spiro pyrrolidineoxindole alkaloids.

VSLURR[LQGROS\UUROL]LGLQHV > @ E\ DSSO\LQJ RSWLFDOO\ DFWLYH FLQQDPR\OR[D]ROLGLQRQH DV FKLUDO DX[LOLDU\ DQG WKH HQDQWLRVHOHFWLYLWLHV ZHUH H[FHSWLRQDOO\ KLJK However, it requires the use of at least one equivalent RI HQDQWLRSXUH DX[LOLDU\ 7R UHVROYH WKLV SUREOHP DQG in continuation of our previous work on the synthesis RI VSLURR[LQGROHV > @ ZH DSSOLHG FRSSHU FRPSOH[ RI F\FORKH[DQH ELV DU\OPHWK\OHQHDPLQH OLJDQGV as a catalyst to synthesis of a small library of this imSRUWDQW FODVV RI VSLURR[LQGROV > @ )LJXUH +HUHLQ ZH ZLVK WR UHSRUW D KLJKO\ H[R DQG HQDQWLRVHOHFWLYH Cl

Cl

N

N

1a

R

R

1,3-dipolar cycloaddition reaction of azomethineyOLGHV GHULYHG IURP LVDWLQ ZLWK HOHFWURQ GHÂżFLHQW dipolarophile by using bidendate bis(imine)-Cu(II) FRPSOH[ WKDW FDQ EH UHDGLO\ DVVHPEOHG IURP FRPPHUFLDOO\ DYDLODEOH WUDQV F\FORKH[DQHGLDPLQH DQG a variety of suitable aldehyde precursors, in optimized UHDFWLRQ FRQGLWLRQ %DVHG RQ H[SHULHQFHV LQ RXU SUHYLRXV ZRUNV DQG OLWHUDWXUH VXUYH\ > @ ,QLWLDOO\ WKH HIIHFWV RI VXEVWLWXHQWV RI ELV LPLQHV OLJDQGV ZHUH H[DPined using 10 mol% [Cu(OTf)2] as catalyst in a typical reaction of azomethineylide 2a with dipolarophile 3a at room temperature in aqueous ethanol as a solvent (Scheme 1). The results are summarized in Table 1.

Cl

1c

1b

R=

2. RESULTS AND DISCUSSION O

1e

S

N

1f

The ligands 1b and 1c bearing the electron-withdrawing and relatively bulky Cl substituents at the 2- or/ and 6-positions of the benzene ring resulted in considerably higher yields and enantioselectivities in

1d

Figure 2: Cyclohexane-1,2-bis(arylmethyleneamine) ligands 1(a-f).

O X O + N H

N R1

CO2H

2

- CO2

H O

H R2

Ligand 1

N H

O

O

EtOH RT, h

N

X

H NH

X

R2

O O N R1

[ +2]

N R1

O N

O 4

Scheme 1: Asymmetric synthesis of new chiral spirooxindolopyrrolizidines 4 with ligand of 1.

504

O

Taghizadeh MJ et al

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 503-510

Table 1: Asymmetric synthesis of new chiral spirooxindolopyrrolizidines with ligand of 1(a- f).

(a)

7 ƒ&

4a

Entry

Ligand

Time (h)

1

1a

25

24

55

2

1b

25

22

3

1c

25

20

63

4

1d

25

Race

5 6 7

1e 1f 1b 1b

25 25 0 -40

32 35

73 35 <10

Race Race n.d

<LHOG b

Ee (%)c

5HDFWLRQ RI D PPRO ZLWK D PPRO ZDV FDUULHG RXW LQ PO RI (W2+ DW URRP WHPSHUDWXUH LQ WKH SUHV-

ence of 10% catalyst [Cu(OTf)2 -1=1.0: 1.1], unless otherwise noted;

(b)

Isolated yield;

(c)

Determined by chiral HPLC

analysis.

comparison with the other ligands [10]. The highest HQDQWLRVHOHFWLYLW\ DQG \LHOG LQ KLJK VHOHFWLYitywere achieved by employing ligand 1b. The yields and enantiomeric ratios of the products showed the temperature dependence of this process. A decrease in WKH UHDFWLRQ WHPSHUDWXUH IURP ƒ& WR ƒ& JUHDWO\ decreased the reaction yield and enantioselectivity HQWULHV &RQVLGHULQJ WKH E DV WKH EHVW OLJDQG we tested the effect of Cu salts (Table 2). In all cases, Cu(OTf)2 proved to be the best copper source while RWKHU &X VDOWV OHG WR D GHFUHDVH LQ WKH HH E\ and longer reaction times (entries 3-4 vs.2). The use of Zn(OTf)2 instead of Cu(OTf)2 gave worse result in term of enantioselectivity (entry1). The effects of catalyst loading were also investigated and the best

results were obtained when 10 mol % catalysts loading was used in the reaction. The ligand-to-metal ratio of 1.1:1 using 20 mol % of ligand was investigated under the similar conditions and the isolated yields DQG HQDQWLRVHOHFWLYLW\ UHPDLQHG WKH VDPH DW UHspectively. Lowering the catalyst loading to less than 10 mol % led to a sharp decrease in the results. It should be noted, the addition of additives such as MS 4A, 3A did not give any observable changes in the results of the reaction and even lead to decreasing yields. Considering the optimized reaction conditions, we QH[W H[DPLQHG WKH VFRSH DQG JHQHUDOLW\ RI WKLV UHDFtion with various types of azomethine ylides and nuPHURXV DONHQR\O R[D]ROLGLQ RQHV DQG V\QWKHVL]HG D VPDOO OLEUDU\ RI QHZ FKLUDO VSLURR[LQGR-

Table 2: Dependence of reaction with Lewis acid.

4a

(a)

Entry

Lewis acid

Time (h)

<LHOG b

Ee (%)c

1

Zn(OAc)2

12

!

Race

2

Cu(OTf)2

22

3

Cu(OAc)2

23

66

4

Cu(Cl)2

76

Race

5

Cu(OTf)2d

22

5HDFWLRQ RI D PPRO ZLWK D PPRO ZDV FDUULHG RXW LQ PO RI (W2+ &+2Cl2 at room tem-

perature in the presence of 10% catalyst [Lewisacid-1=1.0:1.1], unless otherwise noted; (b) Isolated yield; (c)

Determined by chiral HPLC analysis; (d) 20% catalyst is used.

505

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Taghizadeh MJ et al

Table 3: Asymmetric synthesis of new chiral spirooxindolopyrrolizidines derivaitives 4.

X

R1

R2

1

H

H

Me

4a

2

H

H

Ph

4b

3

H

Me

Me

4c

4

H

Et

Ph

4d

5

H

Bn

Me

4e

6

Br

H

Me

4f

7

Br

Me

Me

4g

Br

Et

Me

4h

10

Br

Me

Ph

4i

11

NO2

H

Me

4j

lopyrrolizidines 4a-j (Table 3). The structures of cycloadducts were assigned from their elemental and spectroscopic analyses including IR, 1H NMR, 13C NMR, and mass spectral data. The observation of two characteristic triplets and one doublet in the 1+ 105 VSHFWUD RI SURGXFWV FRQÂżUPHG unambiguously the formation of a new pyrrolizidine ULQJ :H DOVR ZHUH DEOH WR REWDLQ VXLWDEOH FU\VWDOV RI WKH J IRU FU\VWDOORJUDSK\ WR FRQÂżUP WKH DVVLJQHG VWHreochemistry of products 4 that was carried out here using several NMR spectroscopy techniques. The ORTEP view of single crystal X-ray analysis of 4g with atomic numbering is shown in Figure 3. On the basis of X-ray structure of 4, we can now assign the IRXU FKLUDO FHQWHUV LQ VSLURS\UUROL]LGLQHR[LQGROH J WR EH 5 VSLUR FDUERQ & 6 & 5 & 5 (C13). X-ray crystallographic analysis of compound J DOVR FRQÂżUPHG WKLV DEVROXWH FRQÂżJXUDWLRQ Because reactions of most non-stabilized azomeWKLQH\OLGHV ZLWK HOHFWURQ GHÂżFLHQW GLSRODURSKLOHV DUH HOMO (dipole)-LUMO(dipolarophile) controlled [11], thus, in order to obtain an increased reaction rate, the 3-Cu(OTf)2 ZDV FRRUGLQDWHG WR WKH HOHFWURQ GHÂżFLHQW dipolarophileto form square planner geometry [12]. 506

Product

<LHOG (H

Entry

On the other hand, condensation of isatin derivative DQG 6 SUROLQH DIWHU GHFDUER[\ODWLRQ OHG WR WKH QRQ VWDELOL]HG D]RPHWKLQH\OLGH 7KH > @ F\FORDGdition of activated dipolarophileswithazomethineylide UHVXOWHG LQ WKH IRUPDWLRQ RI FKLUDO VSLURR[LQGRORS\Urolizidine 4 which contain contiguous stereogenic FHQWHUV 'HVSLWH WKH IDFW WKDW VL[WHHQ GLIIHUHQW VWHUHRisomers could be prepared theoretically, only diaste-

Figure 3: ORTEP diagram of one of the four crystallographic independent molecules in the asymmetric unit of 4g. Thermal ellipsoids are at 30% probability level.

