Clicking graphene oxide and Fe3O4 nanoparticles together: an efficient adsorbent to remove dyes from aqueous solutions

Nanohybrid of graphene oxide (GO) and azide-modified Fe3O4 nanoparticles (NPs) were fabricated using click reaction. First, Fe3O4 NPs were modified by 3-azidopropionic acid. Then, click-coupling of azide-modified Fe3O4 NPs with alkyne-functionalized GO was carried out in the presence of CuSO4·5H2O and sodium l-ascorbate at room temperature. The attachment of Fe3O4 NPs onto the graphene nanosheets was confirmed by Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy, thermogravimetric analysis, energy dispersive X-ray spectrometry and X-ray diffraction spectrometry. As the FTIR spectroscopy and energy dispersive X-ray spectrometry analysis showed, the final magnetic graphene nanosheets were also reduced by sodium ascorbate which is a merit for click-coupling reactions. The specific saturation magnetization of the Fe3O4-clicked GO was 44.3 emu g−1. The synthesized hybrid was used in the adsorption of methylene blue and congo red (CR). The adsorption capacities in the studied concentration range were 109.5 and 98.8 mg g−1 for methylene blue and CR, respectively.


Introduction
Removing toxic metals and dye from the waste water of the manufacturing industries has been of great concern for decades. For this reason, numerous adsorbent with various compositions have been developed (Gupta et al. 2010a(Gupta et al. , b, c, d, 2012aGupta and Nayak 2012;Gupta et al. 2011Gupta and Saleh 2013;Sanghavi and Srivastava 2013;Ahmaruzzaman and Gupta 2011;Jain et al. 2003;Mittal et al. 2009Mittal et al. , 2010. Among them, there has been an increasing attention toward separation process applying magnetic nano-sized particle for adsorption process. Magnetite (Fe 3 O 4 ) nanoparticles (NPs) have attracted a great deal of attention due to a number of unique properties which makes them promising agents as potential materials for carriers of drugs or gene delivery, biomolecules separation, hypothermal treatment of tumors, contrast agents for magnetic resonance imaging, information storage, catalysis and environmental protections such as removing dye and heavy metals (Indira and Lakshmi 2010;Sanghavi et al. 2013a, b;Sanghavi and Srivastava 2010). Graphene, the parent of all graphitic forms including fullerene, carbon nanotubes and graphite, is a two-dimensional building material for carbon materials of all other dimensionalities. Among them, hybrid of graphene and carbon nanotubes with Fe 3 O 4 NPs have been extensively studied in the fabrication of functional polymer composites, sensors, waste water treatment, catalysis and drug delivery Lim et al. 2013;Yao et al. 2012). It is believed that hybrid of magnetic NPs and graphene/graphene oxide (GO) would have better functionalities and performances in these applications (Georgakilas et al. 2012;Kumar et al. 2012;Zhu et al. 2013). Recently, Yao et al. have reported the synthesis of magnetic Fe 3 O 4 @graphene composite which had great potential as an effective adsorbent for removing cationic and anionic dyes from water solutions (Yao et al. 2012), while Geng et al. have applied a functional hybrid of reduced GO (rGO)-Fe 3 O 4 in the adsorption of Rhodamine B (Geng et al. 2012). The mentioned composites showed high efficiency in both cases. However, there are some disadvantages in the process of production and application of these composites: (1) formation of GO-Fe 3 O 4 is usually achieved by in situ reduction in iron salt precursors or assembly of the magnetic NPs on GO surface and consequently, Fe 3 O 4 NPs are attached to a GO layer only by physical adsorption or electrostatic interaction. Thus, they may be easily leached out from the GO sheets during application (Liu et al. 2008), (2) to prepare Fe 3 O 4 NPs by co-precipitation, high temperature is required. For instance, Cong et al. (2010) have prepared magnetic-functionalized rGO sheets through high-temperature decomposition of Fe(acac) 3 precursor in polyol, (3) precise controlling the loading amount of Fe 3 O 4 , and then, tailoring the properties of resultant hybrids for desired application is a main challenge as well (Yang et al. 2009) and (4) another major challenge is because of easy oxidation/dissolution of the pure Fe 3 O 4 NPs when using these nanomaterials, specially in acidic solutions. To overcome these problems and specifically to protect the magnetic NPs against oxidation, a shell structure is often introduced, including silica (Zhu et al. 2011a, b) polymer (Shin and Jang 2007) and noble metals (Cho et al. 2005;Lu et al. 2005). And recently, covalent attachment of Fe 3 O 4 NPs onto GO has been developed which provides better stability, accessibility, selectivity and less leaching. For example, Fe 3 O 4 NPs modified by tetraethyl orthosilicate and (3-aminopropyl) triethoxysilane was reacted with carboxylic groups of GO with the aid of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to form a GO-Fe 3 O 4 hybrid and was used to remove neutral red from contaminated water ). An easy chemical method to produce superparamagnetic GO-Fe 3 O 4 hybrid composite and its application in the removal of dyes has also been reported by Xie et al. (2012). In a very recent work, dopamine-functionalized Fe 3 O 4 NPs were reacted with GO. The uniqueness of this new and transformative approach was that the NPs provided cellular targeting by an external magnetic field . A covalent bonding technique to obtain magnetic GO composite (Fe 3 O 4 /SiO 2 -GO) decorated with core/shell NPs has also been reported (Li et al. 2013). Herein, for the first time, we report the covalent attachment of Fe 3 O 4 NPs onto GO by powerful and efficient ''click reaction''. The Cu (I)-catalyzed Huisgen 1, 3-dipolar cycloaddition of azides and terminal alkynes (CuAAC) (Kolb et al. 2001) is significantly an outstanding reaction since it is modular, wide in scope, tolerant to other functional groups, insensitive to solvent, moisture and oxygen. It gives very high yields and only inoffensive by-products that can be easily removed by nonchromatographic methods, and be stereospecific, preferably giving only one product. These are the reasons it has extensively been exploited as a novel methodology to advance drug-discovery, functionalize monolayers, synthesize and functionalize different molecules, polymer, dendrimers, NPs, virus, and to post-translationally engineer proteins and modify cell surfaces (Lahann 2009). In the present work, we developed an efficient method to produce superparamagnetic GO-Fe 3 O 4 hybrid using click reaction. Refluxing GO in thionyl chloride (SOCl 2 ) resulted in chlorinated GO (GO-COCl) which was subsequently treated with propargyl alcohol to produce alkyne-GO. Then, 3-azidopropionic acid-functionalized Fe 3 O 4 NPs were easily and efficiently clicked onto GO. Covalent attachment of Fe 3 O 4 NPs onto GO should be an effective way to solve the problems mentioned above. The adsorption properties of the obtained hybrid (Fe 3 O 4 -clicked GO) toward methylene blue (MB) as a cationic dye and congo red (CR) as an anionic dye in aqueous solution were investigated as well.
Date and location of the research February and March of 2013, Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, Iran.

