International Journal of Bio-Inorganic Hybrid Nanomaterials

ABSTRACT: The present study deals with the synthesis andfunctionalization of mesoporous silica nanoparticles as drug delivery platforms. SBA-15 nanorods with high surface area (1010 m2g-1) were functionalized by post grafting method using 3-mercaptopropyl trimethoxysilane (MPTS). The parent and thiol-functionalized SBA-15 nanorods were used as nanocarriers for an anticancer drug (gemcitabine). The characterization of all samples were done by small angle X-ray scattering (SAXS), N2 adsorption/desorption, scanning electron microscopy (SEM), transmission electron microscopy (TEM), FTIR and UV spectroscopies. The adsorption and release properties of all samples were investigated. It was found that the surface functionalization increases the interaction between the carrier and gemcitabine and results in the loading enhancement of the drug. The obtained results reveal that the surface functionalization leads towards significant decrease of the drug release rate. These findings demonstrate that the functionalized mesoporous system is appropriate drug delivery platform due to their loading content and the possibility to modify drug release.

Keywords: Anticancer; Gemcitabine; Nanorods; SBA-15; Thiol-functionalization

(*) Corresponding Author - e-mail: [email protected]

Thiol-functionalized mesoporous silica as nanocarriers for anticancer

drug delivery

Z. Bahrami1*; A. Badiei2; Gh. Mohammadi Ziarani3

1 Faculty of Nanotechnology, Semnan University, Semnan, Iran2 School of Chemistry, College of Science, University of Tehran, Tehran, Iran3 Research Laboratory of Pharmaceutical, Alzahra University, Tehran, Iran

Received: 27 April 2015; Accepted: 30 June 2015

INTRODUCTION

Int. J. Bio-Inorg. Hybr. Nanomater., 4(3): 121-128, Autumn 2015

Recently, mesoporous silica nanoparticles (MSNs) have been emerged as drug delivery systems (DDS) because of several unique properties such as high sur-face area, large pore volume, uniform and tunable pore sizes, very high thermal and chemical stability, control-lable morphology, modifiable surfaces, lack of toxic-ity and well biocompatibility (Vallet-Regi, et al., 2001, Tourne-Peteilh, et al., 2003, Doadrio, et al., 2006, Pro-kopowicz and Przyjazny 2007, Nunes, et al., 2007).

The porous system architecture, thickness of walls,

chemical modification of the surface, chemical char-acteristics of the adsorbed drug, morphology of meso-porous silica particles and many other parameters can influence on the drug loading and release into and out of MSNs (Tozuka, et al., 2010, Izquierdo-Barba, et al., 2005, Munoz, et al., 2003, Song, et al., 2005, Heikkila, et al., 2007). It was demonstrated that the surface mod-ification with appropriate functional groups is an im-portant method of varying the drug loading and release properties of mesoporous silica materials. For exam-ple, MSNs modified with amine groups were applied as

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carriers for adsorption of drugs with acidic character (Halamova, et al., 2010). In contrary, modification of the surface with acids (e.g. with carboxylic groups) increases the adsorptive properties of drugs with ba-sic properties (Doadrio, et al., 2004). Several research groups investigated the effect of the morphology of MSNs on the drug adsorption and release. Tsai, et al. found that rod-like MSNs have greater potential than that of spherical ones in monitoring the cell trafficking, cancer cell metastasis, and drug/DNA delivery (Tsai, et al., 2008).SBA-15 with highly ordered 2D-hexago-nal mesostructure with large pore size of 4.6–10.0 nm is attractive for the easy modification and application as drug delivery system. The performance improve-ment of SBA-15 particles affected by their size and morphology. The rod-like morphology of SBA-15 is more interesting due to its small dimensions, which results in short diffusion paths and therefore the pos-sibility of fast adsorption and mass transfer (Lai, et al., 2003, Qu, et al., 2006, Tang, et al., 2006).

Gemcitabine (Gem) (Fig. 1) is a water-soluble low-molecular-weight anticancer drug that is commonly used for the treatment of several kinds of cancers in-cluding colon, pancreatic, lung, breast, ovarian, and bladder. Different drug delivery systems such as li-posomes and polymeric nanoparticles were designed in order to protect Gem from rapid metabolization, overcome drug resistance, target drug delivery, and improving its anticancer efficacy (Braakhuis, et al., 1991, Burris and Storniolo 1997, Patra, et al., 2010, Sevimli and Yilmaz 2012).

This paper describes the adsorption and release of gemcitabine on the thiol-functionalized SBA-15 nanorods. Investigation of the effect of the surface functionalization on the adsorption capacity and re-lease behavior of Gem is the main aim of this study.

MATERIALS AND METHODS

MaterialsPoly (ethylene glycol) block poly (propylene glycol) block poly (ethylene glycol), P123, (EO20PO70EO20, MW = 5800 g/mol) was obtained from Aldrich. Tetraethyl orthosilicate (TEOS), hydrochloric acid (35 %), ethanol and 3-mercaptopropyl trimethoxysi-lane (MPTS) were purchased from Merck. Gem-citabine was obtained from Lilly France S.A.S. Com-pany. All chemical reagents were used without further purification.

Synthesis of SBA-15 nanorodsSBA-15 nanorods were synthesized according to the procedure that described in our previous work (Bah-rami, et al., 2014). In a typical synthesis, 23.4 g of non-ionic triblock copolymer (Pluronic P123) was dissolved in the mixture of deionized water (606.8 g) and hydrochloric acid (146.4 g, 35 %). Then, 50 g of TEOS was added to this clear solution while stir-ring at the rate of 500 rpm at 55°C. The reaction batch was maintained at static conditions for 24 h (hydro-lysis time) at this temperature and then for another 24 h at 100°C for further condensation. The precipi-tate was obtained after filtration, and ethanol extrac-tion. A soxhlet extractor was used for removing the triblock copolymer template.

Preparation of thiol-functionalized SBA-15 nanoro-ds (SH-SBA-15)To functionalize the surface of SBA-15 nanorods with mercapto groups, 0.1 g of SBA-15 nanorods was dispersed in 20 mL of anhydrous ethanol. Following the addition of 3-mercaptopropyl trimethoxysilane (MPTS) (1 mL), the mixture was stirred moderately at

Z. Bahrami et al.

Fig. 1: Chemical structure of gecitabine Fig. 2: Thiol-functionalization of SBA-15 nanorods

123


80°C for 6 h. The precipitate was collected by cen-trifugation, washed with ethanol and dried at room temperature to give the thiol-functionalized SBA-15 nanorods as white powder. The obtained modified sample was denoted as SH-SBA-15 (Fig. 2).

Adsorption of gemcitabine on SBA-15 nanocarriers20 mg of the non-modified or thiol-functionalized SBA-15 nanorods was dispersed in 8 mL of gem-citabine solution (2.5 mg mL-1). The mixture was stirred at room temperature for 24 h in order to load Gem on SBA-15 nanorods. The gemcitabine-loaded samples were collected by centrifugation and rinsed several times with distilled water to remove the physi-cally adsorbed Gem. The prepared samples were la-beled as G@SBA-15 and G@SH-SBA-15. UV/Vis spectrophotometer was used to measure the amount of the loaded drug at the wavelength of 269 nm. The drug loading content and the entrapment efficiency were calculated using the following equations:

Gemcitabine releasePhosphate-buffered saline (PBS) solutions with the pH values of 5.6 and 7.4 were chosen for the drug re-lease experiments. The certain amount of Gem-loaded samples was suspended in 4 mL of PBS solutions. The solution was kept at 37°C using an incubator in or-der to simulate body temperature. At scheduled time points, suspension was centrifuged at 13000 rpm for 15 min. The amount of released drug was measured by a UV/Vis spectrophotometer at the wavelength of 269 nm. The measurement was repeated three times for each sample to ensure the accuracy.

InstrumentationThe small angle X-ray scattering (SAXS) patterns were recorded with a model Hecus S3-MICROpix SAXS diffractometer with a one-dimensional PSD detector using Cu Kα radiation (50 kV, 1 mA) at the wavelength 1.542 Å. The SEM and transmission elec-tron microscopy (TEM) images were taken using Ox-ford LEO 1455 V STEM and Philips EM-208 100 kV,

respectively. Nitrogen physisorption isotherms were obtained on a BELSORP mini-II at liquid nitrogen temperature (77 K). The specific surface areas were measured using multiple point Brunauer–Emmett–Teller method. The pore size distributions were cal-culated using desorption branches of the isotherms by Barrett–Joyner–Halenda (BJH) method. The FTIR spectra were recorded using Equinox 55 spectrometer in the range between 400 and 4000 cm-1. The UV/Vis absorption spectra were recorded using a Ray light, UV 1600 spectrophotometer.

RESULTS AND DISCUSSION

Small angle X-ray scattering analysis (SAXS)The SAXS patterns of the non-modified and thiol-functionalized SBA-15 nanorods were displayed in Fig. 3. All the samples have a single intensive reflec-tion at around 2θ = 0.72° and two additional peaks related to the higher ordering (110) and (200) reflec-tions similar to the typical SBA- 15 materials, that is reported in the literature (Bahrami, et al., 2015a). These three peaks are characteristic of the ordered structure of hexagonal lattice (Bahrami, et al., 2015b).The existence of three main diffraction peaks in the patterns of the functionalized sample confirms that the introduction of the organic functionalities does not de-stroy the mesoporous structure of the parent SBA-15. However, due to the existence of organic groups in the mesochannels, a decrease in the intensity of peaks is observed.

Morphology studyThe scanning electron microscopy (SEM) image of SBA-15 matrix (Fig. 4a) is shown that the synthesized particles having rod-like shape. As it can be observed in the transmission electron microscopy (TEM) image (Fig. 4b), the width and length of these rods are about 100 nm and 1 μm, respectively. In addition, these nanorods have highly ordered 2D-hexagonal meso-channels along the length of the rods.

Nitrogen adsorption/desorption analysisN2 adsorption/desorption measurements of the non-modified SBA-15 nanorods, thiol-functionalized and

100)materialmesoporousloadedGemfoWeight

materialmesoporousinGemfoWeight((%)contentLoading ×=

100)GemfoweightInitial

materialmesoporousinGemfoWeight((%)efficiencyEntrapment ×=

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Gem-loaded samples exhibiting a similar trend. A typ-ical irreversible type IV nitrogen adsorption isotherm with an H1 hysteresis loop were observed for the all samples (Fig. 5a) (Horcajada, et al., 2004). The shape and the position of hysteresis loop (at p/p0 from 0.60 to 0.85) indicate cylindrical mesopores with very nar-row pore size distributions (Andersson, et al., 2004). The lower amount of the adsorbed nitrogen and the shift of hysteresis loop of the functionalized and Gem loaded samples toward lower relative pressures are the indication of the presence of organic functionalities groups and drug molecules in the pores. The textural properties of the all samples are summarized in Table 1. As it can be observed, there is a decreasing trend in the specific surface area (SBET), pore volume (Vp), and

pore diameter (Dp) after the surface functionalization and drug loading. The pore size distribution was calcu-lated by BJH method based on the desorption branch of N2 adsorption/desorption isotherms. It is observed that all of the samples have narrow pore size distribu-tion with uniform mesochannels (Fig. 5b).

FTIR measurement Infrared spectra of the SBA-15 nanorods, thiol-func-tionalized and the Gem loaded samples were illus-trated in Fig. 6. For all samples, the band at around 3400 cm-1 can be assigned to the stretching vibration of Si-OH groups. The stretching vibrations of Si-O-Si

Thiol-functionalized mesoporous silica as nanocarriers

Fig. 3: SAXS patterns of SBA-15 nanorods and SH-SBA-15 sample

Fig. 4: (a) SEM and (b) TEM images of SBA-15 nanorods

samplesTextural properties

SBET (m2g-1) Vp(cm3g-1) Dp (nm)

SBA-15 nanorods 1010 1.27 7.06

SH-SBA-15 598 1.02 5.40

G@SBA-15 nanorods 449 0.811 6.18

G@SH-SBA-15 358 0.826 5.40

Table 1: Specific surface area (SBET) (m2g-1), pore volume (Vp) (cm3g-1) and pore diameter (Dp) (nm) from N2 adsorption/desorption for SBA-15 nanorods, thiol-functionalized and Gem loaded samples

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appear as two peaks: one broad and strong peak cen-tered at around 1100 cm-1; one narrow and relatively weak peak near 800 cm-1 (Bahrami, et al., 2015b). In addition, the small peaks observed at around 2800 and 2900 cm-1 result from the CH stretching vibrations. Although, the characteristic peak (related to the –SH groups) could not be observed at around

2580 cm-1 due to the sample containing trace amounts of the mercaptopropyltrimethoxysilane(Qu, et al., 2006). The new adsorption band is observed in the spectra of Gem loaded samples. The peak at around 1480 cm-1 is due to N-H bending of Gem molecules (Bahrami, et al., 2014). These results indicate that the Gem loading was done successfully.

(a)

(b)

Fig. 5: (a) N2 adsorption/desorption isotherms and (b) pore

size distribution of SBA-15 nanorods, thiol-functionalized

and Gem loaded samples

Fig. 6: FTIR spectra of (a) SBA-15 nanorods, (b) SH-SBA-15, (c) G@SBA-15 and (d) G@SH-SBA-15

Gem loaded samples Loading content (%) Entrapment efficiency (%)

G@SBA-15 nanorods 7 7.90

G@SH-SBA-15 12.96 14.90

Table 2: Gemcitabine loading content and entrapment efficiency of the non-modified and thiol-functionalized SBA-15 nanorods

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Gemcitabine loading and releaseSuccessful modification was also evidenced by the drug adsorption experiments. The amount of adsorbed gemcitabine of the non-modified and thiol-function-alized SBA-15 nanorods was displayed in Table 2. It was found that the non-modified SBA-15 nanorods can adsorb 7 wt.% of Gem. In contrast, the surface modification resulted in higher amount of adsorbed Gem. This is due to increasing the interaction between the carrier and the drug.

The results of the gemcitabine release from the Gem loaded samples are plotted in Fig. 7. The drug release behaviour was studied in PBS buffers with dif-ferent pH values of 5.6 and 7.4 at 37°C. As shown, in contrast with G@SBA-15, slower release rate was observed from the thiol-functionalized sample. This fact could be explained by taking into account that the interaction between Gem and the functionalized sam-ple is stronger than that of between Gem and silanol groups of the non-modified SBA-15 nanorods. There-fore, by controlling the surface properties of SBA-15 nanorods it should be possible to prepare novel drug carriers with the desired release rate.

CONCLUSIONS

In the present work, the non-modified and thiol-func-tionalized SBA-15 nanorods were used as the carriers

for the adsorption and release of anticancer drug such as gemcitabine. The surface functionalization leads towards formation of the carriers that have strong interaction with the drug. In contrast with SBA-15 nanorods, the drug release rate of the thiol-functional-ized sample is not pH dependent. In addition, slower release rate was observed from this functionalized sample. Therefore, it is demonstrated that the thiol-functionalized SBA-15 nanorods can be suitable carri-ers for gemcitabine. The obtained results give promis-ing perspectives for future application of mesoporous materials in drug delivery systems.

ACKNOWLEDGMENT

The authors thank the financial support from the Uni-versity of Tehran.

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AUTHOR (S) BIOSKETCHES

Zohreh Bahrami, Assistant Professor, Faculty of Nanotechnology, Semnan University, Semnan, Iran. E-mail: [email protected]

Alireza Badiei, Professor, School of Chemistry, College of Science, University of Tehran, Tehran, Iran

Ghodsi Mohammadi Ziarani, Professor, Research Laboratory of Pharmaceutical, Alzahra University, Tehran, Iran

Thiol-functionalized mesoporous silica as nanocarriers



INTRODUCTION

ABSTRACT: Sphere-like shaped cobalt oxide nanoparticles (Co3O4) were synthesized by a simple wet chemical method using cobalt chloride as precursor and cetyl trimethylammonium bromide (CTAB) as surfactant. Their structural and surface morphologial properties were characterized by high resolution transmission electron microscopy (HRTEM), field emission scanning electron microscopy (FESEM) and X-ray diffraction (XRD). XRD measurement exhibited the structure of Co3O4 nanocrystals for annealed samples. The TEM results showed the sphere-like shaped cobalt oxide nanoparticles with good uniformity in the presence of CTAB surfactant. The SEM images revealed that the particles changed to spherical shape and the size of cobalt oxide nanoparicles increased in the range of 25-45 nm with increasing annealing temperature.

Keywords: Cobalt oxide; CTAB surfactant; Nanocrystals; Synthesis; Wet chemical

Chemical synthesis of the Co3O4 nanoparticles in presence

of CTAB surfactant

M. Farahmandjou*; S. Shadrokh

Department of Physics, Varamin Pishva Branch, Islamis Azad University, Varamin, Iran

Received: 9 May 2015; Accepted: 13 July 2015

Metal nanoparticles have been studied for decades as they have properties not found in the same mate-rial in bulk form. The nanosize affects greatly the ra-tio of surface atoms to atoms deep in the crystal ma-trix. Therefore, surface effects are observed to a much greater extent (Lu, et al., 2007). A notable example is catalysis, where a large surface area to volume ratio is a key parameter. Special nanoeffects are seen in nano-magnets as explained below. In addition, processing techniques like printing are not available or reasonable with micron-sized particles (Gu, et al., 2003, Xu, et al., 2004). Magnetic nanoparticles have received a great

deal of attention because of their potential use in vari-ous biomedical applications, including contrast agents in magnetic resonance imaging, the magnetic separa-tion and sorting of cells and proteins, immunoassay in pathology laboratories, hyperthermia treatment for cancerous tumors, and the controlled and targeted de-livery of pharmaceuticals and therapeutic genes (Gu, et al., 2006, Lee, et al., 2006, Lee, et al., 2007).

Ferromagnetic materials are divided into hard and soft materials. Soft magnetic materials have a low re-manent magnetization, a low coercive field, and a high permeability. Hard ferromagnetic materials have a high remanent magnetization, a high coercive field, and a low permeability. Soft ferromagnetic materials are used

130

for recording heads and as inductor cores. Hard mag-netic materials are used for permanent magnets and recording media.

The magnetic properties change as the particle size decreases. For large particles, magnetic properties are similar to bulk magnetic properties. As particle size decreases, the particles become single domain, as it is not energetically favorable to have a domain wall within the particles. For Co, the single domain di-ameter is theoretically 58 nm, but the exact particle shape and crystal structure have large effects (Yi, et al., 2005). The coercivity for single domain particles is higher than for the bulk, and saturation magnetiza-tion is the same. Very small particles may be in su-perparamagnetic state. Then, magnetization can ran-domly change direction due to thermal fluctuations. The time for this direction change is called the Néel relaxation time. In the absence of an external magnetic field, the time used to measure the magnetisation of the nanoparticles is typically much longer than the Néel relaxation time. Then, the magnetization appears to be zero and the particles are said to be in a super-paramagnetic state. In this state, an external magnetic field is able to magnetize the nanoparticles. The criti-cal radius for superparamagnetic behaviour is 16 nm for cobalt (Stoeva, et al., 2005).

Even for the particles with the smallest number av-erage particle diameter, the mass fraction of larger particles is significant. The increased saturation mo-ment of Co nanoparticles as compared to the bulk is reported several times for particles less than 10 nm, for example by Chen et al. (Chen, et al., 1995). Nano-materials properties are strongly dependent on the size particle. Consequently, properties of the nanoparticles such as magnetic, optical, thermal or catalytic are dif-ferent from bulk materials. Chemical solution meth-ods have been widely used to produce nanostructured materials, and different strategies have been applied to achieve monodisperse nanoparticles with controlled size and shape.

However, there is no general strategy to make nanoparticles with narrow size distribution, tailored properties, and desired morphologies, which could be universally applied to different materials. In recent years, much attention has been focused on the synthe-sis of uniformly sized magnetic nanoparticles (Park, et

al., 2007, Dumestre, et al., 2003, Hyeon, et al., 2001, Sun, et al., 2000). However, most of these nanopar-ticles have been synthesized in organic solvents using hydrophobic capping reagents. Very recently, there have been several reports on the synthesis of water-dispersible magnetic nanoparticles (Wang, et al., 2003, Li, et al., 2004, Kandpal, et al., 2014, Salman, et al., 2014) in which magnetic nanoparticles are func-tionalized with water-compatible chemical reagents (Pellegrino, et al., 2004).

In the present work, we focused on synthesis of cobalt oxide (Co3O4) nanoparticles system by wet chemical route. This method has novel features which are of considerable interest due to its low cost, easy preparation and industrial viability. Synthesis of Co3O4 samples by wet synthesis technique is reported by CoSO4.7H2O precursor and calcined at 600°C. The structural and optical properties of cobalt oxide have been studied by XRD, HRTEM and FESEM analyses.


Cobalt oxide nanoparticles were successfully synthe-sized according to the following manner. First 2 g of cetyl trimethylammonium bromide (CTAB) surfac-tant was dissolved into 70 mL de-ionized water with stirring. Then, 1gr of cobalt chloride (CoCl2.6H2O) was slowly added to the solution and stirred for 5 min at room temperature. By adding the solution of NaOH (2 M) the solution changed from pink color to blue color and the volume of the solution reached to 100 mL. The pH was maintained at 6.5 during the process. Resulting Co solution were dried at 85°C for 2 hours and cooled to room temperature and then calcined at 600°C for 3 hours. The Cobalt oxide nanocrystals powder was later obtained. The sam-ples were characterized without any washing and purification.

