Spinach assisted green reduction of graphene oxide and its antioxidant and dye absorption properties

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CERAMICS INTERNATIONAL

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Spinach assisted green reduction of graphene oxide and its antioxidant and dye absorption properties D. Suresha,n, P.C. Nethravathia, Udayabhanua, H. Nagabhushanab, S.C. Sharmac,d a Department of Chemistry, Tumkur University, Tumkur 572103, India Prof. CNR Rao Centre for Advanced Materials, Tumkur University, Tumkur 572103, India c Academic Mentor and Honorary Professor of Eminence, Dept. of Mechanical Engineering, Siddaganga Institute of Technology, B. H. Road, Tumkur 572103, Karnataka, India d Vice Chancellor, Chattisgarh Swami Vivekananda Technical University, Bhilai 493441, Chattisgarh, India b

Received 21 October 2014; received in revised form 6 December 2014; accepted 7 December 2014

Abstract Spinach (Spinacia oleracea) leaves mediated efficient green reduction of graphene oxide (GO) was achieved to produce multifunctional reduced graphene oxide (rGO). GO was mixed with spinach juice and refluxed for 30 min at 100 1C. Powder XRD, TEM and UV–visible techniques demonstrate the formation of well-organized layered rGO. Industrially important carcinogenic Methylene blue (MB) and Malachite green (MG) dyes absorption experiments with rGO were performed under dark condition. Complete removal of MB and MG occurs in spans of 60 and 40 min respectively in the presence of 20 mg rGO. Potential antioxidant activity was exhibited by the rGO with 50% inhibitory concentration of 1590 mg/mL against the scavenging of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. Environment friendly, economical and facile reduction method is suggested for the efficient reduction of GO. & 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Keywords: B. X-ray methods; D. Carbon; E. Functional applications; Graphene oxide; TEM

1. Introduction The synthesis of graphene has been one of the most exciting benchmark scientific advancements in recent years due to its outstanding properties and applications [1–3]. A variety of attempts have been made for the synthesis of graphene such as mechanical routes [1], chemical vapor deposition [4], epitaxial growth [5] and chemical reduction of graphene oxide (GO). Currently, solution-based chemical reduction of GO presents very easier route for the production of good quality graphene [6]. The resultant graphene could be utilized in a variety of applications [7,8]. However, the solution-based chemical reduction methods employ hazardous chemicals such as Sodium borohydride, n

Corresponding author. Mobile: þ 91 9886465964. E-mail address: [email protected] (D. Suresh).

hydrazine and dimethyl hydrazine which are highly toxic and trace amounts of these in final product could pose harmful effects. Superior quality reduced graphene oxide (rGO) was obtained using Hydriodic acid [9], but it is highly corrosive in nature. Handling of hazardous waste generated out of these processes could counter the environment protection and add up to the production cost. Further, the graphene produced by these methods lead to aggregation of graphene sheets and an additional step needs to be incorporated to avoid these aggregations [10]. A solvothermal method of reduction has been one of the recent approaches for the synthesis of graphene. But it involves elevated temperatures during reduction that could produce hazardous gases and wastes [11]. Recently, green approaches have employed natural products in place of toxic reducing agents. Vitamin C was shown to be efficient for reduction of GO; however, the product had highly agglomerated morphology and the reduction process

http://dx.doi.org/10.1016/j.ceramint.2014.12.036 0272-8842/& 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Please cite this article as: D. Suresh, et al., Spinach assisted green reduction of graphene oxide and its antioxidant and dye absorption properties, Ceramics International (2014), http://dx.doi.org/10.1016/j.ceramint.2014.12.036

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involve high temperatures that results in increased defects in graphene [12]. Bovine serum albumin [13] was utilized in reducing GO, but an alkali is needed as a coreductant. Therefore an efficient, economical and eco-friendly reducing agent is highly desirable for reduction to obtain soluble graphene in bulk quantity. Spinach (Spinacia oleracea), an edible plant belongs to Amaranthaceae family is native to central and southwestern Asia and consumed in most of the countries. It is a very rich source of essential nutrients such as vitamin A, vitamin C, vitamin K, magnesium, manganese, folate and iron. Spinach leaves consist of glucuronic acid derivatives of flavonoids, tartarate derivatives of p-coumaric acid and found to be antioxidant [14]. The proximate composition studies reveal that crude protein and ash contents were 2.89% and 1.96% and total lipid content was 0–61% [15]. In view of the presence of considerable amounts of antioxidant phytochemicals in spinach, it may act as good reducing agent for the reduction of GO. Therefore, an attempt has been made for the first time to reduce GO using spinach leaves.

