International Journal of Bio-Inorganic Hybrid Nanomaterials

Int. J. Bio-Inorg. Hybd. Nanomat., Vol. 2, No. 3 (2013), 407-422

International Journal of Bio-Inorganic Hybrid Nanomaterials

Photocatalytic Coating Using Titania-Silica Core/Shell Nanoparticles Hossein Abdollahi1, Amir Ershad Langroudi2*, Ali Salimi3, Azam Rahimi4, Elham Pournamdari5 1

Ph.D. Student, Color, Resin & Surface Coating (CRSC) Department, Faculty of Polymer Processing, Iran Polymer and Petrochemical Institute (IPPI), 14965/115, Tehran, Iran 2

Associate Professor, Color, Resin & Surface Coating (CRSC) Department, Faculty of Polymer

3

Assistant Professor, Color, Resin & Surface Coating (CRSC) Department, Faculty of Polymer

Processing, Iran Polymer and Petrochemical Institute (IPPI), 14965/115, Tehran, Iran Processing, Iran Polymer and Petrochemical Institute (IPPI), 14965/115, Tehran, Iran 4

Professor, Polymer Science Department, Faculty of Science, Iran Polymer and Petrochemical Institute

5

Assistant Professor, Department of Chemistry, Islamshahr Branch, Islamic Azad University, Islamshahr,

(IPPI), 14965/115, Tehran, Iran Iran Received: 30 April 2013 ; Accepted: 13 July 2013

ABSTRACT The photocatalytic coatings were prepared via incorporating the modified titania nanoparticles into epoxy-based inorganic-organic hybrid coatings. Titania nanoparticles were first synthesized from tetra-n-butyl titanate using sol-gel methods by two different calcination treatments, i.e., in mild condition (80째C) and 500째C. The formed anatase nanoparticles were further modified as Titania-Silica (TS) core/shell structure. Characterization of the samples was carried by X-ray diffraction (XRD) analysis, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), Energy-Dispersive X-ray (EDX) spectroscopy and UV-Vis spectroscopy. The decomposition of methylene blue (MB) exhibited high photocatalytic activity for the titania nanoparticles embedded with silica phase in core/shell structure. In a comparative study using a commercial P25 titania from Evonik, the synthesized titania samples showed lower size nanoparticles and more uniform distribution of nanoparticles into hybrid binder. However, the degree of anatase crystalline was lower which results to lower photocatalytic activity.

Keyword: Photocatalytic; Hybrid coatings; Titania; Core/shell nanoparticles; Sol-gel process.

(*) Corresponding Author - e-mail: A.Ershad@ippi.ac.ir

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1. INTRODUCTION Recently, the titanium dioxide (TiO2) or titania organic based films have been intensively investigated for their biological and chemical stability, special optical properties, non-toxicity and low cost [1, 2]. In particular, great attention has been devoted to the study of the photocatalytic properties of titania powders and thin films useful for the removal of pollutants from air or water as self-cleaning surfaces, environmental purification, anti-algal and anti-bacterial coatings [3]. This activity can be obtained due to its ability to mineralize a wide range of organic contaminants and environmental toxins [4]. Under ultraviolet (UV) exposure, the titania nanoparticles can be photo-excited to produce a negative electron in the conduction band and a positive hole in the valence band. In an aqueous environment, the photoinduced electrons and hole pairs react with oxygen or water to generate reactive oxygen species (such as hydroxyl and superoxide radical), which powerfully oxidative and can destroy the structure of various organic molecules [5, 6].

The photocatalytic activity of the titania particles mainly reflects from the physical properties of the titania, such as the crystal structure (amorphous, anatase, rutile, or brookite), the surface area, the particle size, the surface hydroxyls, degree of crystallinity and so on [7, 8].

Different methods have been used to prepare the nanoparticles, such as the chemical precipitation, micro emulsion, hydrothermal crystallization and the sol-gel process [8, 9]. The most common method to form the inorganic core is the hydrolysis of the inorganic alkoxides under mild reaction conditions in the sol-gel process [10]. In the sol-gel processes, titania is usually prepared by hydrolysis and condensation reactions of titanium alkoxides (Ti(OR)n) to form oxide network. Due to high reactivity of titanium alkoxides [11], some chelating reagents, such as Acetyl Acetone (AcAc) and Ethyl Acetoacetate (EAcAc) are often necessary during hydrolysis step. After the condensation step, a calcination treatment (above 400째C) is then required for completing the

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crystallization [12]. It has been reported that the simple blending of some nano particles such as ZnO, SiO2 and even the core/shell structure showed enhancement in photocatalytic activity of the titania nanoparticles [13]. The nanoparticles tendency to aggregation in aqueous media and hence reduction of the particles surface area may be diminished by the nanoparticle surface modification [14]. The inorganic-organic hybrids systems combine the advantages of inorganic and organic polymers [15].

The flexibility of the material design, synthesis and properties has gained enhanced research interests in this field [16, 17]. The organo silica-networks may be generated by hydrolysis and condensation reactions of 3-glycidoxypropyl-trimethoxysilane (GPTMS) in water based sol-gel process [18]. The optical properties of the modified GPTMS sols with anatase titania were studied to obtain photocatalytic active coatings for the development of self-cleaning textiles [7]. It was found that the ability of the GPTMS sol in decomposition of astrazone red corresponds to the GPTMS modification method. The incorporation of silica and Tetraethylorthosilicate (TEOS) has been reported to improve the dispersion of various nanoparticles in a water based environment [19].

One of the most common ways to prepare titania-silica materials is through a sol-gel process where the preparation of titania sol and silica sol is performed separately and then they are mixed to obtain the desired outcome [20]. The method is simple and can be operated at mild condition. The resultant nanoparticles include a core made of the base nanoparticle (titania) and a shell of silica known as a core/shell structure. The silica layer can act as a protector to decrease the effect of the outer environment on the core nanoparticles. Also, the silica surface is electrostatically stable and it improves the dispersion of the core nanoparticles [6]. Although the particle stability and dispersibility can be enhanced after silica coating, some properties of the core component, such as reactivity and thermal stability, also may be altered [21, 22].

Therefore, a mixture of titania-silica is used not

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only as a good photocatalyst but also as a support material for chemical catalysts reactions with good dispersion in the water based environments.

A titania-silica core/shell nanostructure, synthesized in sol-gel process, confirmed the synergistic effects of silica on the titania functionality on cotton fabrics [23, 24]. In another study, the water-based colloidal titania-silica core/shell nanoparticles were prepared using aqueous colloidal nano titania and TEOS sol via sol-gel process. The prepared photoactive nanocomposite films from the titania-silica core/shell nanoparticles and poly(MMA-coMSMA) showed homogenous dispersion of the colloidal titania-silica core/shell nanoparticles and better thermal stability and higher glass transition temperature over pristine binder [25]. A comparative study on the photo degradation of different inorganic, organic and inorganic-organic hybrid binders showed the highest activity of P25 titania from Evonik and tendency to degradation in

organic binder [26]. In this research, an inorganicorganic hybrid polymer network was developed as self-cleaning coating. Here, the titania nanoparticles were prepared from tetra-n-butyl titanate at two different calcination treatments. In addition to the photoactivity of the as-prepared nanocomposite coating, detailed characteristics of the coating such as the chemical structure and morphology were investigated and compared with a commercial titania.

2. EXPERIMENTAL 2.1. Materials and Reagents The Tetraethylorthosilicate (TEOS, ≼98%), tetran-butyl titanate (TBT, 97%) from Merck and 3-glycidoxypropyl-trimethoxysilane (GPTMS, 97%) from Alfa Aesar were used as starting materials.

Bisphenol A (BPA, 97%, Merck), N-methylimidazol (MI, 99%, Fluka), Acetyl Acetone (AcAc, 99%,

Figure 1: Diagram of the two methods used in preparation of anatase titania.

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Merck), and Ethanol (99%, Merck) were used as received. The distilled water was used for all experiments. Hydrochloric and nitric acids were purchased from Merck. The titania AEROXIDE® P25 from Evonik was used as a standard reference.

The photocatalytic activity of all nanocomposite coatings was investigated using a 40 μM aqueous solution of Methylene Blue (MB, ≥95%, Sigma Aldrich).

2.2. Preparation of sol phase and hybrid coatings 2.2.1. Preparation of titania sol In sol-gel method, the titania sol phase was prepared from TBT as a precursor in ethanol. The sol phase undergone a hydrolysis/condensation reaction using an acidic water (pH= 2). In order to control the rate of hydrolysis in titanium alkoxides, 0.6 cc of the AcAc was used in 2 cc of TBT. The molar ratios of the reactants were as follows: TBT: HNO3: Ethanol: AcAc: H2O = 1:0.018:40:1:100.

Then, the mixture was stirred vigorously with a magnetic stirrer at room temperature for 3 h. At next step, two different treatments were employed to obtain the anatase crystal phase. In one method, the prepared sol was refluxed for 8h at 80°C to convert the amorphous titania to anatase crystalline phase (as sample T1). In second method, the prepared sol was heated up to 500°C for 1h in a furnace (as sample T2). Note that the descending rate for annealing temperature was chosen as 10 C/min. The procedure was shown schematically in Figure 1 for the two methods used in preparation of anatase titania.

In the case of using commercial P25 (as sample T3) and sample T2, the particles were dispersed in ethanol at a molar ratio of titania: ethanol = 1:45 using a magnetic stirrer followed by sonication for 10 min on a Bandelin, HD3200, sonicator (KE-76 probe, pulse mode of 0.7 s on and 0.3 s off, 70 W power). Then the sample T2 in powder form and the dried sample T1 (at 60°C over night) were analyzed

by XRD technique.

2.2.2. Preparation of titania-silica core/shell nanoparticles The silica sol was prepared by hydrolysis and

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condensation of TEOS as a precursor. In this method, 1.3 cc TEOS was first dissolved in 10 cc ethanol and then an acidic water (pH= 2) was added into the solution drop wise. The as-prepared mixture was stirred to precede the hydrolysis reaction at 25°C for 1h. Then, the as-prepared titania sol (sample T1) or titania powders dispersed in ethanol (samples T2 and T3) were added in the silica sol at a molar ratio of (titania:silica 1:1). The resulting mixture underwent two individual hydrolysis/condensation reactions at 25°C for 32 h and at 50°C for 8 h to obtain the titania-silica (TS) core/shell solution. The TS nanoparticles were formed through a strong covalent bonding between OH groups of the titania particles and the OH groups of the silica sol.

2.2.3. Preparation of Epoxy-Silica-Titania hybrid nanocomposite The series of Epoxy-Silica-Titania (EST) hybrid nanocomposite were prepared by mixing the as-prepared TS core/shell in GPTMS sol followed by stirring vigorously for 25 min at ambient temperature. Wherein, the GPTMS sol was prepared by hydrolysis and condensation of GPTMS in ethanol as solvent, and an acidic water (pH= 2) as a catalyst of hydrolysis-condensation reaction. Then, the BPA as a crosslinker was added to the above solution and stirred vigorously for 4h at ambient temperature. The methyl imidazol as a catalyst (1% wt vs. GPTMS) was used for increasing the cross-linking process. The procedure for the preparation of the EST hybrid nanocomposite was shown in Scheme 1. The molar ratio of reagents in the EST hybrid nanocomposite samples were shown in Table 1.

2.2.4. Film preparation of the nanocomposites samples The thin films of EST hybrid nanocomposite were fabricated on glass slides (soda lime glass 25.4× 76.2×1.2 mm3) by a dip coating at ambient conditions. The glass slides were previously cleaned repeatedly in a solution containing 0.1 M HCl and methanol/hexane (40:1) followed by washing with distilled water. Then, the EST thin

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Scheme 1: Preparation schematic of EST nanocomposite coatings.

Table 1: The sol compositions of EST nanocomposites (based on mol fraction).

Sample

GPTMS

TEOS

TBT

Titania

Ethanol

BPA

H2O

P25 ES

1

0

0

0

5

0.5

3

EST1

1

0.125

0.125

0

0.625

0.5

3.5

EST2

1

0.125

0.125

0

0.625

0.5

3.5

EST3

1

0.125

0

0.125

0.625

0.5

3.5

All mol fraction are based on 1 mol GPTMS.

films were dried at room temperature for 24 h and subsequently were heated in an air-circulating oven at 130째C for about 120 min.

2.3. Instrumentation The Fourier transform infrared, FT-IR spectra were obtained by using a Bruker (IFS 484, Germany) spectrometer, in the range 400-4000 cm-1. FT-IR

spectroscopy was carried out using KBr pellet on a spectrometer, collecting 16 scans at 4 cm-1 resolution. The particle size distribution and the average particle size were determined by dynamic light scattering measurements, DLS (SEMA Tech 39 chem du terron 06200, France). Also, the transmission electron microscopy, TEM using a PHILIPS (Model CM120, Netherlands) at an

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accelerating voltage 120 kV was used to investigate the average particle size.

The morphological properties of the fractured surface of the EST hybrid nanocomposite were investigated at the by scanning electron microscopy (VEGA\\TESCAN, Czech Republic) at an accelerating voltage of 1500 kV. The distributions of each of the Si and Ti atoms in thin film were investigated by Energy-Dispersive X-ray spectroscopy, EDX mapping (INCA Penta FET×3, Oxford, England).

The titania crystalline phase was investigated by X-ray diffraction measurement, XRD (PW 1800 PHILIPS, Netherlands). The diffraction pattern were obtained using a Cu Kα incident beam (λ= 1.542 Å) at 2θ = 10°-80° and a scanning speed of 4°/min. The voltage and current of X-ray tubes were 40 kV and 30 mA, respectively.

The Brunauer, Emmett and Teller (BET) measurements were carried out to evaluate the specific surface area from nitrogen adsorptiondesorption data (CHEMBET-3000 Quantachrom, USA).

The cross-cut adhesion test of the coatings was carried out on 7×5 cm2 glass substrate using the Scratch-Adhesion Test (Neurtek, Spain). The strength of adhesion of above films coated on glass

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substrates was measured according to the ASTM D 3359B-02 protocol for adhesion tests (B5 and B0 has best and weak adhesion, respectively).

The ultraviolet-visible (UV-Vis) absorption spectra of the films, deposited on silica glass slides were measured from 200 to 800 nm using a Shimadzu UV-Vis spectrophotometer (UV-3101 PC, Japan). The surfaces of the organic-inorganic hybrid films were cleaned using acetone with a cotton tissue.

The photocatalytic activity of the EST hybrid nanocomposite films coated on glass slide was characterized by inserting coated glass slide into a Petri dish filled with a MB solution. Then, the degradation of MB was evaluated in an aqueous solution (40 μM) under UV illumination at room temperature for 3 h. By measuring the decomposition of MB with the spectrophotometer, the extinctions of the differently treated solutions give information about the photocatalytic activity. The power of the UV lamp was 8 W and the main wavelength of the UV lamp was 254 nm.

Degradation of coating samples was investigated under UV irradiation after 1080h in QUV/Spray chamber (Q-Panel, USA) according to the ASTM D4587 standard. According to the standard procedure, the samples were alternately exposed to

Figure 2: FT-IR spectrum of the (a) TBT and (b) amorphous titania sol.

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UVA irradiation (340 nm, 0.89 Wm-2) at 60°C for 8 h followed by 4 h of water condensation at 45°C periodically. Effect of accelerated weathering conditions on the degradation of nanocomposite coatings and surface morphology of the epoxysilica hybrids containing TS nanoparticles was studied using SEM analysis.

3. RESULTS AND DISCUSSION 3.1. FT-IR characterization of titania particles The FT-IR spectra of the TBT and the titania sol (after hydrolysis reaction) were shown in Figure 2.

As shown in Figure 2a, the characteristic absorption peaks for the alkoxy (OR) group of TBT appeared at 1040, 1070 and 1125 cm-1 wave numbers [27].

The spectrum of titania sol after hydrolysis reaction indicates for the conversion of titanium tetrabutoxide to titania particles. A broad peak between 3100 and 3600 cm-1 in both FT-IR spectra corresponds to the stretching vibration of different O-H groups (free or bonded). The presence of acidic water was confirmed by absorption in the 1600-1640 cm-1 region that is assigned to physically adsorbed water (H-O-H bending) [27].

As shown in Figure 2b, there is no absorption peaks related to OR groups. It seems that all four OR groups of TBT were replaced with OH groups of water indicating for a full conversion during the hydrolysis reaction. In fact, the characteristic peaks of OR group were replaced by C-O stretching absorption peaks of the ethanol.

The FT-IR absorption spectrum of the neat titania (AEROXIDE® P25 from Evonik) and the sample TS3 were shown in Figure 3a and b, respectively.

As shown in both spectra in Figure 3, the broad and intensive bands with two overlapping components were appeared in the 400-800 cm-1 region assigning for the Ti-O and Ti-O-Ti groups [28]. In Figure 3b, the peaks at about 800-810 cm-1 and 1080-1105 cm-1 correspond to the symmetric vibration of Si-O-Si and asymmetric (Si-O-Si) stretching vibration, respectively [29]. Besides the absorption band at about 950 cm-1 corresponds to Ti-O-Si bond in TS nanoparticles [30]. Therefore, the FT-IR spectrum of T3 and TS3 samples indicates for a combination of titania and silica nanoparticles.

3.2. SEM observations of titania particles The SEM images of the neat titania (sample T3) and sample TS3 nanoparticles were shown in Figure 4.

Figure 3: FT-IR spectrum of the (a) sample T3 and (b) sample TS3.

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Figure 4: The SEM images of the neat titania (sample T3) and after silanization (sample TS3).

The pure titania particles exhibited a somewhat irregular morphology due to the aggregation of primary particles. In comparison with sample T3, the sample TS3 exhibited more porous morphology.

The silica surface is electrostatically stable which prevents the aggregation of the titania nanoparticles [6].

3.3. Crystal structure and the specific surface area measurement of titania particles The XRD patterns of the prepared particles in two individual methods are shown in Figure 5.