Taghizadeh MJ et al

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 503-510

O OH

H N CO2H

N H

-CO2

N O

O

O

N

R

N

R

Cu O O

O O

N H

N R

N R Cl

N R

N

N

Cu TfO OTf

H

N

R Cl

Cl

Cu Cl O O N O

Re-faced attack Repulsive R Si-faced attack favorable N

R

O

N

N H

O N H

O N

O O

Scheme 2: Propose of the transition state and the reaction pathway.

reoisomer 4 was obtained in high yield in all the cases that we present in this article (Scheme 2). Based on WKH VWHUHRFKHPLVWU\ RI WKH F\FORDGGXFW WKDW FODULÂżHG E\ VLQJOH FU\VWDO ; UD\ DQDO\VLV DQG ' 105 VSHFtroscopic techniques, the transition state and the reaction pathway were proposed as below:

3. EXPERIMENTAL General procedure: To a magnetically stirred solution of an isatin derivatives (1 mmol), proline (1 mmol) DQG FKLUDO R[D]ROLGLQRQH PPRO DV GLSRODURSKLOH LQ 10 mL EtOH was added dropwise at room temperaWXUH 7KHQ WKH UHDFWLRQ PL[WXUH ZDV VWLUUHG IRU K The solvent was then removed under reduced pressure and the residue was separated by recrystalization in CHCl3.

4. CONCLUSIONS 6LPSOH F\FORKH[DQH ELV DU\OPHWK\OHQHDPLQH

OLJDQGV ZLWK FRSSHU ,, WULĂ€DWH FDWDO\]HG GLSRlar cycloaddition reaction of azomethineylides with HOHFWURQ GHÂżFLHQW GLSRODURSKLOH WR JLYH VSLURS\UUROL]LGLQHR[LQGROHV LQ JRRG \LHOG ZLWK KLJK UHJLR GLDVWHUHR DQG HQDQWLRVHOHFWLYLW\ XS WR HH LQ optimized condition. The reaction was accomplished with 10% catalyst at room temperature in environmentally friendly aqueous ethanol. The structures of the products were elucidated using IR, mass, one and two dimensional NMR techniques, and X-ray single FU\VWDO GLIIUDFWLRQ 7KH UHDFWLRQ PHFKDQLVP LV EULHĂ€\ discussed on the basis of the assignment of the absoOXWH FRQÂżJXUDWLRQ RI WKH F\FORDGGXFW 6 6 5 D 5 PHWK\O R[R D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH D ZKLWH SRZGHU PS ƒ& \LHOG >Îą@' F &+2Cl2), IR(KBr)(Ď…PD[, cm-1 & 2 & 2 (C=O), 3430(NH); 1+105 0+] &'&O3); 1.17 (3H, d, 3JHH=6.3 Hz, CH3 + P &+2), + P &+ + P &+ (2H, m, CH2 + P &+ + 507

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 503-510

m, CH and CH2), 4.13-4.21 (1H, m, CH), 4.31 (1H, d, 3 JHH +] &+ + P $U + + s, NH); 13&105 0+] &'&O3 & &+3), & &+2 & &+ & & &+ & & 3C=O); MS, 352 (M 5 6 5 D 5 R[R SKHQ\O D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH E ZKLWH SRZGHU PS & \LHOG >α@' F &+2Cl2), IR(KBr)(υPD[, cm-1 & 2 & 2 (C=O), 3430(NH); 1+105 0+] &'&O3); 1.77-2.02 (4H, m, 2CH2), 2.67 (1H, m, CH), 3.15 + P &+ + P &+ + P OCH2 &+ + P &+ + G 3JHH +] &+ + P $U + + V 1+ 13 &105 0+] &'&O3 (4C, 4CH2 & &+ & OCH2 & & &+ & &+ & & & 2 06 (M ,7), 200 (100), 131 (70). 6 6 5 D 5 GLPHWK\O R[R D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH F ZKLWH SRZGHU PS & \LHOG >α@' F &+2Cl2), IR(KBr) (υPD[, cm-1 & 2 & 2 (C=O); 1+105 0+] &'&O3); 1.16 (3H, d, 3 JHH=6.3 Hz, CH3), 1.72-2.13 (4H, m, 2CH2), 2.57 + P &+ + P &+ + P &+ 3.16 (3H, s, NCH3 + P &+ + m, CH and CH2), 4.11 (1H, m, CH), 4.21 (1H, m, 3 JHH +] &+ + G 3JHH +] &+ (1H, m, CH), 7.14 (1H, d, 3JHH +] &+ + m, CH); 13&105 0+] &'&O3); 16.4 (1C, CH3), 24.7, 26.4, 27.6, 43.1, 62.7 (5C, 5CH2), 41.2 (1C, NCH3 & &+ & & &+ & & & 2 06 0 , 5 6 5 D 5 HWK\O R[R SKHQ\O D KH[DK\GURVSLUR>LQGROLQH

Taghizadeh MJ et al

S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH G <HOORZ SRZGHU PS & \LHOG >α@' (c 0.01, CH2Cl2) IR(KBr) (υPD[, cm-1): 1613(C=O), & 2 & 2 1HNMR (300.1 MHz, &'&O3 + W 3JHH=7.2 Hz, CH3), 1.76-2.01 (4H, m, 2CH2), 2.67 (1H, m, CH), 3.13 (1H, m, CH), 3.61 + P &+ + P &+ &+2), 4.46 + P &+ + G 3JHH +] &+ + P $U + 13&105 0+] &'&O3 (1C, CH3 & &+2), 35.0 (1C, NCH2 & &+ & OCH2 & & &+ & &+ & & & 2 06 (M 6 6 5 D 5 EHQ]\O PHWK\O R[R D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH H ZKLWH SRZGHU PS & \LHOG >α@' F 0.01, CH2Cl2), IR(KBr) (υPD[, cm-1): 1616(C=O), & 2 & 2 1HNMR (300.1 MHz, &'&O3); 1.15 (3H, d, 3JHH=6.6 Hz, CH3 (4H, m, 2CH2), 2.65(1H, m, CH), 3.16 (1H, m, CH), + P &+ + P &+2 4.11(2H, m, CH2), 4.52(1H, m, CH), 4.76 (1H, d, 3 JHH +] &+ + G 3JHH +] &+ 5.13 (1H, d, 3JHH +] &+ + P Ar-H); 13&105 0+] &'&O3); 15.5(1C, CH3), & &+2 & 3CH), 54.3 (1C, NCH2 & 2&+2), 72.1(1C), & &+ & &+ & & & 2 06 0 6 6 5 D 5 EURPR PHWK\O R[R D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH I <HOORZ SRZGHU PS & \LHOG >α@' (c 0.01, CH2Cl2), IR(KBr) (υPD[, cm-1): 1614(C=O), & 2 & 2 1+ 1HNMR (300.1 0+] &'&O3); 1.15 (3H, d, 3JHH=6.7 Hz, CH3), 1.76 + P &+2), 2.07-2.17 (1H, m, CH2), 2.57 (1H, P &+ + P &+2), 3.56 (1H, dt, 2JHH=12 Hz, 3JHH +] &+ + P &+ DQG &+2),