Materials and methods
Graphite (average particle size 30 lm) is commercially available, and it was used without further purification. SOCl 2 was distilled from boiled linseed oil prior to use. Triethylamine (Et 3 N) and dichloromethane (CH 2 Cl 2 ) were dried over calcium hydride (CaH 2 ) and then distilled. Tetrahydrofuran (THF) was dried over sodium. 3-bromopropionic acid was recrystallized from carbon tetrachloride. Propargyl alcohol, sodium azide, ferrous chloride tetrahydrate (FeCl 2 Á4H 2 O), ferric chloride hexahydrate (FeCl 3 Á6H 2 O), N,N-dimethylformamide (DMF), ethanol, copper sulfate pentahydrate (CuSO 4 Á5H 2 O) sodium L-ascorbate, MB and CR. All the reagents and solvents employed for the synthesis were commercially available and used as received without further purification unless mentioned. All the reagents, except graphite, were purchased from Merck. Deionized water (DI water) that was used in all experiments.

Instrumentation
Infrared spectra were obtained on a Fourier-transform infrared (FTIR) spectrometer (Bruker Instruments, model Aquinox 55, Germany) in the 4,000-400 cm -1 range at a resolution of 0.5 cm -1 as KBr pellets. The pattern of X-ray diffraction (XRD) of the samples was obtained by Siemens diffractometer with Cu-ka radiation at 35 kV in the scan range of 2 h from 2 to 70°and scan rate of 1°/min. Room temperature magnetic properties of the composite were characterized using vibrating sample magnetometer (Iran). Energy dispersive X-ray (EDX) spectrum and scanning electron micrographs (SEM) were obtained using a fieldemission scanning electron microscopy (FESEM, MIRA3 FEG-SEM) operating at 10 kV. Thermo gravimetric analysis (TGA) was performed with a TGA-PL thermal analyzer under air atmosphere from room temperature up to 700°C at a heating rate of 10°C/min. UV-Vis absorption spectra were recorded on a Shimadzu 1700 Model UV-Vis spectrophotometer.