As shown in Figure 5a, the diffraction angle at 25.3°, 37.8° and 53.9° correspond to anatase crystal phase for the sample annealed at 500°C (calcinated powder). As shown in Figure 5b, the diffraction angles of the anatase phase (the major peak at 2θ= 25.3°) [31] are evident for the particles formed in sol phase after refluxing. According to the Figure 5, it can be clearly understood that the anatase structure in titania sol phase after refluxing (sample T1) was much less than the calcinated powder (sample T2). Thus, the prepared coatings from these nanoparticles contain the anatase structure. While the coating was prepared from T3, indicated the existence of anatase (70%) and rutile (30%) structures, simultaneously [32].

414

Figure 5: XRD pattern of the sample (a) T2 and (b) T1.

The letters on peaks corresponds for each kind of crystal form in titania.

The crystallite size of T1 and T2 samples was measured by XRD line profile analysis using the DebyeScherrer equation (Eq. 1) [33]. d = kλ/ B cosθ

(Eq. 1)

Where d is the crystallite size in nm, k a constant (0.9),λ the X-ray wavelength of Cu (1.5406 Å), θ the Bragg's angle in degrees and B is the full width at half maximum of the peak. The results are shown in Table 2. The specific surface area of as-prepared nanoparticles before and after the silanization reaction of titania nanoparticles was measured by

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BET test. The BET results were also tabulated in Table 2. According to the BET results for T1 and T2 samples, the decrease in BET surface area from 278.5 m2/g to 78.3 m2/g may correspond to an increase in crystal size from 30 nm to 50 nm.

Moreover BET results exhibited the amount of specific surface area was considerably increased from 278.5 m2/g to 325.2 m2/g for T1 and TS1 samples, respectively, may be due to the formation a shell layer of silica on the titania core. These results are similar to the results of other samples as shown in Table 2. This can be attributed to the fact that surface treatment may provide a porous layer on the surface through grafting. It is believed that grafting on the surface of particles by silanization reaction makes it somehow irregular and rougher and obviously this leads to an increase in the specific surface area [33]. Therefore, silanization reaction increases the specific surface area of nanoparticles, while the crystalline size is constant.

Table 2: Crystal size and specific surface area for different titania nanoparticles.

Sample

Crystal size (nm)

crystalline phase

surface area (m2/g)

T1

30

Anatase

278.5

T2

50

Anatase

78.3

T3

---

Anatase/Rutile

TS1

30

Anatase

325.2

TS2

50

Anatase

136.5

TS3

---

Anatase/Rutile

96.3

*55.2

* BET surface area of P25 (m2/g) 50 Âą 15 (product information)

3.4. Particle size measurements of the titania particles Dynamic light scattering (DLS) technique was employed to measure size distribution of nanoparticles in sample T1 and sample TS1. As shown in Figure 6, the particle size of the titania and TS core/shell were in the range of 5-100 nm (average size of 40 nm) in sample T1 and 9-200 nm (average size of 70 nm) in sample TS1.

Figure 6: Particle size distribution of (a) sample T1 and (b) sample TS1 sol.

3.5. Morphological studies of the hybrid coating 3.5.1. The TEM studies on core/shell structure The TEM images of the amorphous titania in the sol solution and TS core/shell nanoparticles in EST1 and EST2 nanocomposites were represented in Figure 7a, b and d, respectively. Note that the Figure 7c represents a magnification of core/shell structure of the Figure 7b.

As seen in Figure 7a, titania sol shows irregular shape particles with particle size of about 35 nm.

While in Figure 7b and 6c, the titania particles have a regular five-sided shape, which probably reflects the crystalline state of particles and confirmed that anatase crystals formation during refluxing.

Furthermore, Figure 7b shows the formation of silica layer on the titania particles with a thickness of about 15 nm. The TEM images were in good agreement with those of DLS analysis. The difference between the average size of sample T1 and sample TS1 in DLS measurements refers as twice of shell thickness in TEM images, i.e., 15 nm.

Figure 7d shows the inorganic particle sizes in EST2 nanocomposites. As seen in Figure 7d, the TS2 nanoparticles had irregular shape particles with average particle size of about 80 nm while, the particle distribution of TS2 in the hybrid coating was not as well as TS1.

3.5.2. The scanning electron microscopy studies on nanoparticles dispersion After addition of the TS nanoparticles in the

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(a)

(b)

(c)

(d)

Figure 7: TEM images of (a) the amorphous titania sol, (b) TS1 core/shell nanoparticles in sample EST1, (c) a magnification of part (b) and (d) TS2 nanoparticles in sample EST2.

GPTMS sol, these coating solutions were applied onto the glass slides by dip coating method. The map of Si atoms for EST1 was shown in Figure 8a and maps of Ti atoms were shown for EST1, EST2 and EST3 in Figure 8b to d.

It can be seen from the SEM micrographs that distribution of these nanoparticles in the organicinorganic hybrid coatings was uniform. The uniform distribution of TS core/shell nanoparticles is due to the presence of the silica which prevents agglomeration of titania and inhibits the tendency of titania for phase separation. Since the inorganic parts of polymeric hybrid and particle's shell consists similar atoms and silica surface is electrostatically-stable good distribution of core/shell nanoparticles was resulted in the epoxy-silica hybrid system. Nonetheless, the Ti maps in Figure 8 shows that distribution and dispersion of nanoparticles is not similar in all samples.

The sample prepared at mild condition (TS1 sol)

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shows a better distribution and dispersion than other sample (TS2 and TS3 powders). The distribution and dispersion quality of nanoparticles in EST nanocomposites can influence on the material properties such as optical property which will be discussed in next section [34].

3.5.3. Optical quality of the hybrid coating The UV-Vis spectra of the as-prepared thin films were shown in Figure 9.

As shown in Figure 9, The ES hybrid thin film demonstrates the prominent peak at ~320 nm attributed to π→π* transition of the benzene rings of BPA curing agent. The bare titania shows the typical absorption band at 300-400 nm [35]. The photocatalytic activities of titania are directly related to the intensity of UV absorption.

In the visible region (λ>400 nm), the ES film shows good transmission of light. Three phenomena can be considered for the incident beam as

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(a)

(c)

(b)

(d)

Figure 8: Inorganic-Organic Hybrid films on glass by sol-gel processing. (a) Si-mapping of EST1, and Ti-mapping of (b) EST1, (c) EST2 and (d) EST3.

absorption, diffraction and transmission. The sample EST1 was yellowish in color due to complexion of TBT-AcAc. Therefore, in the visible region a higher absorption can be attributed to the color of sample EST1, as seen in Figure 9. While EST2 and EST3 (particularly EST3 due to poor dispersion of TS3 nanoparticles) caused the scattering of light in the visible region. So, the sample EST2 and EST3 were semi-transparent (see Figure 10 c and d) due to the presence of titania nano powder. However, the differences in absorption spectra for the samples EST2 and EST3 can be related to the differences in diffraction of the incident beam. Since these samples have poor distribution (EST2) or poor dispersion (EST3) of TS nanoparticles. These results were also in agreement with Ti mappings and confirmed by transparency measurements of the samples which were presented in Table 3.

Absorption of light below at 300-400 nm is due

to the excitation of electrons from the valence band (VB) to the conduction band (CB) of titania. The sample EST1 shows slightly red-shift compared to the EST3 film. A red-shift of the absorption edge indicates a decrease in the band gap of titania when annealing at mild condition. The absorption edges of the EST2 films were significantly blue-shifted compared to the EST3. The shift may be attributed to a "size-quantization effect" [27]. As seen in Figure 9, EST1 and EST2 films show the both differences in intensity and width of the absorption peak. Sample EST2 exhibits the higher absorbance in compared to sample EST1 which can be attributed to high crystallinity and larger particle size of titania in it. The difference in the absorption spectra between EST1 and EST2 films indicates that the particles size and thereby optical quality of titania changes by crystallization temperatures, which was in good agreement with XRD investigation. Therefore, Figure 9 demonstrates that the larg-

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Figure 9: UV-Vis spectra of the hybrid films.

er crystals increase the absorption of the light in UV region.

The transparency of the hybrid films were shown in Figure 10. The results were also tabulated in Table3. It was observed that the transparency of ES film was higher than other samples.

Figure 10: The pictures of the hybrid films of the (a) ES, (b) EST1, (c) EST2, and (d) EST3.

3.5.4. Photocatalytic behavior of the hybrid coating The photocatalytic activities of as-prepared

coatings were characterized by the degradation of MB in aqueous solutions which was investigated under an 8 W Xe-UV lamp using a 100 cc reactor.

This property of titania depends on its crystallinity, crystalline structure, particle size and surface area of the titania in the coatings [36].

The photocatalytic activity results from the formation of active species that can initiate photo-oxidative degradation of the organic materials by reaction with water or oxygen [37].

The following reaction is photochemical degradation of MB by O2 in the presence of titania under UV light irradiation (3.2 eV is the band gap energy of the titania), i.e. Figure 11a and b shows the percentage of MB degraded as a function of irradiation time for the as-prepared films.

In Figure 11a, the photocatalyst activity of a thin layer of sample T2 and sample TS2 on glass slide were shown. As seen in Figure 11a, the photocatalyst activity of sample TS2 was more pronounced than sample T2, may be due to the increased specific surface area and the higher concentration of hydroxyl groups on the surface in which transfer electrons more easily. The increase in surface area was observed in BET test results as in Table 2, i.e.,

hν (3.2 eV) C16H18N3SCl + 25 1/2O2

418

TiO2

HCl + H2SO4 + 3HNO3 + 16CO2 + 6H2O

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78.3 m2/g for sample T2 to 136.5 m2/g for sample TS2. Another reason is that more negative CB in the presence of silane increases the number of electrons available to titania for initiating decomposition [38].

Table 3: Transparency measurement of samples. sample

Transparency

Uncoated glass slide

92

ES

90

EST1

82

EST2

76

EST3

62.5

Figure 11b shows the MB degradation rate of the coatings on glass slides. It is found that coating prepared from calcinated titania in 500째C (sample EST2) exhibits the highest degree in MB degradation compared with other samples, so that the degradation of MB reached 99% after irradiation for 150 min. It should be pointed out that the particles (T2) in sample EST2 were smaller than particles (T3) in the sample EST3, so the specific surface area is larger as BET results. The specific surface area differs from 78 m2/g for sample T2 to 55 m2/g for sample T3. Therefore, the average path length of a charge carrier to the surface gets longer and hence emerges a negative effect on the photo activity for sample EST3. As the particle size decreases, the combination of electron-hole decreases and as a result the photocatalytic effect is increases [37]. In addition, the sample EST2 shows higher anatase crystals relative to the sample EST3.

Thus, the sample EST2 exhibits higher photo activity relative to sample EST3.

Besides, the sample EST1 shows lower MB degradation than sample ETS2. As mentioned earlier the effective factor on photocatalytic activity is crystallinity and the low photo activity of EST1 is due to its low crystallinity. Simultaneously the higher photocatalytic activity found for particles of sample TS in the sample EST2 compared to sample EST1, which was in good agreement with the XRD results. The XRD test indicated the

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higher crystallinity for sample T2. However, preparation of the hybrid coatings based on EST1 have some advantages such as simple process without any extensive powder dispersing which can eliminates the probability of agglomeration of titania particles in the coating process and as a result can increase transparency.

(a)

(b) Figure 11: Photo degradation of methylene blue.

3.5.5. Durability of the nanocomposite coatings SEM micrographs of sample EST1 and sample EST2 after 1080 h exposure to accelerated weathering conditions are shown in Figure 12.

As seen in Figure 12a and b, the coating surface damage was a function of photocatalytic activity of the nanoparticles in the coating. The photo degrada-

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(b)

(a)

Figure 12: Photo degradation of the EST1 (a) and EST2 (b) coatings under 1080h UV irradiation.

tion was more severe in sample EST2 than sample EST1 which corresponds to higher photocatalytic activity in epoxy hybrid coating containing calcinated titania nanoparticles.

The comparison study on photocatalytic behavior of coating using titania nanoparticles prepared of two different synthesis method showed a challenging decision. In comparison with the sample containing calcinated titania nanoparticles, the use of titania nanoparticle prepared at mild conditions into the epoxy based coating (as sample EST1) resulted to ease of synthesis, lower size nanoparticles, more uniform distribution of nanoparticles into binder, lower anatase crystalline and finally lower photocatalytic activity. However, the photocatalytic behavior of hybrid coating was rather adequate in comparison with the sample EST2.

These findings advise the use of titania nanoparticles which were prepared at mild conditions into the epoxy based coating to reach a hybrid coating of rather good photocatalytic behavior.

3.5.6. Coating adhesion measurements The adhesion of the films on glass surface is based on the condensation of hydroxyl groups of the glass surface and the coating solution. The results were specified as a 5B class (the highest adhesion

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strength) for the ES and EST1 series and 4B for the EST2 and EST3 series.

Figure 9 shows SEM micrographs of the cross section of coating prepared from Epoxy-Silica hybrid (ES) on the glass slide. The thickness of the films was evaluated to be about ~15 Č?m. The adhesion test was indirectly supported by the SEM observations on the cross cut section of above film-glass interfaces. In fact, the elongated fibrils were observed in the cross section of coating-glass interfaces showing for high coating adhesion.

Therefore, according to the results of cross-cut test and SEM cross-section, a good level of adhesion was observed between the coatings and the glass substrate. The good coating adhesion may be attributed to the appearance of Si-O-Si covalent bonds at the glass-coating interface [39].

4. CONCLUSIONS The titania-silica core/shell nanoparticles were successfully prepared from TBT and TEOS via sol-gel method. The crystalline phase of titania sol annealed in low (T1) and high (T2) temperatures were investigated by X-ray diffraction measurement. The results of XRD showed the appearance

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of anatase phase in titania nanoparticles. The TEM images exhibited a regular five-sided shape with particle size of about 35 nm forTS1 core/shell nanoparticles, while image of TS2 showed irregular shape particles with particle size of about 80 nm.

Furthermore, TEM images showed the formation of silica layer on the titania particles with thickness of about 15 nm. The EDX mapping of Si and Ti showed the sample prepared at mild condition (TS1 sol) has a better nanoparticles distribution and dispersion than other sample (TS2 and TS3 powders) in the organic-inorganic hybrid coatings.

For this reason, the transparency of the hybrid coating contain TS1 core/shell nanoparticles was higher than other samples with TS2 and TS3 (i.e., EST2 and EST3). The photocatalytic activity of the prepared titania-silica core/shell nanoparticles as studied by decomposition of MB aqueous solution showed higher photocatalytic behavior. Besides, the decomposition of MB showed the highest photocatalytic activity for EST2 compared to EST1 and EST3 samples mainly due to the lower crystal size and higher surface area of TS2 than TS1 and TS3. In comparison with the coating containing calcinated titania nanoparticles (sample EST2), the use of titania nanoparticle prepared at low temperature into the epoxy based coating (sample EST1) resulted to ease of synthesis (i.e., at mild condition), more uniform distribution of nanoparticles into binder, lower size nanoparticles, lower anatase crystalline and finally lower photocatalytic activity. The Durability of sample EST1 hybrid coating was rather good in comparison with the sample EST2. These findings favor the use of titania nanoparticles prepared at mild conditions into the epoxy based coating to get a hybrid coating of rather good photocatalytic behavior.

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14. Pazokifard S., Mirabedini S.M., Esfandeh M., Mohseni M., & Ranjbar Z., Surf. Interface.

Anal., 44 (2012), 41.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

A Facile Microwave Method to Produce High Crystalline CoFe2O4 Nano-particles Qazale Sadr Manuchehri1*, Hediyeh Bakhtiari2, Navid Assi3 1

M.Sc., Young Researchers Club, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran 2

3

M.Sc., Department of Chemistry, Azad University, South Tehran Branch, Tehran, Iran

Ph.D., Student, Department of Chemistry, Azad University, Science and Research Branch, Tehran, Iran Received: 26 May 2013; Accepted: 1 August 2013

ABSTRACT CoFe2O4 have been synthesized via a surfactant assisted gel microwave route with a molar ratio of Fe/Co= 2 and oleic acid (OA) was used as a surfactant. Fourier Transform Infrared spectroscopy (FTIR), X-Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) were used to consider the structural and morphological properties of CoFe2O4 nano-particles. Results demonstrated that oleic acid is an effective surfactant for producing high crystalline powders. The average particle size and the percentage of crystallinity were calculated 34.7 nm and 90%, respectively.

Keyword: Nanomaterial; Size Distribution; Cobalt Ferrite; Crystallinity; Microwave; Oleic Acid.

1. INTRODUCTION During the last few decades, more attention has been focused on the preparation and characterization of super-paramagnetic metal oxide nano-particles such as spinel ferrites, MFe2O4 (M = Co, Zn, etc.) [1]. Among this material, cobalt ferrite has very specific properties, such as its high coercivity, moderate saturation magnetization, high chemical stability [2], good photo-catalytic activity [3] and high absorption capacity to remove heavy metal or toxic material from water [4].

Magnetic nano-structures have the ability to (*) Corresponding Author - e-mail: gmanuchehri@yahoo.com

affect the relaxation process and thus may be used as contrast agents upon accumulation in tissue [5].

In addition of role of coating agent to provide a stable suspension, coating of magnetic nanoparticles with a suitable biocompatible material is essential for in vivo applications. The coating is essential to prevent the formation of large aggregates, changes of the original structure and sedimentation and bio-degradation when exposed to the biological system [6].

Different methods have been developed to

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synthesize ferrite nano-particles such as solvothermal [7-9], low temperature combustion [10], microwave [11, 12], sol-gel [13] and coprecipitation [14-16]. Generally, in most types of nano-synthesis routes, control of size, distribution and crystallinity are not possible [17]. Among them, microwave synthesis has been shown not only to increase the rate of chemical reactions, but also to give better yields in some cases [11].

The purpose of this work was to develop a simple one-pot surfactant assisted-microwave route in aqueous media to produce a narrow size distribution and uniform nano-cobalt ferrite particles by using surfactant. This method is very attractive for its versatility, since it has been shown to be suitable to prepare high-quality CoFe2O4 nano-crystals [18].