Taghizadeh MJ et al

4.16 (1H, dt, 2JHH=12 Hz, 3JHH=6 Hz, CH), 4.31 (1H, d, JHH +] &+ + G 3JHH +] $U + (1H, m, Ar-H), 7.47 (1H, d, 3JHH +] $U + (1H, s, NH); 13&105 0+] &'&O3); 16.1(1C, CH3), 24.2, 27.4, 41.2, 42.7, 62.5 (5C, 5CH2 & &+ & & &+ & & & 2 06 0 , M 3

6 6 5 D 5 EURPR GLPHWK\O R[R D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH J ZKLWH SRZGHU PS & \LHOG >α@' (c 0.01, CH2Cl2), IR(KBr) (υPD[, cm-1): 1614(C=O), & 2 & 2 1HNMR (300.1 MHz, &'&O3); 1.16 (3H, d, 3JHH=5.1 Hz, CH3), 1.76-2.17 (4H, m, 2CH2 + P &+ + P &+2), 3.14(3H, s, NCH3), 3.66 (1H, m, CH), 3.04 (3H, m, CH and CH2), 4.17 (1H, m, CH), 4.37 (1H, d, 3JHH Hz, CH), 6.67 (1H, d, 3JHH +] $U + + s, Ar-H), 7.43 (1H, d, 3JHH +] $U + 13CNMR 0+] &'&O3); 16.4(1C, CH3), 24.5, 27.7, 41.3, & &+2), 42.4(1C, NCH3 & &+ & & &+ & & & 2 06 0 , M (M , M 3-((1'S,2'S,3R,7a'R)-5-bromo-1-ethyl-1'-methyl R[R D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH K <HOORZ SRZGHU PS & \LHOG >α@' (c 0.01, CH2Cl2), IR(KBr) (υPD[, cm-1): 1611(C=O), & 2 & 2 1HNMR (300.1 MHz, &'&O3); 1.15 (3H, d, 3JHH +] &+3 + W 3 JHH=7 Hz, CH3 + P &+2 (1H, m, CH2 + P &+ + P CH2 + T 3JHH=7 Hz, CH2), 3.56 (1H, dt, 2 JHH=12 Hz, 3JHH +] &+ + P &+ and CH2), 4.16 (1H, dt, 2JHH=12 Hz, 3JHH=6 Hz, CH), 4.31 (1H, d, 3JHH +] &+ + G 3JHH Hz, Ar-H), 7.27 (1H, m, Ar-H), 7.47 (1H, d, 3JHH Hz, Ar-H); 13&105 0+] &'&O3); 12.4 (1C, CH3), 16.1(1C, CH3 & 5CH2), 35.1 (1C, NCH2 & &+

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& & &+ & & & 2 MS, 462, 464 (M , M 0 , M 60), 131 (100). 5 6 5 D 5 EURPR PHWK\O R[R SKHQ\O D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH L ZKLWH SRZGHU PS & \LHOG >α@' (c 0.01, CH2Cl2), IR(KBr) (υPD[, cm-1): 1614(C=O), & 2 & 2 1HNMR (300.1 MHz, &'&O3); 1.77-2.02 (4H, m, 2CH2), 2.67 (1H, m, CH), 3.15 (1H, m, CH), 3.24 (3H, s, NMe), 3.61 (1H, m, &+ + P 2&+2, 2CH), 4.46 (1H, m, &+ + G 3JHH +] &+ + m, Ar-H); 13&105 0+] &'&O3); 24.4, 27.4, & &+2), 42.1 (1C, NCH3 & &+ & 2&+2), 72.1(1C), 110.6, & &+ & &+ & & & 2 06 0 , M 0 , M 6 6 5 D 5 PHWK\O QLWUR R[R D KH[DK\GURVSLUR>LQGROLQH S\UUROL]LQH@ \OFDUERQ\O R[D]ROLGLQ RQH M <HOORZ SRZGHU PS & \LHOG >α@' (c 0.01, CH2Cl2), IR(KBr)(υPD[, cm-1): 1615(C=O), & 2 & 2 1+ 1HNMR (300.1 0+] &'&O3 + G 3JHH=6.6 Hz, CH3), 1.67 + P &+2 + P &+2) 2.52 (1H, m, CH), 2.66 (2H, m, CH2 + P &+ + P &+ + P &+ + P CH2 + G 3JHH +] &+ + G 4 JHH +] &+ + GG 3JHH +] 4JHH=3 +] &+ + V 1+ 13CNMR (300.1 MHz, &'&O3 & &+3 (5C, 5CH2 & &+ & & &+ & & & 2 06 0 ,

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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 511-515

ISSN: 2251-8533

International Journal of Bio-Inorganic Hybrid Nanomaterials The Effect of Resistance and Progressive Training on HSP 70 and Glucose Farah Nameni Assistant Professor, Department of Physical Education, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran

5HFHLYHG FWREHU $FFHSWHG 'HFHPEHU

ABSTRACT Skeletal muscle may develop adaptive chaperone and enhancementdefense system through daily exercise stimulation. The present study investigated resistance and exhaustion training alters the expression of chaper one proteins. These proteins function to maintain homeostasis, facilitate repair from injury and provide protection. Exercise-induced production of HSPs in skeletal muscle and peripheral leukocytes and, it may provide insight into the mechanisms by which exercise can provide increased protection against stressors. The aim of this study was WR H[DPLQH WKH HIIHFW RI W\SHV H[HUFLVH WUDLQLQJRQ +63 H[SUHVVLRQ 1LQHWHHQ WUDLQLQJ IHPDOH LQ JURXSV WDNLQJ part in the intervention volunteered to give blood samples. Levels of chaperone proteins weremeasured in response WR UHVLVWDQFH DQG H[KDXVWLRQ WUDLQLQJ +63 OHYHOV ZHUH LQFUHDVHG LPPHGLDWHO\ DQG K DIWHU 3URJUHVVLYH WUDLQLQJ but decreased after resistance training. The data showed that human skeletal muscle responds to the stress of a single period of Progressive trainingby up regulating and resistance training by down regulating expression of +63 3K\VLFDO H[HUFLVH FDQ HOHYDWH FRUH WHPSHUDWXUH DQG PXVFOH WHPSHUDWXUHV DQG WKH H[SUHVVLRQ SDWWHUQ RI +63 GXH WR WUDLQLQJ VWDWXV PD\ EH DWWULEXWHG WR DGDSWLYH PHFKDQLVPV Keyword: +HDW VKRFN SURWHLQV ([HUFLVH WUDLQLQJ ,PPXQH V\VWHP 6WUHVVRU ,QĂ€DPPDWLRQ ,QMXULHV FHOO

1. INTRODUCTION Living cells are continually challenged by conditions which cause acute and chronic stress. Heat shock proteins (HSP) are a class of functionally related proteins ZKRVH H[SUHVVLRQ LV LQFUHDVHG ZKHQ FHOOV DUH H[SRVHG to elevated temperatures or other stress. Molecular chaperones such as heat shock proteins (HSPs) are (*) Corresponding Author - e-mail: farahnameni@yahoo.com.

known to contribute to reducing cellular damage. HSPs havemultiple functions in maintaining intracellular integrity viaprotection, repair and even control of cell death signaling [1-3]. They play an important role in proteins interactions such as folding and assisting in the establishment of proper protein conformation and pre-