2.3. Characterization The obtained GO and rGO were subjected for characterization using powder X-ray diffractometer (Shimadzu – 7000 with monochromatized CuKα radiation), UV–visible Spectrophotometer (Thermo Evolution—220) and TEM (TECNAIF-30). 2.4. Dye elimination activity 20 mg of rGO was mixed with 100 mL of 5 ppm MB and MG separately and stirred under dark condition. Known volume of the reaction mixture was drawn at specific intervals of time and absorbance was recorded (663 and 617 nm for MB and MG respectively). The percentage of absorption of the dye was determined using the formula Ci  Cf % of absorption ¼  100 Ci where Ci and Cf are initial and final dye concentrations respectively.

2. Experimental

2.5. Antioxidant activity

AR grade conc. Hydrochloric acid, conc. Sulfuric acid, Hydrogen peroxide, Potassium permanganate and Sodium nitrate were procured from S. D. Fine Chemicals Company, Mumbai, India and used without further purification. Graphite flakes were from SigmaAldrich Company (Catalog no.—332461, 490% purity). Fresh leaves of spinach were purchased from local market, thoroughly washed in distilled water, chopped in to small pieces and grinded well with water to obtain homogeneous juice.

DPPH (oxidized form) is a stable free radical with purple color. In the presence of an antioxidant which can donate an electron to DPPH radical decays, and the change in absorbance at 520 nm is followed which can be measured spectrophotometrically [18]. 39.4 mg of DPPH was dissolved in 100 mL of methanol to get concentration of DPPH in the assay which was 0.14 mM. 2360 ul of 50% methanol solutions containing

2.1. Preparation of GO

graphite

001

Intensity (a.u)

Graphite (5 g) and Sodium nitrate (2.5 g) were thoroughly mixed and treated with 120 mL of H2SO4 (95%) [16]. The mixture was vigorously stirred (Remi 1 MLH magnetic stirrer) for 30 min under ice cold condition. Potassium permanganate (15 g) was added with continued vigorous stirring and the temperature was maintained below 20 1C. After overnight stirring, 150 mL of distilled water was slowly added with continued vigorous stirring. The reaction temperature was rapidly increased to 98 1C and 50 mL of 30% Hydrogen peroxide was added. The product was allowed to cool and washed with 5% Hydrochloric acid, then with distilled water and dried [17].

002

GO

2.2. Reduction of GO GO (80 mg) was added to 50 mL of distilled water and subjected to sonication (40 min) (Elma Ultrasonic bath, 3 l capacity) for uniform dispersion. The suspension was uniformly mixed with spinach juice in a round bottomed flask and refluxed for 30 min. The resulting product settled at the bottom was separated and rinsed with distilled water and centrifuged (Remi swing out centrifuge) until colorless, clear supernatant was obtained. The rGO was dried and stored in an airtight container until further use.

002

rGO 10

20

30

40

50

60

70

2θ (degrees) Fig. 1. X-ray diffraction patterns of graphite powder, GO and rGO sheets.

Please cite this article as: D. Suresh, et al., Spinach assisted green reduction of graphene oxide and its antioxidant and dye absorption properties, Ceramics International (2014), http://dx.doi.org/10.1016/j.ceramint.2014.12.036

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0.8

617 nm

Absorbance (a.u)

0.6

5 ppm dye

10 min

0.4

30 min 0.2

40 min 60 min

0.0 400

500

600

700

Wavelength (nm) 1.2

663 nm 1.0

Fig. 2. TEM image of rGO.

Absorbance (a.u)

5 ppm dye

Absorbance (a.u)

225 nm

0.8

0.6

10 min 0.4

20 min

1.5

30 min

0.2

40 min 0.0

GO

500

600

rGO

0.0 200

300

700

Wavelength (nm)

282 nm

400

500

600

700

Fig. 4. Degradation of (a) Malachite green and (b) Methylene blue under dark condition with reduced graphene oxide.