The specification of the size, structure and surface morphological properties of the as-synthesized and annealed nanoparticles were carried out to study of the morphology. X-ray diffractometer (XRD) was used to identify the crystalline phase and to estimate the crystalline size. The XRD pattern were recorded with 2θ in the range of 4-85° with type X-Pert Pro MPD,

M. Farahmandjou & S. Shadrokh

131


Cu-Kα: λ = 1.54 Å. The morphology was character-ized by field emission scanning electron microscopy (FESEM) with type KYKY-EM3200, 25 kV and field emission transmission electron microscopy (FETEM) with type Zeiss EM-900, 80 kV.


X-ray diffraction (XRD) at 40Kv was used to identify crystalline phases and to estimate the crystalline sizes. Fig. 1 shows the XRD morphology of Co3O4 nanopar-ticles and indicates the nanostructure of Co3O4. Well-defined diffraction peaks at about 19.52°, 31.50°, 37.05°, 38.77°, 44.96°, 55.80°, 59.53°, 65.30°, 74.55°, 77.50°, and 78.60° are observed, corresponding to the (111), (220), (311), (222), (400), (422), (511), (440), (620), (533) and (622) planes of Co3O4 crystals. The mean size of the ordered Co3O4 nanoparticles has been estimated from full width at half maximum (FWHM) and Debye-Sherrer formula according to equation the following:

(1)

where, 0.89 is the shape factor, λ is the x-ray wave-length, B is the line broadening at half the maximum intensity (FWHM) in radians, and θ is the Bragg angle. The size of annealed Co3O4 nanoparticles was in the

range of 20-50 nm from this Debye-Sherrer equation.Scanning electron microscope (SEM) was used for

the morphological study of nanoparticles of Co3O4. These Figures show high uniformity emerged in the samples surface by increasing annealing temperature. Fig. 2(a) shows the SEM image of the as-prepared cobalt oxide nanoparticles prepared by wet chemical method. It can be seen that the particles were aggre-gated together with particle size in the range of 25-45 nm. Fig. 2(b) shows the SEM image of the annealed Co3O4 nanoparticles at 600°C for 3 hours in presence of CTAB surfactant. Fig. 2(c) shows the SEM image of the annealed Co3O4 nanoparticles at 600°C for 3 hours without CTAB surfactant. It can be seen the sphere-like shaped Co3O4 nanocrystals were formed with good uniformity when CTAB surfactant was used. The aver-age crystallite size of annealed nanocrystals is about 35 nm. In fact surfactants have capping agent to stabilize the nanoparticles to prevent agglomeration. Repulsive Steric hindrance is appeared between particles in atom-ic scale to prevent attractive inter-atomic interaction for stabilization of the particles.

The transmission electron microscopic (TEM) anal-ysis was carried out to confirm the actual size of the particles, their growth pattern and the distribution of the crystallites. The TEM sample was prepared by dispersing the powder in ethanol by ultrasonic vibra-tion. It can be seen that the product was formed from extremely fine spherical particles which were loosely

Fig. 1: XRD pattern of annealed Co3O4 nanoparticles after annealing at 600oC for 3 hours

0.89DBcos

λ=

θ

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aggregated. The uniform Co3O4 particles have sphere-like shaped with less agglomeration. As can be seen in the inset of Fig. 3, the particle sizes possess a narrow distribution in a range of 25 to 55 nm, and the mean particle diameter is about 35 nm. In fact, the mean particle size determined by TEM is very close to the average particle size calculated by the Debye-Scherer formula from the XRD pattern.

CONCLUSIONS

Cobalt oxide (Co3O4) nanoparticles were success-fully made by simple wet synthesis method using co-balt sulfate as precursor and CTAB as surfactant. The particle size of Co3O4 was measured in the range of 25-45 nm for as-prepared particles and 35 nm for of annealed one. FESEM images revealed that the particles changed to spherical shape with less agglomeration by increasing annealing temperature. XRD pattern of cobalt oxide samples nanoparticles exhibited the structure of Co3O4 nanoparticles. TEM image revealed high uniformity of the sphere-like shaped cobalt oxide nanoparticles by increasing an-nealing temperature

ACKNOWLEDGMENT

The authors are thankful for the financial support of Varamin Pishva Branch at Islamic Azad University.

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Lu, A.; Salabas, E.L.; Schuth, F., (2007). Magnetic Nanoparticles: Synthesis, Protection, Functional-ization, and Application. Angew. Chem. Int. Ed., 46: 1222-1244.

Gu, H.; Ho, P.L.; Tsang, K.W.T.; Wang, L.; Xu, B.,

(a)

(b)

(c)

Fig. 2: SEM images of the as-prepared and annealed Co3O4

nanoparticles: (a) as-prepared cobalt oxide nanoparticles

(b) annealed Co3O4 in presence of CTAB surfactant (c) an-

nealed one without surfactant

Fig. 3: TEM image of the as-prepared Co3O4 nanoparticles

Chemical synthesis of the Co3O4 nanoparticles

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(2003). Using Biofunctional Magnetic Nanoparti-cles to Capture Vancomycin-Resistant Enterococ-ci and Other Gram-Positive Bacteria at Ultralow Concentration. J. Am. Chem. Soc., 125: 15702-15703.

Xu, C.; Xu, K.; Gu, H.; Zhong, X.; Guo, Z.; Zheng, R.; Zhang, X.; Xu, B., (2004). Nitrilotriacetic Ac-id-Modified Magnetic Nanoparticles as a General Agent to Bind Histidine-Tagged Proteins. J. Am. Chem. Soc., 126: 3392-3393.

Gu, H.W.; Xu, K.M.; Xu, C.J.; Xu, B., (2006). Bio-functional Magnetic Nanoparticles for Protein Separation and Pathogen Detection. Chem. Com-mun, 55: 941-949.

Lee, I.S.; Lee, N.; Park, J.; Kim, B.H.; Yi, Y.W.; Kim, T.; Kim, T.K.; Lee, I.H.; Paik, S.R.; Hyeon, T., (2006). Ni/NiO Core/Shell Nanoparticles for Se-lective Binding and Magnetic Separation of His-tidine-Tagged Proteins. J. Am. Chem. Soc., 128: 10658-10659.

Lee, J.H.; Huh, Y.M.; Jun, Y.W.; Seo, J.; Jang, J.T.; Song, H.T.; Kim, S.; Cho, E.J.; Yoon, H.J.; Suh, J.S., (2007). Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imag-ing. J. Cheon, Nat. Med., 13: 95-99.

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Dumestre, F.; Chaudret, B.; Amiens, C.; Respaud, M.; Fejes, P.; Renaud, P.; Zurcher, P., (2003). Unprec-edented Crystalline Super-Lattices of Monodis-perse Cobalt Nanorods. Angew. Chem. Int. Ed., 42: 5213-5216.

Hyeon, T.; Lee, S.S.; Park, J.; Chung, Y.; Na, H.B., (2001). Synthesis of Highly Crystalline and Monodisperse Maghemite Nanocrystallites with-out a Size-Selection Process. J. Am. Chem. Soc., 123: 12798-12801.

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Majid Farahmandjou, Associate Professor., Department of Physics, Varamin Pishva Branch, Islamis Azad University, Varamin, Iran, E-mail: [email protected]

Somayeh Shadrokh, M.Sc., Department of Physics, Varamin Pishva Branch, Islamis Azad University, Varamin, Iran

M. Farahmandjou & S. Shadrokh

ABSTRACT: Antibacterial activity of Fe3O4 nanoparticles was investigated in three microorganisms including P. aeruginosa, E. Coli and Staphylococcus aureus. Fe3O4 nanoparticles were synthesized by chemical precipitation method (20 nm). The Fe3O4 nanoparticles antibacterial effect against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were studied by the culture method. First of all, each bacterium was grown in nutrient broth culture environment aerobically for 24 h at 37°C. After that, about 1 mL of each bacterium was placed onto a plate which was previously prepared by Mueller Hinton Agar culture environment. Their characteristics and physical properties were evaluated by X-ray diffraction (XRD), Alternating Gradiant Force Magnetometer (AGFM) and Transmission Electron Microscope (TEM). Antibacterial effects of Fe3O4 nanoparticles (1.56 to 25 mg/mL concentrations) against three pathogenic bacteria including Pseudomonas aeroginosa, Escherichia coli and Staphylococcus aureus were studied using well diffusion bacteriological test. The inhibition zone diameter was directly and strongly correlated to the nanoparticles concentration and Pseudomonas aeroginosa was the most sensitive bacterium to the Fe3O4 nanoparticles. The maximum diameter of inhibition zone was occurred for P. aeroginosa bacteria for both Fe3O4 samples. The most sensitive bacterium among three pathogenic bacteria tested was P. aeroginosa.

Keywords: Antibacterial effect; Escherichia coli; Fe3O4 nanoparticles; Pseudomonas aeroginosa; Staphylococcus aureus

* Corresponding Author - e-mail: [email protected]

Nanoparticles are relatively new materials which have a more effective interaction with biological systems due to their high proportion of surface to volume ratio in comparison with bulk ones. This characteristic has made them appropriate to be used in many medical and biological applications. This fact also has made them as high risk materials and increases their toxicity risk for biological systems usage. On the other hand, toxic-ity could be useful in some cases. For instance, some

Antibacterial activity of Fe3O4 nanoparticles

Z.S. Shahzeidi; Gh. Amiri*

Department of Physic, Falavarjan Branch, Islamic Azad University, Isfahan, Iran


INTRODUCTION


bacteria may be affected by nanoparticles which may lead to decrease of their harmful effects.

Super paramagnetic property is a behavior which is only could be found in nano size particles. This prop-erty exists in particles which are less than a specific size (critical size). The critical size is different for ev-ery nanoparticle which could be a super paramagnetic material. Iron oxide nanoparticles are among those nanoparticles which exhibit the super paramagnetic behavior at 30 nm critical size and below. Super para-magnetic iron oxide nanoparticles (SPION) with ap-

136

propriate surface chemistry can be used for numerous applications such as in vitro enhancing resolution in MRI, tissue repair, detoxification of biological fluids and hyperthermia (Amiri, et al., 2010, Amiri, et al., 2012).

There are many methods which can be used for syn-thesizing nanoparticles such as co-precipitation, low temperature solid state reaction and sol gel. One of the most commonly used methods for synthesizing SPI-ON is wetted chemical method which is used in this study (Rizwan, et al., 2010, Sobha, et al., 2010, Amiri, et al., 2014, Fashen, et al., 2007, Amiri, et al., 2013).

Nanoparticles have a great range of probable in-teraction with cell surface due to the large surface to volume ratio in comparison with bulk ones (Rizan, et al., 2010). Some nanoparticles like ZnO, CdSe, TiO2, ZnS and SiO2 have considerable antibacterial activ-ity and selective toxicity in biological systems which were previously reported (Sobha, et al., 2010). In this study, an attempt was made in order to investigate the antibacterial effect of Fe3O4 nanoparticles on three pathogen bacteria including Pseudomonas aeroginosa, Escherichia coli, and Staphylococcus aureus).


Synthesis and assessment of propertiesWetted chemical method was used in order to syn-thesize Fe3O4 nanoparticles (Amiri, et al., 2013). For this purpose, three solutions of FeCl2 (0.01 M), FeCl3 (0.02 M) and NaOH (0.08 M) (Merck, Germany) were prepared in the distilled deionized water.

First, FeCl2 solution was poured into a triple-neck round bottom flask and FeCl3 solution in water was added to the same flask under vigorous stirring. Then, 50 µl mercaptoethanol (ME) was added. Then, the NaOH solution was added to the mixture and finally, the prepared solution was washed by deionized water. In order to remove any impurity aggregate, the solu-tion was centrifuged. All of the mentioned processes were repeated when starch (S) was used instead of mercaptoethanol. Antibacterial activity assayThe antibacterial effect of Fe3O4 nanoparticles against P. aeruginosa, E. coli and S. aureus were studied by

culture method. First of all, each bacterium was grown in nutrient broth culture environment aerobically for 24 h at 37°C. After that, about 1 mL of each bacte-rium was placed onto a plate which was previously prepared by Mueller Hinton Agar culture medium. Then, a well was created in every plate by the Pasteur pipette. The concentrations of 1.56 to 25 mg/mL of both Fe3O4 (ME) and Fe3O4 (S) were adjusted in wells separately. After inoculation and culturing of differ-ent target bacteria on the top of nutrient agar, wells were made in selected areas on different plates. The diameter of inhibition zone was assessed 24 h post-incubation.

Measurement of properties Both samples of synthesized Fe3O4 nanoparticles using either mercaptoethanol or starch were assessed by XRD (X-ray Diffraction, Bruker D8 ADVANCE λ= 0.154 nm Cu Kα radiation). The accelerating volt-age and the applied current were 40 kV and 40 mA, respectively. Data were recorded at a scan rate for two seconds in steps of 0.04° for 2q. The crystalline size was calculated from X-ray line broadening analysis by the Debye-Scherer equation for the full-width at half-maximum of the strongest reflection where, D is the crystalline size in nm, λ is the Cu-Kα wavelength (0.154 nm), β is the half-maximum breadth, and θ is the Bragg angle of the (311) plane (Fashen, et al., 2007):

(1)

For more accuracy TEM (Mode: BF, HT: 150 kV) was prepared from the Fe3O4 (S) sample. Moreover, the AGFM (Alternating Gradient Force Magnetome-ter, Meghnatis Daghigh Kavir Co, Iran) was prepared from both samples in order to assess the magnetic properties.


Physical properties investigationXRD patterns of both Fe3O4 nanoparticle samples are shown in Figs. 1 and 2. It can be seen that both samples are single phase and have the ferrite spinel

qβλ

=cos

9.0D

Z.S. Shahzeidi & Gh. Amiri

137


structure. The size of the samples can be calculated by Scherer equation which is 23 nm for Fe3O4 (S) and 22 nm for Fe3O4 (ME).

In order to measure the magnetic properties of fer-rite nanostructure samples, magnetic hysteresis curve prepared using AGFM. Figs. 3 and 4 illustrate the hysteresis curve for both Fe3O4 (S) and Fe3O4 (ME) samples. As can be seen, both plots show the super-paramagnetic property and the saturation magnetiza-tion (around 15 emu/gr) occurred at 4000 Oe.

Fig. 5 shows the TEM micrograph of the Fe3O4 samples produced by starch as inhibitor. It is clear that TEM is one of the most important and widely used methods for determining particles size and size distri-bution with an accuracy of less than a nanometer. The

size of the Fe3O4 (S) nanoparticles is around 20 nm from the TEM photograph.

Antibacterial assessmentWell diffusion agar method was used in order to in-vestigate the antibacterial activity of Fe3O4 nanopar-ticles. The results are shown in Tables 2, 3 and Fig. 6. The diameter of the inhibition zone shows the an-tibacterial effect activity of the Fe3O4 nanoparticles. It can be seen that, the inhibition zone has increased due to increase the Fe3O4 concentration in wells. Moreover, the diameter of the inhibition zone was completely different according to the different type of bacteria.

According to the results obtained from Tables 2 and

Fig. 1: XRD pattern of Fe3O4 samples produced by mercap-toethanol as inhibitor.

Fig. 2: XRD pattern of Fe3O4 samples produced by starch as inhibitor.

Fig. 3: Magnetic hysteresis curve of samples produced by mercaptoethanol inhibitor Fe3O4 (ME).

Fig. 4: Magnetic hysteresis curve of Fe3O4 samples pro-duced by starch as inhibitor.

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3 the maximum inhibition activity is happened for P. aeroginosa bacteria in 25 mg/mL concentrations. Fig. 6 demonstrated the similar results which are com-

pletely agreed with Tables 2 and 3.As it was also shown in the study of (Senthil et

al., 2012) it has been seen in this study that by in-creasing the concentration of Fe3O4 nanoparticles in wells and discs, the growth inhibition has also been increased. The size of inhibition zone was differ-ent according to the type of bacteria, the size and the concentrations of Fe3O4 nanoparticles (Reddy et al., 2007) have reported the same results, empha-sizing on the higher susceptibility of Gram-positive bacteria in comparison with Gram-negative bacte-ria. In the study done by (Selahattin et al.,1998), it has been proposed that the higher susceptibility of Gram-positive bacteria could be related to dif-ferences in cell wall structure, cell physiology, me-tabolism or degree of contact.

Fig. 5: TEM micrograph of Fe3O4 samples produced by starch as inhibitor.

Specimen Size by

XRDSize by TEM Hc (Oe) Mr (emu/g) M (emu/g)

Fe3O4(ME) 22 - 20 0.5 13.2

Fe3O4(S) 23 20 80 3.25 15

Concentration

(mg/mL)

P. aeroginosa

(mm)

E. coli

(mm)

Staphylococcus aureus

(mm)

25 24 6 5

12.5 7 5 5

6.25 5 5 53.125 5 5 51.56 5 5 5

Table 1: Structural and magnetic properties of the synthesized nanoparticles.

Table 2: The diameter of inhibition zone for Fe3O4 samples produced by mercap-toethanol as inhibitor.

Concentration

(mg/mL)

P. aeroginosa

(mm)

E. coli

(mm)

Staphylococcus aureus

(mm)25 23 5 5

12.5 7 5 56.25 5 5 5

3.125 5 5 5

1.56 5 5 5

Table 3: The diameter of inhibition zone for Fe3O4 samples produced by starch as inhibitor.

Antibacterial effect assessment of Fe3O4 nanoparticles

139


CONCLUSIONS

In this research, Fe3O4 nanoparticles were produced by wetted chemical method (20 nm) using ME and starch as an inhibitor separately. It can be concluded that the size of Fe3O4 nanoparticles can be controlled by the type of inhibitor and inhibitor concentration. It is also concluded that the size of the nanoparticles can affect on their antibacterial properties and the diam-eter of inhibition zone.

ACKNOWLEDGEMENT

Authors are grateful from department of Basic Scienc-es, Falavarjan Islamic Azad University for their co-operation and supplying the experimental equipments.

REFERENCES

Amiri G.R.; Yousefi M. H.; Aboulhassani M. R.; Kes-havarz M. H.; Shahbazi D.; Fatahian S.; Alahi M.; (2010). Badr absorption of Ni 0.7, Zn 0.3 Fe2O4 nanoparticles, Dig J Nanomater Biostruct., 5(3): 1025.

Amiri G.R.; Yousefi M.H.; Fatahian S.; (2012). Pre-paratin and investigation of Ni1-xZnxFe2O4 nanoparticles: the effect of x parameter on the

magnetic properties, Optoelectron Adv, Mat., 6(5-6): 564-567.

Rizwan W.; Amrita M.; Soon-Il Y.; Young-Soon K.; Hyung-Shik Sh.; (2010). Antibacterial activity of ZnO nanoparticles prepared via non-hydrolytic solution route, J. Appl. Microbial. Biotechnol., 87(5): 1917-1925.

Sobha K.; Surendranath K.; Meena V.; Jwala K.T.; Swetha N.; Latha K.S.M.; (2010). Emerging trends in nanobiotechnology. J. Biotech. Mol. Bio. Rev., 5(1): 001-012.

Amiri G.R.; Fatahian S.; Kianpour N.; (2014) Inves-tigation of ZnS nanoparticle antibacterial effect, Curr. Nanosci., 10(6): 796-800.

Fashen W.H.; Wang L.; Wang J.; (2007). Magnetic properties and crystal structure of the hydrides of ferromagnetic, J. Magn. Magn. Mater., 295-309.

Amiri G.R.; Fatahian S.; Mahmoudi S.; (2013). Prepa-ration and optical properties assessment of CdSe quantum dots, J. Mater. Sci. Appl., 4: 134-137.

Senthil M.; Ramesh C.; (2012). Biogenic synthesis of Fe3O4 nanoparticles using tridax procumbens leaf extract and its antibacterial activity on pseu-domonas aeruginosa. Dig J Nanomater Biostruct., 3: 1655-1660.

Reddy K.M.; Kevin F.; Jason B.; Denise G.W.; Cory H.; Alex P.; (2007). Selective toxicity of zinc ox-ide nanoparticles to prokaryotic and eukaryotic systems, J. Appl. Phys. Lett., 90(21): 1-3.

Fe3O4 (ME) Fe3O4 (S)Fig. 6: Antibacterial activity of both Fe3O4 samples against P. aeroginosa (25 mg/mL).

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Zeinabosadat Shahzeidi, M.Sc., Department of Physic, Falavarjan Branch, Islamic Azad University, Isfahan, Iran

Gholamreza Amiri, Assistant professor, Falavarjan Branch, Islamic Azad University, Isfahan, Iran, E-mail: [email protected]

Selahattin A.; Kadri Ramazan G.; (1998). The effect of zinc on microbial growth, Tr. J. Med. Sci., 28:

595-597.