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 511-515

vention of unwanted protein aggregation. Heat shock proteins (HSP) are increasingly seen as important players in the response of our biochemistry to stresses and damage. Few data have been reported concerning H[SUHVVLRQ RI +63V LQ KXPDQ WLVVXH DIWHU H[HUFLVH > @ HSP70 is associated with the protection of striated muscle from injury and the attenuation of skeletal muscle atrophy. Therefore enhancementof potenWLDOO\ FRQWULEXWHV WR WKH SURWHFWLRQ RI P\RÂżEHUV DQG WKH PDLQWHQDQFH RI PXVFOH PDVV 7KH H[SUHVVLRQ RI HSP70 is reported to be enhanced by thermalstress DQG H[HUFLVH > @ ,QKXPDQ OHXNRF\WHDQG VNHOHWDO PXVFOH H[KDXVWLYH HQGXUDQFH H[HUFLVH > @ DQG UHVLVWDQFH H[HUFLVH > @ PDUNHGO\ LQFUHDVH +63 H[SUHVVLRQ 7RJHWKHUWKHVH VWXGLHV VXJJHVW WKDW WKH H[SUHVVLRQ of molecular chaperone proteins may play important roles in protection and repair of skeletal muscle IURP H[HUFLVH LQGXFHG VWUHVV > @ 3XQWVFKDUW H[DPLQHG WKH HIIHFW RI DQ DFXWH ERXW RI H[HUFLVH RQ +63 H[SUHVVLRQ DQG IRXQG WKH FRQWHQW RI +63 P51$ increased, but there was no change in the HSP70 protein content. HSP70 was selected because this protein typically demonstrates very large increases after YDULRXV IRUPV RI FHOOXODU VWUHVV > @ 'HWHFWDEOH OHYHOV of HSP70 are present in the systemic circulation of healthy individuals. Previous studies have shown that the systemic concentration of HSP70 is elevated after H[HUFLVH > @ Several investigators have demonstrated the inGXFWLRQ RI +63 V\QWKHVLV LQ VSHFLÂżF UHJLRQV LQ response to hyperthermia [10], ischemia, and hySR[LD DQG HQHUJ\ GHSOHWLRQ +63V DUH TXLQWHVVHQWLDOO\ viewed as intracellular proteins with a vital role in PDLQWDLQLQJ FHOOXODU KRPHRVWDVLV LPSRUWDQW H[WUDFHOOXODU UROHV IRU +63V KDYH EHHQ LGHQWLÂżHG 7KH GHSOHWLRQ RI HQHUJ\ VWRUHV K\SR[LD DQG LVFKHPLD KDV EHHQ shown to induce the synthesis of HSP70 [11]. Prior H[HUFLVH WUDLQLQJDWWHQXDWHV FRQWUDFWLRQ LQGXFHG LQMXU\ LQ VNHOHWDO PXVFOH IROORZLQJDQ DFXWH VLQJOH ERXW RI H[ercise. The acquisition of muscle tolerance to contracWLRQ LQGXFHG PXVFOHGDPDJH WKURXJK H[HUFLVH WUDLQing appears to be partially associatedwith molecular mechanisms including chaperone functions in additionto neuromuscular and morphological adaptations. 6HYHUDO VWXGLHVKDYH UHSRUWHG WKDW SURORQJHG H[HUFLVH training increases several molecular chaperone pro512

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teins in skeletal muscle, such as HSP25, HSP70 and glucose-regulated protein (GRP) [2, 5 and 12]. HSPs KDYH SURWHFWLYHIXQFWLRQV DQWLR[LGDWLRQ DQWLDSRSWRVLV and helping protein formation [3]. These functions of HSPs may potentially contribute to acquisition of a muscledefense system with training [2]. HSPs are increasingly seen as important players in the response of our biochemistry to stresses and damage. 'HWHFWDEOH OHYHOV RI +63 DUH SUHVHQW LQ WKH V\Vtemic circulation of healthy individuals. Previous studies have shown that the systemic concentration of +63 LV HOHYDWHG DIWHU H[HUFLVH > @ 7KH GHSOHWLRQ RI HQHUJ\ VWRUHV K\SR[LD DQG LVFKHPLD KDV EHHQ VKRZQ WR LQGXFH WKH V\QWKHVLV RI +63 > @ 3ULRU H[HUFLVH training attenuates contraction-induced injury in skelHWDO PXVFOH IROORZLQJ DQ DFXWH VLQJOH ERXW RI H[HUFLVH

2. MATERIAL AND METHODS 1LQHWHHQ KHDOWK\ IHPDOHV SDUWLFLSDWHG LQ WKH VWXG\ on Progressive training group with mean age 23 Âą 2 \HDUV >“6(0@ ERG\ PDVV “ NJ DQG PD[LPDO R[\JHQ XSWDNH >92 PD[@ “ / PLQ DQG RQ UHVLVWDQFH WUDLQLQJ JURXS ZLWK DJH “ \HDUV [ÂąSEM], weight 57.3 Âą 6.53, height 161.1Âą 3.21 cm, ERG\ PDVV “ NJ DQG PD[LPDO R[\JHQ uptake[VO PD[] 5.1 Âą 0.4 L /min). Subjects were informed as to the potential risks associated with participation in the study before obtaining their written informed consent to participate.The study was carried out in accordance with the IAU and approved by the (WKLFDO &RPPLWWHH 7KH LQĂ€XHQFH RI H[KDXVWLRQ WUDLQing on HSP70 and glucose in peripheral leukocytes HYDOXDWHG EHIRUH DQG DW DQG K DIWHU E\ Ă€RZ F\tometry and RT/PCR, respectively and glucocard 01. Progressive training subjects performed Bruce 3URWRFRO RQ D WUHDGPLOO 7 +57 86$ 7KH ODERUDWRU\ WHPSHUDWXUH GXULQJ DOO WULDOV ZDV “ ƒ& Resistance training Subjects performed 30 minute resistance training. The laboratory temperature during DOO WULDOV ZDV “ ƒ& 3DLUHG VDPSOHV RI DQWLFXELWDO venous blood were collected in heparinized syringes and kept on ice until analysis for hematocrit on ABL 700 apparatus. To determine the serum HSP70 protein concentra-

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tion were obtained and placed in a tube containing a clot-inducing plug. This tube spun in a centrifuge at ĂŽ J DW ƒ& $ KLJKO\ VHQVLWLYH HQ]\PH OLQNHG immune sorbent assay method was used to determine the concentration of HSP70 protein in serum. All samSOHV ZHUH WHVWHG LQ GXSOLFDWH $OO GDWD DUH H[SUHVVHG DV means Âą SEM. Statistical analysis was conducted by using analysis of variance (ANOVA) repeated meaVXUHV DQG /6' WHVW ZDV XVHG IRU SRVW KRF PXOWLSOH comparisons among means. A P value < 0.05 was conVLGHUHG VWDWLVWLFDOO\ VLJQLÂżFDQW

3. RESULTS Figure 1: Mean HSP70 before exercise and in recovery in

After Progressive, the anticubital serum HSP70 concentration was elevated but after resistance training serum HSP70 concentration was decreased (Table and Figure 1). Repeated Measurers test were sigQLÂżFDQWO\ LQ WZR JURXSV 7DEOH 3! 7KH FKDQJHV VHUXP JOXFRVH FRQFHQWUDWLRQ )LJXUH ZHUH VLJQLÂżFDQWO\ LQ WZR JURXSV 7DEOH 3! %XW /6' WHVW ZHUH QRW VLJQLÂżFDQWO\ $QDO\VLV RI YDULDQFH $129$ ZHUH QRW VLJQLÂżFDQWO\ LQ WZR JURXSV 7DEOH 3!

2 groups.

evation in constitutive levels of this protective protein LQ OHXNRF\WH > @ 'DWD VXJJHVW WKDW +HDW VKRFN SURteins (HSP) possibly have a systemic function, includLQJ PHGLDWLRQ RI WKH HIIHFWV RI H[HUFLVH RQ LPPXQH IXQFWLRQ > @ ,QFUHDVHG +VS LQGLFDWHG WKDW WKH H[HUFLVH VWUHVV FDXVHG VXIÂżFLHQW LQWUDFHOOXODU GLVUXSWLRQ WR WULJJHU DQ +63 UHVSRQVH > @ $Q LQFUHDVHG FRQWHQW of HSPs will facilitate any cellular remodeling, that RFFXUV DIWHU XQDFFXVWRPHG H[HUFLVH > @ 2[LGDWLYH and other stresses are an important component of the cellular protective response. These proteins facilitate successful repair from injury and to aid adaptation and remodeling of the cell to prevent the damage [4].Ac-

4. DISCUSSION ([HUFLVH LV DQ LPSRUWDQW LQWHUYHQWLRQ LQ PDLQWDLQLQJ KHDOWK +RZHYHU H[KDXVWLRQ WUDLQLQJ UHVXOWV LQ DQ HO-

Table 1: MeanÂąStd. deviation HSP70 and glucose before exercise and in recovery. MeanÂąStd. Deviation Variables

Time

N

+63 (Ng/ml)

Glucose (Mg/dl)

“

“

“

“

2h after

“

“

before

“

“

“

“

“

“

before Progressive training

Resistance training

after

after 2h after

9

10

513

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 511-515

Nameni F

Table 2: The results of (ANOVA) repeated measures HSP70 and glucose in 2 groups. Time

Sig.

F

before

after

0.099

2h after

Table 3: Table 3: The results of ANOVA HSP70 and glucose in 2 groups.