Preparation of alkyne-GO
The as-prepared GO by modified Hummers' method (Zhang et al. 2010b), (100 mg) was well dispersed in 1 mL of dry DMF by sonification for 1 h and then was treated with SOCl 2 (20 mL) at 80°C for 3 days. After removing the excess of SOCl 2 under reduced pressure, propargyl alcohol (2 mL), distilled CH 2 Cl 2 (2 mL) and Et 3 N (1 mL) were added dropwise to GO at 0°C. The mixture was stirred at 0°C for 1 h, at room temperature for 6 h and reflux for 24 h. The powder was washed with an excess amount of ethanol and DI water and dried in vacuo overnight.
Preparation of azide-modified Fe 3 O 4 NPs 3-azidopropionic acid was prepared according to the literature (Grandjean et al. 2005) and 1 mL of 3-azidopropionic acid, 10 mg of Fe 3 O 4 NPs (Yao et al. 2012) and 5 mL of CHCl 3 were added to a centrifuge tube. The resultant mixture was sonicated for 10 min until the NPs were dispersed completely. THF was then added to remove excess of 3-azidopropionic acid. The mixture was washed with ethanol, and the precipitate was collected and dried in vacuo overnight.

Synthesis of Fe 3 O 4 -clicked GO hybrid by CuAAC reaction
About 10 mg of azide-modified Fe 3 O 4 NPs was introduced into 5 mL of mixed solvent of DMF and H 2 O (DMF: H 2 O 4: 1). Following sonication, 20 mg of alkyne-GO, about 1 mg of CuSO 4 .5H 2 O and sodium L-ascorbate were added and the mixture was stirred at room temperature overnight. The product was recovered by a magnet and washed with excess of water and ethanol and dried in vacuo overnight.

Adsorption experiments
Effects of contact time and the related isotherms were studied individually. Typically, adsorption experiments were carried out in glass bottles at 25°C. 25 mL of dye solution of a known initial concentration was shaken with 0.025 g of Fe 3 O 4 -clicked GO on a shaker at 200 rpm at 25°C. To evaluate the time effect, at the completion of preset time intervals, a 5 mL dispersion was drawn and separated immediately by the aid of a magnet to collect the adsorbent. The equilibrium concentrations of dyes were measured with a UV-Vis spectrophotometer at the appropriate wavelength corresponding to the maximum absorbance, 664 and 499 nm for MB and CR, respectively. The amount of dye adsorbed was calculated using the following equation: where, q e is the concentration of dye adsorbed (mg g -1 ), C o and C e are the initial and equilibrium concentrations of dye in mg L -1 , respectively, V is the volume of dye solution (L) and m (g) is the weight of the adsorbent used. For adsorption equilibrium experiments, fixed adsorbent dose (25 mg) was weighed into 50 mL conical flasks containing 25 mL of different initial concentrations (20-120 mg L -1 ) of the related dye. The mixture was shaken for 5 h at 25°C until equilibrium was obtained. Then, the adsorbent was separated from solution by an external magnet. The concentration of the dye in the solution was measured using a UV-Vis spectrophotometer.