Z.S. Shahzeidi & Gh. Amiri


INTRODUCTION


Recently, electrocatalytic oxidation of small organ-ic molecules such as CH3OH, C2H5OH, HCHO and HCOOH, on the surface of different modified elec-trodes has received special attention. They have great potential for utilization as electron donors in fuel cells (FCs) and production of high current and power densi-ty (Ojani, et al., 2013, Mai, et al., 2005). Formaldehyde (HCHO) is toxic material and is not very suitable elec-tron donors for FCs but study of its electrochemical oxi-dation is important for a full understanding of methanol

oxidation in FCs. HCHO is also one of the intermediate products in methanol oxidation in FCs (Samjeske, et al., 2007). HCHO is also used in textile industry and thus, its oxidation is relevant to waste water treatment (Santos and Bulhoes, 2004). Therefore, HCHO oxida-tion has been performed on different electrodes such as platinum and platinum alloys (Santos and Bulhoes, 2004, Korzeniewski and Childers, 1998, Miki, et al., 2004, De Lima, et al., 2007, Jiang, et al., 2009, Habibi and Delnavaz, 2010, Mascaro, et al., 2004, Li, et al., 2011), copper and copper alloys (Ramanauskas, et al.,

ABSTRACT: A novel modified Ni-ZSM-5 nanozeolite was fabricated via an organic template-free hydrothermal route. The average particle size of Ni-ZSM-5 nanozeolite was calculated to be 85 nm by scanning electronic microscopy. Then, Carbon paste electrode (CPE) was modified by Ni-ZSM-5 nanozeolite and Ni2+ ions were then incorporated to the nanozeolite matrix. Electrochemical behavior of this electrode was investigated by cyclic voltammetry that exhibits stable redox behavior of Ni(III)/Ni(II) couple in alkaline medium. It has been shown that Ni-ZSM-5 nanozeolite at the CPE surface can improve catalytic efficiency of the dispersed nickel ions toward formaldehyde (HCHO) electrocatalytic oxidation. The values of electron transfer coefficient, charge-transfer rate constant and the electrode surface coverage were obtained to be 0.49, 0.045 s−1 and 4.11×10−8 mol cm−2, respectively. Also, the catalytic rate constant for HCHO and redox sites of electrode and diffusion coefficient were found to be 9.064×103 cm3 mol−1 s−1 and 8.575 ×10−6 cm2 s−1, respectively.

Keywords: Carbon paste electrode (CPE); modified electrode; Electrocatalysis; Formaldehyde; Ni-ZSM-5 nanozeolite; Template-free synthesis

Organic template-free synthesis of Ni-ZSM-5 nanozeolite: a novel cata-

lyst for formaldehyde electrooxidation onto modified Ni-ZSM-5/CPE

M. Rahimnejad1*; S.K. Hassaninejad-Darzi2

1 Biofuel & Renewable Energy Research Center, Faculty of Chemical Engineering, Babol Univer-sity of Technology, Babol, Iran.P.O.Box: 47148-71167

2 Research Laboratory of Analytical & Organic Chemistry, Department of Chemistry, Faculty of Science, Babol University of Technology, Babol, Iran


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1997, Raoof, et al., 2012, Brunelli, et al., 2002, Raoof, et al., 2011), gold (Vaskelis, et al., 2007, Yahikozawa, et al., 1992, Yang, et al., 1999), palladium nanoparti-cles (Safavi, et al., 2009, Zhu, et al., 2009, Gao, et al., 2006, Yi, et al., 2011, Zhang, et al., 1997), gold alloy (Enyo, et al., 1985), nickel based electrodes (Ojani, et al., 2009, Aal, et al., 2008, Raoof, et al., 2011, Raoof, et al., 2012, Raoof, et al., 2009), nickel and copper alloy (Enyo, 1986).

Fabrication of modified electrodes for electro catal-ysis has been extensively developed and investigated by several researchers. Improvement in electrodes provides an excellent way to accelerate charge trans-fer, decrease the over potentials as well as increase the intensity of the corresponding voltammetric responses (Samadi-Maybodi, et al., 2013). Zeolites and nano-zeolites are ordered porous crystalline materials with different practical applications (Nejad-Darzi et al., 2013). Surface areas for these materials is high, with strongly organized microporouse channel systems that exhibits an advantage over other classical support ma-terials which are of high interest in FC technology (Li, et al., 2002). Certain organic templates are usually necessary to be used as charge balancing and structure directing agent to obtain highly crystallized zeolites (Cundy and Cox, 2005). However, there are clear dis-advantages for the organic template-contained syn-thesis, such as relatively high cost and environmen-tal pollution (Song, et al., 2006, Zhang, et al., 2012). Improvement of new ways for fabrication of zeolites in absence of organic template is attractive (Nejad-Darzi, et al., 2013). It is pointed out that zeolites and nanozeolites have utilized for zeolite modified elec-trodes (ZMEs) and applied in electrocatalysis reaction (Walcarius, 1999). Nickel is a relatively abundant and low cost material that is widely utilized in industrial applications. It is well understood that nickel can be used as an active catalyst due to its surface oxidation properties and has long-term stability in alkaline so-lutions (Raoof, et al., 2011). Conversion of alcohols to carboxylic acids using a nickel electrode was de-scribed by Fleischmann (Fleischmann, et al., 1971). Thereafter, nickel is widely applied for enhancement of electrode performance towards electrooxidation of HCHO (Raoof, et al., 2012, Raoof, et al., 2009, Enyo, 1986, Samadi-Maybodi, et al., 2013, Fleischmann, et

al., 1971).This study is an attempt to present a new, low cost

and efficient catalyst for electrocatalytic oxidation of HCHO. Firstly, organic template-free hydrothermal synthesis of Ni-ZSM-5 and ZSM-5 nanozeolites has been performed and characterized. Then, these novel nanozeolites were used for modification of carbon paste electrode (CPE) and applied for electrocatalyti-coxidation of HCHO in the alkaline medium by using cyclic voltammetry technique.


Reagents and materialsHCHO, Tetraethylorthosilicat (TEOS), sodium alu-minate, sodium hydroxide, potassium chloride, po-tassium hexacyanoferrate and NiCl2.6H2O were of analytical reagent grade and were prepared from Merck Company and were used without any purifica-tion. Graphite powder and paraffin oil (density 0.88 gcm−3) as the binding agent (both from Dayjung com-pany) were used for preparing the pastes. Also double distilled water was used throughout.

Preparation of Ni-ZSM-5 nanozeoliteNi-ZSM-5 nanozeolite has been synthesized using sodium aluminate (NaAlO2) and tetraethylorthosilicat (TEOS) as aluminum and silica sources, respectively. In a typical synthesis, sodium aluminate was dissolved in sodium hydroxide solution under stirring, followed by successive addition of double distilled water and NiCl2.6H2O. Then, TEOS was added to the solution-and stirred until a homogeneous gel was obtained. The prepared gel was transferred in a Teflon lined stain-less steel reactor and heated at 180°C for 24 h. The fabricated nanocrystals were centrifuged, washed sev-eral times with distilled water to remove impurity and dried at 90°C overnight. The molar ratio of the above reactants was as follows: 0.5 Al2O3: 0.5 NiO: 30 SiO2: 3.3 Na2O: 1350 H2O. Also, ZSM-5 nanozeolite was prepared by a similar technique without NiCl2 and the mole ratio of Al2O3 in the batch composition was 1.0.

ApparatusXRD pattern was prepared by X-ray diffractometer

S.K. Hassaninejad-Darzi & M. Rahimnejad

143


(XRD, GBC−MMA) with Be Filtered Cu Kα radiation (1.5418 Å) operating at 35.4 kV and 28 mA. The 2θ scanning range was set between 5º and 50º with scan rate of 0.05 degree/second. Scanning electron micros-copy (SEM, VEGA2−TESCAN) was used to study morphology and size of fabricated crystal. Bruker FT-IR spectrometer (Vector 22) was employed for record-ing the fourier transform infrared (FT-IR) spectrum at ambient temperature. Energy-dispersive X-ray spec-troscopy (EDX) spectra were recorded on an EDX Genesis XM2 attached to SEM. The electrochemical experiments were performed at ambient temperature by using potentiostat/galvanostat electrochemical an-alyzer (Ivium, Netherlands, V11100) with a voltam-metry cell in a three electrodes configuration. The Ag|AgCl|KCl (3 M) and platinum wire were used as reference and auxiliary electrodes, respectively. The CPE, and modified carbon paste electrodes with ZSM-5 and Ni-ZSM-5 nanozeolites were used as working electrodes.

Preparation of working electrode A suitable amount of Ni-ZSM-5 nanozeolite (5–30% wt with respect to graphite) was mixed with 200 mg of graphite powder to prepare Ni-ZSM-5/CPE and then paraffin oil (35% wt) was blended with the prepared mixture in a mortar by well hand mixing for 30 min until a homogeneously wetted paste was obtained. This ready paste was filled into the end of a glass tube (ca. 0.35 cm i.d. and 10 cm long). Copper wire was selected for electrical contact in prepared electrode. By pushing an excess of the paste out of the tube and

polishing with a weighing paper, a new surface was obtained. For comparison, unmodified CPE and modi-fied ZSM-5/CPE was also fabricated in the same way.


Characterization of ZSM-5 nanozeoliteFig. 1 shows XRD powder pattern with SEM image of Ni-ZSM-5 nanozeolite. Comparison of the main peaks at 2θ = 7.9, 8.9, 23.2 and 24.5 degrees with the refer-ence sample (Gurses, et al., 2006, Nejad-Darzi, et al., 2013) indicated that pure phase of Ni-ZSM-5 nano-zeolite was fabricated in this research. It is clear that when TEOS is applied as source of silicon in the gel composition, it can play as structure directing agent (SDA) in system and it can effect on the formation of Ni-ZSM-5 nanozeolite due to its hydrolyzation and production of alcohol (Zhang, et al., 2012).

The SEM image of crystalline phase provides useful approach to determination of size and morphology of the prepared crystals. Inset in Fig.1 illustrates SEM image of synthesized Ni-ZSM-5 nanozeolite, which indicates the formation of spherical nano-sized parti-

Fig. 1: The XRD pattern of Ni-ZSM-5 nanozeolite.Inset

shows SEM image of this sample.

Fig. 2: The FT-IR spectrum of synthesized Ni-ZSM-5 nano-

zeolite.

144

cle. The average size of nanoparticle is around 85 nm.FT-IR spectrum of as-synthesized Ni-ZSM-5 nano-zeolite is presented in Fig. 2. The bands located at 1070-1250 cm−1 are related of SiO4 tetrahedron units. Difference between Ni-ZSM-5 nanozeolite and other types of zeolites can be found by the absorp-tion bands at 1225 and 545 cm−1. The external asym-metric stretching vibration near absorption bands at 1225 cm−1 is due to the presence of structures con-taining four chains of four-member rings between SiO4 or AlO4 tetrahedral of Ni-ZSM-5 structure and is a structure sensitive IR band of ZSM-5 zeolite (Mohamed, et al., 2005, Abrishamkar, et al., 2011). The bands near absorption bands at 1072 cm−1 and 837 cm−1 are assigned to the internal asymmetric stretching and external symmetric stretching of exter-nal linkages, respectively. Also, the absorption band about 545 cm−1 is attributed to a structure-sensitive vi-bration caused by the double four-member rings of the external linkages (Li and Armor, 1992).

The EDX spectroscopy analysis was also provided. This technique provides useful information about the presence of elements and their composition in the syn-thesized nanozeolites. Fig. 3 represents the EDX spec-trum of Ni-ZSM-5 nanozeolite. Extracted data from this Fig. are listed in Table 1. The obtained data in Fig. 3 and Table 1 demonstrated that silicon, aluminum, nickel and sodium are presented in the synthesized Ni-ZSM-5 nanozeolite.

Electrochemistry of the modified electrodePotassium ferricyanide was nominated as a probe to evaluate the performance of the fabricated electrodes. Also, cyclic voltammetry technique was employed for detection of electrochemical properties of the unmodi-fied CPE, modified ZSM-5/CPE and Ni-ZSM-5/CPE electrodes. Fig. 4 represents the CVs of Fe(CN)6

3−/4− at the surface of three electrodes in 10 mM of K3Fe(CN)6/ K4Fe(CN)6 + 2 M of KCl solution. As can be seen in Fig. 4, the anodic peak current for Ni-ZSM-5/CPE is much larger than that ZSM-5/CPE and bare CPE. The anodic peak current was obtained to be 217, 159, 92 µA for Ni-ZSM-5/CPE, ZSM-5/CPE and bare CPE, respectively. The experimental results at slow

Element NaK AlK SiK NiK

Wt. % 7.45 12.32 67.43 12.80

Table 1: Amounts of elements in Ni-ZSM-5 obtained by EDX analysis.

Fig. 3: The EDX spectrum of synthesized Ni-ZSM-5 nano-

zeolite.

Fig. 4: CVs of (a) bare CPE, (b)ZSM-5/CPE and (c) Ni-

ZSM-5/CPE in a solution of 10 mM K3Fe(CN)6]/ K4[Fe(CN)6]

(1:1) + 2 M KCl at a scan rate of 25 mV s−1.

Fig. 5: CVs of (a) bare CPE, (b) ZSM-5/CPE and (c) Ni-

ZSM-5/CPE at 0.1 M NaOH after immersed in 0.1 M NiCl2

solution. Inset shows CVs of bare CPE, ZSM-5/CPE and Ni-

ZSM-5/CPE at 0.1 M NaOH before immersed in 0.1 M NiCl2

solution, the scan rate is 25 mV s−1.

Organic template-free synthesis of Ni-ZSM-5 nanozeolite

145


scan rates of 25 mVs−1 at the surface of Ni-ZSM-5/CPE showed reproducible cathodicand anodic peaks related to Fe(CN)6

3−/Fe(CN)64− redox couple with

quasi-reversible behavior with a peak separation po-tential, ΔEp(Epa − Epc), of 255 mV. The ΔEp is greater than that of 59 mV expected for a reversible system. It can be noted that the electrochemical properties of the Fe(CN)6

3−/Fe(CN)64− redox couple in the Ni-ZSM-5/

CPE electrode surface are pH independent.

Electrochemical behavior of Ni-ZSM-5/CPE in al-kaline solutionInset in Fig. 5 shows CVs of bare CPE, ZSM-5/CPE and Ni-ZSM-5/CPE in 0.1 M NaOH solution at the potential range between 0.2 to 0.7 V vs. Ag|AgCl|KCl (3 M) at scan rate of 20 mV s−1. The obtained results showed that no current can be obtained with these electrodes and the background current for Ni-ZSM-5/CPE is much larger than that on bare CPE andZSM-5/CPE because the surface area is bigger than two others (Safavi, et al., 2009).

In the next experiment, three fabricated electrodes (bare CPE, ZSM-5/CPE and Ni-ZSM-5/CPE) were immersed in the 0.1 M NiCl2 solution with stirring for five minutes at 150 rpm, and then each three electrodes washed completely with distilled water to remove the surface adsorbed species. The main panel of Fig. 5 il-lustrates CVs of these electrodes in 0.1 M NaOH so-lution after immersion of these electrodes in 0.1 M NiCl2 solution at a potential sweep rate of 20 mV s−1. It can be deduced that the electrochemical behavior of Ni-ZSM-5/CPE modified electrode in alkaline solution is similar to that of Ni anode (Fleischmann, et al., 1971, Nagashree and Ahmed, 2010). A probable mechanism is that at Ni-ZSM-5 nanozeolite/electro-lyte interface, Ni2+ ions react with OH− ions to create nickel hydroxide (Ni(OH)2). During the anodic direc-tion, the Ni(OH)2 at the surface of Ni-ZSM-5/CPE is converted to the nickel oxy-hydroxide (NiOOH) and in cathodic direction NiOOH is reduced to Ni(OH)2 based on the following reaction (Nagashree and Ahmed, 2010, Fleischmann, et al., 1971, Samadi-Maybodi, et al., 2013):

(1)

A pair of redox peaks appears at 0.560 and 0.335 V vs. Ag|AgCl|KCl (3 M) is assigned to Ni2+/Ni3+ redox couple. As can be seen in Fig. 5, the peak current of Ni(OH)2 oxidation at the surface of Ni-ZSM-5/CPE is 2.65-fold greater than that at the ZSM-5/CPE and is about 25.5-fold greater than that at the bare CPE. These observations can explain clearly the role of the Ni-ZSM-5 nanozeolite on the enhancement of the oxi-dation currents. The separation of anodic and cathodic peak of Ni(OH)2 to NiOOH and reversed conversion is obtained to be 225 mV (i.e. 0.560 and 0.335 mV for anodic and cathodic peaks, respectively) for Ni-ZSM-5/CPE modified electrode (see Fig.5c). It can be realized that this phenomena is a quasi-reversible pro-cedure because the ΔEp is greater than that of 59/n mV expected for a reversible system. The peak-to-peak potential separation is deviated from the theoretical value of zero and increased at higher potential sweep rates. The variation between the voltammetric behav-iors of Ni2+ ions in Ni-ZSM-5/CPE, ZSM-5/CPE and bare CPE electrodes appeared to be due to the differ-ent coordination and mobility of Ni2+ ions in various site of CPE, ZSM-5 and Ni-ZSM-5 nanozeolites. It can be expected that diffusion of Ni2+ ions in Ni-ZSM-5 nanozeolite is much faster owing to their coor-dination to the nanozeolitestructure, the bigger cages and channels and fast migration of Ni2+ ions from the cages (Li and Calzaferri, 1994). These results are in excellent agreement with earlier observations (Ojani, et al., 2008, Mojovic, et al., 2007, Fleischmann, et al., 1971, Samadi-Maybodi, et al., 2013) although the ex-act potential values may change due to experimental conditions.

Fig. 6: Variation of anodic peak current with Ni-ZSM-5 per-

centage in preparation of modified electrodes.

( ) ( )2

2

Ni ZSM 5 / CPE Ni(OH) OH Ni ZSM 5 / CPE NiOOH

H O e

−

−

− − − + ↔ − − − + +

146

Voltammetric responses of the modified electrodes with Ni-ZSM-5 nanozeolite ratio of 5, 10, 15, 20, 25 and 30% (w/w) with respect to the graphite powder were studied by CV technique at 0.1 M of NaOH so-lution. The CVs of Ni(OH)2/NiOOH oxidation on dif-ferent modified electrodes showed that higher anodic current is obtained in 15% of Ni-ZSM-5 nanozeolite-with respect to the graphite powder (see Fig.6). It is recommended that at low ratio of Ni-ZSM-5 nano-zeolite, the amount of nanozeolite is so low in the modified electrodes that the available pores for Ni2+ insertion can be decreased. Also, with increasing the Ni-ZSM-5 nanozeolite over 15% in the modified elec-trodes, the resistance of the electrode increased lead-ing to a decrease in anodic current and sensitivity of the electrode response (Samadi-Maybodi, et al., 2013, Raoof, et al., 2011, Azizi, et al., 2013).

Effect of different scan rates on modified fabricated electrode was investigated. Fig.7A illustrates the CVs of the Ni-ZSM-5/CPE electrode in 0.1 M NaOH at different scan rates. Obviously, the height of the an-odic current enhanced with increasing of scan rate and

potential also moved to positive values. This positive shift may be due to the ohmic drop which generated at high current density. The ΔEp increased with scan rate that indicated a limitation in charge transfer kinetics due to the chemical interactions between the electro-lyte ions and the modified electrode. For ΔEp>200/n mV, ordinarily obtained at higher scan rates, the fol-lowing relations existed for the linear potential sweep voltammetric response proposed by Laviron (Laviron, 1979).

(2)

(3)

where X = RT/(1 −α)nF, m = (RT/F) (ks/nv), Y = RT/αn F, Epa and Epc are the anodic and cathodic peak po-tentials, respectively. Also, α, ks, and υare the elec-tron-transfer coefficient, apparent charge-transfer rate constant and scan rate, respectively. From these ex-pressions, the α can be determined by measuring the

Fig. 7: (a) CVs of the Ni-ZSM-5/CPE in 0.1 M NaOH at various scan rates from inner to outer: 0.005, 0.010, 0.015, 0.025, 0.040,

0.060, 0.075, 0.100, 0.125, 0.150 and 0.200 V s−1. (b) Plot of Ep vs. log υ for CVs showed in the (a) for anodic peaks (a) and

cathodic peaks (b). (c) The dependency of Ipa (a) and Ipc (b) on lower values of υ (0.01–0.075 V s−1) and (d) Plot of Ipa (a) and Ipc

(b) on υ1/2 at higher values of υ (υ> 0.075 V s−1).


0pa

1E E XInnF−α = +

0pcE E YIn

mα = +

147


variation of the peak potential with respect to the scan rate, and ks can be determined for electron transfer between the electrode and the surface-deposited layer by measuring the Ep values.

Fig. 7B displays the plot of Ep with respect to the logarithm of scan rate from CVs at the ranges of 0.005–0.20 V s−1 for both anodic and cathodic peaks. It can be seen that Ep is proportional to log υ at υ> 0.075 V s−1 proven by Laviron (Laviron, 1979). The value of anodic electron transfer coefficient (α) is obtained 0.49. These values suggest that the activa-tion energy for the reduction and oxidation processes might be the same (Luo, et al., 2001, Zheng, et al., 2009). The mean value of charge-transfer rate con-stant (ks) is found to be 0.045 s−1.