Figure 2: Mean Glucose before exercise and recovery in 2 groups.

FXPXODWLQJ HYLGHQFH VWURQJO\ VXJJHVWV WKDW H[WUDFHOOXlar Hsp72 has potent immune regulatory effects [15]. 7HPSHUDWXUH LQFUHDVH R[LGDWLYH VWUHVV DQG LQĂ€DPPDWRU\ UHDFWLRQV DIWHU H[KDXVWLRQ H[HUFLVH ZHUH H[SHFWHG to stimulate synthesis of HSPs in peripheral blood leuNRF\WHV 6WUHQXRXV H[HUFLVH LQFUHDVHG +63 H[SUHVVLRQ in blood immediately at the end of running, which shows a positive function of HSP in leukocytes of athOHWHV WR PDLQWDLQ IXQFWLRQ DIWHU KHDY\ H[HUFLVH > @ +63 H[SUHVVLRQ LV DOVR DOWHUHG GXULQJ JOXFRVH GHSOHWLRQ DQG R[LGDWLYH VWUHVV &HOOV WKDW DUH VWDUYHG IRU glucose over produce a set of proteins called glucoseregulated proteins (GRP). The functions of HSP, GRP and OSP are incompletely understood, but evidence suggests that many stress proteins are enzymes that either provide immediate stress protection or conduct cellular repair processes. Sustained physical activity results in the progressive depletion of glucose and glycogen stores, a phenomenon that is highly correlated ZLWK IDWLJXH ,Q FHOOVGHSULYHG RI JOXFRVH RU R[\JHQ RU treated with agents that perturb calcium homeostasis, synthesis GRP and HSP70 [17]. The elevation of body temperature and depletion of glycogen [11] are all regarded as factors that induce +63 H[SUHVVLRQ LQ VNHOHWDO PXVFOH GXULQJ H[HUFLVH > @ :KHQ ERG\ WHPSHUDWXUH ZDV PDLQWDLQHG GXULQJ H[KDXVWLRQ H[HUFLVH +63 DFFXPXODWHG LQ SHULSKHUDO OHXNRF\WHV 7KHVH ÂżQGLQJV VXJJHVW WKH SRVVLELOLW\ WKDW the treadmill running, as used in the present study, PD\ VWLPXODWH VWUHVV UHVSRQVH WR LQGXFH +63 :H 514

Group

Factor

Sig.

F

Progressive training

+63

0.04*

glucose

.000*

+63

0.002*

glucose

.000*

Resistance training

FRQFOXGHG WKDW H[KDXVWLRQ H[HUFLVH HOHYDWHV WKH UHVWing level of peripheral leukocytes HSP70 and that the resultant accumulation of HSP70 helps to protect stress-loaded cells from injury due to the elevation of FKDSHURQH DFWLYLW\ > @ However,comparison amongst studies is comSOLFDWHG E\ YDULDWLRQV LQ H[HUFLVH SURWRFRO PRGH intensity, durationand damage), muscle group, and differences in subject characteristics (training and nuWULWLRQDO VWDWXV DJH VH[ ,Q UHVLVWDQFH WUDLQLQJ JURXS the down regulation of HSP-positive cells seems to EH D UHVXOW RI DGDSWDWLRQ PHFKDQLVPV WR WUDLQLQJ > @ 6R WKH UHGXFWLRQ RI +63 DV DQ LQGLFDWLRQ WKDW H[HUFLVH WUDLQLQJ UHGXFHV LQĂ€DPPDWLRQ 7KH PHFKDQLVP DUH LQYROYHG LQ V\VWHPLF ORZ JUDGH LQĂ€DPPDWLRQ LV QRW NQRZQ ,W LV SRVVLEOH +63V DUH OHDNHG LQWR WKH H[WUDFHOOXODU FRPSDUWPHQW GXH WR QHFURWLF FHOO GHDWK > @ HSP70 can be released independently of necrotic cell death in response to a number of stressful conditions LQFOXGLQJ H[KDXVWLYH H[HUFLVH > @ )XWXUH LQYHVWLJDWLRQV ZLOO H[SORUH WKH SK\VLRORJLFDO VLJQLÂżFDQFH RI H[WUDFHOOXODU +63V

5. CONCLUSIONS The data presented show that human leukocyte re-

Nameni F

VSRQGV WR WKH VWUHVV RI D VLQJOH SHULRG RI H[KDXVWLRQ training by up regulating and resistance training by GRZQ UHJXODWLQJ H[SUHVVLRQ RI +63 ,W LV SRVVLEOH WKH +63 UHVSRQVH WR H[HUFLVH LQ UHODWLRQ WR WKH WLVsue assayed (skeletal muscle, lymphocyte, venous and arterial serum). The differences observed when HSP70 in the present study may be related to the mode RI H[HUFLVH DQG WKH DPRXQW RI SURWHLQ GDPDJH DVVRFLDWHG ZLWK WKH H[HUFLVH

ACKNOWLEDGEMENTS :H WKDQN WKH VXEMHFWV IRU SDUWLFLSDWLQJ LQ WKLV GHPDQGing study and we acknowledge the technical assistance of Noor Lab. This study was supported by VaraminPishvaBranch Islamic Azad University.

REFERENCES 1. (a) Kregel K.C., J Appl. Physiol., 92 (2002), 2177; E /DWFKPDQ ' 6 Cardiovasc Res., 51 (2002), 637. 2JDWD 7 2LVKL < +LJDVKLGD . +LJXFKL 0 0Xraoka I., Am J Physiol. RegulIntegr Comp Physiol., 296 5 3. Sreedhar A.S., Csermely P., Pharmacol Ther., 101 (2004), 227. 4. Khassaf M., Child R.B., McArdle A., Brodie ' $ (VDQX & -DFNVRQ 0 - J. Appl. Physiol., 90 (2001), 1031. 5. Gonzalez B., Hernando R., Manso R., 3ÀXJHUV Arch., 440 (2000), 42. 2LVKL < 7DQLJXFKL . 0DWVXPRWR + ,VKLKDUD $ 2KLUD < 5R\ 55 J. Appl. Physiol., 92 (2002),

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7. Febbraio M., Koukoulas I., J. Appl. Physiol., 89 (2000), 1055. 7KRPSVRQ + 6 6FRUGLOLV 6 3 &ODUNVRQ 3 0 /RKUHU : $ Acta Physiol. Scand., 171 (2001), :DOVK 5 & .RXNRXODV , *DUQKDP $ 0RVHOH\ P.L., Hargreaves M., Febbraio M.A., Cell Stress Chaperones, 6 (2 /HRQL 6 %UDPELOOD ' 5LVXOHR * 'H )HR * Scarsella G., Mol. Cell Biochem., 204 (2000), 41. )HEEUDLR 0 $ 6WHHQVEHUJ $ :DOVK 5 .RXNRXlas I., Van Hall G., Saltin B., and Pedersen B.K., J Physiol., 53 12. Murlasits Z., Cutlip R.G., Geronilla K.B., Rao . 0 :RQGHUOLQ : ) $OZD\ 6 ( Exp. Gerontol., 41 0HOOLQJ - & : 7KRUS ' % 0LOQH . - .UDXVH M.P., Noble E.G., Am. Physiol. Lung Cell Mol. Physical, 293 + 0RUWRQ - 3 .D\DQL $ & 0F$UGOH $ 'UXVW % Sports Med., 39 15. Lancaster G.I., Moller K., Nielsen B., Secher N.H., Febbraio M.A., Nybo L., Cell Stress Chaperson, 9 (3) (2004), 276. *KRVK $ ([HUFLVH WUDLQLQJ KRZHYHU XSUHJXODWHV HQGRJHQRXV DQWLR[LGDQW GHIHQVHV DQG KHDW VKRFN SURWHLQ +63 H[SUHVVLRQ 30,' [PubMed-indexed for MEDLINE], 2003. 17. Fehrenbach E., Passek F., Niess A.M., Pohla H., :HLQWRFN & 'LFNKXWK + 1RUWKRII + Med. Sci. Sports Exerc., 32 0LNDPL 7 6XPLGD 6 ,VKLEDVKL < 2KWD 6 J. Appl. Physiol., 96 (5) (2004),1776. 2JDZD . 6DQDGD . 0DFKLGD 6 2NXWVX 0 Suzuki K., 0HGLDW,QÀDPP $UWLFOH ,' (2010), 7.