In this work, OA was used for synthesis of CoFe2O4 nano-particles with simple gelmicrowave technique. Characterization of nanoparticles showed the high crystallinity and good average size by using the OA as surfactant.

2. EXPERIMENTAL 2.1. Materials Iron (III) nitrate nona-hydrate (Merck 99-101%), cobalt nitrate hexa-hydrate (Merck 99%), urea ((CO(NH2)2), Merck 99%), and special-grade of oleic acid (C18H32O4, Merck 88%), ammonia (NH4OH, Merck 25%) used without any purification.

2.2. Synthesize of CoFe2O4 Nano-particles synthesis in the presence of OA as a surfactant carried out as follow: Appropriate amounts of Fe(NO3)3.9H2O, Co(NO3)2.6H2O and urea, in a molar ratio of 1:2:3 were first dissolved in a minimum amount of deionized water. Ammonia was added drop by drop to the mixed solution to adjust pH around 7. Then 0.3 mmol of OA added to the solution. During water evaporation, the solution was continuously stirred using a magnetic agitator and heated at 60°C to transform into a gel. Obtained viscous gel was poured into the wide head beaker

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and was irradiated at 500 W for 5 min in a microwave oven.

After solution reaches the point of spontaneous combustion, it began burning and released a great amount of heat, vaporized all the remaining water and became a solid burning at the high temperature.

The nano-powders were calcined in 600°C with an electrical furnace for 2 hours.

2.3. Characterization process X-ray diffraction (XRD) pattern was measured by a "Philips X'pert", using Cu Kα radiation at 40 kv and 30 mA. A "Philips XL-30" scanning electron microscope was used to characterizing the morphologies and microstructure of the samples.

Fourier transform infrared spectra (FTIR) were measured with a "Thermo Nicolet Nexus 470 ESP" FT-IR within the wave number range of 5000-400 cm-1 using KBr pressed pellet technique.

3. RESULTS AND DISCUSSION Figure 1 illustrates X-ray powder diffraction pattern of obtained nano-powders. It is evident that produce powder contains only the spinel cubic ferrite. All the peaks in the pattern well matched with JCPDS (No. 22-1086) card.

Average crystallite sizes calculated from the full-width at half-maximum (FWHM) of diffraction peaks using Scherrer's formula (Eq. 1): d= 0.9 λ/ βcosθ

(Eq.1)

Where d is the grain diameter, β is half-intensity width of the relevant diffraction; λ is X-ray wavelength and θ the diffraction angle.

The percentage of crystallinity of sample calculated from ratio of net area to total area.

Results shown that the good crystallinity about 90% was obtained. Using the Bragg law, for any cubic system, the lattice parameter calculated from Eq. 2.

Sin2θ= λ2 (h2 + k2 + l2)/ 4a2

(Eq. 2)

Where λ is the lamp wavelength, (h k l) are the

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miller planes, θ is the maximum peak position and a is the lattice parameter. The measured value of this parameter for the sample is in good agreement with the reported value [19].

Figure 2 shows the SEM images of cobalt ferrite nano-particles. According to the images, cobalt ferrite nano-particles lead to formation of fine and uniform particles with a spherical shape. Some soft agglomeration was observed due to magnetic nature of the sample. The observed particle size, 35 nm, in these images confirmed with Scherrer's calculation

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from XRD data series.

Figure 3 illustrates the FTIR spectrum of un-calcined cobalt ferrite. Spectrum has a broad band around 3422.45 cm-1 which attributed to asymmetric and symmetric OH stretching vibration of lattice water. Characteristic vibrations around 2926 and 2853 cm-1 are indicated the stretching of asymmetric and symmetric vibrations of (-CH2-) group of oleic acid, respectively. The weak peak at 2337.14 cm-1 confirmed by observed peaks at Limaye and et al. [20] work corresponds to

Figure 1: X-ray diffraction pattern of CoFe2O4 synthesis in present of OA as a surfactant.

Figure 2: SEM imagining of CoFe2O4 nano-particles synthesized with OA as a surfactant.

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Figure 3: FTIR spectrum of un-calcined CoFe2O4 nano-particles.

gas-phase CO2. This phenomenon may be due to adsorption of gas in the porous structure of cobalt ferrite from air. Also the peak at 1635.27 cm-1 has been assigned to H-O-H vibration bending of the absorbed water. In the region of 1000-1400 cm-1 some peaks overlapped. OA coated CoFe2O4 nanoparticles show two distinct vibrations at 1583 and 1413 cm-1 and are attributed to asymmetric and symmetric vibrations of COO, receptively [21]. The peak at 1108.97 cm-1 characteristic of C-O, C-C or C=O Vibration bands. At last, the sharp peak at 577.58 cm-1 demonstrate Metal-O (Fe-O or Co-O) stretching band.

4. CONCLUSIONS Cobalt ferrite nano-particles were produced successfully in microwave system using the oleic acid as a surfactant. Results demonstrated that obtained CoFe2O4 nano-particles by combination of applied surfactant and microwave irradiating, have high crystallinity and uniform morphology. In addition, FTIR spectrum of the microwave irradiated sample has an oleic-cape layer that

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allows homogenous particle size and uniform shape. The average particle size and percent crystallinity were calculated from X-ray powder diffraction data 34.7 nm and 90%, respectively.

REFERENCES 1. Liu C., Rondinone A.J., Zhang Z.J., Pure Appl.

Chem., 72 (2000), 37.

2. Kim C.H., Myung Y., Cho Y.J., Kim H.S., Park S.H., Park J., Kim J.Y., Kim B., J. Phys. Chem.

C, 113 (2009), 7085.

3. Casbeer E., Sharma V.K., Li X.Z., Sep. Purif.

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4. Zhang S., Niu H., Cai Y., Zhao X., Shi Y., Chem.

Eng. J., 158 (2010), 599.

5. Joshi M., Lin H., Aslam Y.P., Prasad M., P.V., Schultz-Sikma, E.A., Edelman, R., Meade, T., Dravid, V.P., J. Phys. Chem. C, 113 (2009), 17761.

6. Terreno E., Castelli D.D., Alessandra Viale A., Aime S., Chem. Rev., 110 (2010), 3019.

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8. Yanez-Vilar S., Sanchez-And ujar M., GomezAguirre C., Mira J., Senaris-Rodriguez M.A., Castro-Garcia, S., J. Solid State Chem., 182 (2009), 2685.

9. Repko A., Niznansky D., Poltierova-Vejpravova J., J, Nanopart Res, 13 (2011), 5021.

10.Xiao S.H., Jiang W.F., Li L.Y., Li X.J., Mater.

Chem. Phys., 106 (2007), 82.

11.Bensebaa F., Zavaliche F., L'Ecuyer P., Cochrane R.W., Veres T., J. Colloid Interf. Sci., 277 (2004), 104.

12.Ibrahima A.M., Abd El-Latif M.M., Mahmoud M.M., J. Alloy. Compd., 506 (2010), 201.

13.Gopalan E.V., Joy P.A., Al-Omari I.A., Sakthi Kumar D., Yoshida Y., Anantharaman M.R., J. Alloy. Compd., 485 (2009), 711.

14.Chena Y., Ruan M., Jiang Y.F., Cheng S.G., Li W., J. Alloy. Compd., 493 (2010), 36.

15.Tartaj P., Morales M.P., Veintemillas-Verdaguer S., Gonzalez-Carreno T., J Serna C., J. Phys. D. Appl. Phys., 36 (2003), 182.

16.El-Okr M.M., Salema M.A., Salim M.S., El-Okr R.M., Ashoush M., Talaat H.M., J. Magn.

Magn. Mater, 323 (2011), 920.

17.Maaz K., Mumtaz A., Hasanain S.K., Ceylan A., J. Magn. Magn. Mater, 308 (2007), 289.

18.Khorrami S.A., Sadr Manuchehri Q., Sadeghipour S., Int. J. Bio-Inorg. Hybd.

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19.Ayyappan S., Philip, J., Raj, B., Mater. Chem.

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20.Limaye M.V., Singh S.B., Date S.K., Kothari D., Reddy V.R., Gupta A., Sathe V., Choudhary R.J., Kulkarni S.K., J. Phys. Chem. B, 113 (2009), 9070.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

Determination of Potassium Sorbate and Sodium Benzoate in "Doogh" by HPLC and Comparison with Spectrophotometry Neda Bahremand1*, Soheil Eskandari2 1 2

M.Sc. Student, Varamin Pishva Branch, Islamic Azad University, Varamin, Iran

Assistant Professor, Food and Drug Organization, Ministry of Health and Medical Education, Tehran, Iran Received: 4 June 2013; Accepted: 17 August 2013

ABSTRACT There are various methods for the analysis of Potassium Sorbate and Sodium Benzoate in food products, but a rapid and reliable method for identification of these preservatives in Doogh (an Iranian traditional dairy drink) is a procedure, in which high performance liquid chromatography (HPLC) utilized and followed by UV diode array detection of the two preservatives. The aim of this case study was determination of Potassium Sorbate and Sodium Benzoate in Doogh, Samples consumed in the city of Tehran, Iran by HPLC in compare of Spectrophotometry method. In this study, 27 samples were analyzed. The HPLC determination of the preservatives was performed reversed-phase; C18 column and UV detected at 225 nm for sodium benzoate and 255 nm for potassium sorbate. In Spectrophotometry method, Sodium benzoate and Potassium sorbate were detected in 228 nm and 250 nm, respectively. The results of spectrophotometry in low concentrations, showed high values in comparison to what had been mention by HPLC. In high concentration, spectrophotometry showed the low value in comparison to HPLC. In conclusion, spectrophotometry could not detect and determine the Potassium sorbate and Sodium benzoate in a sample at the same time with reliable and exact results.

Keyword: Preservatives; Sodium benzoate; Potassium sorbate; Doogh; Detection; Reversedphase.

1. INTRODUCTION Chemical preservation has become an increasingly important practice in modern food technology with the increase in the production of processed foods. (*) Corresponding Author - e-mail: bahremand_neda@yahoo.com

These preservatives are added to stop or delay nutritional losses due to microbiological, enzymatic or chemical changes of foods and to extend shelf

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life and quality of foods; they also prevent hazards for consumers due to the presence of microbial toxin or pathogenic microorganisms and economic losses due to spoilage. The most commonly used preservatives in many types of foods are Benzoic and Sorbic acids, Nitrate and Nitrite (Kucukcetin et al., 2008; Santini et al., 2009). Benzoic and Sorbic acids and their respective sodium, Potassium and Calcium salts are the most commonly used preservatives in food stuffs. They are generally used to inhibit Yeast and Mould growth and being also effective against a wide range of bacteria. These compounds are most active in foods with low pH value and ineffective at neutral pH value (Santini et al., 2009; Tfouni and Toledo, 2002). At acidic pH, where Sorbic and Benzoic acids and their relative salts are so effective, the lipophilic undissociated molecule is freely permeable across the cell membrane. Subsequently upon encountering the higher pH inside the cell, the molecule dissociates resulting in the release of changed anions and protons, which cannot cross the plasma membrane (Cigic et al. 2000). The importance of food preservatives for consumers has always been a health safety issue (Kucukcetin et al., 2008). Although Benzoic and Sorbic acids and their salts are generally recognized as safe (GRAS) but the development of allergic reactions to Benzoate in humans, such as Uriticaria, non-immunological contact Urticaria, metabolic acidosis, convulsions, hyperpnoea, weak clastogenic activity and asthma has been reported in some studies (Tfouni and Toledo, 2002; Wen et al., 2007; Santini et al., 2009; Lino and Pena, 2010). Further studies showed that Sorbic acid has a relatively low toxicity to humans, explained by the fact that it is rapidly metabolized by path ways similar to those of other fatty acids. In humans a few cases of idiosyncratic intolerance to Sorbic acid and Sorbate salts have been reported (non-immunological contact Urticaria and pseudoallergy) (Santini et al., 2009; Tfouni and Toledo, 2002). According to aforementioned reasons, Sorbic acid and Sorbate salts (especially Potassium sorbate) have become the leading preservatives for a wide variety of food products (Santini et al., 2009). For these reasons, the uses of food additives

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in different countries have been limited by specific regulations. These preservatives are allowed by legislation but their use demands special care. Iran follows regulations of Institute of Standard and Industrial Research of Iran (ISIRI) on the safe use of food additives (Kucukcetin et al., 2008). The acceptable daily intake (ADI) values, determined by the joint FAO/WHO expert committee on food additives (JECFA) is 25 mg/Kg of body mass for Sorbic acid and Sorbates salts. According to ISIRI, Potassium sorbate and Sodium benzoate usage in dairy products is prohibited.

The analytical determination of these preservatives is not only important for quality assurance purposes but also for consumer interest and protection. The most common analytical method for the determination of Benzoic acid (BA) , Sorbic acid (SA) or Sodium benzoate (E211) and Potassium sorbate (E202) has been reversed-phase HPLC (Saad et al., 2005; Theron and Rykerslues, 2011), although other analytical methods such as Capillary Electrophoresis (Tang and Wu, 2007), Spectrophotometry (Hofer and Jenewein, 2000), Gas Chromatography-Mass Spectrometry (Galli and Barabas, 2004), Thermal description Gas Chromatography (Wang et al., 2006), HPLC (Ferreira et al., 2000; Pylpiw and Grether, 2000; Chen and Wang, 2001; Cigic et al., 2001; Tfouni and Toldo, 2002; Saad et al., 2005; Chinnici et al., 2005; Kucukcetin et al., 2008) and SPME-HPLC (Wen et al., 2007) have also been reported. Such a method become so important as there seem to be an increasing trend in using combination of preservatives in food stuff. Here we report on a simplified procedure of separation Sodium benzoate (E211) and Potassium sorbate (E202) mixture followed by HPLC. The method was applied to the analysis of these preservatives in 27 samples.

Doogh as a traditional drink in Iran with a high value of nutrients (same as fermented milk and Yoghurt) and remedial property has a numerous effects on human's healthiness such as: Improving lactose digestion; lowering serum cholesterol levels and stimulating the immune system (Olson and Aryana, 2007).

The purpose of this study was to investigate the

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existence of Sodium benzoate, Potassium sorbate, Nitrate and Nitrite in strained yoghurt, kasar cheese, tulum cheese and ayran which were commercially available on the local markets in Antalya, Turkey in order to compare their levels to allowable ones.

2. MATERIALS AND METHODS 2.1. Sampling The samples of Doogh with different brands were purchased from vendors in Tehran, Iran. A total of 27 samples were chosen as a representative of what a consumer would find in market-basket. Samples' sizes ranged from 500 mL to 1 Liter. Each sample was tested for the two preservative, Sodium benzoate and Potassium sorbate.

2.2. Standards and chemicals HPLC grade acetonitrile and other reagents such as Ammonium acetate, Glacial acetic acid, Chloridric acid and Petroleum benzene (analytical grade) were purchased from Merck (Darmstradt, Germany).

Commercial standards of Sodium benzoate and Potassium sorbate were used (Sigma chemical).

Deionised water used for chromatography processing was obtained from a Millipore Milli-Q water purification system (ELGA, UHQ-II-MK3 and UK). For the filtration of sample prior to injection, a Millex HV0.45ȝm filter (Millipore) was used.

2.3. Mobile phase preparation The mobile phase consists of 90% ammonium acetate buffer with 10% HPLC-Grade acetonitrile was prepared in two steps (Pylypiw and Grether, 2000): Step1: Acetate Buffer: exactly 0.30 gr of ammonium acetate were dissolved in approximately 900 mL of deionised water in a 1 L beaker. Then approximately 0.5 mL of Glacial acetic acid added to this solution acid and the pH adjusted to 4.2.

After that, buffer solution was transferred to 1 L volumetric flask, brought to the volume and filtered through a 47 mm × 0.45 µm nylon filters.

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Step2: Completion: Exactly 900 mL of the Acetate buffer solution was mixed with 100 mL of HPLC grade Acetonitrile. This was mixed, degassed in degasser (ultrasonic clear sweep system) and used for sample dilution, (standard dilution and HPLC mobile).

2.4. Analysis of sodium benzoate and potassium sorbate 2.4.1. Sample preparation 2.4.1.1. HPLC method The Liquid Chromatography Technique was used to determine the concentrations of Sodium benzoate and Potassium sorbate in the samples, following the procedures described by Pylypiw and Grether, 2000. Each of Doogh samples degassed in an ultrasonic bath and 1.0 mL of sample was diluted (1:10) with mobile phase. After that, the obtained aqueous phase solution transferred into dry falcon and put in centrifuge (biofuge primco 6000 Heraeus) for 6000 rpm/15 min. The clear aqueous solution on top of samples in falcons were caught with pipettes and filtered through a 25 mm × 0.45 ȝm nylon Acrodisk filter in order to remove particulate matter from the samples and prevent these particles from damaging the pumping or injection system or clogging the column. After that, aqueous phase solution was transferred to dry vials of HPLC and put on Auto sampler of HPLC for determination and detection.

2.4.1.2. Spectrophotometry method Firstly, Doogh samples degassed in ultrasonic bath and then filtered with Watman paper No.42. , 5 mL of clear solution were caught and added 0.4 mL Chloridric acid 6 N and brought to 50 mL with petroleum Benzene and shake vigorously for 1 min.

Sodium benzoate and Potassium sorbet detected in 228 nm and 250 nm, respectively.

2.4.2. Apparatus 2.4.2.1. Spectrophotometry conditions Shimadzu, UV visible spectrophotometer Pharma Spec UV-17000, with UV detection at 228 nm for Sodium benzoate and 250 nm for Potassium sorbate were used. Correlation coefficient value was 0.994 for either preservative.