800

Wavelength (nm)

different concentrations of rGO (2, 4, 6 and 8 mg) were mixed individually with 140 ul of 1 mM DPPH solution and incubated at 37 degree Celsius for 30 min. The absorbance was measured at 520 nm against 50% methanol blank using a spectrophotometer; a control sample was maintained without addition of the test sample. The actual absorbance was taken as the absorbance difference of the control and the test sample and IC50 value was determined. 3. Results and discussion The XRD patterns of graphite, GO and layered rGO were examined. The diffraction peak (2θ) of graphite powder was observed at  261, which corresponds to interlayer distance

% Inhibition

Fig. 3. UV–visible spectra of GO and rGO.

50

IC50 = 1590 μg/mL

0 0

1000

2000

3000

4000

5000

Concentration (μg) Fig. 5. DPPH free radical scavenging activity of rGO.

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(d-spacing) of 0.33 nm. The diffraction peak of GO was observed at  101 due to (002) plane, which corresponds to interlayer distance of 0.76 nm (Fig. 1). This increase in interlayer distance is due to the incorporation of many oxygenated functional groups between the carbon layers. The synthesized GO was subjected for the reduction using spinach juice. The reduction has resulted in the shift of diffraction from  101 to  261. This peak is very close to the peak in the diffraction pattern of graphite. However, it appears to be broadened. This clearly reveals the formation of highly organized few graphene layers with an interlayer spacing of 0.33 nm. This diffraction peak is characteristic of graphene and is consistent with the thickness of a single layer; the interlayer spacing in single layered graphene sheet has a structure similar to that of normal graphite [7]. TEM analysis undoubtedly indicates the formation of transparent few layered graphene due to the reduction employing spinach leaves (Fig. 2). The layers were wrinkled and exfoliated during the reduction process indicating the complete reduction. UV–visible absorption spectra of GO and the rGO are shown in Fig. 3. The absorption peak of GO at 225 nm is due to the π-πn transitions of aromatic CQC bonds [19]. Upon reduction, the absorption peak red-shifted to 282 nm indicating the revival of the conjugated CQC bonds [20]. The dyes such as MB and MG are extensively used in various industries for a variety of applications. However, these dyes were found to be carcinogenic. rGO prepared by green approach was employed for the removal of these dyes. A load of 20 mg rGO was used for the elimination of MB and MG having 5 ppm concentration under dark condition. The rate of absorption was followed using the spectrophotometer at 663 and 617 nm respectively for MB and MG. Fig. 4 illustrates the removal of these dyes. It was observed that MB was removed completely in a span of 40 min and MG in 60 min. The efficient dye riddance activity may be due to the presence large surface area. DPPH, a stable free radical with a characteristic absorption at 520 nm was used to study the radical scavenging effects. The decrease in absorption at 526 nm was taken as a measure of the extent of radical scavenging. The radical scavenging activity values were expressed as the ratio percentage of sample absorbance decrease and the absorbance of DPPḢ solution in the absence of test sample at 520 nm (Fig. 5). The rGO proved to be potent at inhibiting the DPPH free radical scavenging activity with IC50 value of 1590 mg/mL. 4. Conclusion The investigation illustrates a facile green reduction method of graphene oxide from a very common leafy vegetable for the first time. Crucial advantages of the method are abundance of the reducing agent, eco-friendly, cost effective and the ease of product separation from the mixture. The carcinogenic dye elimination and antioxidant properties of the prepared rGO were found to be significant. The method is a very economical and greener approach which produces reduced graphene oxide

having a variety of applications. Therefore, the method may be utilized for large scale production of reduced graphene oxide.