Fig. 7C illustrates plots of anodic and cathodic peak currents for oxidation–reduction of the NiOOH/Ni(OH)2 redox couple versus scan rate at low values from 0.01 to 0.075 V s−1. This dependence is prob-ably due to electrochemical activity of immobilized redox species at the surface of modified electrode. The electrode surface coverage (Γ*) can be calculated from linear section of the plot and using the following equation which correspond to reversible process with adsorbed species (Bard and Faulkner, 2006).

(4)

Where Ip, n, A and Γ* be the peak current, the number of electrons involved in the reaction (n=1), the surface area of the electrode (0.0962 cm2) and the surface cov-erage of the redox species, respectively. From plot of

anodic current vs. scan rate and the above equation, the total surface coverage of the Ni-ZSM-5/CPE was calculated to be 4.11×10−8 mol cm−2, considering the mean of both anodic and cathodic currents.

Fig. 7D shows plots of anodic and cathodic peak currents vs.υ1/2 at υ>0.075 V s−1. It can be concluded that the charge-transport through the Ni-ZSM-5/CPE modified electrode is controlled by diffusion. This limiting-diffusion process may be due to the charge neutralization of the electrode surface during the oxi-dation/reduction process (Taraszewska and Rosłonek, 1994, Azizi, et al., 2013).

Electrocatalytic oxidation of HCHO at the surface of Ni-ZSM-5/CPEThe electrochemical oxidation of HCHO at the sur-face of Ni-ZSM-5/CPE was investigated in 0.1 M NaOH solution.The electrochemical response of the Ni-ZSM-5/CPE in 0.1 M NaOH solution exhibited well-defined cathodicand anodic peaks associated with Ni(III)/Ni(II) redox couple (see Fig. 8a).

Fig. 8b demonstrates the CV for electrocatalytic oxidation of the HCHOon the surface of Ni-ZSM-5/CPE in 0.01 M HCHO + 0.1 M NaOH at scan rate of 20 mV s−1. Addition of HCHO to the electrolyte solution caused an enhancement in the anodic peak current. Comparison of curves (a) and (b) in Fig. 8 demonstrated that utilization of Ni-ZSM-5 as incor-porated materials into a carbon paste electrode im-proved the electrochemical signal of HCHO oxida-tion. The Ni(OH)2 layer at the electrode surface can act as a catalyst for oxidation of HCHO. As can be seen in this Fig., the pair of peaks around0.560 and 0.335 V that seen in Fig. 8a (corresponding to the NiOOH/Ni(OH)2 conversion) were vanished in the presence of HCHO, meanwhile a new oxidation peak appeared around 0.71 V. A comparison between the CVs of Ni-ZSM-5/CPE modified electrode in 0.1 M NaOH solution in the absence (Fig. 8a) and presence (Fig. 8b) of HCHO specified that anodic current for HCHO oxidation is 575 µA, meanwhile anodic cur-rent is 256 µA for Ni(OH)2/NiOOH conversion in the same scan rate (i. e. 20 mV s−1). It can be concluded that the applied modifier in this process contributed directly to the electrocatalytic oxidation of HCHO with ECʹ mechanism.

TR4AFnI

22

pυΓ

=∗

Fig. 8: CVs of Ni-ZSM-5/CPE in the (a) absence and (b)

presence of 0.010 M HCHO in 0.1 M NaOH at scan rate of

20 mV s−1.

148

HCHO molecule is almost hydrated and converted to the methylene glycol (CH2(OH)2) (Koper, et al., 1996). Due to its pKa of ca. 12.8, the CH2(OH)2 exists predominantly in its ionized form (CH2(OH)O−) in 0.1 M NaOH solution. When CH2(OH)O− diffuses from the bulk mediaon surface of electrode and is quickly oxidized to CH2(O)O− by the NiOOH species on the electrode surface that generated according equation 1. Hence, the amount of NiOOH species decreases due to its chemical reaction with HCHO. Mechanism of HCHO oxidation at the surface of Ni-ZSM-5/CPE can be described by the following equation (Yang, et al., 1999, Raoof, et al., 2012, Ciszewski and Milczarek, 1999, Zhao, et al., 2006):

(5)

Effect of HCHO concentrationFig. 9 illustrates the effect of HCHO concentration on its electrooxidation current onto Ni-ZSM-5/CPE at scan rate of 20 mV s−1. It is clearly observed that the anodic peak current increased with increasing of HCHO up to the concentration of 50.0 mM. In the concentrations above 50.0 mM, no remarkable in-crease in the anodic peak current was observed (data not shown). It can be stated that this effect may be due to the saturation of active sites and/or poisoning the electrode surface with adsorbed intermediates. Thus, 50.0 mM of HCHO represented the optimumconcen-tration after which the adsorption of the oxidation products at the electrode surface may cause the stop-page of further oxidation.

The effect of scan rate on the electrocatalytic oxida-tion of HCHO (0.01 M) was also studied. The peak currents (Ip) increased and the anodic peak potentials shifted to more positive directions by an increase in the scan rate (data not shown). It can be regarded to a kinetic limitation in the reaction between the redox sites of the HCHO and Ni-ZSM-5/CPE. Moreover, linear dependency of anodic peak currents versus the square root of the scan rate (ʋ1/2) indicated that the electrode reaction is a diffusion controlled process.

Fig. 9: Current-potential curves of different HCHO concen-

trations: (a) 0.0, (b) 3.0, (c) 10.0, (d) 25.0 and (e) 50.0 mM at

the Ni-ZSM-5/CPE and scan rate of 20 mV s−1.

Fig. 10: :(a) Double step chronoamperograms of Ni-ZSM-5/CPE in 0.1 M NaOH solution in the absence (a) and presence of

(b) 0.003, (c) 0.005, (d) 0.01 and (e) 0.03 M of HCHO. The potential steps were 0.730 and 0.350 V vs. Ag/AgCl/ KCl (3 M). (b)

Charge-time curves in the absence (aʹ) and presence of 0.03 M of HCHO (eʹ). (c) Dependence of IC/IL on t1/2, derived from the

data of chronoamperograms (a) and (e). (d) Dependence of I on t−1/2, derived from the data of chronoamperogram (c).


( )[ ]2

Ni ZSM 5 / CPE NiOOH HCHO Pr oducts

(Ni ZSM 5 / CPE) Ni(OH)

− − − + ↔ + − − −

149


Chronoamperometric studiesThe electrocatalytic oxidation of HCHO at the sur-face of modified electrode was also studied using chronoamperometry method. Fig. 10A shows dou-ble step chronoamperometric measurements at the surface of the Ni-ZSM-5/CPE at different concen-trations of HCHO such as 0.00, 0.003, 0.005, 0.01 and 0.03 M. The applied potential steps were 0.73 and 0.35 V vs. Ag|AgCl|KCl (3 M) in the anodic and cathodic direction, respectively.The forward and backward potential step chronoamperometry of the Ni-ZSM-5/CPE electrode in the blank solution showed an almost symmetrical chronoamperogram, which shows that almost equivalent charges were consumed for the oxidation and reduction of surface confined Ni(OH)2/NiOOH sites. However, in the presence of HCHO, the charge value (Q) associated with the forward chronoamperometry is greater than that observed for the backward chronoamperometry (see Fig. 10B).

Chronoamperometry can be applied for the approxi-mation of the catalytic rate constant (k) between the HCHO and redox sites of electrode based on equation (7) (Bard and Faulkner, 1980):

(6)

where, IC and IL are the currents in the presence and absence of HCHO, respectively. Symbol γ =kc0t is the argument of error function, k is the catalytic rate constant (cm3mol−1 s−1), c0 is the bulk concentration of HCHO (molcm−3) and t is the elapsed time (s). When γ exceeds 1.5, erf (γ1/2) is nearly equal to 1 and the above equation can be summarized to:

(7)

Fig. 10C shows plot of IC/IL versus t1/2 derived from the data of chronoamperograms in the absence of HCHO (a) and in the presence of 0.03 M HCHO (e). From the slopes of the IC/IL versus t1/2 for all concentrations, the mean value of k was found to be 9.064×103 cm3mol−1 s−1. An exponential behavior of I–t curves shows that a diffusion-controlled process has occurred according to Cottrell equation (Bard and Faulkner, 1980). From the chronoamperometric study, the diffusion coefficient, D, of HCHO was determined in aqueous solution by the following equation (Hassaninejad-Darzi and Ra-

γ

γ−+γπγ= 2/1

2/12/12/1

L

C )exp()(erfII

Electrode Electrolyte

Saturation limit of

HCHO for current

(mol cm-3)

Scan rate

(mV s-1)

Ep/V vs.

(Ag/AgCl)

Current

density

(mA cm-2)

Ref.

Cu/P(2ADPA)/MCNTPE 0.2 M NaOH 0.17 20 0.63 25.56 1

Pt/SWCNT/PANI 0.5 M HClO4 0.50 50 0.66 90.0 8

Pt/PAANI/MWNTs/GCE 0.5 M H2SO4 0.50 50 0.45 7.32 10

Pt/Carbon-Ceramic 0.1 M H2SO4 0.75 20 0.85 31.40 11

Pd–CILE 0.1 M NaOH 0.30 100 0.15 9.40 20

Ni/P(1,5-DAN)/MCPE 0.1 M NaOH 0.17 10 0.80 0.76 27

Ni(OH)2/POT

(TX-100)/MCNTPE0.1 M NaOH 0.048 20 0.70 12.70 29

Ni/P(NMA)/MCPE 0.1 M NaOH 0.07 20 0.74 4.10 30

Ni-ZSM-5/CPE 0.1 M NaOH 0.050 20 0.71 13.02 This work

Table 2: Comparison of the electrocatalytic behavior of Ni-ZSM-5/CPE for oxidation of formaldehyde with some of the previously reported electrodes.

1/2 1/2 1/2 1/2C0

L

I (kc t)I

= γ π = π

150

himnejad, 2014, Bard and Faulkner, 1980):

(8)

Where F is the faraday number, A is the area of the electrode, C is the known concentration of compound and D is the apparent diffusion coefficient. Fig. 10D demonstrates experimental plots of I vs. t−1/2 for 0.005 M of HCHO at the surface of Ni-ZSM-5/CPE. The same curves were plotted for all concentrations and then the slopes of the resulting straight lines were plot-ted vs. the HCHO concentration. From the slope of the resulting plots and using the Cottrell equation, the mean value of the D was achieved to be 8.575 ×10−6 cm2 s−1 (with n=1, F=96485 C mol−1, and A=0.0962 cm2). A linear relationship in the plot I vs. t−1/2 indicat-ed that the electrode reaction is a diffusion controlled process and this result is in good agreement with cy-clic voltammetric experiments (see previous section).

Stability of the Ni-ZSM-5/CPEIn the practical view, stability of the modified elec-trode was examined by measuring its response to HCHO oxidation after 1 and 3 months of storage in the laboratory atmosphere condition using CV tech-nique. The electrode response to electrocatalyticoxi-dation of HCHO retains 94% and 86% of initial value, respectively. In comparison with some other previous works, it seems clearly that nickel hydroxide in the Ni-ZSM-5/CPE can act as a comparable catalyst in the oxidation of HCHO. Table 2 exhibits peak potential, peak current density and saturation limit of HCHO for current of the Ni-ZSM-5/CPE modified elec-trode toward the electrooxidation of HCHO in 0.1 M NaOH with some modified electrode reported in the literature. In comparison with some previous reported works, it seems that Ni-ZSM-5/CPE can act as an ef-ficient electrocatalyst in HCHO oxidation process. It can be noted that the comparison of the efficiency in terms of anodic peak potential and stability for HCHO oxidation at low cost modified electrode (EP= 0.71 V) shows that this value is less than that previous works and comparable with the precious metal (i.e., Pt, Pd and Cu) modified electrodes. Besides, the surface modification of the electrode is very simple and repro-ducible compared to other modified electrodes.

CONCLUSIONS

In this done research,an organic template-free meth-od was developed to the synthesis of Ni-ZSM-5 and ZSM-5 nanozeolites. Then, a novel modified CPEs were prepared by Ni-ZSM-5 and ZSM-5 nanozeolites. The electrocatalytic oxidation of HCHO onthe surface of Ni-ZSM-5/CPE was investigated by using of CV techniques. The Ni-ZSM-5/CPE modified electrode had large electrochemical surface area, and exhibited the superior electrocatalytic performance foroxida-tion of HCHO with decreasing over potential versus bare CPE and ZSM-5/CPE and showed good electro-catalytic activity toward HCHO compared to many of the previously reported electrodes. Results revealed that the NiOOH species formed during the oxidation of Ni-ZSM-5/CPE is found to be a good catalyst for oxidation of HCHOand the modified electrode can overcome the kinetic limitation by a catalytic process. However, this non-noble catalyst has some advantages such as low cost and stability, ease of preparation and regeneration, stable response and very low ohmic re-sistance in fuel cell.

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Mostafa Rahimnejad, Ph.D., Assistant Professor, Biofuel & Renewable Energy Research Center, Faculty of Chemical Engineering, Babol University of Technology, Babol, Iran.P.O.Box: 47148-71167, E-mail: [email protected]

Seyed Karim Hassaninejad-Darzi, Ph.D., Assistant Professor, Research Laboratory of Analytical & Or-ganic Chemistry, Department of Chemistry, Faculty of Science, Babol University of Technology, Babol, Iran

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INTRODUCTION



ABSTRACT: Magnetic nanoparticles are the good choice for using in MRI as the contrast agent. Iron oxide particles such as magnetite (Fe3O4) or its oxidized form maghemite (γ-Fe2O3) are the most commonly employed in biomedical applications. In this study, we synthesized and optimized the preparation of chitosan manganese-ferrite nanoparticles (CMn-Fe nps) and evaluated its ability for the mice macrophage cells imaging with MRI. The core and hydrodynamic size of the nanoparticles was 54±27 nm and 149±48 nm respectively. The magnetization reached a saturation magnetization value of 95 emu/g. The MRI images showed that the liver and some other organs were accumulated the CMn-Fe nps and produced a negative contrast in images. CMn-Fe nps demonstrated that it is a good candidate for using as a contrast agent material in macrophage cells imaging with MRI.

Keywords: Chitosan; Contrast agent; Manganese ferrite nanoparticles; MRI imaging

Synthesis and evaluation of chitosan manganese-ferrite nanoparticles as

MRI contrast agent

S. Rasaneh1*; M. Raheleh Dadras2

1 Nuclear Sciences and Technology Research Institute, Tehran2 International Campus, Iran University of Medical Sciences, Tehran

Received: 6 June 2015; Accepted: 9 August 2015

Magnetic particles from nanometer to micrometer size have been widely used in biological and medical fields (Ito, et al., 2005). Magnetic particles have good re-sponse to magnetic force and magnetic nanoparticles show superparamagnetic behavior at room temperature that does not retain any magnetism after removal of the magnetic field. The applications of magnetic nanopar-ticles for imaging are: gastrointestinal tract imaging, detecting liver and spleen disease, kidney imaging, de-tecting lymph node metastases, blood pool characteris-tics, phagocytosis imaging, and etc. (Ito, et al., 2005). Furthermore, the particles must have combined proper-ties of high magnetic saturation, biocompatibility and

interactive functions at the surfaces (Thorek, et al., 2006). Iron oxide particles such as magnetite (Fe3O4) or its oxidized form maghemite (γ-Fe2O3) are the most commonly employed in biomedical applications since their biocompatibility has already been proven (Gup-ta and Gupta 2005). The applications of MRI have steadily increased over the past decade. MRI offers the advantage of high spatial resolution and contrast dif-ferences between tissues. Due to the unique function of this imaging modality, there is a need to develop effective contrast agents that will enhance and widen its diagnostic utility (Thorek, et al., 2006). Manganese ferrite (MnFe2O4) nanoparticles are useful for remark-able soft-magnetic properties (low coercivity, moder-ate saturation magnetization) accompanied by good

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chemical stability and mechanical hardness (Rasaneh and Dadras 2014). Uncoated magnetic nanoparticles aggregate in biological solutions due to their large sur-face area to volume ratio, forming large clusters and rending them unsuitable for biomedical applications. Therefore, an appropriate coating is needed to reduce the surface free energy in order to protect the nanopar-ticles aggregate against agglomeration. The polymer coating not only inhibits aggregation and increases stability but also leads to the creation of more hydro-philic nanostructures and provides a variety of surface functional groups to bind drug molecules. Non-toxic and biodegradable polymers such as chitosan have been used extensively in biomedical fields (Wang, et al., 2011, Dung, et al., 2009). Chitosan is a linear polysaccharide comprised of two monosaccharides: N-acetyl-D-glucosamine and D-glucosamine linked together by glucosidic bonds. Chitosan has 1 primary amino and 2 free hydroxyl groups for each C6 building unit (Banerjee, et al., 2002). In recent researchs, the magnetite chitosan NPs were obtained by crosslinking of chitosan amino groups using glutaraldehyde (Pa-til, et al., 2014, Qu, et al., 2010). The disadvantage of this method is the toxicity of this crosslinker (Hritcu, et al., 2009). Ionic gelation (polyionic coacervation) is an interesting technique that uses non-toxic poly-anions, such as sodium tripolyphosphate (TPP) as non-toxic ionic crosslinker. This method is simple and reproducible and NPs are encapsulated in a chitosan shell by ionic interactions (Hritcu, et al., 2009). In this study, we synthesized and optimized the preparation of chitosan manganese-ferrite nanoparticles (CMn-Fe nps) and evaluated its ability for the mice macrophage cells imaging with MRI.


MaterialsFerric chloride (FeCl3), manganese chloride (MnCl2•4H2O), ammonia solution (25% wt), Water-soluble chitosan (Low molecular weight, 97% deacet-ylated) and Sodium tripolyphosphate (TPP) were pur-chased from Sigma Co. All salts and buffers used in this study were of reagent grade and purchased from Fluka chemical corp.

Synthesis50 mL of FeCl3•6H2O (0.32 M) and 50 mL of MnCl2•4H2O (0.2 M) were mixed in the reactor. Then, 0.125 gr of chitosan was added to the solution and the temperature was raised to 70°C. Precipitation was ini-tiated by addition of 20 mL of the aqueous NH4OH at 0.5 mL/min. Then, the completion reaction was al-lowed to proceed for 60 min. The nanoparticles were washed with Q water several times by using a perma-nent magnet, separated in desired size by centrifuga-tion in 10000 rpm and lyophilized to obtain the final product (Rasaneh, et al., 2011, Kueny-Stotz, et al., 2012).

Physical characteristicsFor determining the amounts of iron content in the Mn-Fe nps used colorimetric assay. A sample (100 µL) of nanoparticles was mixed with HCl (100 µL) and H2O2 (10%, 100 µL) to allow the iron content of nanopar-ticles be dissolved and oxidized to Fe3+. After adding potassium thiocyanate (3%, 1 mL), the Fe3+ formed a red complex with the thiocyanate which could be measured by a spectrophotometer at 480 nm (2100 UV spectrophotometer, UNICO Instruments, China). The standard curve for calculation of iron content was obtained measuring different concentrations (0, 250, 500 and 1,000 µg/mL) of ferric chloride (Rasaneh, et al., 2011). The hydrodynamic sizes of the particles were measured using a dynamic light scattering (DLS) device from Malvern Instruments (model HPPS). The mean sizes were determined with independent mea-surements from three drawn samples of the prepara-tion with 5 runs per sample, 15 determinations in total. The core sizes of nanoparticles were also determined by Transmission Electronic Microscopy (TEM) (JEM 2010, JEOL, JAPAN). Magnetization measurements were performed using an MPMS-XL superconducting quantum interference device (SQUID) magnetometer (Quantum Design Inc., San Diego, CA). The freeze-dried samples of Mn-Fe nps (5-6 mg Fe) were ana-lyzed in a DC magnetic field range of 0–55 kG.

MRI studyThe male bulb/c mice (n= 6, 6-8 weeks old, 25-30 g) were obtained and housed under pathogen free condi-tions and fed with autoclaved food and water. Mice

S. Rasaneh and M. Raheleh Dadras

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received a dose of 100 µg (100 µL) of Mn-Fe nps by the tail vein injection. MR imaging of the animals (n= 3) were performed before and 4 hours after injection of Mn-Fe nps. The animals were anesthetized and fixed in the same position. Imaging was performed with 1.5 Tesla MR imaging (SIEMENS, Symphony) and a knee coil. Pulse sequence comprised coronal T2-weighted three-dimensional fast field-echo, 32/10 sequences with a flip angle of 250 and effective sec-tion thickness of 650 µm. For better comparison, at before, 10 minutes and 4 hours after injection, the mice were scarified, organs removed, washed and put in saline buffer and imaged with MRI.


Particle characterizationThe magnetic responsiveness of Mn-Fe nps in solu-tion was visualized by a simple experiment in which a 0.40 T magnet was placed near the glass vials (Fig. 1). The Mn-Fe nps deposited notably on the wall adjacent to the magnet within 1 minute.

The average core size of nanoparticles measured by TEM was 54±27 nm that is shown in Fig. 2-A. The hydrodynamic size of the Mn-Fe nps that determined by DLS technique was 149±48 nm and the particles distribution is presented in Fig. 2-B.