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2.4.2.2. HPLC conditions The chromatographic analysis was carried out in a high-performance liquid chromatography from Dionex, equipped as follows: ultimate 3000 pump, ASI-100 Automated sample injector, UVD 170U detector, thermostatted column compartment oven TCC-100. The HPLC operating mode was isocratic, the injection volume was 20 ȝL and the column temperature 20°C (room temperature). The chromatography column was a Supelcosil LC-18: 25 cm X 4.6 mm, 5 mm, Supelco, Bellefonte, PA, USA. Sample data collection was optimized to 30 min per sample with UV detection at wavelength of maximum absorption of the compounds, 225 nm for Sodium benzoate and 255 nm for Potassium sorbate, with the detector wavelength switched between analyses during each run. The optimal flow rate was determined 0.8 mL/min. Correlation coefficients value was 0.996 for either preservative.

2.4.3. Preparation of the standard curve The External Standard Plot method was used.

Duplicate injections of 20 µL Sodium benzoate and Potassium sorbate standard solutions were done to make linear regression lines (peak area versus concentration). The peaks were identified based on the retention time. The standard curves were obtained from five points for both of Sodium benzoate and Potassium sorbate. Concentrations values were 5, 10, 20 and 40 mg/L. 2.4.4. Recovery study In order to verify the accuracy and precision of the analytical procedure, the recovery studies were carried out. The recovery of Sodium benzoate and Potassium sorbate added to the samples free of the two preservatives. Samples of Doogh were analyzed before and after addition of 100 and 200 mg of sodium benzoate and potassium sorbate to 100 mL of the samples.

3. RESULTS AND DISCUSSION The Recent research which has been done in accordance to the legal obligations of preservatives

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usage in Doogh, legislated by authorized organizations, shows that HPLC method is more applicable in compression to the other ones. In this case study, HPLC method compared with spectrophotometry method's results. The analytical method used for extraction of Sodium Benzoate and Potassium Sorbate in samples was based on (Pylypiw and Greyher, 2000). Spiked samples were chosen as a prototype to validate this procedure. Recovery for sodium benzoate was 83-96% and 82-93% for potassium sorbate. The correlation coefficients of Sodium Benzoate and Potassium Sorbate were 0.9968 for either preservative.

In HPLC method, values found in the separation and the resolution of the peak, indicate that the analytical method proposed in this work completely separates the analytes. The approximate retention time was 9.80 min for Sodium benzoate and 26.50 min for Potassium sorbate. The limit of detection (LOD) for Sodium benzoate and Potassium sorbate were 0.15 mg/kg and 0.24 mg/kg in the samples, respectively. The limit of quantitation (LOQ) for Sodium benzoate and Potassium sorbate were 0.5 mg/kg and 0.8 mg/kg in the samples, respectively. Recoveries ranged from 93.1-96.3% and 92.9-99.7% respectively. The mean regression equations for concentrations of Sodium benzoate and Potassium sorbate versus arbitrary units of peak area were Y= 48.92 X + 34.51 (Y represents peak area, X represents concentration in mg/L) and Y= 134.04 X + 11.30, respectively. The correlation coefficients for standard curves of Sodium benzoate and Potassium sorbate were 0.9993 and 0.9988, respectively. Table 1 shows mean concentrations (mg/kg) of Sodium benzoate and Potassium sorbate in Doogh samples, whereas the typical chromatogram of standard of Sodium benzoate and Potassium sorbate are shown in Figure 1.

100% of Doogh samples contained Sodium benzoate in the range of 18.3-2345.16 mg/kg, which are not acceptable according to Institute of Standard and Industrial Research of Iran (ISIRI).

25.92% of samples contained concentrations of Potassium sorbate between 0~ 4961.3 mg/kg, which was not in compliance with the ISIRI

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Table 1: Concentrations (mg/kg) of sodium benzoate and potassium sorbate in Doogh.

Sodium benzoate

Potassium sorbate

Method Mean

Range

Mean

Range

HPLC

12.008

53.71

13.07

54.07

Spectrophotometry

195.893

1270.52

198.8

4961.3

legislations. Only 25.9% of Doogh samples contained both of Sodium benzoate and Potassium sorbate.

Our results were complying with previous studies. Results of HPLC were more exact in compression to spectrophotometry's results. Results of some samples in HPLC and spectrophotometry were so different. Achieved Results of spectrophotometry were varying, especially in samples which were spiked with two preservatives at same time.

Statistical analysis and spearman post hoc test was used to evaluate the correlation. A significant correlation was observed (P<0.05). Statistical calculation showed that in medium concentrations,

there were overlap between HPLC and spectrophotometry's results and determined values became closer to each other. There were huge difference in high value and low value. The results of spectrophotometry in low concentration, showed high values in comparison to what had been mention by HPLC and it means that in low concentration of potassium sorbate, there were lots of differences in results of spectrophotometry. In high concentration, spectrophotometry showed low value in comparison to HPLC. In conclusion, spectrophotometry could not detect and determined the Potassium sorbate and Sodium benzoate in a sample at the same time and also does not have

Figure 1: Potassium sorbate's results comparison in samples. The x axis represents the number of sample; y axis represents the measure (maybe ppm or mg/dl).

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Figure 2: eak of sodium benzoate achieved from HPLC. The x axis represents Time (min); The y axis represents Reflect Wavelength (nm).

Figure 3: Peak of sodium benzoate achieved from HPLC. The x axis represents Time (min); The y axis represents Reflect Wavelength (nm).

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accurate results because potassium sorbate and sodium benzoate peaks over lapped. For this reason, in a Doogh sample with mixed of these two preservatives, HPLC must be used. In flavored Doogh, essence and flavors caused difficulties in detection and determination.

4. CONCLUSIONS Many report's methods use complicated and labor-intensive pre-treatment procedures such as Steam Distillation Multiple Steps and Solid-Phase Extractions. Comparing to the previous methods (Tfouni and Toledo, 2002), the present analytical method simplifies the analysis considerably, reduces its cost and time also encompasses higher level of sensitivity. HPLC method is preferred one in using to quantitative determination of Sodium Benzoate and Potassium Sorbate in Doogh.

The extracted information about general detections of Sodium Benzoate and Potassium Sorbate in most samples shows that they are commonly used as a preservative in Doogh. The Sodium Benzoate and Potassium Sorbate usage in Doogh is prohibited by the Institute of Standard and Industrial Research of Iran (ISIRI). Therefore, using of Sorbate and Benzoate should be regulated and on the other hand more cooperation between producers, processors and the regional administration seems to be essential.

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6. Ferreira I.M.P.L.V.O., Mendes E., Brito p., & Ferreira M.A., Food Res. Intl., 33 (2000), 113.

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8. Hattingh L.N., and Viljoen C.B., Int. Dairy. J., 11 (2001), 1.

9. Kucukcetin A., Sik B., and Demir M., Aras.

Makal, 33 (4) (2008), 159.

10. Olson D.W., & Aryana K.J., Sci. Direct, 41 (2007), 911.

11. Pylypiw H.M., and Grether M.T., J. Chromatogr. A, 883 (2000), 299.

12. Saad B., Bari M.F., Saleh M.I., Ahmad K., and Talib M.K., J. Chromatogr. A, 1073 (2005), 393.

13. Tang Y., Wu M., Food Chem., 103 (2007), 243.

14. Tfouni S.A.V., Toledo M.C.F., Food Control, 13 (2002), 117.

15. M. Theron, F.J. Maria Rykerslues, 2011.

Organic acid and food preservation, the academic division.

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17. Wen Y., Wang Y., and Fng Y.Q., Anal. Bio. Anal Chem., 388 (2007), 1779.

REFERENCES 1. Castineira A., Pena R.M., Herrero C., and Garcia-Martin S., J. Chromatogr. A, 23 (2000), 647.

2. Chen Q.C., and Wang J., J. Chromatogr. A, 973 (2001), 299.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

Surface-Modified Superparamagnetic Nanoparticles Fe3O4@PEG for Drug Delivery Afsaneh Sharafi1*, Mirabdullah Seyedsadjadi2 1

Ph.D., Department of Chemistry, Science and Research Branch, Islamic Azad University, Tehran 14778

2

Associate Professor, Department of Chemistry, Science and Research Branch, Islamic Azad University,

92855, Iran Tehran 14778 92855, Iran Received: 12 June 2013; Accepted: 19 August 2013

ABSTRACT In this work, we report on the synthesis of superparamagnetic iron oxide nanoparticles at room temperature using microemulsion template phase consisting of cyclohexane, water, CTAB as cationic surfactant and butanol as a cosurfactant. Surface modification have been carried out by using poly(ethyleneglycol) (PEG). The structure,morphology, and magnetic properties of the products were characterized by X-ray powder diffraction (XRD), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and vibrating sample magnetometer (VSM) at room temperature. The results revealed formation of iron oxide nanoparticles, with an average size of 8.8-12 nm, a superparamagnetism behavior with fast response to applied magnetic fields and zero remanence and coercivity.

Keyword: Inverse micelle; Surface modification; Superparamagnetism; Functionalize; Poly(ethyleneglycol); Nano magnetic.

1. INTRODUCTION The relatively large surface area and highly active surface sites of nanoparticles enable them to have a wide range of potential applications magnetic iron oxide nanoparticles as a new kind of nanometersized material, have multiple practical applications, such as physics, medicine, and biology due to their multifunctional properties such as small size, superparamagnetism and low toxicity, etc [1-3].

In recent years, ferrofluids (FFs), colloidal (*) Corresponding Author - e-mail: af.sharafi@yahoo.com

suspensions of magnetic nano-ferroparticles stabilized by polymer coating,have attracted a great attention in the biomedical fields related to magnetic resonance imaging (MRI) [4-6], hyperthermia therapy of cancers [7, 8], and targeted drug delivery [9, 10]. A particular interest is placed on the superparamagnetic nano-ferroparticles (with a diameter < 10 nm) of magnetite (Fe3O4) because of their high magnetic saturation, negligible

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toxicity, and easier surface modification properties.

These polymers include dextran [11, 12], poly(ethylene glycol) (PEG) [13-18], and poly(vinylpyrrolidone) (PVP) [19]. In particular, PEG exhibits great predominance with improved biocompatibility, biodegradability and blood circulation times.

In this paper, we Fe3O4 nanoparticles coated with poly(ethylene glycol) (PEG) were synthesized.

Despite the use of large quantities of poly(ethylene glycol) (PEG) is high Mghnatysty property. And also due to be dissolved in water is suitable for drug delivery.

of Fe3O4 and silica coated Fe3O4 nanoparticles.

Figure 1(a) shows that standard Fe3O4 crystal with spinal structure has six diffraction peaks ((220), (311), (400), (422), (511), and (440)). The presence of diffused broad peaks of Fe3O4@PEG indicates lower crystalline order owing to the formation of larger fraction of PEG. The preponderance of amorphous peaks of PEG indicates that the crystalline behavior of Fe3O4 is suppressed due to the presence of large fraction of PEG in comparison to Fe3O4 nanocrystals. The average crystalline size of Fe3O4 nanostructures at the characteristic peak (311) were calculated by using Scherer formula:

2. EXPERIMENTAL

D = kλ/βcosθ

2.1. Preparation of magnetite iron oxide nanoparticles The magnetic nanoparticles were prepared by the reverse microemulsion method. First 3gr of cetyltrimetyl ammonium bromid (CTAB) and 10 mL n-butanol were added in 60 mL of n-hexane.

The mixture was stirred at 100 rpm for 20 min and was added dropping aqueous solution of FeCl2/FeCl3 (0.14 g / 0.06 g, 2.7 mL water) under nitrogen (N2) atmosphere and purging with N2 for 20 min. An ammonium hydroxide solution (16% NH4OH in water, 0.7 mL) was finally dropped in the solution under N2 protection. The nanoparticles were isolated by centrifugating and washed with ethanol [20].

Where, D is the mean grain size, k is a geometric factor, λ is the X-ray wavelength, β is the FWHM of diffraction peak and θ is the diffraction angle.

The results of D values, using the peak (311) planes of the spinel structures was 35 nm for uncoated.

2.2. Preparation of Fe3O4@PEG nanoparticles Fe3O4@PEG nanoparticles were prepared by the stober method. The magnetic nanoparticals Fe3O4 (0.01 g) was dissolved in mixed solution of water (20 mL). (0.1 g) PEG was added to the mixed solution with stirring and reactant for 15 h. The nanoparticles were isolated by centrifugating and washed with ethanol.

3. RESULTS AND DISCUSSION 3.1. X-Ray study Figure 1 (a, b) shows the X-ray diffraction pattern

438

(1)

Figure 1: X-ray powder diffraction patterns of: a) Fe3O4 nanoparticles and (b) Fe3O4@PEG composite particle.

3.2. Edx study The surface composition of PEG coated sample was qualitatively determined by energy dispersion spectrum (EDS) as shown in Figure 2. It shows that Fe and C and O peak are obtained and atomic (%) ratio of O/ Fe /C = 66.88/2.33/5.25. It is therefore assumed that PEG is coated onto the surface of Fe3O4 nanoparticles.

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Table 2: Assignment of FTIR spectra of Fe3O4 and Fe3O4@PEG shown in Figures 3 (a, b).

Description

630-568

629

ν (HO-H) stretching

3422

3415

3446

ν (HO-H) bending

1636

1632

1643

1107

1110

1342

1351

stretching ν (C-O) asymmetric stretching

Table 1: EDAX quantification element normalized.

Element

[Wt.%]

[At%]

O

54.07

66.88

Mg

24.27

19.76

Br

9.46

2.34

Fe

6.59

2.33

C

3.19

5.25

N

2.43

3.43

ν (C-O-O) ν (C=O) symmetric

1453

1806

stretching ν (C=O) asymmetric

FTIR spectra of unmodified and PEG modified magnetite nanoparticles and PEG is shown in Figure 3 (a, b). The strong broad peaks at about 630 cm-1 and 568 cm-1 (in Figure a) are due to the stretching vibrations of Fe-O bonds. As shown in the (Figure b), strong peaks in the 957cm-1 corresponding to the stretching mode of vinyl double bonds disappeared in the spectrum of PEG-coated particles indicating that polymerization has taken place. The C-O-C ether stretch band at 1107 cm-1 and vibration band at 1342 cm-1 (antisymmetric stretch) appear in the FTIR spectrum of the nanoparticles after PEG modification. The bands around 2912 and 955 cm-1 correspond to CH2 stretching vibrations and CH out- of -plane bending vibrations, respectively. The C-O-C, CH2, and CH peaks are strong evidence that PEG-coated nanoparticles surface.

PEG

ν (Fe-O)

ν (C-O) symmetric

Figure 2: Edx result of PEG coated Fe3O4 nanoparticles.

Fe3O4 Fe3O4@PEG

1944

stretching ν (CH vinyl)

956

ν (CH2)

2886

3.3. Morphological study Figure 4 (a, b) shows the SEM image of the Fe3O4 nanoparticles (a), the Fe3O4 nanoparticles Coated with PEG (b). According to the SEM image the agglomeration is very strong in the Fe3O4 nanoparticles. After surface modification, there is a little aggregation. This indicated that PEG molecules may help to decrease the size of adsorption of PEG chain onto the Fe3O4 nanoparticle surface.

3.4. Magnetic study Figure 5 (a, b) represents magnetic-field-dependent magnetization parameters, M(H) for Fe3O4; and Fe3O4@PEG at room temperature, using vibrating sample magnetometer with a peak field of 15 kOe. The hystersis loops for Fe3O4 ; and Fe3O4@PEG with coercivity (Hc= 0.0 Oe) and remanence (Mr= 0) indicate a superparagnetism properties at 300 K with a saturation magnetization of 65 emu/g for Fe3O4; and 50 emu/g for Fe3O4@PEG.

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Figure 3 (a, b): FT.IR Spectra a) Fe3O4 b) Fe3O4@PEG c) PEG.

(a)

(b)

Figure 4 (a, b): a) SEM image of Fe3O4 ; b) SEM image of Fe3O4@PEG Core-Shell nanostructures.

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Figure 5: Magnetization vs. applied magnetic field for a) Fe3O4; b) Fe3O4@PEG, at room temperature.

4. CONCLUSIONS 3 Fe3O4 nanoparticles were prepared by the microemulsion technique using Fe3+ and Fe2+ and surface modification have been carried out by using poly(ethyleneglycol) (PEG).

3 X-ray diffraction pattern showed formation of spinal structure for prepared Fe3O4.

3 FTIR spectra showed formation PEG on to the surface of Fe3O4 nanoparticles.

3 Edx analysis data showed the presence of PEG in our prepared sample.

3 Microemulsion (inverse micelle) is a suitable way for obtaining the uniform and size controllable nanoparticles.

3 Preparation of homogeneous particle size and particle distribution has been possible using microemulsion method.

REFERENCES 1. Chomchoey N., Bhongsuwan D., Bhongsuwan1 T., Kasetsart J., Nat. Sci., 44 (2010), 963.

2. Hoa L.T.M., Dung T.T., Danh T.M., Duc N.H., Chien D.M., J, Phys., 187 (2009), 012048.

3. Acar H.Y.C., Garaas R.S., Syud F., Bonitatebus P., Kulkarni A.M., J. Magn. Magn. Mater, 293 (2005), 1.

4. Herve K., Douziech-Eyrolles L., Munnier E.,

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Cohen-Jonathan S., Souce M., Marchais H., Limelette P., Warmont F., Saboungi M.L. ,Dubois P., and Chourpa I., Nanotechnology, 19 (2008), 465608.

5. Feng B., Hong R.Y., Wang L.S., Guo L., Li H.Z., Ding J., Zheng Y., Wei D.G., Colloids Surf., A, 328 (2008), 52.

6. Chertok B., Moffat B.A., David A.E., Yu F.Q., Bergemann C., Ross B.D., Yang V.C., Biomaterials, 29 (2008), 487.

7. Zhang L.Y., Gu H.C., Wang X.M., J, Magn Magn Mater, 311 (2007), 228.

8. Hergt R., Dutz S., J, Magn Magn Mater, 311 (2007), 187.

9. Alexiou C., Arnold W., Klein R.J., Parak F.G., Hulin P., Bergemann C., Erhardt W., Wagenpfeil S., Lubbe A.S., Cancer Res., 60 (2000), 6641.