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ISSN: 2251-8533

International Journal of Bio-Inorganic Hybrid Nanomaterials 7KH 'HWR[LÂżFDWLRQ RI 0HWKDPLdophos as an Organophosphorus Insecticide on the Magnetite (Fe3O4) Nanoparticles/Ag-NaY Faujasite Molecular Sieve Zeolite (FMSZ) Composite Meysam Sadeghi1*, Mirhassan Hosseini2 1

M.Sc., Young Researchers and Elite Club, Islamic Azad University of Ahvaz Branch, Ahvaz, Iran 2

M.Sc., Payame Noor University, Germi Moghan, Ardebil, Iran & Nano Center Research, Imam Hossein Comprehensive University (IHCU), Tehran, Iran 5HFHLYHG 2FWREHU $FFHSWHG 'HFHPEHU

ABSTRACT 7KLV ZRUN XQGHUWDNHV WKH VWXG\ RI WKH GHWR[LÂżFDWLRQ UHDFWLRQV RI 2 6 GLPHWK\O SKRVSKRUDPLGLWKLRDWH PHWKDPLGRSKRV DV DQ RUJDQRSKRVSKRUXV LQVHFWLFLGH RQ WKH ZW PDJQHWLWH )H O4) nanoparticles/Ag-NaY IDXMDVLWH PROHFXODU VLHYH ]HROLWH )06= FRPSRVLWH 7KH LQĂ€XHQFH RI VROYHQW PHWKDQRO DFHWRQLWULOH DQG Q KH[DQH RQ WKH GHWR[LÂżFDWLRQ SRWHQWLDO RI FRPSRVLWH ZDV LQYHVWLJDWHG )RU WKLV VXUYH\ LQ WKH ÂżUVW VWHS VRGLXP W\SH < 1D< zeolite was synthesized by hydrothermal method (HM). Then, Ag-NaY zeolite was prepared from NaY zeolite by ion-exchange method (IEM). In the next step, Fe O4 nanoparticles (NPs) were incorporation and deposited on the Ag-NaY zeolite structure by using precipitation method (PM). The synthesized samples were characterized by SEM, EDAX and XRD techniques. The GC-FID and IR analysis results demonstrated that about 99% of methamidophos ZDV GHWR[LÂżHG DGVRUEHG YLD WKLV FRPSRVLWH DW Q KH[DQH VROYHQW DIWHU K 2Q WKH RWKHU KDQG WKH UHVXOWV IRU WKH acetonitrile and methanol solvents were lower. It seems that a nonpolar solvent transfer to the reactive surface site on the composite without occupying and blocking of these sites. Keyword: 'HWR[LÂżFDWLRQ 2 6 GLPHWK\O SKRVSKRUDPLGLWKLRDWH PHWKDPLGRSKRV ,QVHFWLFLGH PDJQHWLWH )H O4) nanoparticles/Ag-NaY faujasite molecular sieve zeolite (FMSZ); Composite.

1. INTRODUCTION O, S-dimethyl phosphoramidithioate or methamidophos with molecular formula C2H NO2PS is an organophosphorus insecticide (Figure 1), poses inevitable threat to persons who make contact, thereby causing health haz(*) Corresponding Author - e-mail: meysamsadeghi45@yahoo.com.

ards. Its mode of action in insects and mammals is by decreasing the activity of an enzyme important for nervous system function called acetylcholinesterase. This enzyme is essential in the normal transmission of nerve

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impulses. Methamidophos is a potent acetylcholinesterase inhibitor [1, 2]. Recently, there has been growing interest in the development of novel methods and PDWHULDOV IRU WKH GHFRQWDPLQDWLRQ DQG GHWR[LÂżFDWLRQ of organophosphorus pollutants (OPPs). Inorganic PHWDO R[LGHV DUH ZHOO NQRZQ IRU WKHLU XVH LQ FKHPLFDO industry as adsorbents, sensors, catalyst, etc. Because of their unique morphological features and high surIDFH DUHD QDQRFU\VWDOV RI PHWDO R[LGHV ZHUH XVHG DV adsorbents for decomposition or detection of variety of pollutants and harmful substances, including organophosphorous compounds [3]. On the other hand, zeolites are widely used in industry for water and waste water treatment, waste gas treatment, as catalysts, as molecular sieve, in the production of laundry detergents, nuclear processing medicine and in agriculture purposes for the preparation of advanced materials and recently to produce the nanocomposites > @ ,Q D VWXG\ GRQH YLD ZDJQHU DQG EDUWUDP FRPPHUFLDOO\ DYDLODEOH 1D< DQG $J< ]HROLWHV ZHUH XVHG to investigated the reactivity of the actual chemical DJHQWV > @ =HROLWHV RU PROHFXODU VLHYHV DUH FU\VWDOline aluminosilicates containing pores and channels of molecular dimensions that are widely used in industry DV LRQ H[FKDQJH UHVLQV PROHFXODU VLHYHV VRUEHQWV DQG catalysts. A representative empirical formula of a zeolite is: M2/n.Al2O3.ySiO2.wH22 :KHUH 0 UHSUHVHQWV WKH H[FKDQJHDEOH FDWLRQ RI YDOHQFH Q 0 LV JHQHUDOO\ a Group I or II ion, although other metal, non-metal and organic cations may also balance the negative charge created by the presence of Al in the structure > @ < ]HROLWH H[KLELWV WKH )$8 IDXMDVLWH VWUXFWXUH (Figure 2). It has a 3-dimensional pore structure with

Sadeghi M et al

Figure 2: Structure of type-Y faujasite zeolite.

SRUHV UXQQLQJ SHUSHQGLFXODU WR HDFK RWKHU LQ WKH [ \ and z planes similar to LTA, and is made of secondary building units 4, 6, and 6-6 [11, 12]. The methods for modifying zeolites are usually by LPSUHJQDWLRQ > @ DQG LRQ H[FKDQJH > @ ,URQ DQG its compounds had been important in human life due WR LWV ELRORJLFDO DQG FKHPLFDO SURSHUWLHV ,URQ R[LGHV DUH ZLGHVSUHDG LQ HDUWK FRUWH[ DQG SOD\ DQ LPSRUWDQW role in nature. Recently they have widely utilized in QDQRFRPSRVLWH IRU YDULRXV DSSOLFDWLRQ > @ ,Q WKLV UHVHDUFK W\SH < ]HROLWH KDV EHHQ VHOHFWHG DV WKH KRVW material for the incorporation of Fe3O4 as the guest due to its three dimensional channels which limits the particle size of Fe3O4 during the growth. Then, the deWR[LÂżFDWLRQ VWXGLHG RI PHWKDPLGRSKRV ZDV FDUULHG RXW by 14.7 wt% magnetite (Fe3O4 QDQRSDUWLFOHV $J 1D< faujasite molecular sieve zeolite (FMSZ) composite at room temperature.

2. EXPERIMENTAL All chemical are purchased from Merck and Alfa Aesar German Fluka was used as received.

Figure 1: Structure of O, S-dimethyl phosphoramidithioate (methamidophos).

2.1. Preparation of NaY zeolite by hydrothermal method (HM) $W ÂżUVW J RI VRGLXP K\GUR[LGH ZDV PL[HG ZLWK 10 mL of distilled water until being dissolved. J RI DOXPLPLXP WULK\GUDWH ZDV GLVVROYHG LQ WKH VRGLXP K\GUR[LGH VROXWLRQ ZKLFK ZDV SUHYLRXVO\ KHDWHG WR ƒ& J RI WKH SUHSDUHG VROXWLRQ ZDV PL[HG

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ZLWK P/ RI GLVWLOOHG ZDWHU DQG J RI VRGLXP K\GUR[LGH XQWLO EHLQJ GLVVROYHG VROXWLRQ $ 7KH VRlution of 22 g of sodium silicate was slowly added to WKH VROXWLRQ FRQWDLQLQJ J RI VRGLXP K\GUR[LGH DQG PO RI GLVWLOOHG ZDWHU WKHQ ZHUH PL[HG XQWLO EHing dissolved (solution B). Solution A was slowly addHG WR VROXWLRQ % DQG PL[WXUH ZDV ZHOO DJLWDWHG IRU min. The solution was transferred to a stainless steel DXWRFODYH OLQHG ZLWK 37)( 7HĂ€RQ DQG NHSW LQ D VWDWLF DLU RYHQ DW ƒ& IRU K 7KH FU\VWDOOLQH PDWHULDO ZDV VHSDUDWHG E\ ÂżOWUDWLRQ DQG ZDVKHG ZLWK GLVWLOOHG ZDWHU XQWLO WKH S+ ZDV QHXWUDO S+ )LQDOO\ WKH PDWHULDOV ZHUH GULHG DW ƒ& > @

QDQRSDUWLFOHV $J 1D< ]HROLWH FRPSRVLWH DQG PHWKamidophos. the samples were prepared according to the following method: 5 mL of methanol, acetonitrile RU Q KH[DQH DV WKH VROYHQW ¾L of methamidophos, 10 ¾L of octane as internal standard and 0.35 g of Fe3O4 QDQRSDUWLFOHV $J 1D< ]HROLWH FRPSRVLWH ZHUH DGGHG WR WKH P/ (UOHQPH\HU ÀDVN 7R do a complete reaction between composite and organophosphorus compound, all samples were attached to a shaker and were shaken for 10 h under N2 atmosphere and in room temperature. Then, by micropipette H[WUDFWHG ¾L of solution and injected to GC instrument.