The CMn-Fe nps magnetization, induced by an ap-

plied magnetic field, increased with increasing applied field strength and reached a saturation magnetization value of 95 emu/g. The results were shown in Fig. 3.

MRI ImagingThe MRI images (T2 imaging) of the mice before and 4 hours after injection of CMn-Fe nps were shown in Fig. 4. The liver and some other organs were accumu-lated the CMn-Fe nps particles and produced a nega-tive contrast in images. The imaging from the extrac-tion tissues showed that nanoparticles were existed in blood and liver at 10 minutes after injection. At 4 hours after injection, the nanoparticles were seen in liver, spleen and kidneys but washed from the blood.

Iron determination was performed in organs by tis-sue extraction. The results are presented in Fig. 5 as percent of iron (g) injected per gram of dry tissues. At

Fig. 1: Mn-Fe nps samples before (A) and after imposing

an external magnetic field in the left (B) and under (C) of

vial wall

Fig. 2: TEM image shows the core size of Mn-Fe nps is 54±27 nm (A). Mn-Fe nps hydrodynamic size distribution

(mean 149±48 nm) determined by dynamic light scattering technique (B)

158

48 h after administration of the HMNs, the difference of iron concentration in organs and tumor was com-pared with and without external instant magnetic field. The results are shown in Fig. 5. The iron concentration in tumors was significantly increased (p-value < 0.05) by external magnetic field. While in other group, the higher concentration of iron was distributed in livers and spleens.

DiscussionIn this work, CMn-Fe nps were synthesized, deter-mined the physical characteristics and considered their biodistribution in some organs of normal mice such as liver, spleen, blood and kidneys with MRI imaging. The core size of CMn-Fe nps that measure by TEM was 54±27 nm and the hydrodynamic size was 149±48, that determined by DLS technique. This size is appro-priated for phagocyte by kupffer cells in liver. The nanoparticles with hydrodynamic radius larger than 80 nm are non-specifically taken up by kupffer cells in the healthy liver, allowing for hepatic imaging and diag-

n o - s i s o f a b -

normality in liver, spleen and macrophage cell. The magnetization reached a saturation magnetization val-ue of 95 emu/g. The MRI imaging results showed that the spleen and liver kupffer cells can absorb CMn-Fe nps very easily and produce a negative contrast in im-ages. In 2013, Lopez and et al., synthesized chitosan iron oxide nanoparticles in Microemulsion method. The core size of their magnetic nanoparticles was 50 nm with TEM images and the magnetization value was 49–53 emu/g (Lopez, et al., 2013). In a study, the potential of chitosan coated ferrite nanoparticles were examined as an MRI contrast agent. The coating of chitosan was formed simultaneously with the synthe-sis of ferrite nanoparticles. A dynamic light-scattering spectrometer determined the average diameters of the coated nanoparticles at 67.0 nm. The animal experi-ment was performed in abdomen of a New Zealand rabbit with and without the injection of the aqueous solution of chitosan-coated nanoparticles by MR images. The ratio of the signal intensity showed the presence of the particles in the kupffer cell positions (Hong, et al., 2010).

CONCLUSIONS

In this study, chitosan manganese-ferrite nanoparticles

Fig. 3: CMn-Fe nps magnetization curve measured by

SQUID exhibiting magnetic saturation

Fig. 4: The Comparison of mice liver before (A) and 4 hours

after (B) CMn-Fe nps injection. The liver after absorption

particles produced a negative contrast in MR images

Fig. 5: The Comparison of mice liver, blood, kidneys and

spleen before (A) 10 minutes (B) and 4 hours (C) after CMn-

Fe nps injection

Synthesis and evaluation of chitosan manganese-ferrite nanoparticles

159


were synthesized easily with good properties. The re-sults showed that these nanoparticles can specially lo-calize in the liver and spleen and produced a negative contrast in MRI images. Therefore we can conclude it is a good candidate for using as a contrast agent mate-rial in macrophage cells imaging with MRI.

REFERENCES

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Agents: From Small Chelates to Nanosized Hy-brids. Eur. J. Inorg. Chem., 2012(12): 1987-2005.

Lopez, R.G.; Pineda, M.G.; Hurtado, G.; Diaz de Leon, R.; Fernandez, S.; Saade, H.; Bueno, D., (2013). Chitosan-Coated Magnetic Nanoparticles Prepared in One Step by Reverse Microemulsion Precipitation. Int. J. Mol. Sci., 14(10): 19636-19650.

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Qu, J.; Liu, G.; Wang, Y.; Hong, R., (2010). Prepa-ration of Fe3O4–chitosan nanoparticles used for hyperthermia. Adv. Powder Technol., 21(4): 461-467.

Rasaneh, S.; Dadras, M.R., (2014). Detection of Her2 levels in cancerous cells based on iron oxide nanoparticles. Int. J. Bio-Inorg. Hybr. Nanomater., 3(3): 157-162.

Rasaneh, S.; Rajabi, H.; Babaei, M.H.; Akhlaghpoor, S., (2011). MRI contrast agent for molecular im-aging of the HER2/neu receptor using targeted magnetic nanoparticles. J. Nanopart. Res., 13(6): 2285-2293.

Thorek, D.L.J.; Chen, A.K.; Czupryna, J.; Tsour-kas, A., (2006). Superparamagnetic Iron Oxide Nanoparticle Probes for Molecular Imaging. Ann. Biomed. Eng., 34(1): 23.

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Samira Rasaneh, Assistant Professor, Nuclear Sciences and Technology Research Institute, Tehran, E-mail: [email protected]

Maryam Raheleh Dadras, M.Sc., International Campus, Iran University of Medical Sciences, Tehran


INTRODUCTION


ABSTRACT: In this study, catalyst of rutile titanium dioxide nanoparticles (rTiO2-NP) has been investigated for the removal and reduction of unburned hydrocarbons as benzopyran. To evaluate and calculate the thermodynamic properties of this aim, pollutants are closed to the nanoparticles and converted them into other products and the carbon dioxide molecules are simulated in the 12th steps. The geometrical structure of all stages is optimized by Density Functional Theory (DFT) method based on B3LYP/6-31G. The structure of rutile titanium dioxide nanoparticles, there are several different locations on it as the cross bridge Ti-O and Ti-Ti, the thermodynamic properties of these conversions and locations are calculated by a semi empirical method (ZINDO/S). The results shown these interactions are exothermic and spontaneous. The total energy (kcal/mol) for conversion benzopyran on Ti-O bond is lower than Ti-Ti bond. Therefore the probability of interaction with Ti-O is more. This phenomenon dramatically increases the electrical conductivity of the nano-particles, suggesting that the rTiO2-NP may be potential sensor for benzopyran gaseous molecule detection.

Keywords: Benzopyran; Density Functional Theory (DFT); Empirical method (ZINDO/S); Rutile titanium dioxide nanoparticles; Unburned hydrocarbons

Theoretical study of reduced benzopyran to CO2 by rTiO2-NP

L. Mahdavian

Department of Chemistry, Doroud Branch, Islamic Azad University, P.O. Box: 133,Doroud. Iran

Received: 17 June 2015; Accepted: 20 August 2015

The unburned hydrocarbons are toxic and carcinogenic to humans and can react with components of air (NOx, O2 and H2O) and can create ozone in these reactions with presence of sunlight in the troposphere (Chmie-larz, et al., 2011, Kwon and Min 2010 and Bhandark-ar 2013). Ways and methods have been developed to eliminate and reduce them; the catalyst is neutralized to 90% of toxic gases and unburned hydrocarbons emitted from the engine to convert carbon dioxide and water (farayedhi 2002 and Kirkpatrick, et al., 2011). Gasoline consumption in Iran has a high percentage of

unburned hydrocarbons, which have caused the pollution of the air and the environment (Shamekhi, et al., 2008). The use of a catalytic converter to reduce pollutants at source shall be considered an essential requirement.

In this study, reduced of benzopyran (is an unburned hydrocarbon) by rTiO2-NP is simulated and calculated. The rTiO2-NP size range between clusters and colloids, powders and single crystals of large are an ideal photo catalyst. The nano scale titanium dioxide is one of the most widely used industrial materials in the produc-tion of cosmetics, laboratory photosynthesis, refin-ing of water and air, is a synthesis of pigments and so on (Diebold 2003, Leavy, et al., 2006 and Xia, et al.,

162

2007). Titanium dioxide (titania) can be appeared in three crystalline form, rutile (tetragonal), anatase (te-tragonal), brookite (orthorhombic) and so on (Dhage and Ravi 2004, Torres, et al., 2007, Jones, et al., 2007 and Yang and Parr 1988). The structure of the rutile is the most common form of titania, that system is the most resistant phase thermodynamically.

COMPUTATIONAL METHODS

Chemical computations involving different math-ematical methods are divided into two groups: 1- Mo-lecular mechanics, 2- Quantum mechanics.

In this research, the simulation of nano-surfaces re-action of titanium dioxide and benzopyran are done Gauss View 5.0 software and its geometrical shape and structure is improved by GAMESS-US program package (Becke 1993, Caricato, et al., 2006 and Rid-ley and Zerner 1973), and changing benzopyran into less dangerous products on the connection of differ-ent rTiO2 nanoparticles in different states is studied

and calculated. Fig. 1 shows the ball and stick model of a rutile TiO2-NP, shown all probability of the pol-lutants approaching. In this research, the pollutants approaching to Ti-O connection, the first and second probability (there are two connections for Ti-O) and Ti-Ti connection, the third probability (Fig. 1) were calculated and studied. After all geometrical structures are optimized by B3LYP/6-31G. Their thermodynam-ic properties were assessed by the ZINDO/S in semi-empirical method. ZINDO/S method is extensively used to compute heat formation, geometric form of the molecule, ionization energy, electron adherence, and other features (Zerner 1991).


In this study, the interaction of benzopyran on rTiO2 nanoparticles have been simulated in twelve steps (Fig. 2), from 1st until 2nd steps entry benzopyran on rTiO2-NPs then in 3th and 4th steps are transition state conversion of them to o-cresol and ethylene and in 5th

step, product generated from the surface is removed. In 6th step, o-cresol is closed on surface and from 8th

step is transiting state conversion of them to propa-nol and butane in 9th steps. Then new produces are adsorbed on the surface and 10th step is its transition state. The conversion was completed in 11th step and CO2 molecules are produced, then it is excretion from rTiO2-NPs in 12th step. The electronic structure and the thermodynamic properties are calculated for all steps by ZINDO/S-DFT.

ZINDO/S has been widely used to calculate the heat of formation, molecular geometry, dipole moment,

Fig. 1: The ball and stick model of a) benzopyran and rutile TiO2-NP, b) front view and c) top view.

L. Mahdavian

Scheme 1: Oxidation of benzopyran into carbon dioxide and

water

163


ionization energy, electron affinity, and other proper-ties. The corresponding calculated thermodynamic data (ΔEtotal, ΔHele, ΔSele, and ΔGele) were determined. After simulation and writing files, for the perform of calculation, their Z- matrix files, send on the site: High performance computing research center (http://hpcrc.aut.ac.ir) by using special software file (Open VPN, VNC Viewer and …). This institute has computers 48 cores and GAMESS-US program package for Linux.

All thermodynamic properties shown in the Tables 1-3 except the dipole moment and RMS gradient are obtained by the following:

(1)

The thermodynamic properties of these interactions on Ti-O (2 locations) and Ti-Ti cross bridges are shown

Fig. 2: Ball-and-stick models configuration of benzopyran interaction on cross bridge of Ti-O in rutile TiO2 nanoparticle.

The benzopyran converted to CO2 on Ti-O(1)

Steps Etotal

(kcal/mol)Dipol

Moment (D)RMS

kcal/mol.oAEbin

(kcal/mol)H

(kcal/mol)Gele

(kcal /mol)Enuc

(kcal/mol)Eelec

(V)

rTiO2-NP -67934.53 3.81 415.1 -12183.09 -10815.04 -237906.98 169972.45 10354.50

Benzopyran -42736.33 2.00 190.8 -7866.31 -5851.92 -153227.46 110491.13 6668.97

Steps

ΔEtotal

(kcal/mol)

Dipol

Moment (D)

RMS

kcal/mol.oA

ΔEbin

(kcal/mol)

ΔH

(kcal/mol)

ΔGele

(kcal/mol)

ΔEnuc

(kcal/mol)

ΔEelec

(V)

1 -1730.78 4.43 311.5 -1730.79 -1730.79 -154822.85 153092.07 6738.402 -2694.03 10.21 313.4 -2694.04 -2694.03 -183210.49 180516.47 7973.93

3 -3189.59 28.99 321.5 -3189.59 -3189.60 -190346.32 187156.74 4142.25

4 -3292.95 5.64 323.5 -3292.96 -3292.97 -190010.41 186717.46 4134.94

5 -4179.13 20.01 319.8 -3576.79 -3472.59 -202479.79 198300.65 8812.59

6 -3664.55 6.69 306.0 -2459.85 -2251.45 -167947.62 164283.07 3654.82

7 9844.98 9.69 335.3 768.95 152.08 -63225.54 73070.51 1375.89

8 9803.6 11.16 333.2 727.57 110.70 -64163.04 73956.64 2792.59

9 8255.61 17.31 336.6 -820.41 -1437.28 -90893.11 99148.72 3955.97

10 5507.25 20.45 323.7 -2364.06 -2772.53 -123820.35 129327.61 5389.0711 5626.31 12.20 321.8 -1642.65 -1946.91 -117004.07 122630.09 5092.41

12 -70462.81 5.03 460.0 -3910.02 -4184.36 -511283.58 440820.77 22252.76

Table 1: The thermodynamic properties of adsorption benzopyran on Ti-O (1) bridge cross of rTiO2 nanoparticle and converted to CO2 at 298K (ZINDO/s)

Fig. 3: The total energy of benzopyran interaction on rTiO2-

NP by ZINDO/S method.

( ) ( )

( with )

initial clean surface molecule

final surface molecule

final initial

T T T

T T

ads T T T

E E E

E E

E E E E

= +

=

= ∆ = −

164

in Tables 1-3. The Eads (kcal/mole) is shown in Fig. 3, that change the total energy of this interaction on all locations of rTiO2-NP can be observed and com-pared. The Eads of this interaction for all locations of rTiO2-NP is same. Only minor changes are observed in the transition states (3rd, 8th and 10th steps). The most changing of Eads is for across bridge of Ti-O (1), which is related spatial structure of this location on rTiO2-NP.

Dipole moment for all steps are calculated and are shown in Tables 1-3, which its most changes are in the transition state of intermediates and maximum moment of it is 28.99D for 3rd in across the bridge of Ti-O(1).

As the results show, compared of data in Tables 1-3, the probability of pollutant interaction on cross bridge of Ti-Ti is more than other locations. The bond dis-tance between Ti-Ti, Ti-O (1) and Ti-O (2) are 2.95, 2.02 and 1.62 oA respectively. Therefore distance, the location and spatial structure of titanium, titanium bond is more suitable for close pollutants.

In order to access to the electrical properties these interactions by using the electrical energy and the fol-lowing equation (Lee 2005):

(2)

where R is the electrical resistance and I is the current intensity per (A), that I can be found from I=q/t the

The benzopyran converted to CO2 on Ti-O(2)

StepsEtotal

(kcal/mol)

Dipol

Moment (D)

RMS

kcal/mol.oA

Ebin

(kcal/mol)

H

(kcal/mol)

Gele

(kcal/mol)

Enuc

(kcal/mol)

Eelec

(V)

rTiO2-NP -67934.53 3.81 415.1 -12183.09 -10815.04 -237906.98 169972.45 10354.50

Benzopyran -42736.33 2.00 190.8 -7866.31 -5851.92 -153227.46 110491.13 6668.97

StepsΔEtotal

(kcal/mol)

Dipol

Moment (D)

RMS

kcal/mol.oA

ΔEbin

(kcal/mol)

ΔH

(kcal/mol)

ΔGele

(kcal/mol)

ΔEnuc

(kcal/mol)

ΔEelec

(V)

1 -1730.78 4.43 311.5 -1730.79 -1730.79 -154822.85 153092.07 6738.40

2 -2368.58 14.69 310.9 -2368.59 -2368.59 -174859.15 172490.57 7610.45

3 -3624.51 14.15 328.9 -3624.52 -3624.52 -194604.90 190980.39 8469.85

4 -3664.54 7.83 341.0 -3664.55 -3664.56 -194874.88 191210.34 8481.60

5 -5021.39 12.16 341.2 -4419.05 -4314.84 -213623.17 208601.78 9297.59

6 -4799.58 16.26 304.0 -3594.88 -3386.48 -209434.96 204635.38 9115.30

7 9779.46 4.69 337.4 703.44 86.56 -64972.05 74751.51 2827.80

8 8981.34 12.87 343.2 -94.68 -711.56 -80746.30 89727.65 3514.35

9 7836.38 19.41 341.9 -637.29 -1149.97 -95319.52 103155.89 4148.62

10 6280.35 9.84 314.0 -988.61 -1292.88 -114418.43 120698.78 4979.87

11 6350.61 21.81 318.7 -918.36 -1222.62 -96478.53 102829.14 4199.07

12 -70462.81 5.03 460.0 -3910.02 -4184.36 -511283.58 440820.77 22252.76

Table 2: The thermodynamic properties of adsorption benzopyran on Ti-O(2) bridge cross of rTiO2 nanoparticle and converted to CO2 at 298K (ZINDO/S).

Fig. 4: The electrical resistance of benzopyran interaction on

aTiO2-NP by ZINDO/S method.

Theoretical study of reduced benzopyran to CO2 by rTiO2-NP

elecE RI

165


equation so:

(3)

The electrical resistance of all steps and conversions are computed based on electrical data, (2) and (3) equations, as can be seen in Fig. 4.

The electrical resistances of all steps for different lo-cations rTiO2-NP are same, except for 2nd and 5th steps. In 2nd step, electrical resistance converted pollutant to CO2 on Ti-Ti is lower than in other locations and in 5th

step, Ti-O (1) is lower than. Equation constant and other thermodynamic param-

eters such as Gibbs free energy, enthalpy and entropy of the whole reactions were computed through the fol-lowing equation:

(4)

That T is the reaction temperature that in these com-putation is considered 298K. In order to compute the

entropy reactions:

(5)

The Gibbs free energies (ΔGele) for Ti-Ti location in this interaction is negative than in other locations for all Intermediates (Table 4). The function mechanism for this conversion on rTiO2-NT can be as follows:

TiO2 + hν → TiO2 (ecb− +holevb

+)

H2O → OH− +H+

Oxidative reaction:

•OH + benzopyran + 3/2H2 → C7H8O + C2H2

•OH + C7H8O + C2H2 + 23/2O2 → 9CO2 + 6H2O

Titanium dioxin molecules on the metal surface acti-vate by ultraviolet radiation and during some chemical processes make bacteria, algae and fungi disappeared (Varghese, et al., 2009). When ultraviolet ray radiates

Table 3: The thermodynamic properties of adsorption benzopyran on Ti-Ti bridge cross of rTiO2 nanoparticle and converted to CO2 at 298K (ZINDO/s).

The benzopyran converted to CO2 on Ti-Ti

StepsEtotal

( kcal /mol)

Dipol

Moment (D)

RMS

kcal/mol.oA

Ebin

( kcal /mol)

H

( kcal /mol )

Gele

( kcal /mol )

Enuc

(kcal/mol)

Eelec

(V)

rTiO2-NP -67934.53 3.81 415.1 -12183.09 -10815.04 -237906.98 169972.45 10354.50

Benzopyran -42736.33 2.00 190.8 -7866.31 -5851.92 -153227.46 110491.13 6668.97

StepsΔEtotal

( kcal /mol)

Dipol

Moment (D)

RMS

kcal/mol.oA

ΔEbin

( kcal /mol)

ΔH

( kcal /mol )

ΔGele

( kcal/mol )

ΔEnuc

(kcal/mol)

ΔEelec

(V)

1 -1594.49 4.19 311.2 -1594.49 -1594.50 -147313.24 145718.75 6411.56

2 -1869.53 8.90 372.3 -1869.54 -1869.53 -155320.09 153450.56 6760.04

3 -3553.68 12.67 323.0 43652.49 -3553.70 -185463.87 181910.18 4036.00

4 -3658.12 6.32 323.3 -3658.14 -3658.13 -184843.73 181185.60 4022.51

5 -5099.25 9.07 316.5 -4496.91 -4392.70 -204822.13 199722.88 8914.54

6 -4257.8 7.52 306.7 -3053.11 -2844.70 -189596.47 185338.66 8251.87

7 9594.48 8.75 328.7 518.45 -98.42 -62074.47 71668.95 2701.69

8 8496.39 5.77 345.8 -579.63 -1196.5 -76377.83 84874.22 1662.11

9 7316.12 18.48 342.5 -1157.55 -1670.22 -90868.57 98184.69 1977.45

10 5508.97 12.86 315.1 -1759.99 -2064.25 -114269.07 119778.04 4973.37

11 6663.07 4.77 320.6 -605.89 -910.16 -107249.62 113912.69 2333.93

12 -70462.81 5.03 460.0 -3910.02 -4184.36 -511283.58 440820.77 22252.76

elecE tRnF

=

exp eleGKRt∆ = −

eleele

HST

∆∆ =

166

to titanium dioxide, and electrochemical reactions happen and it frees the radical, free radicals confront to pollutants and destroy them. After attracting the sun's ultraviolet by rTiO2-NP the electrons of capacity balances displace to guide band balance, and an empty hole appears in capacity balance.