10. Son S.J., Reichel J., He B., Schuchman M., Lee S.B., J, Am Chem Soc., 127 (2005), 7316.

11. Kaufman C.L., Williams M., Ryle L.M., Smith T.L., Tanner M., Ho C., Transplantation, 76 (2003), 1043.

12. Berry C.C., Wells S., Charles S., Aitchison G., Curtis A.S., Biomaterials, 25 (2004), 5405.

13. Zhang Y, Kohler N, Zhang M, Biomaterials, 23 (2002), 1553.

14. Butterworth MD, Illum L, Davis SS, Colloids Surf A, 179 (2001), 93.

15. Xie J, Xu C, Kohler N, Hou Y, Sun S, Adv Mater, 19 (2007), 3163.

16. Mondini S, Cenedese S, Marinoni G, Molteni G, Santo N,Bianchi CL, Ponti A, J, Colloid Interface Sci, 322 (2008), 173.

17. Zhang Y., Kohler N., Zhang MQ, Biomaterials, 23 (2002), 1553.

18. Peng J., Zou F., Liu L., Tang L., Yu L., Chen W., Liu H., Tang J.B., Wu L.X., Trans Nonferrous Met Soc China, 18 (2008), 393.

19. D'Souza A.J., Schowen R.L., Topp E.M., J, Controlled Release, 94 (2004), 91.

20. Sajadi M.S., Sharafi A., Farhadyar N., J, Nano Research, 21 (2013), 37.

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International Journal of Bio-Inorganic Hybrid Nanomaterials

Synthesis and Morphology of Face Centered Cubic (FCC) Fe-Pt Nanoparticles Majid Farahmandjou Assistant Professor, Department of Physics, Varamin Pishva Branch, Islamic Azad University, Varamin, Iran Received: 19 June 2013; Accepted: 23 August 2013

ABSTRACT FePt nanoparticles with thermally stable room-temperature ferromagnetism are investigated. The monodisperse nanoparticles are prepared by chemical synthesis and a salt-matrix annealing technique. Structural and magnetic characterizations confirm the phase transition from the disordered face-centered cubic structure. In this paper, 3 nm FePt nanoparticles are first synthesized by superhydride reduction of Fe and Pt. Transmission Electron Microscopy (TEM) images show that the hard magnetic FePt are agglomerated after annealing at 675째C. Scanning Electron Microscopy (SEM) images indicate that the size of FePt nanoparticles increase by increasing of the annealing temperature.

Keyword: FePt nanocrystals; Anisotropy; Hard magnetic nanoparticles; Chemical synthesis; Sintering; Recording media.

1. INTRODUCTION Development of new high density magnetic media for data recording has become a key issue for data storage industry. In the near future current magnetic media will reach the areal density beyond of 1 Tbit/in2 imposed by the phenomenon known as superparamagnetism [1]. Generally, the FePt films deposited at room temperature are face-centered cubic (fcc) and show a soft-magnetic behavior [2].

The fcc FePt phase can be transformed to a hard-magnetic face-centered tetragonal (fct) FePt (L10 FePt) ordered phase after annealing at high temperature [3]. In L10 alloy, the FePt system (*) Corresponding Author - e-mail: farahmandjou@iauvaramin.ac.ir

presents highly coercivity, good corrosion resistance, large magnetic energy product but high temperature annealing is required to transform fcc disordered structure to L10 ordered FePt phase.

Recently, the areal density of recording media is approaching to 1 Tbits/in2. Such a high density would require a grain size of only a few nanometers in diameter [4, 5]. For high density magnetic recording media, the magnetic grains must be small enough to be nanoparticle size and also requires that uniform size and isolated magnetic particles to reduce inter-grain interaction, which leads to lower

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media noise [6-8]. However, small particle size will result in smaller KuV/KBT value (where Ku is the uniaxial magneto-crystalline anisotropy, V is the particle volume, KB is the Bolzmann's constant and T is the absolute temperature), which leads increasing the thermal fluctuation of magnetization [9, 10]. It well know that large Ku can resist thermal fluctuation of magnetization even the particle size is very small. The Ku value of FePt alloy is as high as 7×107 erg/cm3 and the saturation magnetization, Ms is about 680 emu/cm3.The large magneto-crystalline anisotropy energy, may be due to the spin-orbit coupling of Pt atom in which Pt atoms can have some induced moment in ferromagnetic state [11].

The chemical synthesis of very small FePt nanoparticles was reported by Sun et al. [12]. Due to the organic coating, particle size and geometry is well preserved even after thermal treatments at high temperatures. As-made particles crystallize in a disordered fcc phase with a relatively small anisotropy. The order-disorder transformation can be obtained by thermal annealing [13]. In stoichiometric FePt complete order of the high anisotropy L10 tetragonal phase can be only achieved with a temperature annealing higher than 600°C which undesirably lead to the particle agglomeration and sintering [14].

In this paper, the colloid fcc FePt nanoparticles are fabricated with 3 nm diameter. The size and structure of the FePt have been studied by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) and vibrating sample magnetometer (VSM) analyses before and after heat treatment.

Farahmandjou M.

mixture was cooled to room temperature and then combined with methanol to remove the impurity.

The fcc FePt nanoparticles was stirred until all the solvent evaporates at room temperature and then annealed to complete the fcc to fct transition.

The annealing temperature fixed at 675°C for 3.5 hours. Morphology of the FePt nanoparticles before and after annealing was observed by TEM analysis using a Philips EM 208 TEM (100kV) with resolution 200 kX. To determine the nanoparticle's structure, XRD measurement was prepared after evaporation of hexane on a Silicon wafer using a Seifert with Cu-Kα (wavelength= 1.54 Å) radiation. SEM analysis was done using VEGA (15 kV) 50 Kx. The magnetization of FePt samples in a variable magnetic field was measured using vibrating sample magnetometer (VSM).

3. RESULTS AND DISCUSSION Figure 1 indicates the as-synthesized colloidal FePt nanoparticles. It is realized that the color of the samples changes from light to dark with increasing temperature from 110°C to 210°C. It is because that the monomers are released from precursors with increasing temperature and then the number of nanoparticles increases.

2. EXPERIMENTAL DETAILS Synthesis of the nanoparticles, involves the reduction of Pt(acac)2 and FeCl2, in phenyl ether solvent. The oleic acid and oleylamine surfactants were added to solvent, at 110°C, as a protective agent in order to prevent agglomeration and oxidation. By adding superhydride at 210°C, the FePt nanoparticles were formed. The black reaction

444

Figure 1: As-synthesized colloidal FePt nanoparticles.

Figure 2a shows (TEM) image of the as-synthesized fcc FePt nanoparticles in presence of oleic acid and oleylamine surfactant stabilizers. The samples were refluxed at 250°C for 20 min. As you

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(b)

(a) Figure 2: TEM image of the (a) as-synthesis and (b) annealed FePt nanoparticles.

Figure 3: SEM image of the annealed FePt nanoparticles.

can see from the picture, the 3 nm FePt nanoparticles are dispersed on the TEM grid because of surfactant stabilizers. Figure 2b indicates TEM image of the salt-matrix FePt nanoaprticles with size of 50 nm in diameter annealed at 675°C for 3.5 hours. It is observed that the nanoparticles agglomerate to each other and the size of them increases [15].

Figure 3 shows the SEM image of the FePt nanoparticles without salt-matrix annealing after

heat treatment. SEM image indicate the size of annealed FePt increases to 1 µm in diameter. In fact, by increasing annealing temperature the surfactants are removed and the FePt agglomerated.

Figure 4 indicates X-ray diffraction (XRD) pattern of the FePt nanopartcles annealed at 675°C for 3.5 hours. The sharp peaks indicate that size and crystallity of FePt nanoparticles increase after annealing process. These peaks provide evidence of chemical ordering phase fcc to fct transition.

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Figure 4: XRD patterns of the annealed FePt nanoparticles.

Figure 5 shows the results of magnetic measurements after heat treatments by vibrating sample magnetometer (VSM). It is understood that after annealing process the saturation magnetization increases to 50 emu/g and the coersivity increases to 6.5 kOe. This high coercivity of fct FePt nanoparticles is because of the high magnetocrystalline anisotropy in L10 phase.

treatment. The size and crystallity of FePt nanoparticles were increased with increasing annealing temperature. Phase transition from fcc to fct were done after heat treatment.

ACKNOWLEGMENTS This work was supported by the Physics Research Center, Science and Research Campus at Islamic Azad University.

REFERENCES

Figure 5: Magnetic hysterisis loops of annealed FePt nanoparticle.

4. CONCLUSIONS Fcc and Fct FePt nanoaprticles were fabricated by chemically route. It was found that by salt matrix annealing the size of FePt nanoparticles increased from 3 nm to 50 nm in diameter after heat

446

1. Bertram H.N., Zhou H., Gustafson R., IEEE Trans. Magn., 34 (1998), 1845.

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3. Daniil M., Farber P.A., Okumura H., Hadjipanayis G.C., Weller D., J. Magn. Magn.

Mater., 246 (2002), 297.

4. Joseyphus J., Shinoda K., Sato Y., Tohji K., Jeyadevan B., J, Mater Sci., 43 (2008), 2402.

5. Tournus F., Sato K., Epicier T., Konno T.J., Dupuis V., Phys. Rev. Lett., 110 (2013), 055501.

6. Ding Y., Majetich A.S., Kim J., Barmak K., Rollins H., Sides P., J. Magn. Magn. Mater., 284 (2004), 336.

7. Ranjan R., Christner J.A., Ravipati D.P., IEEE Trans. Magn., 26 (1990), 322.

8. Lee Y.H., Wang J.P., Lu L., J. Appl. Phys., 87 (2000), 6346.

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9. Gittleman J.I., Abeles B., Bozowski S., Phys.

Rev. B, 9 (1974), 3891.

10. Shimatsu T., Lodder J.C., Sugita Y., Nakamura Y., IEEE Trans. Magn., 35 (1999), 2697.

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12. Sun S., Adv. Mater., 18 (2006), 393.

13. Wanga H.L., Huanga Y., Zhanga Y., Hadjipanayisa G.C., Wellerb D., Simopoulosc A., J. Magn. Magn. Mater., 310 (1) (2007), 22.

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15. Farahmandjou M., Sebt S.A., Chin. J. Phys., 47 (2009), 561.

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Improvement Physical Properties of Pullulan-Whey Protein Biocomposite Films with Nanoclay Mahboobeh Hasannia Kolaee1*, Faramarz Khodaiyan2, Rezvan Pourahmad3 1

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

2

Associate Professor, Department of Food Science, Engineering and Technology, Faculty of Agricultural

Branch, Iran Engineering and Technology, Campus of Agriculture and Natural Resources, University of Tehran, Iran 3

Assistant Professor, Department of Food Science and Technology, Islamic Azad University, VaraminPishva Branch, Iran Received: 23 June 2013; Accepted: 1 September 2013

ABSTRACT In the current study, whey protein- pullulan- clay nanocomposite films are prepared by casting method. The effect of nanoclay at three concentrations (1%, 3% and 5%) on physical properties such as moisture content, solubility in water, water vapor permeability and transparency of whey protein- pullulan composite films investigated. The results show that the effect of nanoparticles on composite depends on kind of nanoparticle and level of incorporation. Nanoclay particles changed solubility, water absorption and moisture content of the films but did not influence on transparency.

Keyword: Edible films; Whey protein; Pullulan; Nano clay; Biodegradable; Biocomposite.

1. INTRODUCTION Approximately 125mt plastic is produced in the world yearly. 41% of plastic consumption is related to packaging industry of which food packaging accounts for 47%. Pollution resulted from packaging materials produced by oil derivatives as well as problems resulted from different methods of fumigation (such as burning, burning and recycling) have made researchers to find appropriate substitutes for these packaging materials. One of proposed solutions is to use from (*) Corresponding Author - e-mail: mh.hasannia@gmail.com

edible and biodegradable films as a substitute for petroleum undegradable polymers [11]. In this research pullulan and whey protein were used as biodegradable biopolymer. Pullulan is an extracellular microbial polysaccharide that produces by Aureaobasidium pullulans. Pullulan films are biodegradable, transparent, highly impermeable to oil and impermeable to oxygen [11]. Whey protein is the byproduct of cheese industry which is left after case in has been curd at 20째C and pH= 4.64.

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Use of whey protein for film production seems desirable because of high nutritional value of whey, optimal use of cheese making wastes and decrease of environmental problems [7]. As it said biopolymers have advantages than synthetic polymers among which biodegradability and renewability are the most important. However mechanical and barrier properties are two basic drawbacks of biopolymers which limit industrial use of these materials for packaging. To solve this problem use of nanoparticles has been proposed since it improves biopolymer efficiency as well as mechanical, thermal and barrier properties.

Therefore a nanoparticle has an important role at improvement of biopolymer properties and can be used for food packaging [16, 20]. Capability of nanoclay at diffusing as separate layers, changing surface characteristics of these substances, compatibility with different kinds of polymers and biopolymers. Lower price and easier availability are among causes that have enhanced use of nanoclay for producing biopolymer nanocomposites [22].

Various natural biopolymer, including carbohydrate such as cellulose [14] starch [4] and chitosan [17] and protein such as soy protein [9, 10] gelatin [15] and whey protein [19] have been tested to exploit the property enhancement through the formation of nanocomposites. The main purpose of this study is improve barrier properties of WPI with PUL and nanoclay and investigated physical properties of these films.

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2.2. Method 2.2.1. Film preparation 5 g WPI dissolved in 100 mL distilled water and heated at 90°C for 30 min and stirred with magnetic stirrer. Then 5 g pullulan dissolved in 100 mL distilled water and mixed with WPI solution (1:1) w/w after that 30% w/v glycerol added into the solution. Three clay suspension prepared by ultrasonic bath (Elma, s 60 h, Germany) at ambient temperature for 1 h. So that the concentration of clay in WPI/PUL filmogenic solution was 1%, 3% and 5% wt (dray base). Finally after drying the solution at 25°C, the films peeled out and conditioned at 25°C and relative humidity 50% for 48 h before all tests.

2.2.2. Moisture content Moisture content (MC) of film specimens were determined by weight of sample before (m1) and after (m2) oven drying (Shimaz co., Heraeus, Germany) at 105°C (Eq. 1).

MC =

m1 − m2 ×100 m2

(1)

2.2.3. Moisture absorption The film specimens have conditioned by calcium sulphate at RH= 0% for 24 h then weight (m1) and placed them in desiccator containing saturated solution of calcium nitrite at -25°C in order to achieve 55% relative humidity. After equilibrium state the samples weighed (m2) and Moisture absorption (MA) calculated by Eq. 2.

2. MATERIALS AND METHODS 2.1. Material WPI was purchased from (Arla food ingredient, Denmark), pullulan was provided from Hayashibara (Hayashibara Co LTD, Japan) and nanoclay was supplied by TECNAN (Tecnologia Navarra de Nanoproductos S.L, Spain). The analytical grade chemicals including sodium chloride (NaCl), calcium chloride (CaCl2) and calcium nitrite (Ca(NO2)2) were purchased from Merck (Merck Co, Germany).

450

MA =

m1 − m2 ×100 m2

(2)

2.2.4. Water vapor permeability Water vapor permeability was measured according to ASTM E96 [1]. The films were fixed on the top of glass cup containing CaCl2 and sealed with melted paraffin then the cups placed in a desiccator contain saturated NaCl solution and the cups weighted every hour for a period of 24 hour. The

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slope (S) of time-weight was calculated by linear regression model (R2≥0.986). WVTR and WVP were calculated by Eq. 3 and Eq. 4.

WVTR =

WVP =

S A

WVTR × X ∆P

(3)

(4)

3. RESULTS 3.1. Moisture content Moisture content of WPI-PUL and nanocomposite films observed in Figure 1. Moisture content of WPI-PUL film is 16.93% and decreased slowly and reaches to 14.85%. Lowest moisture content is 12.99% at 3% clay concentration increasing nanoclay content up to 5% increased moisture content. However this enhancement was not si gnificantly difference.

Where A is the effective film area (m2), x is the average film thickness (m) and ∆P is the driving force (1753.55 Pa).

2.2.5. Solubility in water The film specimens were dried at 105°C, and weighed (mL) then dried samples immersed into 50 ml of distilled water for 6 h. The remained films were dried at 105°C and weighed (m2). Solubility of the film (SW) in water calculates by Eq. 5.

SW =

m1 − m2 × 100 m2

(5) Figure 1: Effect of clay content on moisture content of WPI-PUL film.

2.2.6. Transparency Transparency of the films was determined by (Testo 540 pocket sized lux meters, UK).

2.2.7. Scanning electron microscopy The morphology of the surface and the crosssection of films were observed by field emission scanning electron microscopy (FE-SEM) KYKYEM3200 (KYKY, China) with the accelerating beam at a voltage 5 kV. The film specimens were sputtered with a thin layer of gold using a KYKYSBC-12 sputter coater (KYKY, China).

2.2.8. Statistical analysis Statistics on a completely randomized design were performed with the analysis of variance (ANOVA) procedure using SPSS software (Version 20; SPSS Inc., USA). Duncan's multiple range tests were used to compare the difference among mean values of film specimen’s properties at the level of 0.05.

Figure 2: Effect of clay content on moisture absorption of WPI-PUL film.

3.2. Moisture absorption One of important problems at using biopolymers is their tendency to absorb water. Results related to

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amount of water absorption by WPI-PUL film in Figure 2 indicated that moisture absorption of WPI-PUL is 15.48% and reduced significantly by addition of nanoclay so that it reaches to 9.81% at concentration of 3% clay concentration.

Figure 4: Effect of clay content on solubility in water of WPI-PUL film.

Figure 3: Effect of clay content on water vapor permeability of WPI-PUL film.