2.2. Preparation of Ag-NaY zeolite by ion exchange method (IEM) In a typical preparation procedure, 2 g of the syntheVL]HG 1D< ]HROLWH FDOFLQHG DW ƒ& IRU K 7KH FDOFLQHG 1D< ]HROLWH ZDV WKHQ DGGHG WR D P/ RI D 0.15 M silver nitrate (AgNO3 VROXWLRQ DQG WKH PL[WXUH ZDV VWLUUHG DW ƒ& IRU K WR SHUIRUP LRQ H[FKDQJH SURFHVV 7KH UHVXOWLQJ ]HROLWH ZDV ÂżOWHUHG DQG ZDVKHG ZLWK GHLRQL]HG ZDWHU WKHQ GULHG DW ƒ& IRU 16 h. Finally, the clean and dry zeolite was calcined in D IXUQDFH IRU K DW ƒ& > @

2.5. Characterization of samples The morphology and particle size of the crystalline zeolites and composite were analyzed using SEM images. Semiquantitative analysis were carried out RQ DQ HQHUJ\ GLVSHUVLYH [ UD\ VSHFWURPHWHU ('$; connected to LEO-1530VP XL30 Philips scanning electron microscope. Prior to the measurement, the samples were coated with a thin layer of gold. 3RZGHU ; UD\ GLIIUDFWLRQ ;5' SDWWHUQV ZHUH UHcorded at room temperature using a Philips X’Pert Pro 'LIIUDFWRPHWHU ZLWK *H PRQRFKURPDWHG &X .Îą1 raGLDWLRQ ZLWK D ZDYHOHQJWK RI c 'DWD ZHUH FROOHFWHG RYHU WKH UDQJH ƒ LQ θ with a scanning VSHHG RI ƒ PLQ 7KH *& ),' DQG ZDV XVHG IRU WKH GHWR[LÂżFDWLRQ UHactions. A Varian Star 3400CX series gas chromatoJUDSK HTXLSSHG ZLWK Ă€DPH LRQL]DWLRQ GHWHFWRU ),' DQG DQ 29 &:+3 VLOLFD FDSLOODU\ FROXPQ P ĂŽ PP LQQHU GLDPHWHU L G Č?P ÂżOP thickness) was used to monitor the decontamination reactions. The GC conditions used were as follows: WKH FROXPQ WHPSHUDWXUH ZDV LQLWLDOO\ KROG DW ƒ& IRU PLQ DQG SURJUDPPHG DW ƒ& PLQ-1 WR ƒ& WR UHDFK WKH ÂżQDO WHPSHUDWXUH ZKLFK ZDV WKHQ KHOG IRU 13 min. The injector, MS quad and source temperatures ZHUH Âż[HG DW ƒ& ƒ& DQG ƒ& UHVSHFWLYHO\ +HOLXP SXULW\ ZDV VHOHFWHG DV WKH FDUULHU JDV ZLWK WKH Ă€RZ UDWH RI P/ PLQ-1. The IR spectrum was scanned using a Perkin-Elmer FTIR (Model 2000) in the wavelength range of 450 to 4000 cm-1 with KBr pellets method.

2.3. Preparation of Fe3O4 nanoparticles/Ag-NaY zeolite composite by precipitation method (PM) For the synthesis of Fe3O4 QDQRSDUWLFOHV $J 1D< ]HROLWH FRPSRVLWH DW ÂżUVW J RI $J 1D< ]HROLWH ZDV added to 40 mL of distilled water and slowly stirs for 10 min until homogenate suspension was obtained. After that a desired quantity of FeCl3 and FeCl2 solutions with molar ratio of 2:1 was added into the suspension DQG VWLU IRU PLQ 7KHQ ZKLOH WKH PL[WXUH ZDV EHLQJ stirred vigorously about 20 mL of 1 M NaOH solution was slowly added and vigorous stirring was continued for another 30 min. The synthesized product (Fe3O4 QDQRSDUWLFOHV $J 1D< ]HROLWH ZDV WKHQ ÂżOWHUHG ZDVKHG ZLWK GLVWLOOHG ZDWHU DQG ÂżQDOO\ GULHG LQ RYHQ DW ƒ& > @ 2.4. Reaction procedure of Fe3O4 nanoparticles/AgNaY zeolite composite with methamidophos (composite/methamidophos sample) For the investigation of the reaction between Fe3O4

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3. RESULT AND DISCUSSION 3.1. Scanning electron microscope (SEM) study The morphology and crystallite size of the as-synthesized samples were investigated by SEM images (Figure 1). Comparison between the morphologies

(a)

(b)

Sadeghi M et al

RI 1D < DQG $J 1D< ]HROLWHV VKRZV WKDW WKH FXELF morphology and crystalline size are retained with ion H[FKDQJH DQG ZHUH LQ WKH UHJLRQV RI WKH PLFUR VFDOH (Figure 3a and 3b). On the other hand, as it is indicated by image with high resolution, the Fe3O4 nanoparWLFOHV KDYH DSSHDUHG DQG GHSRVLWHG RQ WKH H[WHUQDO VXUIDFH RI $J 1D< ]HROLWH )LJXUH F 7KH FU\VWDOOLWH size for the Fe3O4 nanoparticles was less than 100 nm. Also, some particles aggregation has occurred due to synthesize of nanoparticles of Fe3O4 on the surface of zeolite. 3.2. Energy-dispersive x-ray spectrometer (EDAX) analysis Figure 4 give the composition elements present in Fe3O4 QDQRSDUWLFOHV $J 1D< ]HROLWH FRPSRVLWH ZDV LQYHVWLJDWHG E\ HQHUJ\ GLVSHUVLYH ; UD\V ('$; DQDO\VLV ,Q WKH ('$; VSHFWUXP RI FRPSRVLWH WKH DSSHDUHG SHDNV LQ WKH UHJLRQV RI DSSUR[LPDWHO\ DQG DQG NH9 DUH FRUUHVSRQGHG WR WKH ELQGLQJ HQHUJLHV RI R[\JHQ 2 VRdium (Na), aluminum (Al), silicon (Si) and silver (Ag) UHVSHFWLYHO\ WKDW DUH UHODWHG WR WKH $J 1D< ]HROLWH 2Q the other hand, in this spectrum, the appeared two SHDNV LQ WKH UHJLRQV RI DQG NH9 DUH UHODWHG to the binding energies of iron (Fe) which reveals the SUHVHQFH RI )H LQ WKH FRPSRVLWH 7KHVH UHVXOWV FRQÂżUP FRH[LVWHQFH RI ZW DQG ZW VLOYHU DQG LURQ in the prepared sample, respectively. 3.3. X-Ray diffraction (XRD) patterns 7KH ;5' SDWWHUQV RI WKH V\QWKHVL]HG 1D< DQG $J 1D< ]HROLWHV DQG ZW )H3O4 nanoparticles/

(c) Figure 3: The SEM images of the samples: (a) NaY, (b) Ag-

Figure 4: The EDAX analysis of the 14.7 wt% Fe3O4

NaY, c) 14.7 wt% Fe3O4 nanoparticles/Ag-NaY.

nanoparticles/Ag-NaY.