Results and discussion
In this study, we report the covalent attachment of superparamagnetic Fe 3 O 4 NPs onto GO through click reaction between 3-azidopropionic acid coated-Fe 3 O 4 NPs and propargyl-functionalized GO as described in Fig. 1. The application of the powerful and efficient click reaction in covalent bonding of NPs onto GO is reported for the first time in this paper. Nano-sized particles tend to aggregate to minimize their surface energy due to their large ratio of surface area per unit volume. Therefore, to achieve stability, NPs are usually functionalized with specific groups, for instance surfactants, polysaccharides, zeolite, activated carbon and cyclodextrin. The coating method can also hamper the aggregation of the particles at a distance where the attraction energy between the particles is larger than the disordering energy of thermal motion (Nigam et al. 2011). Carboxylates such as citric acid (Namazi et al. 2011;Namazi and Adeli 2003) can suitably stabilize Fe 3 O 4 NPs (Cheraghipour et al. 2012). Thus, we decided to use 3-azidopropionic acid to cap the NPs which was achieved by ultrasonication. Then, azide-modified NPs were clicked onto the edges of GO to result the final product (Fe 3 O 4clicked GO).
The superparamagnetic behavior of Fe 3 O 4 -clicked GO was verified using an external magnet as shown in Fig. 2.
When a magnet is placed close to the vial containing Fe 3 O 4 -clicked GO, the nanohybrid readily aggregates to the wall of the vial which proves that the azide-modified Fe 3 O 4 NPs have successfully attached onto GO and it is consequently magnetically responsive.
Characterization of the synthesized materials by FTIR was performed to confirm the accomplishment of each step. The FTIR spectra of GO, alkyne-GO, 3-azidopropionic acid, azide-modified Fe 3 O 4 NPs and Fe 3 O 4 -clicked GO are shown in Fig. 3. The presence of several characteristic peaks of GO such as C=O (1,735 cm -1 ), aromatic C=C (1,626 cm -1 ) and alkoxy C-O (1,074 cm -1 ) stretching confirms the successful oxidation of graphite and is in good agreement with previous reports ). In the FTIR spectrum of alkyne-GO, the new signals at 1,745 cm -1 (C=O of ester), 2,150 cm -1 of (CC stretch of alkyne), 2,874 cm -1 (aliphatic hydrogens) and 3,027 cm -1 ðC-H stretch of alkyne) prove the attachment of alkyne groups onto the edges of GO. In addition, there is no sign of the stretching signals of CO-H which indicates that the carboxylic acid groups have changed into ester functionality (Ryu et al. 2013). A sharp absorption band at 2,105 cm -1 in the FTIR spectrum of azide-modified Fe 3 O 4 NPs, is attributed to azide stretching signal that shows the formation of 3-azidopropionic acid. Fe 3 O 4 NPs were attached to 3-azidopropionic acid with ultrasonication. The appearance of a signal at 570 cm -1 which is attributed to Fe 3 O 4 NPs and more importantly, the absence of peaks at 1,719 and 3,374 cm -1 , assigned to stretching of the C=O and CO-H of carboxyl groups, respectively, in the FTIR spectrum of azide-modified Fe 3 O 4 NPs is strong confirmations to the attachment of Fe 3 O 4 NPs to 3-azidopropionic acid. The disappearance of the characteristic peaks related to alkyne and azide moieties in the FTIR spectrum of Fe 3 O 4 -clicked GO indicates that the starting materials no more exist. Besides, the upcoming new band at 1,646 cm -1 , which is attributed to the triazole group and the presence of the peaks of azide-modified Fe 3 O 4 NPs, prove that the click reaction was carried out and the Fe 3 O 4 NPs were successfully attached covalently onto GO. Besides, ascorbic acid has been reported to be an effective reducing agent for GO than can compete with hydrazine (Zhang et al. 2010a). As it is obvious from the FTIR spectrum of the nanohybrid, the stretching band of O-H and epoxy are not present which indicates that during click reaction, GO has been reduced as well. This is an added advantage for click-coupling reactions.
The XRD patterns of GO, alkyne-GO, azide-modified Fe 3 O 4 and Fe 3 O 4 -clicked GO are presented in Fig. 4. Oxidation generates oxygen-containing groups on the originally atomically flat graphene sheets which makes individual GO sheets thicker than individual pristine graphene sheets (Shen et al. 2010) thus, GO shows a sharp (0 0 1) peak at 10.44°. Alkyne-functionalized GO shows a XRD pattern similar to GNS, and the related peak is seen at 2h = 24.11°. Besides, the (0 0 1) peak of GO is barely there. This indicates that layered GO has been exfoliated (Shen et al. 2010). For azide-functionalized Fe 3 O 4 , diffraction peaks with 2h at 30.21°, 35.58°, 43.35°, 53.30°, 57.10°and 62.57°were observed and are attributed to the (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) planes of the Fe 3 O 4 . The pattern was in accord with pure spinel Fe 3 O 4 (ICDD file no. 65-3107), indicative of a cubic spinel structure of the magnetite. Compared with azide-modified Fe 3 O 4 , the same set of characteristic peaks was also observed for Fe 3 O 4 -clicked GO indicating the stability of the crystalline phase of Fe 3 O 4 NPs in the nanohybrid (Li et al. 2013) and confirming the attachment of NPs onto and confirming the attachment of NPs onto GO. The diffraction peak of alkyne-GO has disappeared which suggests that alkyne-GO layers have been exfoliated and less agglomerated graphene sheet are present in the nanohybrid (Yao et al. 2012).
The magnetization curve of Fe 3 O 4 -clicked GO was measured at room temperature and is presented in Fig. 5. The magnetic hysteresis loop is S-like curve. The saturation magnetization (Ms) is 44.3 emu g -1 . The prepared hybrid exhibited zero coercivity and permanence indicating its superparamagnetism.
To investigate the morphology and structure of the products, FESEM images were taken for the azide-modified Fe 3 O 4 NPs and Fe 3 O 4 -clicked GO. Figure 6a and b shows the FESEM images of azide-modified Fe 3 O 4 NPs in which the granular nature of the NPs is clearly observed with gray/white color on their surface indicating the azidopropionic acid coating. Homogeneous distribution of the Fe 3 O 4 NPs is obvious. Figure 5c and d confirms the attachment of Fe 3 O 4 NPs onto GO. The layerby-layer assembly of magnetic GO sheets is clearly observed.
Furthermore, the composition of this heterostructure is confirmed by the EDX spectroscopy experiment (Fig. 7) which reveals the presence of intense peaks for Fe along with carbon and oxygen peaks. This clearly shows the grafting of Fe 3 O 4 onto GO. In addition, low content of oxygen given by this analysis also proves the reduction in GO sheets by sodium ascorbate.
TG analysis was also conducted on GO, azide-modified Fe 3 O 4 , and Fe 3 O 4 -clicked GO (Fig. 8) to distinguish and access thermal stability as well as to confirm the attachment of azide-modified Fe 3 O 4 NPs onto GO. GO exhibits two steps of mass loss, first one before 120°C which is attributed to the evaporation of adsorbed water and the main step up to 400°C is related to the decomposition of oxygen-containing functional groups such as carboxyl, hydroxyl and epoxy . In the TGA diagram of azide-modified Fe 3 O 4 NPs, weight loss of 1.2 %  GO (c, d) can be assigned to the removal of water which is seen below 140°C and the weight loss of 5.0 % between 140 and 418°C can be due to the loss of 3-azidopropionic acid groups. The pattern and the percentage of weight loss of azide-modified Fe 3 O 4 NPs totally differ from the pristine Fe 3 O 4 NPs , and this confirms that the azidopropionic acid molecules have well covered the surface of Fe 3 O 4 NPs which prevents the exposure of thermally labile hydroxyl functional groups of Fe 3 O 4 NPs (Yang et al. 2014). TG analysis of Fe 3 O 4 -clicked GO presents that the nanohybrid undergoes three stages of thermal degradation. The first stage with the weight loss of 7.8 % is observed between 150 and 370°C. The main step which is seen between 370 and 550°C with the mass loss of 31 % can be attributed to the decomposition of triazole group. And the final stage happening after 550°C is 4.9 % and is related to the decomposition of carbon skeleton.
Besides, the mass loss over the whole temperature of 500-700°C is low demonstrating the high efficiency of the covalent functionalization (Shen et al. 2010).