3.3. Water vapor permeability As a result that has shown in Figure 3, Water vapor permeability of WPI-PUL film is 1.62×10-10 g m-1 s-1 pa-1 that indicates weak barrier properties of this biopolymer. By addition of 1% and 3% nanoclay water vapor permeability was reduced to 1.59×10-10 gm-1s-1pa-1 and 1.1×1010 gm-1s-1pa-1 respectively.

3.4. Solubility in water Figure 4 Shows solubility in water of WPI-PUL and its nanocomposite films. Solubility in water related to the clay concentration. Solubility in water of WPI-PUL film is 87.46% and decreased significantly to 62.84% at 1% clay concentration. Lowest solubility is 61.35% and observed at 3% clay concentration.

3.5. Transparency Figure 5 Shows that WPI-PUL film have high transparency 92% and decreased slowly by additional of clay concentration but transparency didn't change significantly for all samples.

Figure 5: Effect of clay content on transparency of WPI-PUL film.

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(a)

(b)

Figure 6: Cross section of WPI-PUL and WPI-PUL - 3% nanoclay composite film.

3.6. SEM micrograph Figure 6 exhibited the cross section of WPI-PUL and nanocomposite film with 3% clay concentration. Cross section of WPI-PUL film is smooth and homogenous. At 3% clay concentration white particle appear on the cross section of the film and the size of nano particle was determined.

4. DISCUSSION 4.1. Moisture content As show in Figure 1, Nanoclay decreased moisture content of nanocomposite films. It may be due to good dispersion of nanoclay particle in polymer matrix and good interaction between polymer and nanoclay particles. These interactions reduce the occupied void volume of polymer matrix by water molecules [7]. Increasing nanoclay content up to 5% increased moisture content. However this enhancement was not significantly difference but presumably it is due to declining homogeneity of nanoclay particles in polymer matrix. Increasing nanoclay content up to 5% increased moisture content. However this enhancement was not significantly different but presumably it is due to declining homogeneity of nanoclay particles in polymer matrix.

4.2. Moisture absorption One of important problems at using biopolymers is their tendency to absorb water. As shown in Figure 2, nanoclay decreased water absorption of biopolymer. It may be attributed to creation of hydrogen bond between hydroxyl group of polymer and oxygen atoms in nanoclay. The occupation of hydroxyl group of polymer with nanoclay decreases absorption of water by polymer matrix [6]. Similar results have been reported in CMC-starch composite films [2].

4.3. Water vapor permeability One of the most important uses of food packaging is reduce moisture between food and atmosphere and water vapour permeability should be at minimum. Reduced water vapour permeability of nanoclay composites is due to presence of silicate impervious layers dispersed into polymeric matrix creating tortuous path way for molecules. Therefore these molecules should go around the layers to pass the film which results in enhancement of time and way length thus reduction in permeability [18]. In addition hydrophilic properties of nanoclay and its compatibility with hydrophilic biopolymer lead to improvement of water vapour permeability.

Reduced water vapour permeability due to addition of nanoclay in other biopolymers such as chitosan

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[17] and thermoplastic starch [13] has been reported.

4.4. Solubility in water As result in Figure 4 clay decreased solubility in water of nanocomposite films. This is mainly due to formation of strong interaction through hydrogen bonds between hydroxyl groups of the biopolymer matrix and nanoclay with high surface area, thus improving the cohesiveness of biopolymer matrix and decreasing the water sensitivity [3]. Decresed in solubility by additional of clay content reported in starch - cmc films [2] and chitosan film [3].

4.5. Transparency The transparency of film is a desirable property once the consumer wishes to see clearly the aspect of the product which the film will cover. The result shows that transparency of nanocomposite films didn't change significantly. When clay platelets well dispersed through the polymeric matrix since clay platelets are less than the wavelength of visible light and didn't hinder light passage [12, 21]. The similar result reported that nanoclay didn't change transparency of film at low concentration but at high concentration nanoclay wasn't completely dispersed and formed agglomeration in the polymeric matrix and decrease transparency of the films [17].

4.6. SEM micrograph As shown in Figure 6. nano clay well disturbed into polymeric matrix and preserve the uniformity of the structure of the films. Previous researcher have been shown, if there is affinity between nano particle and polymeric matrix, nano particle can distribute homogeneously [17].

5. CONCLUSIONS In this study nanoclay was used to improve properties of WPI - PUL film. Nanoclay increased water vapour barrier properties of the biopolymer and reduced water absorption and moisture content.

Also results showed that nanoclay led to reduction

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in water solubility of nanocomposite film.

Transparency of the film didn't change significantly due to dispersion of nanoparticles into polymeric matrix.

ACKNOWLEDGMENTS Authors wish to express their especial thanks to the University of Tehran, Department of Food Science and Technology because of provided facilities for this study in Transfer Phenomena Lab (TPL) and also to Islamic Azad University, Varamin-Pishva Branch for their assistance.

REFERENCES 1. ASTM. 1995. Standard test methods for water vapor transition of material, E 95-96. Annual book of ASTM. Philadelphia, PA: American society for testing and material.

2. Alamsi H., Ghanbarzadeh B., & Entezami A.A., Int. J. Biol. Macromol., 46 (2010), 1.

3. Casariego A., Souza B.W.S., Cerqueira M.A., Teixeira J.A., Cruz L., Diaz R., Food Hydrocolloid, 23 (2009), 1895.

4. Cyras V.P., Manfredi L.B., Ton-That M.T., & Vazquez A., Carbohydr. Polym., 73 (2008), 55.

5. De Wit J.N., J. Dairy Sci., 81 (1998), 597.

6. Ghanbarzadeh B., Almasi H., & Entezami A.A., Innov. Food Sci. Emerg. Technol., (2010), doi:10.1016/j. ifset. 2010. 06.001.

7. Jiang Y., Li Y., Chai Z., & Leng X., J. Agric.

Food Chem., 58 (2010), 5100.

8. Kester J.J., & Fennema O., Food Technol., 40 (1986), 47.

9. Kumar P., Sandeep K.P., Alavi S., Truong V.D., & Gorga R.E., J. Food Eng.,100 (2010a), 480.

10.Kumar P., Sandeep K.P., Alavi S., Truong V.D., & Gorga R.E., J. Food Sci., 75 (2010b), N46.

11.Lazaridou A., Biliaderis G. & Kontogiorgos V., Carbohydr. Polym., 51 (2003), 151.

12.Ogata N., Jimenez G., Kawai H., & Ogihara T., J. Polym. Sci., Part B: Polym. Phys., 35 (1997), 389.

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13.Park H.M., Li X., Jin C.Z., Park C.Y., Cho W.J., & Ha C.S., Macromol Mater Eng., 287 (2002), 553.

14. Park H.M., Liang X., Mohanty A.K., Manjusri M., & Drzal L.T., Macromolecules, 37 (2004), 9076.

15.Rao Y.Q., Polymer, 48 (2007), 5369.

16.Ray S.S., & Bousmina M., Mater. Scie., 50 (2005), 962.

17.Rhim J.W., Hong S.I., Park H.M., Ng P.K.W., J. Agric. Food. Chem., 54 (2006), 5814.

18.Rhim J.W., Carbohydr. Polym., 86 (2011), 691.

19.Sothornvi R., Hong S.I., An D.J., & Rhim J.W., LWT-Food Sci., Technol., 43 (2010), 279.

20. Sozer N., & Kokini J.L., Trends Biotechnol, 27 (2009), 82.

21.Tang S., Zou P., Xiong H., & Tang H., Carbohydr. Polym., 72 (2008), 521.

22.Yano K., Usuki A., & Okai A., J. Polym. Sci., Part A: Polym. Chem., 35 (1997), 2289.

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1,3-Dipolar Cycloaddition Reaction of Dibenzalaceton with Non-stabilized Azomethinylides: Synthesis of New Spirooxindolo(pyrrolizidines/ pyrrolidines) Mohammad Javad Taghizadeh1*, Mir Hasan Hosseini2, Meysam Sadeghi2 1

Ph.D., Organic Chemistry, Department of Chemistry, School of Sciences University of Imam Hossein, Tehran, Iran

2

M.Sc., Inorganic Chemistry, Department of Chemistry, School of Sciences University of Imam Hossein, Tehran, Iran Received:: 2 July 2013 ; Accepted: 8 September 2013

ABSTRACT 1,3-dipolar cycloaddition reaction of 1 mol or 2 mol of dibenzalaceton with 1 mol of non-stabilized azomethinylides generated in situ by decarboxylative condensation of isatin and proline or sarcosine give the novel new spiro-oxindolo(pyrrolizidines/ pyrrolidines) instead of bis-spirooxindolo(pyrrolizidines/ pyrrolidines).

Keyword: 1,3-Dipolar cycloaddition; Azomethinylides; Spiro-oxindolo(pyrrolizidines/ pyrrolidines); Isatin; Proline; Sarcosine.

1. INTRODUCTION 1,3-Dipolarcycloaddition of azomethineylides is a fesible protocol for the synthesis of highly functionalised five-membered ring heterocycles [1].

Azomethineylides, generated in situ from isatin and sarcosine, add to ι,β-unsaturated carbonyl compounds to afford spiropyrrolidines [2-4].

Some spiropyrrolidines are potential antileukemic and anticonvulsant agents [5] and possess antiviral [6] and local anaesthetic [7] activities.

They are found in a number of biologically active compounds [8, 9]. Isatin and its derivatives have

interesting biological activities and are widely used as precursors for many natural products [10-14].

Spiropyrrolidinyloxindoles are also found in a number of alkaloids of biological importance [15]. Gelsemine is representatives of the alkaloids, which incorporate spiro-oxindole ring systems [16-18]. Here in we will report the synthesise a series of new spirooxindoles using a threecomponent reaction involving 1,3-dipolar cycloadditions of appropriate azomethineylides with diolefins which makes possible the simultaneous

(*) Corresponding Author - e-mail: Mohammadjavadtaghizadeh31@yahoo.com

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formation of up to four stereocenters in cycloaddact. Afterwards, the reactions were carried out in various conditions, but only at room temperature, in aqueous ethanol and in the absence of any bidentate chelating Lewis acids through a one-pot threecomponent 1,3-dipolar cycloaddition reaction of the dipolarophiles 1 with non-stabilized azomethineylides which was generated in situ by the decarboxylative condensation of isatins 2 with proline 3 or sarcosine 6 (Scheme 1 and 2).

O NH

N Me

Gelsemine

O

O

H

X O + N H

N R

O

(S)

CO2H

H

H

1

3

2

H

Various conditions

Ă&#x2014; H

H X

H NH

O O

N R

H

X

H

H NH

O H OH

N R

R N

O

N

X

H 5

4

Scheme1: Synthesis of spirooxindolopyrrolidines (4a-i).

O

O

H

X O + N H

N R

CO2H

H

H

1

6

2

H

Various conditions

Ă&#x2014; H H X

O

NH O N R

7

X

O

NH

H OH

H N R

H 8

Scheme 2: Synthesis of spirooxindolopyrrolizidines (7a-i).

458

O

R N N

X

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respectively. The 1HNMR spectrum of 7g exhibits a triplet signal at δ= 3.64 ppm and a doublet at δ= 4.14 ppm which are related to Hb and Ha protons respectively. Stereochemistryand absolute configuration of spirooxindoles 7g was determined by singlecrystal X-ray analysis (Figure 1).

2. RESULTS AND DISCUSSION The dibenzalaceton 1 was prepared based on the literature procedure [19, 20]. Three component reactions between dipolarophile 1, isatin derivatives 2 and L-proline 3 or sarcosine 6 carried out in ethanol at room temperature. As shown in Scheme 1 and 2, condensation of compounds 2 and 3 after decarboxylation leading to the non-stabilized azomethineylides stereogenic centers in one step.

We expected by this method a bis-spirooxindolo (pyrrolizidines/ pyrrolidines) 5 and 8 products would be prepared, but by using the 1 mmol or 2 mmol of the dipolarophileonly the diastereoisomers 4 and 7 were obtained purely in high total yield (Scheme 1 and 2). After this, other derivatives of this new spirooxindolo(pyrrolizidines/ pyrrolidines) were also synthesized. The results are summarized in Table1.

The structures of cycloaducts were assigned by IR, 1HNMR, 13CNMR Observation of three characteristic singlet at about (52.5, 66.5 and 72.6) in the 13CNMR spectra of 4 and two characteristic singlet at about (60.8 and 65.4) in the 13CNMR spectra of 7 is consistent with formation new pyrrolidine cyclic. The stereochemistry and the correct structure of this isomer andother derivatives were determined by 1HNMR, For example, the 1HNMR spectrum of 4g exhibits a triplet signal at δ= 3.83 ppm, a multiple at δ= 4.19 and a doublet at δ= 4.57 ppm which are related to Hb, Hcand Ha protons

Figure 1: Molecular structure of compound 7g (thermal ellipsoids at 50% probability level).

3. EXPERIMENTAL General melting points were recorded on an electrothermal digital melting point apparatus.

Infrared spectra were recorded on a mattson 1000

Table 1: Yield of spirooxindolo(pyrrolizidines/ pyrrolidines) (4a-i) and (7a-i).

Entry 1 2 3 4 5 6 7 8 9

R

X

Product

4 Yield (%)

H H H Me Me Me Et Et Et

H Br NO2 H Br NO2 H Br NO2

a b c d e f g h i

95 90 93 95 93 90 90 93 90

7 Yield (%) 85 83 80 83 80 85 87 85 83

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FTIR. 1H, 13CNMR spectra were measured with a Bruker DRX-250 AVANCE instrument with CDCl3 as solvent at 250.1 MHz. Mass spectra were recorded on a Finnigan MAT 8430 mass spectrometer operating at an electron energy of 70 eV. Isatin derivatives, proline, and sarcosine were obtained from Fluka (Buchs, Switzerland) and were used without further purification, and dibenzalaceton were obtained via synthesized.

General procedure: To a magnetically stirred solution of anisatin derivatives (1 mmol), proline or sarcosin (1 mmol) and dibenzalaceton (1 mmol) as dipolarophile in 10 mL EtOH was added dropwise at room temperature. Then, the reaction mixturewas stirred for 12 h. The solvent was then removed under reduced pressure and the residue was separated by recrystalization in CHCl3.

4. CONCLUSIONS Because of wide distribution in nature and variegated biological activities, pyrrolizidines alkaloids are very attractive synthetic targets. For the reason that a pyrrolizidine can be viewed as a fused pyrrolidine, thus method employed for the formation of pyrrolidine rings can be used to construct the pyrrolizidine ring system. So, the 1,3-Dipolar cycloaddition reaction of azomethine ylides, including pyrrolidine derivatives, with olefins can be useful method for the synthesis of pyrrolizidines. In result of we have found a tricomponent synthetic method for the prepration of some oxindoles derivatives of potential synthetic interest. The present method carries the advantage that not only is the reaction performed under neutral conditions, but also the starting materials and reagents can be mixed without any activation or modification.

2'-cinnamoyl-3'-phenyl-3',3a',4',5',6',6a'-hexahydro-2'H-spiro[indoline-3,1'-pentalen]-2-one (7a): yellow powder, yield 95%; IR(KBr)(νmax, cm-1): 1614(C=C), 1692(C=O), 1722(C=O), 3440(NH); 1HNMR (300.1 MHz, CDCl ); 1.27-2.06 (4H, m, 3 2CH2), 2.58-2.63 (2H, m, CH2), 3.86 (1H, t, 3J =9 Hz, CH), 4.19 (1H, m, CH), 4.58 (1H, d, HH

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3J

CH), 6.27 (1H, d, 3JHH=12.5 Hz, CH), 6.83-7.49 (15H, m, Ar-H), 9.04 (1H, s, NH); 13CNMR (75 MHz, CDCl ); 27.7, 31.2, 48.0 (3C, 3 3CH2), 52.5, 66.5, 72.6 (3C, 3CH), 74.1(1C),110.7(1C, 1CH), 125.4, 125.5, 127.0, 127.4, 129.6, 130.5 (6C, 6CH), 128.0, 128.3, 128.6, 128.8 (4C, 8CH), 122.5, 134.2, 139.8, 140.8 (4C), 143.6 (1C, 1CH), 181.7, 195.0 (2C, 2C=O); MS, 434 (M+, 5), 200 (100), 131 (70).

2'-cinnamoyl-5-nitro-3'-phenyl-3',3a',4',5',6',6a'hexahydro-2'H-spiro[indoline-3,1'-pentalen]-2- one (7b): yellow powder, yield 90%; IR(KBr)(νmax, cm-1): 1610(C=C), 1712(C=O), 3395(NH); 1HNMR (300.1 MHz, CDCl3); 1.26-2.05 (4H, m, 2CH2), 2.57-2.63 (2H, m, CH2), 3.85 (1H, t, 3J =9 Hz, CH), 4.19 (1H, m, CH), 4.59 (1H, d, HH 3J =9Hz, CH), 6.27 (1H, d, 3J =12.5 Hz, CH) HH HH 6.83-7.56 (14H, m, Ar-H), 8.84 (1H, s, NH); 13CNMR (75 MHz, CDCl ); 27.8, 31.1, 47.9 (3C, 3 3CH2), 52.5, 66.4, 72.6 (3C, 3CH), 74.1 (1C), 110.7 (1C, 1CH), 125.4, 125.5, 127.4, 129.6, 130.5 (5C, 5CH), 128.0, 128.3, 128.6, 128.8 (4C, 8CH), 122.5, 134.2, 137.8, 139.8, 140.8 (5C), 143.5 (1C, 1CH), 180.5, 195.0 (2C, 2C=O); MS, 479 (M+, 6), 245 (100), 176 (75).