CONCLUSIONS

Air pollution is 11 times more than water pollution and food contamination are 16 times more dangerous for human. 92% of respiratory diseases and 20% of cardiovascular diseases caused by air pollution. 3 mil-lion people worldwide each year lose their lives due to air pollution. Thus reducing pollution induced fossil fuels by car shall be considered a necessity.

The aim of this study was to find a way to eliminate and reduce unburned hydrocarbons (butadiene, form-aldehyde, etc.) and source control before entering the environment is by TiO2 nanoparticles. TiO2-NP is a strong photocatalytic which caused the loss of pollut-ants and a substance into low-risk (that is environmen-tal friendly). These particles have high potential for detoxification and elimination of industrial pollutants. In this study, benzopyran after absorption on rTiO2 NPs is converted to o-cresol & ethylene and they are reduced to butane & propanol, then are decreased into

the water and carbon dioxide.Locations on the surface of rTiO2 nanoparticles to

absorb pollutants are investigated such as cross bridg-es Ti-O (1), Ti-O (2) and Ti-Ti pollutants close to the locations. After being optimized interaction struc-tures, the thermodynamic properties are calculated ZINDO/S. The results showed that these interactions and conversions are endothermic, and needs to solar or other energy. According to the exhaust gas high temperature of car may be used for this conversion. The results show that the energy of approaching to Ti-Ti cross bridge is the lease other locations on rTiO2-NP. This means that the chance of close pollution to the cross bridge of Ti-Ti is greater because it is more symmetric and electron bond polarity.

ACKNOWLEDGEMENT

I would like to thank from Doroud branch of Islamic Azad University, for providing me with all the neces-sary facilities for the research.

REFERENCES

Al-farayedhi, A.A.; (2002). Effects of octane number on exhaust emissions of a spark ignition engine.

lnKΔSele

kcal/K.mol

ΔHele

kcal/mol

ΔGele

kcal/mol

14202437112.84-1562.18-35187.56Benzopyran to o-cresol & ethylene Cross bridge of

Ti-O(1) 1116721187.51-1589.36-27667.57o-cresol to butane & propanol

270325925.25825.62-6697.52Butane & propanol to CO2

16165839127.91-1933.76-40052.03Benzopyran to o-cresol & ethylene

Cross bridge of

Ti-O(2) 636681850.26-798.12-15774.25o-cresol to butane & propanol

4678013.65-72.65-1159.01Butane & propanol to CO2

15148093119.02-2063.63-37530.49Benzopyran to o-cresol & ethylene Cross bridge of

Ti-Ti 1162190295.03-473.72-28794.10o-cresol to butane & propanol

283319719.68-1154.09-7019.45Butane & propanol to CO2

Table 4: The thermodynamic properties of adsorption benzopyran on rTiO2 nanoparticle and converted to CO2 at 298K (ZINDO/S).

L. Mahdavian

167



Leila Mahdavian, Ph.D., Associate Professor, Department of Chemistry, Doroud Branch, Islamic Azad University, P.O. Box: 133. Doroud. Iran, E-mail: [email protected], [email protected]

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1. INTRODUCTION

ABSTRACT: A simple, highly sensitive, accurate and selective method for determination of trace amounts of Cu2+ in water samples was reported. In this paper, Isopropyl 2-[(isopropoxy carbothioyl)disulfanyl]ethanethioate (IIDE) in order to prepare an effective sorbent for the preconcentration and determination of Cu. The sorption capacity of Isopropyl 2-[(isopropoxy carbothioyl)disulfanyl]ethanethioate (IIDE)-modified Octadecane-functionalized nano graphene (OD-G) (IIDE MS) was 82.34 mg.g-1 and the optimum pH for the quantitative recovery of Cu was found as 5.3. The optimum flow rate, sorbent amount and sample volume were 8 mL.min-1, 300 mg and 50 mL, respectively. 10 mL of 0.1 mol.L-1 HCl was the most suitable eluent. The recommended method is simple and reliable for the determination of Cu without any notable matrix effect and successfully applied to environmental water samples. The limit of detection of the proposed method is 7.5 ng per mL. The method was applied to the extraction and recovery of Cu(II) in different water samples. In the present study, we report the application of preconcentration techniques still continues increasingly for trace metal determinations by flame atomic absorption spectrometry (FAAS) for quantification of Cu in Formalin-fixed paraffin-embedded (FFPE) tissues from Liver loggerhead turtles. This method exhibits the superiority in compared to the other adsorption reagents because of the fact that there is no necessity of any complexing reagent and optimum pH of solution presents in acidic media. In this method is relative standard deviation (R.S.D.) of 2.7%.

Keywords: Cu; Formalin-fixed paraffin-embedded (FFPE); Isopropyl 2-[(isopropoxy carbothioyl)disulfanyl]ethanethioate (IIDE) -modified silica-gel; Octadecane-functionalized graphene (OD-G); Tissues from Liver loggerhead turtles

Extraction sorbent with octadecane-functionalized nano graphene for the preconcentration of Cu(II) in water and tissues from liver

loggerhead turtles specimens

A. Moghimi

Department of Chemistry, Varamin Pishva-Branch Islamic Azad University, Varamin, Iran

Received: 29 June 2015; Accepted: 2 September 2015

Copper, at trace concentrations, acts as both a micronu-trient and a toxicant in marine and fresh water systems (Leyden, et al., 1976; Narin, et al., 2000; Akama, et al., 2000; Ohta, et al., 2001; Cuculic, et al., 1997; Moghi-mi, et al., 2012).The direct determination of trace met-als especially toxic metal ions such as Cu, tin, arsenic,

lead, antimony and selenium from various samples re-quires mostly an initial and efficient pre-concentration step (Leyden, et al., 1976). This pre-concentration is required to meet the detection limits as well as to de-termine the lower concentration levels of the analyte of interest (Jones, et al., 1983). This can be performed simply in many ways including liquid and solid phase extraction techniques (Nambiar, et al., 1998; Caroli, et

170

al., 1991). The application of solid phase extraction technique for preconcentration of trace metals from different samples results in several advantages such as the minimal waste generation, reduction of sample matrix effects as well as sorption of the target spe-cies on the solid surface in a more stable chemical form(Alexandrova, et al., 1993).

The normal and selective solid phase extractors are those derived from the immobilization of the organic compounds on the surface of solid supports which are mainly polyurethane foams (Arpadjan, et al., 1997), filter paper (Leyden, et al., 1975), cellulose (Gennaro, et al., 1983) and ion exchange resins (Grote, et al., 1985). Silica gel, alumina, magnesia and zirconia are the major inorganic solid matrices used to immobilize the target organic modifiers on their surfaces (Unger 1979 ) of which silica gel is the most widely used solid support due to the well documented thermal, chemical and mechanical stability properties compared to other organic and inorganic solid supports (Boudreau, et al., 1989). The surface of silica gel is characterized by the presence of silanol groups, which are known as weak ion exchangers, causing low interaction, binding and extraction of the target analytes (Kvitek, et al., 1982). For this reason, modification of the silica gel surface with certain functional groups has successfully been employed to produce the solid phase with certain se-lectivity characters (Bruening, et al., 1991).

Two approaches are known for loading the surface of solid phases with certain organic compounds and these are defined as the chemical immobilization which is based on chemical bond formation between the silica gel surface groups and those of the organ-ic modifier, and the other approach is known as the physical adsorption in which direct adsorption of the organic modifier with the active silanol groups takes place (Unger 1979). Selective solid phase extractors and pre-concentrators are mainly based on impregna-tion of the solid surface with certain donor atoms such as oxygen, nitrogen and sulfur containing compounds (Mahmoud 1997; Mahmoud, et al., 1997; Tong, et al., 1990; Dadler, et al., 1987). The most successful selective solid phases for soft metal ions are sulfur-containing compounds, which are widely used in different analytical fields. Amongst these sulfur-con-taining compounds are dithiocarbamate derivatives

for selective extraction of Cu(II) (Mahmoud 1998; Mahmoud 1999) and pre-concentration of various cat-ions (Leyden, et al., 1976; Narin, et al., 2000; Akama, et al., 2000; Ohta, et al., 2001; Cuculic, et al., 1997; Moghimi, et al., 2009; Thurman 1998; Pawliszyn 1997; Izatt, et al., 1996; Hagen, et al., 1990; Krueger 1995; Yamini, et al., 1994; Shamsipur, et al., 1999; Shamsipur, et al., 2001; Brunner, et al., 2003; Zelder, et al., 2004; Boll, et al., 2005; Nayebi, et al., 2006; Moghimi, et al., 2007; Moghimi 2007) and 2-mer-captobenzothiazol-modified silica gel for on-line pre-concentration and separation of silver for atomic absorption spectrometric determinations (Qiaosheng, et al., 1998). Ammonium hexa-hydroazepin-1-dithio-carboxylate (HMDC)-loaded on silica gel as solid phase pre-concentration column for atomic absorp-tion spectrometry (AAS) and inductively coupled plasma atomic emission spectrometry (ICP-AES) was reported (Alexandrova, et al., 1993). Mercapto-mod-ified silica gel phase was used in pre-concentration of some trace metals from seawater (Moghimi, et al., 2010). Sorption of Cu(II) by some sulfur containing complexing agents loaded on various solid supports (Tajodini, et al., 2010) was also reported. 2-Amino-1- cyclopentene-1-dithiocaboxylic acid (ACDA) for the extraction of silver(I), Cu(II) and palladium(II) (Moghimi, et al., 2009), 2-[2-triethoxysilyl-ethylthio] aniline for the selective extraction and separation of palladium from other interfering metal ions (Narin, et al., 2000) as well as thiosemicarbazide for sorption of different metal ions (Campderros, et al., 1998) and thioanilide loaded on silica gel for pre-concentration of palladium(II) from water (Narin, et al., 2000) are also sulfur contaning silica gel phases.

This study focuses on the utilization of IIDEMS as an efficient octadecane-functionalized nano graphene (OD-G) in the preconcentration step of Cu(II) ions prior to FAAS determination. The influences of some analytical conditions on the preconcentration proce-dure, such as initial pH, sample volume, eluent type and volume, sorbent amount, flow rate were investi-gated.

The second aim of this study was the selection of an appropriate method for the analysis of FFPE tissue were based on present work with atomic absorption spectrophotometric determination of Cu(II).

A. Moghimi

171


EXPERIMENTAL

ApparatusDetermination of Cu2+ contents in working samples were carried out by a Varian spectra A.200 model atomic absorption spectrometer equipped with a high intensity hallow cathode lamp(HI-HCl) according to the recommendations of the manufacturers. Instru-mental parameters were selected according to the manufacturer’s suggestion. The infrared spectra of the materials were recorded on a Perkin Elmer spec-tra 100-IR spectrometer (Waltham, MA, USA) using KBr disk in the range of 4000-400 cm-1. Heidolph PD 5201 (Schwabach, Germany) model peristaltic pump with eight heads was used for controlling the flow of the liquid into column. The pH measurements were carried out by an ATC pH meter (EDT instruments, GP 353).

Reagents Octadecane-functionalized nano graphene (OD-G) was used as the support material. All the chemicals used in this study were of analytical grade deionized water was used in all experiments. The standard so-lution of Cu(II) (1000 mg.L-1) for the calibration of AAS was purchased from Merck (Darmstadt, Germa-ny). The other concentrations of the standard solutions were prepared by diluting this solution. A stock solu-tion of Cu(II) was prepared by dissolving appropri-ate amount of Cu(NO3)2 (Merck) in doubly distilled deionized water and the other concentrations of the working solutions were obtained by diluting this stock metal solution. The pH of the solutions was adjusted to desired values with 0.1 M HCl and/or 0.1 M NaOH solutions. Working solutions were prepared by appro-priate dilution of the stock solution.

Synthesis of Molecular structure of Isopropyl 2-[(iso-propoxycarbothioyl) disulfanyl] ethanethioateIodine (1 mmol) in CH2Cl2 (10 mL) was added to a stirred solution of potassium o-isopropyl (dithiocar-bomate) (1 mmol) in CH2Cl2 (10 mL) and stirred for 1 h. The reaction mixture was washed with 10% aque-ous Na2S2O3 (2×10 mL) and H2O (2×10 mL). The organic layer was dried over MgSO4 and evaporated under reduced pressure. More purification carried

out with the re-crystallization in hexane so that pale yellow crystals of L were obtained in 90% yield (0.24 g). The structure and purity of L was confirmed by elements analysis, NMR and IR Spectroscopy. 1H NMR (CCl4), δ (ppm): 1.43 (t, 12H, CH3), 5.63 (m, 2H, CH). IR (KBr). νmax (cm-1): 2979.8 (s), 2869.9 (w), 1463.9 (s), 1442.7 (s), 1373.0 (s), 1271.1 (s, b), 1145.6 (s), 1082.2 (s), 1048.0 (s, b), 898.8 (s), 796.5 (s), 690.5 (m) (Scheme 1).

Synthesis of octadecane-functionalized graphene (OD-G)The GO was synthesized according to the modifica-tion of Hummers' methods (Hummers, et al., 1958). In a typical preparation of OD-G, 50 mg of GO and 100 mL of dimethylformamide (DMF) were added to the flask with sonication for 1 hour to get a homogeneous dispersion. Five hundred milligrams BOD and 30 ml pyridine were added into the reaction mixture. The

Scheme 1: Molecular structure of Isopropyl 2-[(isopropoxy-

carbothioyl)disulfanyl]ethanethioate. (Harvey et.al., 1950)

Scheme 2: Schematic drawing of the reaction system for the

synthesis of OD-G, with ethanol and acetone twice, sepa-

rately. The as-prepared OD-G was then dissolved in tetrahy-

drofuran (THF) or dichlorobenzene (DCB) by sonication for

30minutes (Liu, et al., 2010)

172

mixture was then heated to 115°C and kept refluxing for 24 hours. To purify the OD-G, 100 ml ethanol was added to the mixture, followed by filtration with 0.45 μm PTFE membranes. The filter cake was washed.

Preparation of Isopropyl 2-[(isopropoxy carbothioyl)disulfanyl]ethanethioate modified Octadecane-func-tionalized nano graphene (OD-G) All acids were of the highest purity available from Merck and were used as received. Methanol and Chlo-roform were of HPLC grade from Merck. Analytical grade nitrate salts of lithium, sodium, potassium, mag-nesium, calcium, strontium, barium, zinc, cadmium, copper(II) nickel, cobalt(II), and Cu (II) were of the highest purity. Ultra pure organic solvents were ob-tained from E. Merck, Darmstat, Germany. The stock standard solution of Cu (II) was prepared by dissolv-ing 0.1000 g of the Cu(II) powder in 10 mL concen-trated nitric acid and diluted to 1000 mL with water in a calibrated flask. Working solutions were prepared by appropriate dilution of the stock solution. In order to prepare a 0.1% Isopropyl 2-[(isopropoxy carbothioyl)disulfanyl] ethanethioate (IIDE) solution, 0.1 g of the reagent was dissolved in 10 mL of acetone and 8 mL of concentrated ammonia solution was added. The fi-nal volume of this solution was diluted to 100 mL with water. 100 mL of reagent solution was added into four grams of Octadecane-functionalized nano graphene (OD-G) suspended in 100 mL water and then mixed on a magnetic stirrer for 24 h. The final product was filtered, washed with doubly distilled deionized water and then dried at 100°C in an oven overnight.

Column preparationA glass column (5.5 cm height ×9 mm i.d.) was packed with a known amount of IIDEMS between two layers of glass wool into the column. A definite volume of Cu(II) solution (1 µg mL-1) was passed through the column. All the column studies were performed at room temperature of 25°C.

Preconcentration procedureThe preconcentration method was tested with synthet-ic Cu(II) solutions prior to its application to the real samples. For this purpose, an aliquot of 50 mL of stan-dard Cu(II) solution (1 µg.mL-1) was taken and the pH

of the solution was adjusted to desired value with HCl and/or NH3. The model solution was pumped through the column at a flow rate of 8 mL min-1 controlled with a peristaltic pump. The bound metal ions were eluted from IIDEMS with 10 mL of 0.1 mol L-1 HCl solution. The Cu(II) concentration in the eluate was determined by FAAS. The recoveries of Cu(II) were calculated from the ratio of concentration found by FAAS to that calculated theoretically. The general preconcentration procedure described above was carried out to optimize the experimental conditions such as pH, amount of ad-sorbent, flow rate, type, concentration and volume of the elution solutions, etc.

Analysis of sample paraffin-embedded tissues from liver loggerhead turtles specimensSelected areas from fresh frozen tissues from liver log-gerhead turtles specimens were sliced in three pieces (numbered as 1, 2 and 3) of approximately 10 mm × 5 mm × 2 mm each. Sets of pieces of set 1 (controls), were placed into a vacuum chamber at 50°C overnight to dry (until a constant weight was obtained) and the sets 2 and 3 were subjected to the standard 10 % buffered formalin fixation and paraffin embedding31 histological process using a tissue processor (Tissue-Tek VIP, Sakura Finetek USA Inc., Torrance, CA). After the paraffin embedding process, tissues were subsequently excised from the blocks with a titanium knife and deparaffinized in xylene at 55°C for 1 h in the tissue processor (the set 2), or with hexane at 20°C for 1 week with frequent changes of the solvent in handling-based procedure (the set 3). Xylene was of a grade routinely used for the FFPE process and hex-ane was of ''Optima'' grade (Fisher Scientific). Upon deparaffinization, the tissue samples were dried in a vacuum chamber until constant weight was obtained. Each dried sample (of the sets 1-3) was divided into three portions (5-10 mg each) to be further analyzed as triplicates.


Characteristics of the materialThe morphologies of GO and OD-G are observed by TEM. As shown in Fig. 1a, the image of GO sheets is

Extraction sorbent with octadecane-functionalized nano graphene

173


smooth with the average size of about 1 μm. The verge is quite clear while some tend to fold and roll. After functionalized with OD groups, the size of OD-G is about 0.5 μm, which is smaller than that of the original GO (Fig. 1b). During the functionalization, the reac-tion between oxygen groups from GO and BOD could split the larger graphene into smaller graphene sheets. Similar results were reported by (Sun, et al., 2010; Graf, et al., 2007). The introduction of OD groups can effectively prevent the aggregation of GO during re-duction. The FTIR spectra are tested for confirming the effective reduction and ether-functionalization with OD groups of GO. From Fig. 2a, the FTIR spec-trum of OD-G presents the doublet bands at 2854 and

2923 cm-1, which are attributed to the antisymmetric and symmetric C-H stretching vibrations of the -CH2- groups from OD groups (Tuzen, et al., 2009). The band centered at 1200 cm-1 is resulted from the C-O-C asymmetric stretch (Moghimi, et al., 2012; Choi, et al., 2003) this suggests the ether-functionalization oc-curs between GO and BOD. In addition, the band at 1574 cm-1 is connected to the C=C skeletal vibration of reduced graphene sheets (Choi, et al., 2003) .This indicates that GO has been effectively reduced dur-ing the functionalization process in pyridine at 115°C. (McAllister, et al., 2007) reported the deoxygen-ation by the nucleophilic substitution between epoxy groups of GO and alkylamine or diaminoalkane. So

Fig. 1: TEM images of (a) GO sheets and (b) OD-G single sheets. (Saitoh, et al., 2004)

(a) (b)Fig. 2: (a) FTIR spectra of GO and OD-G produced at different temperatures. Normalized according to the intensity of peaks

at 1725 nm, (b) XRD patterns of GO and OD-G produced at 115°C (Saitoh, et al., 2004)

174

we speculate that the reduction is associated with the nucleophilic attack of GO by pyridine. Meanwhile, for the FTIR spectra of OD-G at 50°C, the lack of bands at 1574 cm-1, 2854 cm-1 and 2923 cm-1 suggests that GO is neither functionalized with OD groups nor reduced by pyridine at this temperature. Fig. 2b shows the XRD patterns of GO and OD-G produced at 115°C. It can be seen that the XRD pattern of OD-G produced at 115°C shows a broader peak of graphitic {002} diffraction plane at 2θ = 21.95° which is result-ed from the disordered stacking of reduced graphene sheets (Saitoh, et al., 2004).This further confirms the effective reduction during the ether-functionalization. Additionally, the weak peak at 2θ = 8.76° is associ-ated with the {001} diffraction plane of OD-G. The corresponding interlayer spacing is 10.1 Å, which is larger than that of original GO due to the successful ether-functionalization with OD groups.