References [1] K.S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I.V. Grigorieva, A.A. Firsov, Electric field effect in atomically thin carbon films, Science 306 (2004) 666–669. [2] C. Lee, X. Wei, J.W. Kysar, J. Hone, Measurement of the elastic properties and intrinsic strength of monolayer graphene, Science 321 (2008) 385–388. [3] S.P. Pang, H.N. Tsao, X. Feng, K. Mullen, Patterned graphene electrodes from solution processed graphite oxide films for organic field-effect transistors, Adv. Mater. 21 (2009) 3488–3491. [4] K.S. Kim, Y. Zhao, H. Jang, S.Y. Lee, J.M. Kim, K.S. Kim, J.H. Ahn, P. Kim, J.Y. Choi, B.H. Hong, Large-scale pattern growth of graphene films for stretchable transparent electrodes, Nature 457 (2009) 706–710. [5] P.W. Sutter, J.I. Flege, E.A. Sutter, Epitaxial graphene on ruthenium, Nat. Mater. 7 (2008) 406–411. [6] S. Park, R.S. Ruoff, Chemical methods for the production of graphenes, Nat. Nanotechnol. 4 (2009) 217–224. [7] S. Stankovich, D.A. Dikin, G.H.B. Dommett, K.M. Kohlhaas, E.J. Zimney, E.A. Stach, R.D. Piner, S.T. Nguyen, R.S. Ruoff, Graphene-based composite materials, Nature 442 (2006) 282–286. [8] D.A. Dikin, S. Stankovich, E.J. Zimney, R.D. Piner, G.H.B. Dommett, G. Evmenenko, S.T. Nguyen, R.S. Ruoff, Preparation and characterization of graphene oxide paper, Nature 448 (2007) 457–460. [9] I.K. Moon, J. Lee, R.S. Ruoff, H. Lee, Reduced graphene oxide by chemical graphitization, Nat. Commun. 1 (2010) 1–6. [10] S. Dubin, S. Gilje, K. Wang, V.C. Tung, K. Cha, A.S. Hall, J. Farrar, R. Varshneya, Y. Yang, R.B. Kaner, A one-step, solvothermal reduction method for producing reduced graphene oxide dispersions in organic solvents, ACS Nano 4 (2010) 3845–3852. [11] J. Gao, F. Liu, Y. Liu, N. Ma, Z. Wang, X. Zhang, Environment-friendly method to produce graphene that employs vitamin C and amino acid, Chem. Mater. 22 (2010) 2213–2218. [12] C. Zhu, S. Guo, Y. Fang, S. Dong, Reducing sugar: new functional molecules for the green synthesis of graphene nanosheets, ACS Nano 4 (2010) 2429–2437. [13] J. Liu, S. Fu, B. Yuan, Y. Li, Z.J. Deng, Plasmon-polaritons on graphenemetal surface and their use in biosensors, Am. Chem. Soc. 132 (2010) 7279–7281. [14] M. Bergman, L. Varshavsky, H.E. Gottlieb, S. Grossman, The antioxidant activity of aqueous spinach extract: chemical identification of active fractions, Phytochemistry 58 (2001) 143–152. [15] M. Antonia Murcia, Ana Vera, Francisco Garcia-Carmona, Effect of processing methods on spinach: proximate composition in fatty acids and soluble protein, J. Sci. Food Agric. 59 (1992) 473–476. [16] W.S. Hummers, R.E. Offeman, Preparation of graphitic oxide, J. Am. Chem. Soc. 80 (1958) 1339. [17] Zhigang Xiong, Li Li Zhang, Jizhen Ma, X.S. Zhao, Photocatalytic degradation of dyes over graphene–gold nanocomposites under visible light irradiation, Chem. Commun. 46 (2010) 6099–6101. [18] W. Brand-Williams, M.E. Cuvelier, C. Berset, Use of a free radical method to evaluate antioxidant activity, Food Sci. Technol. 28 (1995) 25–30. [19] V.H. Pham, H.D. Pham, T.T. Dang, S.H. Hur, E.J. Kim, B.S. Kong, S. Kim, J.S. Chung, Chemical reduction of an aqueous suspension of graphene oxide by nascent hydrogen, J. Mater. Chem. 22 (2012) 10530–10536. [20] S. Bose, T. Kuila, A.K. Mishra, N.H. Kim, J.H. Lee, Dual role of glycine as a chemical functionalizer and a reducing agent in the preparation of graphene: an environmentally friendly method, J. Mater. Chem. 22 (2012) 9696–9703.

Please cite this article as: D. Suresh, et al., Spinach assisted green reduction of graphene oxide and its antioxidant and dye absorption properties, Ceramics International (2014), http://dx.doi.org/10.1016/j.ceramint.2014.12.036