520

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$J 1D< ]HROLWH FRPSRVLWH XQGHU VWXG\ DUH VKRZQ LQ )LJXUH 7KH VWUXFWXUH RI WKH 1D< ]HROLWH ZDV UHWDLQHG DIWHU WKH VLOYHU FDWLRQ H[FKDQJHV )LJXUH D DQG E +RZHYHU IRU $J 1D< ]HROLWH D VPDOO ORVV RI crystallinity is observed, associated with the lower intensity of the peaks at 2Theta=10-34. This effect could be related to a dealumination of its structure, possibly DVVRFLDWHG ZLWK WKH ORFDWLRQ RI H[WUD IUDPHZRUN VLOYHU $OVR ;5' SDWWHUQV VKRZHG WKDW WKH $J 1D< ]HROLWH and Fe3O4 QDQRSDUWLFOHV $J 1D< DUH DOPRVW VLPLODU WR WKH SDUHQW 1D< ]HROLWH 7KH ZW )H3O4 deposLWHG LQ WKH SRUHV RI $J 1D< ]HROLWH GRHV VKRZ D VHULHV new diffraction peak. The peaks of Fe3O4 phase appeared at 2θ YDOXHV RI ƒ ƒ ƒ ƒ DQG ƒ FRUUHVSRQGLQJ WR WKH GLIIUDFWLRQ SODQHV ' ' ' ' DQG ' UHVSHFtively [13, 21]. These peaks are illustrated as the red points in Figure 5c. The structures of prepared Fe3O4 QDQRSDUWLFOHV GHSRVLWHG LQ WKH $J 1D< ]HROLWH ZHUH LQYHVWLJDWHG YLD ; UD\ GLIIUDFWLRQ ;5' PHDVXUHment. The average particle size was calculated from line broadening of the peak at 2θ ƒ XVLQJ 'HE\H Scherrer equation: d

O E cos T

(1)

:KHUH G LV WKH FU\VWDOOLQH VL]H Îť is the wavelength

of X-ray source, β LV WKH IXOO ZLGWK DW KDOI PD[LPXP ):+0 DQG θ is the angle of incidence for the selected diffraction peak (Bragg diffraction angle). The average crystallite size of the Fe3O4 nanoparticles were calculated about 31 nm. 3.4. GC-FID analysis 7KH GHWR[L¿FDWLRQ DGVRUSWLRQ GHVWUXFWLRQ UHDFWLRQV between 14.7 wt% Fe3O4 QDQRSDUWLFOHV $J 1D< ]HRlite composite and methamidophos was investigated DQG PRQLWRUHG E\ *& ),' 7R DFFHGH PD[LPXP HI¿ciency, the effects of different solvents and time intervals have been investigated. The GC chromatograms, area under curve (AUC) data and results under different conditions are summarized in Figures 6 and 7 DQG 7DEOH 7R FDOFXODWH WKH DPRXQWV RI GHWR[L¿HG organo-phosphorous insecticide, the integrated area under peak data of two samples, methamidophos and octane as the internal standard have been given for all solvents and times. Subsequently, the ratios of the integrated data (integrated AUC of methamidophos/inWHJUDWHG $8& RI RFWDQH ZHUH GHWHUPLQHG 7KH H[SHUiments were performed at different time intervals from DQG K :LWK LQFUHDVLQJ WKH WLPH $8& DPRXQWV RI PHWKDPLGRSKRV ZHUH ¿UVWO\ GHFUHDVHG XQWLO K DQG then a slight increasing trend was observed. These reVXOWV FRQ¿UP WKDW WKH FRPSRVLWH LV SHUIHFWO\ DEOH WR GH-

Figure 5: The XRD patterns of the samples, (a) Na-Y, (b) Ag-NaY, c) Fe3O4 nanoparticles/Ag-NaY.

521

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Figure 6: The GC chromatograms for methamidophos-Fe3O4 nanoparticles/Ag-NaY sample.

WR[LÂżFDWLRQ PHWKDPLGRSKRV DW Q KH[DQH DIWHU K 2Q WKH RWKHU KDQG WKH GHWR[LÂżFDWLRQ GDWD ZHUH lower for acetonitrile and methanol as the solvents. Notwithstanding the transition state must be involved in the polar reaction, polar solvent hinders the reaction's progress. It could be construed from GC analysis that polar solvent can compete with the reaction sites presented on the surface of the composite including Bronsted K\GUR[\O JURXSV )H 2+

DQG /HZLV DFLG )H )

sites. In particular, the blocking of Lewis acid sites would hinder the coordination of methamidophos. Since methanol is considered as such a strong hindrance to the reaction, this points out to the fact that isopropanol simply blocks access to the surface of the catalyst. 3.5. FT-IR spectrum The FT-IR spectrum of the 14.7 wt% Fe3O4 nanoparWLFOHV $J 1D< ]HROLWH FRPSRVLWH DIWHU WKH UHDFWLRQ

Table 1: The GC analysis results in the presence of different solvents, (a) zero time (the blank solution) and (b) after 10 h.

Solvent

Methanol Acetonitrile Q +H[DQH

522

Sample

AUC/Octane (1)

AUC/ methamidophos (2)

Ratio (AUC 2/AUC 1)

'HWR[LÂżHG (%)

a

356489

333567

100.00

b

356246

234605

70.34

a

397726

332461

100.00

b

a

410687

405992

100.00

b

000000

000000

00.00

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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 517-524

SUHFLSLWDWLRQ PHWKRG 7KH GHWR[LÂżFDWLRQ DGVRUSWLRQ of methamidophos on this composite was investigatHG 7KH UHVXOWV HPSKDVL]HG WKDW DERXW RI PHWKDPLGRSKRV PROHFXOH ZDV DGVRUEHG DW Q KH[DQH VROvent after 10 h.

ACKNOWLEDGEMENTS 7KH DXWKRUV JUDWHIXOO\ DFNQRZOHGJH WKH ÂżQDQFLDO VXSports of Tehran Imam Hossein comprehensive University (IHCU), Iran. Figure 7: The IR spectrum for adsorbed O, S-dimethyl phosphoramidithioate (methamodophos) on the 14.7 wt% Fe3O4 nanoparticles/Ag-NaY zeolite composite.

with methamidophos is shown in Figure 7. The peak at 463 cm-1 is corresponded to the structure insensitive internal TO4 (T= Si or Al) tetrahedral bending peak RI < ]HROLWH 7KH SHDN DW FP-1 is attributed to the GRXEOH ULQJ H[WHUQDO OLQNDJH SHDN DVVLJQHG WR < ]HRlite. The peaks at 676 and 754 cm-1 DUH DVVLJQHG WR H[ternal linkage symmetrical stretching and internal tetUDKHGUDO V\PPHWULFDO VWUHWFKLQJ UHVSHFWLYHO\ ' 5 )XUWKHUPRUH WKH SHDNV DW FP-1 and 1070 cm-1 are assigned to internal tetrahedral asymmetrical stretchLQJ DQG H[WHUQDO OLQNDJH DV\PPHWULFDO VWUHWFKLQJ UHVSHFWLYHO\ DQG SHDNV DURXQG DQG FP-1 are DVVLJQHG WR + 2 + EHQGLQJ DQG K\GUR[\O JURXSV RI WKH ]HROLWH UHVSHFWLYHO\ $OVR WKH QHZ SHDNV DW + 2 + & & DQG )H 2 FP-1 regions are corresponded to the synthesized Fe3O4 nanoparticles. On the other hand, the adsorption of methamidophos on the 14.7 wt% Fe3O4 QDQRSDUWLFOHV $J 1D< ]HROLWH was investigated by IR spectrum. The new peaks at DQG FP-1 are seen. These observed IR data lead to an understanding of the adsorption reaction of methamidophos on the surface of FRPSRVLWH )LJXUH

4. CONCLUSIONS ,Q WKLV SDSHU 1D< DQG $J 1D< ]HROLWHV ZHUH V\QWKHVL]HG E\ K\GURWKHUPDO DQG LRQ H[FKDQJH PHWKRGV UHVSHFWLYHO\ ,Q WKH QH[W VWHS ZW )H3O4 nanoparWLFOHV ]HROLWH $J 1D< FRPSRVLWH ZDV SUHSDUHG XVLQJ

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Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 4 (2013), 517-524

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