Effect of pH on the recovery of CuThe pH of the sample solutions were adjusted to dif-ferent values between 2-8, by addition of hydrochloric acid or a suitable buffer such as sodium acceate-acetic acid or sodium dihydrogen phosphate- disodium hy-drogen phosphate, and then solutions passed through the column. Eventually, the metal ions were stripped by 5 mL of HNO3, 1M followed by flame atomic ab-sorption determination of the eluted Cu(II). Then, percentage recovery at various pH values was deter-mined (Fig. 3). According to the results shown in Fig. 3 up to pH 4.5-5.5, complete recoveries are obtained. However, at higher pH values, percentage recovery decreases. This is due to fact that in an acidic solution the protonation of Isopropyl 2- (isopropoxycarbothio-yl)disulfanyl]ethanethioate (IIDE) occurs and there

is a weak tendency for retention between Cu(II)and 1-nitroso-2-naphthol-3,6-disulfonic acid, whereas at higher values (pH>5.7), Cu(II) reacts with hydroxide ions to produce Cu(OH)2. Therefore, sodium acceate-acetic acid buffer with pH = 5.3 was used for the pre-concentration step. Other solvents used for dissolving Isopropyl 2-(isopropoxycarbothioyl)disulfanyl]eth-anethioate (IIDE) were 5 mL of HNO3 (1M). The in-fluences of these solvents on the recoveries as a func-tion of pH are compared and shown in Fig. 3.

Evaluation of the role of the ligandSome preliminary experiments were performed for in-vestigation of absence or presence of Isopropyl 2-(iso-propoxycarbothioyl)disulfanyl]ethanethioate (IIDE) on the quantitative extraction of Cu(II). It was con-cluded that the surface itself does not show any ten-dency for the retention of Cu(II), but introduction of 100 mL portions of aqueous Cu(II) samples contain-ing 10 µg of Cu(II) and 10 mg of Isopropyl 2-(isopro-poxycarbothioyl)disulfanyl]ethanethioate (IIDE) Cus to satisfactory its retention (Table 1). The latter case is most probably attributed to the existence of a con-siderable interaction between Cu(II) and the Isopropyl 2-(isopropoxycarbothioyl)disulfanyl]ethanethioate (IIDE). It should be mentioned that formation of sta-ble complexes between Cu(II) and Isopropyl 2-(isop-ropoxycarbothioyl)disulfanyl] ethanethioate (IIDE) at pH 5.3 is probably due to an ion pair formation mech-anism. However, at pH higher than 5 the retention and percentage recovery of Cu(II) are negligible.

Choice of eluentIn order to select the most appropriate eluent for the quantitative stripping of the retained Cu(II) on the col-umn, 5 mL of various non organic solvents were test-ed. The results can be seen, the best eluting solvents

Fig. 3: Effect of pH on the recovery of Cu (II) by IIDEMS

IIDE pH Extraction percent of Cu (II)

Absence 2-6 0.07(6.6)b

Presence 2-6 98.8(2.9) to 65(2.0)

Table 1: The effect of presence of IIDE on extraction percent of Cu (II)a

(a) Initial samples contained 10 µg of Cu (II) in 100 mL of water; (b)

Values in parentheses are RSDS based on five individual replicate

analyses

A. Moghimi

175


were found to be 5 mL of 0.1 mol.L-1 HCl, resulting in quantitative elution of Cu(II) from the column.

Effect amount of counter anionIn order to investigate the effect of counter ion on the recovery Cu(II) ions by the modified column, different counter anions were tested Table 2, it is immediately obvious that the nature of the counter anion strongly influences the retention of Cu(II) ions by the column. The results revealed that the Isopropyl 2-(isopropoxy-carbothioyl)disulfanyl]ethanethioate (IIDE) behaves as a neutral ionophore in the pH range 4.5-5.3 compo-nents (Moghimi, et al., 2012; Choi, et al., 2003; Kaiss, et al., 2007) so that the Cu(II) ions are retained as ion pair complexes by the column. As seen, acetate ion is the most efficient counter anion for the SPE of Cu(II) ions. The influence of the concentration of sodium acetate ion on Cu(II) recovery was investigated, and the results are shown in Table 2. As seen, the percent recovery of Cu(II) increased with the acetate concen-

tration until a reagent concentration of about 0.1 M is reached, beyond which the recovery remained quanti-tative. Moreover, acetate ion acts as a suitable buffer-ing agent, while it effectively contributes to the ions-pair formation; thus, in the SPE experiments, there was no need for the addition of any buffer solution.

The influence of flow-rateOne of the most important parameters affecting solid phase extraction is the speed of the process. Hence, the effect of flow-rates on extraction efficiencies was investigated. It was found that in the range of 1-15 mL.min-1, the retention of Cu(II) was not con-siderably affected by the sample solutions flow-rates and Cu to reproducible and satisfactory results (Fig. 4). Thus, the flow-rate was maintained at 8.0 mL.min-1 throughout the experiment.

Quantity of the Isopropyl 2-[(isopropoxy carbothio-yl)disulfanyl]ethanethioate (IIDE) The optimum amount of Isopropyl 2-(isopropoxy-carbothioyl)disulfanyl]ethanethioate (IIDE) for the quantitative extraction of Cu(II) was also investigated by adding various amounts of it to solution (between 2-20 mg). The results are listed in Table 3. The experi-mental results revealed that the extraction of Cu(II)was quantitative using a sample solution containing more than 10 mg Isopropyl 2-(isopropoxycarbothioyl)disulfanyl]ethanethioate (IIDE). Hence, subsequent extractions were performed with 15 mg of Isopropyl 2-(isopropoxycarbothioyl)disulfanyl] ethanethioate (IIDE).

Counter anion Recovery (%)

Cl - 28.8

`Br- 22.0

ClO4 - 30.6

SCN- 42.8

Picrate 74.7

Acetate 92.9

Table 2: Percent recovery of Cu(II) from the modified of Octadecane-functionalized nano graphene (OD-G) in the presence of 0.01 M of different counter anionsa

Fig. 4: Effect of flow-rate of sample solutions on the recovery

of Cu (II) by IIDEMS

IIDE amount

(mg)Recovery (%) of Cu (II)

2 30(2.7)b

5 45(2.6)8 80(2.5)10 95.8(2.4)

15 99.0(2.5)

20 98.1(2. 4)

Table 3: Influence of the IIDE amount on the recovery of Cu (II) ionsa

(a) Initial samples contained 10 µg of each Cu (II) in 100 mL water; (b)

Values in parentheses are RSDs based on five individual replicate

analysis

176

Analytical PerformanceWhen solutions of 10 µg Cu(II) in 10, 50, 100, 500, and 1000 mL solutions under optimal experimental conditions were passed through the column, the Cu(II) was quantitatively retained in all cases. Thus, the breakthrough volume for the method must be greater than 1000 mL, providing a concentration factor of > 200. The limit of detection (LOD) of the method for the determination of Cu(II) was studied under the op-timal experimental conditions. The LOD based on 3σ of the blank is 7.5 ng per mL. In order to investigate the selective separation and determination of Cu(II) ions from its binary mixtures with various metal ions, an aliquot of aqueous solutions (50 mL) containing 10 µg Cu(II) and mg amounts of other cations was taken and the recommended procedure was followed. The results are summarized in Table 4. The results show that the Cu(II) ions in binary mixtures are re-tained almost completely by the modified column, even in the presence of up to about 100 mg of various ions. Meanwhile, retention of other cations by the col-umn is very low and they can be separated effectively from the Cu(II) ion. It is interesting to note that, in

other experiments, we found that in the presence of high enough concentrations NH2OH.HCl as a suitable reducing agent (> 0.5M) (McAllister, 2007).

Analysis of water samplesTo assess the applicability of the method to real sam-ples, it was applied to the extraction and determina-tion of Cu(II) from different water samples. Tap water (Tehran, taken after 10 min operation of the tap), rain water (Tehran, 25 January, 2015), Snow water (Vara-min, 30 February, 2015) and Sea water (taken from Caspian Sea, near the Mahmoud-Abad shore) samples were analyzed. Development of a methodology for the determination of Cu(II) in FFPE tissue was performed in a number of steps to optimize the major factors af-fecting the precision of the analysis (Table 5). As can be seen from Table 5 the added Cu(II) ions can be quantitatively recovered from the water samples used.

Effect of the type, concentration and volume of the elution solutionsIn order to determine the most suitable solution for the elution of Cu, three different types of eluting agents

Diverse ion Amounts taken(mg) Found (%) Recovery of Cu2+ ion (%)

Na+ 92.0 1.19(2.6)b 98.9(1.9)K+ 92.9 1.30(2.0) 98.9(2.1)

Mg2+ 14.2 0.62(1.8) 99.2(2.0)

Ca2+ 20.3 2.20(2.0) 98.5(2.7)

Sr2+ 2.80 2.87(2.2) 98.4(2.0)

Ba2+ 2.90 3.15(2.3) 98.3(2.8)

Mn2+ 2.26 1.73(2.5) 97.4(2.8)

Co2+ 2.33 1.20(2.7) 98.8(2.9)

Ni2+ 1.90 2.13(2.4) 98.0(2.4)

Zn2+ 2.10 1.74(2.0) 98.6(2.2)

Cd2+ 2.35 1.95(2.3) 98.2(2.6)

Pb2+ 1.90 2.75(1.4) 97.7 (2.5)

Hg2+ 0.60 2.81(2.9) 97.7(2.4)

Ag+ 2.45 3.43(2.9) 96.6(2.5)

Cr3+ 1.70 2.93(2.1) 97.8(2.6)

UO2+ 2.60 2.82(2.2) 98.9(2.0)

Table 4: Separation of Cu (II) from binary mixtures a

(a) Initial samples contained 10µg Cu2+ and different amounts of various ions in 100 mL water (0.1 M acetate ion); (b) Values in

parentheses are RSDs based on five individual replicate analysis


177


(HCl, HNO3 and EDTA) were evaluated. HCl was found to be most effective eluent (Table 6). 0.1 mol.L-1 and 0.2 mol.L-1 of HCl solution give quantitative re-coveries (≥ 99%). Various volumes of 0.1 mol.L-1 HCl were also examined as eluent and the results were represented in Figure 5. Since quantitative recovery (≥99%) was obtained with 10 mL of 0.1 mol.L-1 HCl solution, it was selected as an eluent for the recov-ery of Cu by Isopropyl 2-[(isopropoxy carbothioyl)disulfanyl]ethanethioate (IIDE)-modified silica-gel (IIDEMS).

Adsorption capacityAdsorption capacity of IIDEMS for Cu(II) ions was determined by a batch method. The initial Cu(II) ion

concentration was changed from 100 to 1000 mg.L-1 for the investigation of adsorption capacity of modi-fied adsorbent. The pH of 50 mL of Cu(II) solution was adjusted to optimum value and then 300 mg of

Sample Cu2+ added

(µg)

Cu 2+ determined

(ng.mL-1)ICP-AES

Tap water0.0

10.0

1.74(2.0)a

11.95(2.6)

NDb

11.6

Snow water0.0

10.0

4.84(2.1)

14.96(2.4)

ND

14.6

Rain water0.0

10.0

2.65(2.3)

12.46(2.3)

ND

12.3

Sea Water0.0

10.0

12.65(2.4)

22.96(2.0)

12.4

23.1

Development of a methodology for the

determination of Cu2+ in FFPE tissue

0.0

10.0

N.Db

9.95(2.2)

N.Db

2.5))10.05

Table 5: Recovery of Cu(II) added to 1000 mL of different water samples (contaning 0.1Macetate at pH = 5.3)

(a) Values in parentheses are %RSDs based on five individual replicate analysis; (b) Not detected

Fig. 5: Effect of the volume of 0.1 mol L-1 HCl solution on the

recovery of Cu (II) by IIDEMS

Fig. 6: Langmuir isotherm plot for the sorption of Cu (II) by

IIDEMS

Fig. 7: Freundlich isotherm plot for the sorption of Cu (II) by

IIDEMS

178

modified sorbent was added to Cu(II) solutions and mixed on a digitally controlled magnetic stirrer at a rate of 200 rpm. Suspended solids were separated from the adsorption medium by centrifugation at 4500 rpm for 3 min and Cu(II) ion concentrations were then measured using FAAS. The equilibrium data were an-alyzed by Langmuir (Saitoh, et al., 2004) and Freun-dlich (McAllister 2007) isotherm models.

(1)

(2)

Where qe and qmax are the equilibrium and monolayer sorption capacities of the sorbent (mg.g-1), respective-ly, Ce is the equilibrium metal ion concentration in the solution (mg.L-1) and KL is the equilibrium constant (L.mg-1) related to the free energy of biosorption. KF (L.g-1) and n (dimensionless) are Freundlich sorption isotherm constants. The Langmuir and Freundlich iso-therm plots are shown in Figures 6 and 7, respectively and the model constants are presented in Table 7. The Langmuir isotherm model provided a better correla-tion than the Freundlich isotherm model. It could be concluded that the sorption of Cu(II) onto IIDEMS was monolayer and the maximum monolayer sorption capacity of the sorbent was found to be 82.34 mg.g-1. The adsorption capacity of IIDEMS is comparable to and moderately higher than that of many correspond-ing sorbents reported in the literature (Choi, et al., 2003). The dimensionless separation factor, RL, was also evaluated and calculated according to Eq. (3).

(3)

RL values can be used for the interpretation of the sorption type and it was reported that, when 0<RL<1, the sorption process is favorable (Choi, et al., 2003).

Further, the RL value for this study was 1.60×10-3, therefore, adsorption of Cu(II) IIDEMS was favor-able.

Analysis of Cu content in real sampleThe proposed SPE method possesses advantages such as easiness, and considerable selectivity in comparison with the previously reported procedures for isolation and determination of Cu(II) contents (Table 8). The maximum time taken for separation, preconcentration and monitoring of Cu(II) in 50 mL portions of water samples is at the most 10 min. The reproducibility of the procedure is near 2%. The proper preconcentration factor improves the LOD of the method by a factor of about 250. This procedure has the advantage of pre-concentration of Cu(II) depending on the pH of the sample solution (Table 8).

Analytical featuresPrecisionThe precision of the proposed method for the determi-nation of Cu was investigated at the optimum experi-mental conditions (sample volume: 50 mL; pH: 5.3; flow rate: 8 Ml.min-1; eluent: 10 mL 0.1 M HCl). 10

eLmaxmaxe C1

Kq1

q1

q1equationLangmuir

+=

eFe Cnln/1KnlqnlequationFreundlich +=

eLL CK1

1R+

=

Type of the ConcentrationRecovery a %

elution solution (mol.L –1)

HCl 0.05 170.1 990.2 97

HNO3 0.04 28

0.1 560.2 97

EDTA 0.05 980.1 910.2 87

Table 6: Effect of the type and concentration of the elution solutions

(a) Mean of three replicates

Langmuir Freundlich

qmax KL rL2 RL n KF rF

2

(mg.g-1) (L mg-1) (L.g-1)

74.45 0.68 0.9989 1.60×10-3 6.30 29.80 0.850

Table 7: Isotherm model parameters for the adsorption of Cu2+ on to IIDEMS

A. Moghimi

179


independent sorption and elution cycles were carried out by following the recommended procedure. The precision of the preconcentration method was evalu-ated by using the relative standard deviation (RSD) and was found to be 2.7%. The mean recovery of ten replicates was 98±3% at a confidence level of 95%. The precision of the proposed method was good and the recovery of Cu was quantitative.

Calibration graphA linear calibration curve was obtained in the con-centration range of 0.21.0 µg.mL-1. The calibration equation was A = 0.0025 + 0.0398C, where A is the absorbance and C is the Cu concentration in µg.mL-1. Correlation coefficient was 0.9989 and the average values of triplicate readings for each standard solution were used for the calculations.

Detection limitThe value of detection limit based on three times the standard deviation of blank signal (N=20) was 7.5 ng.mL-1. The detection limit of the proposed meth-od is comparable to those obtained by other methods described in the literature (Moghimi, et al., 2012; Choi, et al., 2003; Kaiss, et al., 2007). The corresponding limit of quantification was calculated from ten times the standard deviation of blank signal and found as 20.2 ng.mL-1. As is seen, the recovered Cu ion reveals that the results are quite reliable and are in satisfactory agreement with those obtained by ICPAES.

CONCLUSIONS

A novel and selective method for the fast determina-tion of trace amounts of Cu ions in water samples has been developed. The Cu ions was determined by us-

ing Isopropyl 2-[(isopropoxy carbothioyl)disulfanyl]ethanethioate (IIDE) modified Octadecane-function-alized nano graphene (OD-G) sorbent. The optimum recoveries for Cu were obtained with 300 mg sorbent, at pH 5.3 and 8 mL.min-1 of flow rate. Cu ions were quantitatively recovered (>95%) with 10 mL of 0.1 mol.L-1 HCl and the preconcentration factor was 250 at optimum conditions. The modified Octadecane-functionalized nano graphene (OD-G) has high sorp-tion capacity (82.34 mg .g-1) and the equilibrium data followed by the Langmuir isotherm model. The preci-sion of the proposed method evaluated as the relative standard deviation obtained from ten replicates, was 2.7%. In comparison to other solid phases, high flow rates and large preconcentration factor was achieved using Isopropyl 2-[(isopropoxy carbothioyl) disul-fanyl] ethane thioate (IIDE) modified Octadecane-functionalized nano graphene (OD-G) sorbent. While other advantages over reported methods are the high tolerances for matrix components (Moghimi, et al., 2012; Choi et al., 2003; Kaiss, et al., 2007) superior sorption capacity and good reusability.

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Ali Moghimi, Associate Professor, Department of Chemistry, Varamin Pishva-Branch Islamic Azad Uni-versity, Varamin, Iran, E-mail: [email protected]; [email protected]

A. Moghimi


INTRODUCTION


ABSTRACT: One-pot, four-component procedure for the synthesis of a small library of new chiral spirooxindolopyrrolidines with high regio-, diastereo- (>99:1 dr), and enantioselectivity (up to 80% ee) is described. In this process, the regio- and stereochemical 1,3-dipolar cycloaddition of azomethine ylides, which were generated insitu by the reaction of isatin derivatives and sarcosin,with optically active chiral menthyl cinnamate studied on the basis of the assignment of the absolute configuration of the cycloadducts, and on theoretical calculations. In comparison with active cinnamoyl oxazolidinone, when the reactions were performed with active chiral menthyl cinnamate as dipolarophile, a remarkable unexpected inversion in the regioselectively was observed. The regioselectivity of the reactions was investigated using global and local reactivity indices at the B3LYP/6-311G(d,p) level of theory. The effects of the electronic and steric factors on the regioselectivity of the reactions were discussed. The electronic structures of critical points were studied by the natural bond orbital (NBO) method.

Keywords: Asymmetric 1,3-dipolar, Chiral auxiliaries, chiral non-racemic menthol, Chiral spiro-oxindolopyrrolidines

Computational study in Regioselectivie Synthesis of New

Spiro-oxindolopyrrolidines

M.J. Taghizadeh1*; Kh. Jadidi2; M. Hamzehlooian3

1 1 Department of Chemistry, University of Imam Hossein, Tehran, Iran2 Department of Chemistry, University of Shahid Beheshti, Tehran, Iran

3 Department of Chemistry, Mazandaran University, Babolsar, Iran

Received: 5 July 2015; Accepted: 8 September 2015

Compounds that differ in the position of a substituent are known as regioisomers. Although the regioisomers look very alike, they might possess different properties.Since Padwa and co-workers performed the first dia-stereoselectivity of 1,3-dipolar cycloaddition reaction in 1985, by applying a chiral non-racemic azomethine ylide, (Daly, et al., 1986) their applications has been developed as a cornerstone in organic synthesis (Car-roll and Grieco 1993). One of today’s challenges in this field is to control the regio-, diastereo- and enantiose-lectivities of these reactions. For many years the chi-

ral auxilliary was the only way of asymmetric induc-tion in synthetic organic chemistry. The different types chiral auxiliary were applied in asymmetric synthesis of chiral structure. Two of the most important chiral auxiliary are menthol (in both enantiomer) and chiral oxazolidinone derivatives, which have frequently been used in various asymmetric reactions as reliable meth-od of creating new stereogenic centers Rosenmond, et al., 1994). In compare with oxazolidinones, chiral non-racemic menthol or p-menthan-3-ol have not been as extensively used as chiral auxiliaries in 1,3-dipolar cy-cloaddition reactions. Better results were achieved by

184

Grigg et al. using menthyl acrylate for the construc-tion of substituted pyrrolidines starting from azome-thine ylides (Grigg, 1995). Asymmetric 1,3-dipolar cycloaddition reactions of azomethine ylides offer an effective means to access chiral pyrrolidines substruc-tures containing up to four new stereogenic centres that found in many biologically active compounds (Shi, et al., 2009, Li, et al., 2011, Suresh Babu, et al., 2009).

Systematic investigation has shown that spirooxin-dolopyrrolidines systems, formed from joining spiro-oxindole and pyrrolidine rings at C-3 (spiro carbon), provide more opportunities for the development of a wide spectrum of biologically active compounds (Raj-kumar, et al., 2012). In 2002, Ganguly and co-workers synthesized spirooxindolopyrrolidines 1 with high regioselectivity via stereocontrolled 1,3-dipolar cy-cloaddition reactions of isatin derivative 2, sarcosine and chiral cinnamoyl oxazolidinone as dipolarophile 3 (Scheme 1).

According to the above facts and in continuation of our previous work on the synthesis of spirooxindoles, (Faraji, et al., 2010) herein, we report a facile synthesis of a small library of novel spirooxindolopyrrolidines 4 with the help of menthol as a chiral auxiliary in an asymmetric three-component 1,3-dipolar cycloaddi-tion reaction of azomethine ylides derived from isatin.