2'-cinnamoyl-5-bromo-3'-phenyl-3', 3a', 4', 5', 6',6a'-hexahydro-2'H-spiro[indoline-3,1'-pentalen]2-one (7c): yellow powder, yield 93%; IR(KBr)(νmax, cm-1): 1603(C=C), 1719(C=O), 3422(NH); 1HNMR (300.1 MHz, CDCl3); 1.282.06 (4H, m, 2CH2), 2.57-2.63 (2H, m, CH2), 3.85 (1H, t, 3JHH=9 Hz, CH), 4.21 (1H, m, CH), 4.58 (1H, d, 3JHH=9Hz, CH), 6.27 (1H, d, 3JHH=12.5 Hz, CH) 6.75-7.49 (14H, m, Ar-H), 8.38(1H, s, NH); 13CNMR (75 MHz, CDCl3); 27.7, 31.2, 48.0 (3C, 3CH2), 52.5, 66.5, 72.6 (3C, 3CH), 74.0 (1C), 110.7 (1C, 1CH), 125.4, 125.5, 127.4, 129.6, 130.5 (5C, 5CH), 128.0, 128.3, 128.6, 128.8 (4C, 8CH), 122.4, 134.2, 137.4, 139.7, 140.8 (5C), 143.6 (1C, 1CH), 181.6, 195.1 (2C, 2C=O); MS, 513, 515 (M+, M++2, 5), 278, 280 (100), 131 (40).

2'-cinnamoyl-1-methyl-3'-phenyl-3',3a',4', 5', 6',6a'-hexahydro-2'H-spiro[indoline-3,1'-pentalen]2-one (7d): yellow powder, yield 95%; IR(KBr)(νmax, cm-1):1609(C=C), 1703(C=O), 1726(C=O); 1HNMR (300.1 MHz, CDCl3); 1.28HH=9Hz,

Taghizadeh M. et al

2.06 (4H, m, 2CH2), 2.57-2.63 (2H, m, CH2), 3.58 (3H, s, NMe), 3.85 (1H, t, 3JHH=9 Hz, CH), 4.06 (1H, m, CH), 4.41 (1H, d, 3JHH=9Hz, CH), 6.07 (1H, d, 3JHH=12.5 Hz, CH), 6.82-7.49 (15H, m, Ar-H); 13CNMR (75 MHz, CDCl3); 27.7, 31.2, 48.0 (3C, 3CH2), 42.4 (1c, NMe), 52.5, 66.4, 72.6 (3C, 3CH), 74.1 (1C), 111.2 (1C, 1CH), 125.4, 125.5, 127.1, 127.4, 129.6, 130.5 (6C, 6CH), 128.0, 128.3, 128.6, 128.8 (4C, 8CH), 122.4, 134.2, 139.7, 140.8 (4C), 143.6(1C, 1CH), 181.4, 195.1 (2C, 2C=O); MS, 448 (M+, 8), 214 (100), 131 (77).

2'-cinnamoyl-1-methyl-5-nitro-3'-phenyl3',3a',4',5',6',6a'-hexahydro-2'H-spiro[indoline-3,1'pentalen]-2-one (7e): yellow powder, yield 93%; IR(KBr)(νmax, cm-1): 1603(C=C), 1720(C=O); 1HNMR (300.1 MHz, CDCl ); 1.28-2.06 (4H, m, 3 2CH2), 2.57-2.63 (2H, m, CH2), 3.57 (3H, s, NMe), 3.85 (1H, t, 3JHH=9 Hz, CH), 4.07 (1H, m, CH), 4.41 (1H, d, 3JHH=9Hz, CH), 6.06 (1H, d, 3J =12.5 Hz, CH) 6.82-7.49 (14H, m, Ar-H); HH 13CNMR (75 MHz, CDCl ); 27.7, 31.2, 48.0 (3C, 3 3CH2), 42.4 (1c, NMe), 52.5, 66.4, 72.7 (3C, 3CH), 74.1 (1C), 110.9 (1C,1CH), 125.4, 125.5, 127.4, 129.6, 130.5 (5C, 5CH), 128.0, 128.3, 128.6, 128.8 (4C, 8CH), 122.4, 134.2, 137.2, 139.7, 140.8 (5C), 143.6 (1C, 1CH), 181.4, 195.0 (2C, 2C=O); MS, 493 (M+, 3), 259 (100), 190 (70).

5-bromo-2'-cinnamoyl-1-methyl-3'-phenyl3',3a',4',5',6',6a'-hexahydro-2'H-spiro[indoline-3,1'pentalen]-2-one (7f): yellow powder, yield 90%; IR(KBr)(νmax, cm-1): 1615(C=C), 1717(C=O); 1HNMR (300.1 MHz, CDCl ); 1.27-2.05 (4H, m, 3 2CH2), 2.57-2.64 (2H, m, CH2), 3.50 (3H, s, NMe), 3.73 (1H, t, 3JHH=9 Hz, CH), 4.09 (1H, m, CH), 4.36 (1H, d, 3JHH=9Hz, CH), 6.19 (1H, d, 3J =12.5 Hz, CH), 6.76-7.47 (14H, m, Ar-H); HH 13CNMR (75 MHz, CDCl ); 26.3, 27.3, 30.0 (3C, 3 3CH2), 42.9 (1c, NMe), 48.3, 51.0, 63.4 (3C, 3CH), 71.4 (1C), 110.7 (1C, 1CH), 125.3, 125.5, 127.3, 129.6, 130.5 (5C, 5CH), 128.1, 128.3, 128.6, 128.8 (4C, 8CH), 122.4, 134.2, 137.2, 139.7, 140.8 (5C), 143.6 (1C, 1CH), 181.4, 194.9 (2C, 2C=O); MS, 527, 529 (M+, M++2, 7), 292, 294 (100), 131 (49).

2'-cinnamoyl-1-ethyl-3'-phenyl-3',3a',4',5', 6',6a'-hexahydro-2'H-spiro[indoline-3,1'-pentalen]2-one (7g): yellow powder, yield 90%;

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IR(KBr)(νmax, cm-1): 1615(C=C), 1716(C=O); (300.1 MHz, CDCl3); 1.06 (3H, t, 3JHH=7.5 Hz, CH3), 1.67-2.03 (4H, m, 2CH ), 2 2.55-2.60 (2H, m, CH2), 3.66 (2H, q, 3JHH=7.5 Hz, CH2), 3.83 (1H, t, 3JHH=10 Hz, CH), 4.19 (1H, m, CH), 4.57 (1H, d, 3JHH=10Hz, CH), 6.22 (1H, d, 3J =12.5 Hz, CH), 6.69-7.50 (15H, m, Ar-H); HH 13CNMR (75 MHz, CDCl ); 12.3 (1C, CH ), 27.1, 3 3 30.2, 31.3 (3C, 3CH2), 35.4 (1C, NCH2), 47.2, 51.4, 65.2 (3C, 3CH), 72.4(1C), 108.6 (1C,1CH), 125.5, 125.6, 127.1, 127.4, 129.6, 130.5 (6C, 6CH), 128.2, 128.5, 128.8, 129.0 (4C, 8CH), 122.4, 134.2, 139.7, 140.8 (4C), 142.7 (1C, 1CH), 178.4, 194.7 (2C, 2C=O); MS, 462 (M+, 9), 228 (100), 159 (68).

2'-cinnamoyl-1-ethyl-5-nitro-3'-phenyl3',3a',4',5',6',6a'-hexahydro-2'H-spiro[indoline-3,1'pentalen]-2-one (7h): yellow powder, yield 93%; IR(KBr)(νmax, cm-1): 1613(C=C), 1720(C=O); 1HNMR (300.1 MHz, CDCl ); 1.07 (3H, t, 3 3J =7.5 Hz, CH ), 1.69-2.03 (4H, m, 2CH ), HH 3 2 2.53-2.60 (2H, m, CH2), 3.66 (2H, q, 3JHH=7.5 Hz, CH2), 3.83 (1H, t, 3JHH=10 Hz, CH), 4.19 (1H, m, CH), 4.57 (1H, d, 3JHH=10Hz, CH), 6.19 (1H, d, 3J =12.5 Hz, CH) 6.69-7.50 (14H, m, Ar-H); HH 13CNMR (75 MHz, CDCl ); 12.3 (1C, CH ), 27.1, 3 3 30.2, 31.6 (3C, 3CH2), 35.4 (1C, NCH2), 47.1, 51.4, 65.2 (3C, 3CH), 72.1(1C), 108.6 (1C,1CH), 125.5, 125.6, 127.4, 129.6, 130.5 (5C, 5CH), 128.2, 128.5, 128.8, 129.0 (4C, 8CH), 122.3, 134.1, 137.9, 139.7, 140.3 (5C), 142.7 (1C, 1CH), 178.4, 194.9 (2C, 2C=O); MS, 507 (M+, 4), 273 (100), 158 (73).

5-bromo-2'-cinnamoyl-1-ethyl-3'-phenyl3',3a',4',5',6',6a'-hexahydro-2'H-spiro[indoline-3,1'pentalen]-2-one (7i): yellow powder, yield 90%; IR(KBr)(νmax, cm-1): 1612(C=C), 1715(C=O); 1HNMR (300.1 MHz, CDCl ); 1.06 (3H, t, 3 3J =7.5 Hz, CH ), 1.67-2.03 (4H, m, 2CH ), HH 3 2 2.55-2.60 (2H, m, CH2), 3.64 (2H, q, 3JHH=7.5 Hz, CH2), 3.83 (1H, t, 3JHH=10 Hz, CH), 4.19 (1H, m, CH), 4.57 (1H, d, 3JHH=10Hz, CH), 6.22 (1H, d, 3J =12.5 Hz, CH) 6.69-7.49 (14H, m, Ar-H); HH 13CNMR (75 MHz, CDCl ); 12.3 (1C, CH ), 27.1, 3 3 30.2, 31.3 (3C, 3CH2), 35.5 (1C, NCH2), 47.2, 51.4, 65.2 (3C, 3CH), 72.4 (1C), 108.6 (1C,1CH), 125.5, 125.6, 127.4, 129.6, 130.5 (5C, 5CH), 128.2, 128.4, 128.8, 129.0 (4C, 8CH), 122.4, 134.1, 137.6, 1HNMR

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139.7, 140.7 (5C), 142.7 (1C, 1CH), 179.7, 194.7 (2C, 2C=O); MS, 541, 543 (M+, M++2, 8), 306, 308 (100), 131 (70).

3'-cinnamoyl-1'-methyl-4'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one (4a): yellow powder, yield 85%; IR(KBr)(νmax, cm-1): 1609(C=C), 1711(C=O), 3430(NH); 1HNMR (300.1 MHz, CDCl3); 2.16 (3H, s, NMe), 3.43 (1H, m, CH), 3.64 3J (1H, t, HH=10 Hz, CH), 4.14 (1H, d, 3J =10Hz, CH), 4.43 (1H, m, CH), 6.08 (1H, d, HH 3J =12.5 Hz, CH), 6.63-7.55 (15H, m, Ar-H), HH 8.84 (1H, s, NH); 13CNMR (75 MHz, CDCl3); 35.2 (1C, NCH3), 43.9 (1C, 1CH2), 60.8, 65.4, (2C, 2CH), 74.2 (1C), 110.1 (1C, 1CH), 125.3, 125.5, 127.1, 127.4, 129.6, 130.5 (6C, 6CH), 128.2, 128.7, 128.8, 129.4 (4C, 8CH), 122.5, 134.2, 139.8, 140.8 (4C), 143.0 (1C, 1CH), 180.6, 195.9 (2C, 2C=O); MS, 408 (M+, 5), 147 (100), 131 (68).

3'-cinnamoyl-1'-methyl-5-nitro-4'-phenylspiro [indoline-3,2'-pyrrolidin]-2-one (4b): yellow powder, yield 83%; IR(KBr)(νmax, cm-1): 1615(C=C), 1721(C=O), 3420(NH); 1HNMR (300.1 MHz, CDCl3); 2.16 (3H, s, NMe), 3.43(1H, m, CH), 3.64 (1H, t, 3JHH=10 Hz, CH), 4.14 (1H, d, 3JHH=10Hz CH), 4.43(1H, m, CH), 6.08 (1H, d, 3J =12.5 Hz, CH), 6.63-7.55 (14H, m, Ar-H), HH 8.84 (1H, s, NH); 13CNMR (75 MHz, CDCl3); 35.2 (1C, NCH3), 43.9 (1C, 1CH2), 60.8, 65.4, (2C, 2CH), 74.2 (1C), 110.1 (1C, 1CH), 125.3, 125.5, 127.4, 129.6, 130.5 (5C, 5CH), 128.2, 128.7, 128.8, 129.4 (4C, 8CH), 122.5, 134.2,137.6, 139.8, 140.8 (5C), 143.0 (1C, 1CH), 180.6, 195.9 (2C, 2C=O); MS, 453 (M+, 5), 219 (100), 131 (72).

5-bromo-3'-cinnamoyl-1'-methyl-4'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one (4c): yellow powder, yield 80%; IR(KBr)(νmax, cm-1): 1612(C=C), 1707(C=O), 3427(NH); 1HNMR (300.1 MHz, CDCl3); 2.16 (3H, s, NMe), 3.43 (1H, m, CH), 3.64 (1H, t, 3JHH=10 Hz, CH), 4.14 (1H, d, 3JHH=10Hz CH), 4.43(1H, m, CH), 6.08 (1H, d, 3J =12.5 Hz, CH) 6.63-7.55 (14H, m, Ar-H), HH 8.84(1H, s, NH); 13CNMR (75 MHz, CDCl3); 35.2 (1C, NCH3), 43.9 (1C, 1CH2), 60.8, 65.4, (2C, 2CH), 74.2 (1C), 110.1 (1C, 1CH), 125.3, 125.5, 127.4, 129.6, 130.5 (5C, 5CH), 128.2, 128.7, 128.8, 129.4 (4C, 8CH), 122.5, 134.2,137.4, 139.8, 140.8

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(5C), 143.0(1C, 1CH), 180.6, 195.9 (2C, 2C=O); MS, 483, 485 (M+, M++2, 6), 251, 253 (100), 131 (70).

3'-cinnamoyl-1,1'-dimethyl-4'-phenylspiro [indoline-3,2'-pyrrolidin]-2-one (4d): yellow powder, yield 83%; IR(KBr)(νmax, cm-1): 1609(C=C), 1703(C=O), 1725(C=O); 1HNMR (300.1 MHz, CDCl3); 2.16 (3H, s, NMe), 3.14 (3H, s, NMe), 3.43 (1H, m, CH), 3.64 (1H, t, 3JHH=10 Hz, CH), 4.14 (1H, d, 3JHH=10Hz CH), 4.43 (1H, m, CH), 6.08 (1H, d, 3JHH=12.5 Hz, CH), 6.637.55 (15H, m, Ar-H); 13CNMR (75 MHz, CDCl3); 26.2 (1C, NCH3), 35.2 (1C, NCH3), 43.9 (1C, 1CH2), 60.8, 65.4, (2C, 2CH), 74.2 (1C), 110.1 (1C, 1CH), 125.3, 125.5, 127.1, 127.4, 129.6, 130.5 (6C, 6CH), 128.2, 128.7, 128.8, 129.4 (4C, 8CH), 122.5, 134.2, 139.8, 140.8 (4C), 143.0 (1C, 1CH), 180.6, 195.9 (2C, 2C=O); MS, 422 (M+, 3), 188 (100), 131 (78).

3'-cinnamoyl-1,1'-dimethyl-5-nitro-4'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one (4e): yellow powder, yield 80%; IR(KBr)(νmax, cm-1): 1603(C=C), 1717(C=O); 1HNMR (300.1 MHz, CDCl3); 2.16(3H, s, NMe), 3.15 (3H, s, NMe), 3.43 (1H, m, CH), 3.65 (1H, t, 3JHH=10 Hz, CH), 4.14 (1H, d, 3JHH=10Hz CH), 4.45(1H, m, CH), 6.12 (1H, d, 3JHH=12.5 Hz, CH) 6.63-7.55 (14H, m, Ar-H); 13CNMR (75 MHz, CDCl3); 26.3 (1C, NCH3), 35.1 (1C, NCH3), 43.9 (1C, 1CH2), 60.9, 65.3, (2C, 2CH), 73.8(1C), 107.9 (1C, 1CH), 125.3, 125.5, 127.4, 129.6, 130.5 (5C, 5CH), 128.0, 128.2, 128.6, 128.8 (4C, 8CH), 123.2, 134.2, 137.6, 139.8, 141.9 (5C), 142.5 (1C, 1CH), 177.9, 196.1 (2C, 2C=O); MS, 467 (M+, 6), 233 (100), 131 (81).

5-bromo-3'-cinnamoyl-1,1'-dimethyl-4'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one (4f): yellow powder, yield 85%; IR(KBr)(νmax, cm-1): 1613(C=C), 1714(C=O); 1HNMR (300.1 MHz, CDCl3); 2.16(3H, s, NMe), 3.14(3H, s, NMe), 3.43 (1H, m, CH), 3.64 (1H, t, 3JHH=10 Hz, CH), 4.15 (1H, d, 3JHH=10Hz CH), 4.43 (1H, m, CH), 6.09 (1H, d, 3JHH=12.5 Hz, CH), 6.63-7.50 (14H, m, Ar-H); 13CNMR (75 MHz, CDCl3); 26.2 (1C, NCH3), 35.2 (1C, NCH3), 43.9 (1C, 1CH2), 60.8, 65.4, (2C, 2CH), 74.2 (1C), 110.1 (1C, 1CH), 125.3, 125.5, 127.4, 129.6, 130.5 (5C, 5CH), 128.1,

Taghizadeh M. et al

128.6, 128.8, 129.4 (4C, 8CH), 122.4, 134.2, 137.4, 139.8, 140.8 (5C), 143.1(1C, 1CH), 181.6, 195.8 (2C, 2C=O); MS, 501, 503 (M+, M++2, 4), 266, 268 (100), 131 (75).