Adsorption studies
The adsorption of dyes MB and CR from aqueous solutions using the synthesized hybrid was studied. The effect of contact time on the amount of dye adsorbed was investigated at an initial concentration of 20 mg L -1 of both dyes (Fig. 9a). It only took 20 min for both dyes to reach equilibrium. The short equilibrium time needed indicates that the hybrid has high adsorption efficiency to remove cationic and anionic dyes from contaminated water. The obtained results are better than the ones reported previously (Yao et al. 2012;Xie et al. 2012). The adsorption isotherms of MB and CR for the hybrid are shown in Fig. 9b. To study the distribution of the adsorption molecules between the liquid phase and solid phase at the equilibrium state, the adsorption isotherm is used. The maximum adsorption capacity was attained 109.5 and 98.8 mg g -1 for MB and CR, respectively, at an initial concentration of 20 mg L -1 . The obtained result is comparable with the previous reports (Yao et al. 2012;Xie et al. 2012).
Tables 1 and 2 show the results obtained in this study with those in the previously reported works on adsorption capacities of various adsorbent in aqueous solution for MB.

Conclusion
The covalent attachment of NPs onto the GO surface using the powerful click reaction is reported for the first time. Not only this nanohybrid was prepared in an ambient condition, but also the magnetic GNS were reduced by sodium ascorbate during click-coupling reaction which was confirmed by FTIR and EDX. The covalent attachment of Fe 3 O 4 NPs onto GO, being able to predefine the loading amount of Fe 3 O 4 NPs and less leaching are of other outstanding advantages of this method. The attachment of Fe 3 O 4 NPs onto the GNS was confirmed by FTIR, FESEM, XRD, TGA and EDX. The specific saturation magnetization of the Fe 3 O 4 -clicked GO was 44.3 emu g -1 . The synthesized hybrid was used in the adsorption of MB and CR. The adsorption capacities in the concentration range studied were 109.5 and 98.8 mg g -1 for MB and CR, respectively. High adsorption capacity, compared with other adsorbents, accompanied by the ease of separation by an external magnetic field makes the prepared nanohybrid a powerful separation tool to be utilized in wastewater treatment.