Interestingly, in contrast Ganguly’s report (Ganguly, et al., 2002) and in a same reaction condition when chiral menthyl cinnamate 5 (menthol derived dipola-rophile) was utilized in this reaction as dipolarophile, inversion in the regioselectivity was observed and cor-

responding spirooxindolopyrrolidines 4 was obtained as a sole product in high yield, high diastereo- (>99:1 dr) and enantioselectivity (up to 80% ee) (Scheme 1). This means that the reaction pathways for this reac-tion probably proceed through different intermediate. Thus, the molecular mechanism of this reaction has been investigated by means of a density functional theory (DFT) method.


ExperimentalGeneral melting point were recorded on an elec-trothermal digital melting point apparatus. Infrared spectra were recorded on a mattson 1000 FTIR. 1H, 13CNMR spectra were measured with a Bruker DRX-300 AVANCE instrument with CDCl3 as solvent at 300.1 MHz. Mass spectra were recorded on a Finni-gan MAT 8430 mass spectrometer operating at an electron energy of 70 eV. isatin derivatives, proline, were obtained from Fluka (Buchs, Switzerland) and were used without further purification, and trans-cin-namic acid derived from the menthol were obtained via synthesized.

General procedureTo a magnetically stirred solution of aisatin deriva-tives (2) (1 mmol), sarcosine (6) (1 mmol) and trans-cinnamic acid derived from the menthol (5) (1 mmol), as chiral auxiliaries in 10 mL EtOH was added drop-wise at reflux temperature. Then, the reaction mixture

NH

O

O +HN

O

OH

Ph N

O

O

O

NH

N

O

PhO

N

OO

Ph

O

O

NH

N

OPh

O

O

NH

N

O

PhO

OHNH

N

OPh

O

MeO

Ganguly s workOur work

Rem

o va l o f a ux ilia ry

Rem

oval

of

aux

iliar

y

12

34

5

Scheme 1: Synthesis of chiral spirooxindolopyrrolidines 1, 5.

M. J. Taghizadeh et al.

185


was stirred for 12 h. The solvent was then removed un-der reduced pressure and the residue was separated by column chromatography (silica gel, Merck 230–400 mesh) using n-hexane–ethyl acetate (90:10) as eluent. To a magnetically stirred solution of a 4 derivatives (1 mmol), sodium methoxide (3 mmol) and 10 (mol%) BF3. OEt2 in 10 mL THF at room temperature. Then, the reaction mixture was stirred for 1 h. The solution was quenched with HCl (1N). Then the solution was extracted with 20 mL of EtOAc and the organic phase was separated, washed with 20 mL of brine, dried over Na2SO4, and concentrated in vacuo. The solvent was then removed under reduced pressure.


Chiral menthyl cinnamate 5 is conveniently prepared from the corresponding cinnamoyl chloride (usual-ly obtained from the cinnamic acid after treatment with SOCl2) and chiral with commercially available (E)-(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl after deprotonation of this with a base such as BuLi (Ennis and Womack, 1955). The one-pot three component re-action was carried out by stirring a mixture of 1 equiv of isatin 2 and 1 equiv of sarcosine 6 for 10 min in 10 mL of aqueous ethanol followed by addition of 1 equiv of chiral menthyl cinnamate 5. The [3+2] cy-cloaddition of chiral dipolarophile 5 with azomethine

ylide 7 resulted in the formation of new chiral spiroox-indolopyrrolidines 4 which contains four contiguous stereogenic centers. Despite the fact that sixteen dif-ferent stereoisomers could be prepared theoretically, only diastereoisomer 4 was obtained (Scheme 1).

In order to investigation of solvent effect in stereose-lectivity of the products, the one-pot four component reaction was also carried out in toluene and aqueous dioxane, similar solvent with Ganguly’s report, for the dipolarophiles 3 and it was found that even under re-fluxing condition, there was no effect on regioselectiv-ity and no improvement in result reaction even reac-tion in toluene, led to a decrease in the isolated yield of the cycloadducts. This is attributed to the poor solu-bility of reactantin toluene specially the amino acid, sarcosine 6 which is responsible for the formation of azomethine ylide with isatin derivatives 2.

The structures of cycloadducts were characterized on the basis of spectroscopic data. Thus, the IR spec-trum of spirooxindolopyrrolidines 4a showed two absorption at 1613 cm-1 and 1714 cm-1 indicating the presence of two carbonyl group. In 1HNMR spectrum of the product 7d, the pyrrolidin ring proton attached to the phenyl ring appeared as a multiplet at δ= 4.02. The pyrrolidin –NCH2 proton appeared as a multiplet at δ= 3.66 whereas the pyrrolidin proton attached to the menthyl moiety is more deshielded and exhibited a multiplet at δ= 4.35 (H20). The aromatic protons ap-peared as a multiplet in the region δ= 6.96–7.63.

Entry R X Product

4a 8

Yield (%)b [α]D25c Yield (%)b [α]D

25c

1 H H A 70 -5.4 65 -4.2

2 H Br B 75 -6.2 72 -4.3

3 H NO2 C 72 -5.8 65 -4.7

4 Me H D 65 -5.4 60 -4.4

5 Me Br E 70 -5.2 65 -4.5

6 Me NO2 F 67 -5.7 60 -4.4

7 Et H G 65 -5.2 60 -4.2

8 Et Br H 65 -5.7 62 -4.7

9 Et NO2 I 72 -5.1 65 -4.3

Table 1: Synthesis of chiral spirooxindolopyrrolizidines 4, 8.

(a) The reaction was carried out in the ratio of 1/2/3/ 1:1: 1; (b) Isolated yield based on substituted isatins; (c) [α]

D25 (c 1, CH2Cl2).

186

The off resonance decoupled 13CNMR spectra of 4d exhibited characteristic peaks for the spiro carbon and carbonyl group attached to the menthyloxy moiety at 74.2 and 179.9 ppm respectively and the signals for all other carbons are located at appropriate chemical shifts in agreement with the proposed structure. The formation of the product was confirmed by mass spec-tral and elemental analyses. The mass spectrum of 4d showed a peak at m/z 553 (M+).

We also were able to obtain suitable crystals of the 4e for crystallography to confirm the assigned ste-reochemistry and regionselectivity of products 4, as suggested by NMR spectroscopy. The absolute con-figuration of chiral spirooxindolopyrrolidines 4e was

also determined by single crystal as 5R (spiro carbon C7), 6S (C21), 7R (C14), 8R (C13) (Fig. 1). Particu-larly noteworthy is the fact that all of the covalently attached azomethine ylide auxiliaries require destruc-tive removal. It was in this context that we initiated studies whose goal was to develop a recoverable chi-ral auxiliary. Interestingly, for compounds 6, the chiral auxiliary was removed easily with sodium methoxide and BF3.OEt2 in THF and provides access to a variety of enantiomerically pure products 8a–i in excellent to quantitative yields (Scheme 2). Note that the auxiliary was recovered from this reaction in high yield.

The stereochemistry and the structure of resulted cycloadduct 4a were carried out using 1HNMR and 13CNMR and also 2DNMR spectroscopy techniques. The 13CNMR spectrum displays 30 signals, which are classified into four methylene, sixteen methines, and ten quaternary carbons including to spiro carbon by the DEPT 135° experiments. In the HMQC spectrum of product 4a, the positions of three protons (Ha, Hb, and Hc) that were directly bonded to these carbon at-oms (CH) were assigned. The 1HNMR spectrum dis-plays two signals at δ= 4.02 and δ= 4.35 ppm (Hc, Hb respectively); the Hb could be trans to Hc, because of absence of any correlation between them in the RO-ESY spectrum. This is also confirmed from the weak NOE pattern between them. To determine the exact regioselectivity, the connectivity of the carbons in the molecular structure was obtained by using the analysis of the HMBC spectra. Based on the HMBC spectrum, there is no correlation between Hc and the signal of carbonyl group, which confirms presence of the car-bonyl group at C-2′ position in 4a (instead of C-1′)

Fig. 1: The ORTEP diagram of one of the two crystallo-

graphic independent molecules in the asymmetric unit of 4g

is shown. Thermal ellipsoids are at 30% probability level.

XO

O

NR

O

O

H

H

CO2HNH

NaOMe

NR

NX

O

H

H

OO

Ph

NR

NO

O Ph

NR

NPh

HH

OO

O

BF3.OEt2

NR

XO

N++

ee>99%

EtOH reflux , 12h

- CO2 [3+2]

5

ee>99%8

2

6 7

7

17

3335

4

Scheme 2: Synthesis of chiral spirooxindolopyrrolizidines 4, 8.

Computational study in regioselectivie synthesis

187


(Scheme 2).It is known that chiral cinnamoyl oxazolidinone, in

the absence of Lewis acids, prefer a low energy Z-conformer of S-Cis. So, on the basis of the absolute configuration of the four stereogenic centers in the pyrrolizidine ring, it is assumed that the azomethine ylide approaches endo to the si-face of the dipolaro-phile as a Z-conformer of S-Cis (Scheme 3).

COMPUTATIONAL STUDY

All calculations were performed using Gaussian09 (Frisch, 2009), suite of programs. The full geometri-cal optimization of all structures and transition states (TSs) were carried out with Density Functional The-ory (DFT) using non local B3LYP hybrid functional and 6-311G(d,p) basis set in the gas-phase. No sym-metrical restriction was applied during geometrical optimizations. The nature of stationary geometries has been characterized by calculating the frequencies in order to verify that the transition states have only one imaginary frequency with the corresponding ei-genvector involving the formation of the newly cre-ated C–C bonds. The electronic structures of critical points were studied by the natural bond orbital (NBO) method (Reed, et al., 1988). Furthermore, Zero-point vibrational energies and thermodynamic corrections at 298.15 K were calculated at the same level as the geometry optimization.The atomic Cartesian coordi-nates of optimized structures for all transition states are included in supporting information.

Prediction of regiochemistryFMO, Global and Local Electrophilicity/Nucleophilicity AnalysisThe regio- and stereoselectivity of the reaction of azomethine ylide 7 with chiral non-racemic menthyl cinnamate 1 was investigated by theoretical meth-

ods (Scheme 2). The FMO approach provides the most reliable general explanation of regiochemis-try of the cycloaddition reactions (Fukui, 1981). The HOMO-LUMO energy gap for the reactants gener-ally determines the most significantinteraction. Then the regiochemical preferences of these reactions can be predicted in terms of the maximum overlap of the largest coefficients of the HOMO and LUMO orbitals at the reaction sites (Wang, et al., 2005). According to Houk’s rule, the regioselectivity of 1,3-dipolar cyclo-addition reactions can be explained on the basis that the large-large and small-small FMO interactions are more favoured than the large-small and small-large FMO interactions (Wang, et al., 2005). The calculated frontier orbital energies and the coefficients of 5 and 7 (at DFT/B3LYP/6-311G(d,p) level of theory) are given in Tables 2 and 3, respectively. Obviously, the electron density transfer takes place from the HOMO orbital of azomthine ylide 7 to LUMO orbital of men-thyl cinnamate 5, as a result of the small energy gap (normal electron demand character). The FMO analy-sis was suggested that C7 of the azomethine ylide 7 reacts preferentially with the β-enone carbon atom C35 of the alkene, which is in good agreement with the experimental observation (Scheme 2).

The chemical potential, hardness, softness, global and local electrophilicity and nucleophilicity have been computed for the reagents (Tables 2 and 3). The electronic chemical potential μ is usually asso-ciated with the charge transfer ability of the system in its ground state geometry and it can be defined as the mean value of HOMO and LUMO energies [µ= (εH+εL)/2] (Politzer, et al., 1983, Sanderson, 1983). The chemical hardness η which is describes the re-sistance to this charge transference is the difference between LUMO and HOMO energies. The chemical softness parameter S is strictly related to the chemical hardness and it is due to the inverse of 2η. The global electrophilicity index ω, which measures the stabiliza-

Reactant HOMO (eV) LUMO (eV) µ (a.u) η (a.u) ω (eV) Ѕ(a.u.)

5 -6.584 -1.952 -0.1568 0.1702 1.965 2.938

7 -4.924 -1.637 -0.1206 0.1193 1.660 4.190

Table 2: Global properties of dipole 7 and dipolarophile 5.

188

tion in energy when the system acquires an additional electronic charge ΔN from the environment, was giv-en the following simple expression, ω= µ2/2η, in terms of the electronic chemical potential and the chemical hardness (Parr, 1999).

The electronic chemical potentials, µ, of the azo-methine ylide 7, -0.1206 a.u., is higher than those for menthyl cinnamate 5, -0.1568 a.u., indicatesa net charge transfer from the azomethine ylide to menthyl cinnamate. The electrophilicity of menthyl cinnamate 5 in Table 2 is greater than 1.5 eV (ω= 1.965), thus according to the classification of electrophilicity, this compound can be classified as a strong electrophile (Domingo and Andres 2003). Azomethine ylide 7 also has a large electrophilicity value, ω= 1.660. Since,

the electrophilicity of menthyl cinnamate 5 (dipola-rophile) is greater than that of the azomethine ylide (dipole), the electrondensity transfer takes place from dipole 7 to dipolarophile 5.

After considering the global properties, a local anal-ysis was carried out through Fukui indices calculation. The atomic condensed Fukui functions,based on Mul-liken population analysis and depending on the type of electron transfer, are defined as f-(r)=qk(N)-qk(N-1) and f+(r)=qk(N+1)-qk(N) (Li and Evans 1995). Then the electrophilicity local indexis calculated from Fu-kui index f+ as w=wfk

+. The analysis of the local elec-trophilicity index, ωk at the electrophilic reagent and the nucleophilic Fukui function, fk

- at the nucleophilic compound allows one to explain the observed regi-

Reactant SiteMO coefficient

(HOMO)

MO coefficient

(LUMO)fk

+ fk- s+ s-

ωK

(eV)

dipolarophile 5 C33 0.128 -0.108 0.0750 0.2153 0.2202 0.6324 0.15

dipolarophile 5 C35 0.076 0.122 0.0709 0.0118 0.2084 0.0345 0.14

dipole 7 C7 0.144 0.057 0.0062 0.1083 0.0261 0.4537 0.01

dipole 7 C17 -0.134 0.184 0.1729 0.1373 0.7246 0.5753 0.29

Table 3: Local properties of dipolarophile 5 and dipole 7.

NR

O

N+O

O

H

H

NR

N

O

H

H O

PhO

NR

N

O

H

H

OO

Ph

NR

N

O

H

H

OO

Ph

NR

NPh

HH

OO

(R)

NR

NHPh

OOH

(R)

NR

NH

H PhO

O(R)

NR

NH

H PhO

O(R)

NR

N

O

H

H O

PhO

5 7

9exo-re

+

4endo-re 9exo-si

4endo-si

10exo-re

10exo-si 11endo-re

11endo-si

3335 7

17

Scheme 3: Possible transition structures for the concerted reaction of azomethine ylide


189


( ) ( )2lj2

kiklji ssss +−+− −+−=∆

Sfs kk±± =

η= 2/1S

oselectivity (Domingo and Andres 2003). Thementhyl cinnamate 5 has the largest electrophilic activation at the C33 carbon atom, ωk= 0.15 eV, whereas the azome-thine ylide 7 has the largest nucleophilic activation at the C17 carbon atom, fk

- = 0.1373 (Table 3). Therefore, C33 of menthyl cinnamate 5 will be the preferred posi-tion for a nucleophilic attack by C17 of the dipole 7, which is in good agreement with the experimental ob-servation (Scheme 2). Regioselectivity can be studied in terms of the softness matching index, defined by the following relationship (Chandra and Nguyen 2002).

(1)

where i and j are the atoms of a molecule A involved in the formation of a cycloadduct with atoms k and l of a molecule B, and sis are the appropriate type of atomic softness. si and sj are electrophilic where sk and sl are nucleophilic and they calculated as:

(2)

where S is the global softness and computed as (Cher-mette, 1999):

(3)

According to local HSAB concept, the reaction asso-ciated with a lower Δ value (equation 1) will be the preferred one (Pearson, 1995). In the case of cycload-dition reaction of 7 to 5, Δij

kl for the generation of 4 (0.1861) is smaller than that of the generation of other regioisomer (0.1891). (Table 3, Scheme 2) and hence agrees well with the experimental results.

Energies of transition state structuresPossible transition structures for the concerted reac-tion of azomethine ylide 7 with dipolarophile 5 and their corresponding cycloadducts for both re and si faces of the chiral non-racemic menthyl cinnamate 5 have been optimized and characterized (Scheme 3). The activation energies, enthalpies and Gibbs free en-ergies as well as the reaction energies, enthalpies and Gibbs free energies are reported in Table 4. For each transition state, the most stable conformation has been chosen. The naming symbols are coined according to a particular highlighted combination endo/exo and re/si faces attack at the C33 and C35 of menthyl cinnamate 5.

TS 4endo-re TS 4endo-si

TS 11endo-re TS 11endo-si

Fig. 2: Selected optimized transition structures at the B3LYP/6-311G(d,p) corresponding to the regiosomeric

path of the 1,3-dipolar cycloaddition reaction 5 and 7.

190

The relative activation energies of the transition states in Fig. 3 show that azomethine ylide 7 should undergo 1,3-dipolar cycloadditions with menthyl cin-namate 5 very easily, as predicted by the low activa-tion energy of 8.1 kcal/mol and 8.2 kcal/mol, obtained at the Becke3LYP/6-311G(d,p) level for the 4 endo-re and 4 endo-si channels. The cycloaddition reaction be-tween 5 and 7 is favored in the endo reaction channels. The optimized geometries of endo transition states are shown in Fig. 2.

A theoretical preference for regioisomers 4, Where the carbon bearing the phenyl group attached to the Spiro Center, is observed and the predominance of endo adducts are correctly predicted with a difference of more than 4.0 kcal/mol. The theoretical preference for re and si faces is less pronounced,especially for 4 endo adifference of only 0.1 kcal/mol is observed. Preference based on this energy difference is very weak and not reliable. Rate constants were calculat-ed according with the Eyring transition state theory:

k=TKB/h×e-ΔG /RT where kB is Boltzmann’s constant; h is Planck’s constant; R is the ideal gas constant; T is the temperature (25°C), and, ΔG# is the activation Gibbs free energy of the transition state structures. The unequal new C–C bonds in transition states are consistent with anasynchronous concerted cycloaddi-tion mechanism.

CONCLOUSIONS

Because of wide distribution in nature and variegated biological activities, chiral pyrrolizidines alkaloids are very attractive synthetic targets. Since a pyrrolizidine can be viewed as a fused pyrrolidine, the method em-ployed for the formation of pyrrolidine rings can be used to construct the pyrrolizidine ring system. So, the asymmetric 1,3-dipolar cycloaddition reaction of azo-methine ylides, including pyrrolidine derivatives with olefins, can be the useful method for the synthesis of chiral pyrrolizidines. On the other hand, oxindoles are also structural key moieties in many bioactive sub-stances and it is interesting that systematic investiga-tion has shown that if this moiety is joined to the pyr-rolizidine or pyrrolidine ring through a spiro atom at C-3, the resulting compounds show an increased spec-trum of biological activity. As a result, we have found a tri- component synthetic method for the prepration of some oxindoles derivatives of potential synthetic interest. The present method carries the advantage that

Fig. 3: Relative energy of possible transition structures.

Structure Ea ΔH# ΔG# ΔErxn ΔH ΔG k(25)

4 endo-re 8.1 7.6 21.6 -25.3 -25.8 -11.4 8.85×10-4

4 endo-si 8.2 8.0 22.4 -25.6 -26.5 -11.3 2.29×10-4

9 exo-re 15.3 15.1 28.4 -21.1 -21.9 -6.0 9.09×10-9

9 exo-si 12.2 12.1 26.1 -22.1 -22.6 -8.0 4.42×10-7

11 endo-re 10.9 10.5 26.1 -22.8 -23.3 -8.0 4.42×10-7

11 endo-si 9.9 9.5 25.2 -25.1 -26.0 -9.9 2.02×10-6

10 exo-re 12.4 12.1 25.5 -24.7 -25.3 -9.8 1.22×10-6

10 exo-si 15.6 15.3 30.2 -21.7 -22.4 -6.6 4.34×10-10

Table 4: Calculated electronic activation energies Ea, reaction Gibbs free energies ΔG, reaction enthalpies ΔH, reaction energies ΔErxn, activation Gibbs free energies ΔG#, activation enthalpies ΔH# and Rate constants at the B3LYP/6-311G(d,p), all energies are in kcal/mol.

Computational study in regioselectivie synthesis

#

191


not only the reaction is performed under neutral con-ditions, but also the starting materials and the reagents can be mixed without any activation or modification. The regioselectivity of the reactions was investi-gated using global and local reactivity indices at the B3LYP/6-311G(d,p) level of theory. The effects of the electronic and steric factors on the regioselectivity of the reactions were discussed. The electronic structures of critical points were studied by the NBO method.

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Mohammad Javad Taghizadeh, Ph.D., Department of Chemistry, University of Imam Hossein, Tehran, Iran, E-mail: [email protected]

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