3'-cinnamoyl-1-ethyl-1'-methyl-4'-phenylspiro [indoline-3,2'-pyrrolidin]-2-one (4g): yellow powder, yield 87%; IR(KBr)(νmax, cm-1): 1609(C=C), 1714(2C=O); 1HNMR (300.1 MHz, CDCl3); 1.07 (3H, t, 3JHH=7.5 Hz, CH3), 2.15 (3H, s, NMe), 3.44 (1H, m, CH), 3.64 (1H, t, 3JHH=10 Hz, CH), 3.68 (2H, q, 3JHH=7.5 Hz, CH2), 4.14 (1H, d, 3JHH=10Hz CH), 4.44 (1H, m, CH), 6.11 (1H, d, 3JHH=12.5 Hz, CH), 6.63-7.55 (15H, m, Ar-H); 13CNMR (75 MHz, CDCl3); 12.3 (1C, CH3), 35.2 (1C, NCH3), 35.5 (1C, NCH2), 43.7 (1C, 1CH2), 60.8, 65.3, (2C, 2CH), 74.1 (1C), 110.2 (1C, 1CH), 125.3, 125.5, 127.3, 127.4, 129.6, 130.5 (6C, 6CH), 128.2, 128.5, 128.7, 128.9 (4C, 8CH), 122.4, 134.2, 139.8, 140.8 (4C), 143.1 (1C, 1CH), 180.6, 195.7 (2C, 2C=O); MS, 436 (M+, 4), 202 (100), 131 (65).

3'-cinnamoyl-1-ethyl-1'-methyl-5-nitro-4'phenylspiro[indoline-3,2'-pyrrolidin]-2-one (4h): yellow powder, yield 85%; IR(KBr)(νmax, cm-1): 1609(C=C), 1717(C=O); 1HNMR (300.1 MHz, CDCl3); 1.06 (3H, t, 3JHH=7.5 Hz, CH3), 2.16 (3H, s, NMe), 3.43(1H, m, CH), 3.63 (1H, t, 3JHH=10 Hz, CH), 3.69 (2H, q, 3JHH=7.5 Hz, CH2), 4.13 (1H, d, 3JHH=10Hz CH), 4.43(1H, m, CH), 6.08 (1H, d, 3JHH=12.5 Hz, CH) 6.65-7.49 (14H, m, Ar-H); 13CNMR (75 MHz, CDCl3); 12.3 (1C, CH3), 35.2 (1C, NCH3), 35.4 (1C, NCH2), 43.9 (1C, 1CH2), 61.3, 65.4, (2C, 2CH), 74.2(1C), 110.1(1C, 1CH), 125.3, 125.5, 127.4, 129.6, 130.5 (5C, 5CH), 128.2, 128.6, 128.8, 129.3 (4C, 8CH), 122.5, 134.2,137.6, 139.8, 140.8 (5C), 143.0 (1C, 1CH), 178.9, 195.4 (2C, 2C=O); MS, 481 (M+, 6), 247 (100), 131 (73).

5-bromo-3'-cinnamoyl-1-ethyl-1'-methyl-4'phenylspiro[indoline-3,2'-pyrrolidin]-2-one (4i): yellow powder, yield 83%; IR(KBr)(νmax, cm-1): 1612(C=C), 1719(C=O); 1HNMR (300.1 MHz, CDCl3); 1.06 (3H, t, 3JHH=7.5 Hz, CH3), 2.16 (3H, s, NMe), 3.43 (1H, m, CH), 3.64 (1H, t, 3JHH=10 Hz, CH), 3.64 (2H, q, 3JHH=7.5 Hz, CH2), 4.14 (1H, d, 3JHH=10Hz CH), 4.43(1H, m, CH), 6.08

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(1H, d, 3JHH=12.5 Hz, CH), 6.63-7.55 (14H, m, Ar-H), 8.84 (1H, s, NH); 13CNMR (75 MHz, CDCl3); 12.1 (1C, CH3), 35.2 (1C, NCH3), 35.5 (1C, NCH2), 43.9 (1C, 1CH2), 60.7, 65.4, (2C, 2CH), 74.1(1C), 110.1 (1C, 1CH), 125.4, 125.5, 127.2, 129.6, 131.2 (5C, 5CH), 128.2, 128.7, 128.8, 129.4 (4C, 8CH), 122.5, 134.2,137.4, 139.7, 140.7 (5C), 143.0 (1C, 1CH), 181.2, 195.9 (2C, 2C=O); MS, 515, 517 (M+, M++2, 7), 280, 282 (100), 131 (70).

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International Journal of Bio-Inorganic Hybrid Nanomaterials

The Effect of Hydrophobicity and Hydrophilicity of Gold Nanoparticle on Proteins Structure and Function Khadijeh Eskandari1*, Mehdi Kamali1, Manizheh Ramezani1, Zahra Safiri1, Amir Homayon Keihan1, Jamal Rashidiani1, Hamid Kooshki1, Hajar Zarei2 1Assistant

Professor, Nanobiotecnology Research Center, Baqiyatallah University of Medical Science, Tehran, Iran

2

Ph.D. Student, Persian Gulf Research and Studies Center, Persian Gulf University, Bushehr, Iran Received: 8 July 2013; Accepted: 17 September 2013

ABSTRACT The surface parameter of nanoparticles such as hydrophobicity and a hydrophilicity on protein structure and function is very important. In this study, conformational changes of glucose oxidase (GOx) in the mercaptopurine: GNPs and 11-mercaptoundecanoic acid: GNPs as a hydrophobic and a hydrophilic GNPs surface was investigated by various spectroscopic techniques, including: UV-Vis absorption, fluorescence and circular dichroism (CD) spectroscopies. Moreover, the fluorescence quenching constant and binding parameters after the formation of the GOx: GNPs conjugates follows by Stern-Volmer (S-V) plots. Size of GNPs was determined by Zeta Sizer, which their size is 80 nm. CD and florescence spectroscopy show that the conformational changes in both the secondary and the tertiary structure levels of GOx in conjugate with hydrophobic and hydrophilic-GNPs was occured. Also, Stern-Volmer plots for the binding of hydrophilic-GNPs and hydrophobic-GNPs with GOx was plotted. Stern-Volmer quenching constant, binding constant and the number of binding sites of GOx: GNPs conjugates was determined.

Keyword: Glucose oxidase (GOx); Hydrophobic-GNPs; Hydrophilic-GNPs; Fluorescence and circular dichroism (CD) spectroscopy.

1. INTRODUCTION Nanoparticles have a great role in diagnostic, drug delivery, therapy, biosensing and so on. Therefore, it is necessary to improve the knowledge of the mechanisms of nanoparticles interaction with proteins, cells and tissues, for applying to the design of applicable nanodevice. In drug delivery; (*) Corresponding Author - e-mail: kha_esk@yahoo.com

its necessary nanoparticles are having at least tend to protein. Because when bound to proteins, they may be quickly cleared by macrophages before they can reach target cells [1] and sometimes such as biosensors and bio-fuel cells, high ratio of absorption of protein is needed. Therefore, the study of

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nanoparticle surface for protein immobilization and protein biocompatibility is required. Also, the changes in the structure and function of protein caused to the thoughtful effects biological activity or the activation of immune response [2, 3].

Among the various methods to characterize the protein conformational changes, the spectroscopic method is the most commonly adopted methods, that including circular dichroism (CD), UV-Vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, fluorescence spectroscopy, and so on [4-7].

Glucose oxidase (GOX, β-D-glucose oxygen 1-oxidoreductase, EC 1.1.3.4) is a homodimer flavoprotein containing two active sites per molecule [8-9]. It catalyses the oxidation of β-D-glucose to gluconic acid, concomitant with the reduction of oxygen to hydrogen peroxide. Glucose oxidase as a cheap and available enzyme has been used to test various types of enzyme immobilization, and is the most commonly studied in the construction of biosensors for glucose assay development [10].

In this study, colloidal gold nanoparticles (NC-GNPs) was synthesized by a popular procedure [11]. Also the hydrophobic and hydrophilic GNPs was prepared by mercaptopurine and 11-mercaptoundecanoic acid respectively. Then the effect of nanoparticle coating on protein structure and function as well as protein adsorption were studied.

2. EXPERIMENTAL 2.1. Reagents HAuCl4·3H2O and glucose oxidase, Horse radish peroxidase (HRP), o-dianisidine, glucose, mercaptopurine and 11-mercaptoundecanoic acid were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Hydrogen tetrachloroaurate(HAuCl4·3H2O), cysteamine, trisodium citrate, potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) were purchased from Merck (Darmstadt, Germany) and used as received.

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2.2. Apparatus Circular dichroism spectroscopy was done with Aviv, model 215 spectropolarimeter (Lakewood, NJ, USA), fluorescence spectroscopy with Hitachi spectrofluorimeter (MPF-4 model, Japan) and the UV-Vis spectroscopy by Cary spectrophotometer (100 Bio-model, USA). The hydrodynamic size and the surface charge (zeta potential) of nanoparticle were characterized with a Zeta sizer and Zeta potential analyzer (Zeta Plus, Brookhaven Instruments Corporation, USA).

2.3. Gold nanoparticles synthesis and conjugation forms For preparation of colloidal gold nanoparticles (GNPs), 25 mL of 0.01% (w/v) HAuCl4·3H2O were heated up to 60°C, then 2 mL of 0.1% (w/v) sodium citrate added to it. The final red color nanoparticles was stored in dark glass bottles at 4°C [12]. For preparation of hydrophobic and hydrophilic GNPs, 10 mM of mercaptopurine and 11-mercaptoundecanoic acid was added to GNPs with the ratio of 1/20 respectively.

3. RESULTS AND DISCUSSION 3.1. Size and surface charge analysis The hydrodynamic size and the surface charge (zeta potential) was investigated by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively [13]. The hydrodynamic size of GNPs is 80 nm and their surface charge is -16 mV. Also, the concentration of GNPs is calculated to be approximately 3.5×10-15, assuming that all gold in the HAuCl4 was reduced.

3.2. Characterization of the synthesized GNPs and GOx/GNPs conjugates The GNPs solution exhibits a color of dark red, which is known to arise from the collective oscillation of the free conduction electrons induced by an interacting electromagnetic field. UV-Vis absorption measurements indicated that the maximum wavelength of the surface plasmon resonance (SPR) was 533 nm (Figure 1). The position of this

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peak is almost unchanged in all of the GNPs, but the shape of the peak is different, especially in the hydrophobic GNPs, showing that the hydrophobic surface is susceptible to agglomeration. But hydrophilic GNPs interact with the aqueous surroundings and remain separate particles in solution.Also, in comparison with the peak of the GNPs, the peak intensity of GOx/GNPs conjugates is significantly reduced, indicating that the GOx was binding on the GNPs surface.

bonds and aromatic residues. And in GOx/GNPs, this peak shift to 270 nm, which confirms the GOx binding on the GNPs too.

Figure 2: CD spectra of GOx, 5 µL GOx (30 mg/mL) solutions was added to 245 µL PBS (52 mM), pH 7.4 (a) and the conjugates of GOx: hydrophilic-GNPs (b) and GOx: hydrophobic-GNPs (c). 5 µL (30 mg/mL) GOx solution was mixed with 100 ȝL GNPs and was diluted by 145 µLPBS (52 mM), pH 7.4.

Figure 1: UV-Vis spectra of hydrophilic-GNP (a), GOx: hydrophilic-GNP conjugates (b), hydrophobic-GNP (c), GOx: hydrophobic-GNP conjugates (d) and GOx (e). 20

µL (8 mg/mL) GOx in 200 µL GNPs.

Moreover GOx exhibits an absorbance maximum in 280 nm, which originates from peptide

3.3. Circular dichroism spectroscopy CD spectroscopy is one of the useful and common methods to study of protein conformations in solution or adsorbed onto colloidal surfaces. CD spectroscopy was performed for investigation of secondary structure in GOx/GNPs conjugates.

Figure 2 shows the far-UV CD spectra of native

Table 1: Secondary structures percentage of GOx on different GNPs was obtained by deconvolution. secondary structures

GOx Native

GOx: hydrophilic GOx: hydrophobic -GNPs -GNPs

α-helix

28.9

22.1

17.8

β-sheet

21.3

31.3

32

β-turn

17.6

19.5

20

Random coil

34.8

36.7

40.6

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(a)

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(b)

Figure 3: Effect of hydrophilic-GNPs (A) and hydrophobic-GNPs (A) on the fluorescence spectrum of GOx. 2 µL (30 mg/mL) GOx was added to 0, 10, 20, 40, 60, 80, 100 µL GNPs (from up to down) and the final volume was brought to 200 µL by PBS (52 mM), pH 7.4.

GOx (a) and the conjugates of GOx: hydrophilicGNPs (b), GOx: hydrophobic-GNPs (c).

Deconvolution of the spectra reveals that the % α-Helicity in of native GOx is 28.9%, but decreases to 22.1 and 17.8% and subsequently beta structure increase from 21.3% to 31.3 and 32% for hydrophilic-GNPs and hydrophobic-GNPs respectively (Table 1). On the other hand, the conjugation of GOx on GNPs leads to alpha-beta transition [14]. However, hydrophilic-GNPs are better surface for link of protein.

3.4. Binding property of the GNPs nanoparticle to the GOx Fluorescence spectroscopy is useful to obtain local information about the conformational changes of protein at tertiary structure levels. Typically, from the interpretation of fluorescence parameters, one can obtain information such as the degree of exposure of the fluorophore to the solvent and the extent of its local mobility. For proteins with intrinsic fluorescence, more specific local information can be obtained by selectively exciting the tryptophan (Trp) residues. GOx contains 10 Trp per each subunit, therefore any changes in the

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enzyme conformation and oxidation states have been proved to affect the tryptophan fluorescence of GOx [15]. Figure 3 (A and B) shows the emission spectra of native GOx at different concentrations of GNPs upon excitation at 295 nm.

The choice of 295 nm as the excitation wavelength was to avoid the contribution from tyrosine residues [16]. The fluorescence intensity was found to decrease with increasing the GNPs while the emission maximum shifted from 345 nm at native GOx to 346.5 and 345.5 nm at hydrophilic-GNPs (A) and hydrophobic-GNPs (B) respectively. The shift in the position of emission maximum reflected the changes of the polarity around the Trp residues.

The slightly red shifts on GNPs indicate that Trp residues are partly exposed to the solvent. Figure 3A shows the decrease in fluorescence intensity with the increase of hydrophilic-GNPs, this may be due to the fact that water molecules were placed between the enzyme and the hydrophilic-GNPs [17].

These different fluorescent characteristics reflected different conformational states of GOx on GNPs. Fluorescence intensity data were then analyzed using the Stern-Volmer equation.

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F0 = K sv [Q ]+ 1 F

(Eq. 2)

Where F0 and F are the maximum fluorescence intensities in the absence or presence of quencher, respectively, KSV is the Stern-Volmer quenching constant and [Q] is the concentration of quencher.

The linearity of the F0/F versus [Q] plots is shown in Figure 4. Also The binding constant (K) and the number of binding sites (n) between GNPs with GOx can be calculated using the Eq. 2.

F0 − F  Log   = LogK + nLog [Q ]  F 

respectively. Also the nanoparticle-protein surface ratios, by Assuming they are spherical, are approximately 171, indicating that the protein adsorption on the nanoparticle is multilayer.

Moreover these results clearly indicate that hydrophilic GNPs display good protein adsorption.

So it can be said, although different types of forces such as hydrophobic interactions and coordination binding might also work in the conjugation of protein with nanoparticles but the electrostatic force have a highlight role. By considering the 15 lysine residue in Gox, protein binding to hydrophilicGNPs is possible with electrostatic force too [16].

(Eq. 2)

A plot of log [(F0- F)/F] versus log [Q] gives a straight line, whose slope equals to n (the number of binding sites) and the intercept on Y-axis equals to log K [18]. The results revealed the presence of a single class of binding site on GOx. The values of KSV, K and n are listed in Table 2.

Table 2: Stern-Volmer quenching constant, binding constant and the number of binding sites of GOx:GNPs conjugates interactions.

No.

KSV (×1015 M-1) K (×10-6 M-1)

n Figure 4: Stern-Volmer plots for the binding of

hydrophilic-GNPs

0.53

1.41

1

hydrophobic-GNPs

0.47

0.27

0.7

3.5. GOx absorption studies Due to large surface area/volume ratio nanoparticles tend to high adsorb proteins. GOx concentrations absorbed on the GNPs were determined by the Bradford methods [19]. The amount of GOx adsorbed on hydrophilic-GNPs and hydrophobicGNPs were measured to be 1.83 ± 0.03 and 1.11 ± 0.02 µg/mL, respectively. In the other word, the nanoparticle-protein ratio is one molar of nanoparticle to 3.5 and 2.1×106 M of GOx on hydrophilic-GNPs and hydrophobic-GNPs,

hydrophilic-GNPs (σ) and hydrophobic-GNPs (×) with GOx.

3.6. Enzymatic activity measurements by reaction with a substrate The enzymatic activity (U) represents conversion of 1 µmol of the substrate per minute and the specific activity is defined as the enzymatic activity per mg of the enzyme (U/mg) at 25°C. The activity of GOx was assayed colorimetrically by UV-Vis spectroscopy after 20 min incubation of GOx with GNPs [20]. The activity of native GOx and GOx solution in the presence of on hydrophilic-GNPs and hydrophobic-GNPs were measured to be 180, 87 and 47 U/mg, respectively. These data indicates that in the presence of hydrophilic-GNPs, the

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enzyme activity conserved more than hydrophobicGNPs.

4. CONCLUSIONS In this work, the hydrophobic and hydrophilic GNPs was prepared by mercaptopurine and 11-mercaptoundecanoic acid respectively. Then the effect of nanoparticle coating on protein structure and function as well as protein adsorption was studied by a combination of spectroscopic techniques. In UV-Vis spectroscopy intensity of GOx/GNPs conjugates is significantly reduced, indicating that the GOx was binding on the GNPs. Moreover, CD and florescence spectroscopy show that the conformational changes in both the secondary and the tertiary structure levels of GOx in conjugate with hydrophilic-GNPs are lower than hydrophobic-GNP. Because hydrophobic-GNP is insusceptible for agglomeration and despite the lower absorption of the enzyme, but it have most conformational changes. Therefore, hydrophilic surface is most biocompatible than hydrophobic surface for protein immobilization.

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