Electronic Supplementary Material
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A nanocomposite consisting of graphene oxide and Fe3O4 magnetic nanoparticles for the
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extraction of flavonoids from tea, wine and urine samples
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Jianrong Wu1, 4, Deli Xiao1, 4, Hongyan Zhao1, 3, Hua He1, 2 *, Jun Peng1, Cuixia Wang1, Chan
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Zhang1, Jia He1
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1 Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009,
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China
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2 Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education,
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China Pharmaceutical University, Nanjing 210009, China
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3 Department of Hygienic Analysis and Detection, School of Public Health, Nanjing Medical
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University, Nanjing, Jiangsu 211166, China.
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4 These authors equally contributed to this work and should be considered co-first authors
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Correspondence to: Hua He, E-mail: dochehua@163.com , jcb315@163.com
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Synthesis of Graphene oxide
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Graphene oxide was synthesized according to the modified Hummers method.[30] Briefly,
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Graphite powder (300 meshes, 1.0 g) and sodium nitrate (1.5 g) were mixed in sulfuric acid
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(75 mL, 98 wt %) under stirring and cooled by using an ice bath. Then KMnO4 (6.0g, 6wt
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equiv) was added gradually under stirring and the temperature of the mixture was kept to be
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below 10 oC by cooling. The reaction mixture was maintained at approximately 4 oC in an ice
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bath for 4h. Next, 100 mL of distilled water was added (gas evolved) and the temperature was
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increased to 90 oC in an oil bath. As the reaction progressed, the mixture gradually became
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pasty. Afterward, the reaction was cooled to room temperature and poured onto ice (150mL)
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with 30 % H2O2 (10mL) until gas evolution ceased. The colour of the mixture turned to
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yellow. Finally, the mixture was filtered and washed with 10 % HCl aqueous solution to
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remove metal ions followed by water until the pH was nearly neutral. The solid was obtained
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by centrifugation and washed thoroughly with deionized water, dried in a vacuum at room
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temperature.
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HPLC analysis
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The three flavonoids (luteolin, quercetin and kaempferol) were separated and quantified by
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using a liquid chromatography–spectrophotometry system with an automatic sample injector
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(Agilent, USA).The analytical column was a ZORBAX Eclipse XDB-C18column
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(4.6mm100mm, 5-Micro) supplied by Agilent. The mobile phase consisted of
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methanol-0.2 % aqueous phosphoric acid solution (48:52, V/V) and the flow-rate was set at 1
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mL/min. Spectrophotometry detection of analytes was performed at 360 nm wavelengths. The
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injection volume was 10µL.
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BET
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Fig. S1. BET surface area measurement shows surface area of 325.9 m2/g for GO and 115.7
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m2/g for GO/Fe3O4.
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Effect of the amounts of sorbent
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The adsorbent amount had a significant effect on extraction efficiency. In order to choose the
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optimum amount of the adsorbent (GO/Fe3O4) for the extraction of the target analytes in
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different matrices. The different amounts of the magnetic graphene (2, 4, 6, 8 and 10mg) were
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used for the extraction the analytes. As shown in Fig. S2, the recoveries of three flavonoids
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increased rapidly as the amount of adsorbent increased in both three matrices, the maximum
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extraction efficiency was obtained at 8 mg of GO-Fe3O4 in green tea (a) and red wine (b) and
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in urine (c). According to the result, 8 mg of GO-Fe3O4 was employed in the following
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studies in three matrices.
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Fig. S2. Effect of the amount of adsorbent on the adsorption of three flavonoids in three
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matrices. Operation in the batch mode. (a) green tea;(b)red wine;(c)urine.
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Effect of extraction time
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The effect of extraction time from 0 to 90 min on the recoveries of the target analytes was
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investigated while other parameters being held at
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the extraction efficiency increased with the increased extraction time from 0 to 20 min in both
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three matrices, and then remained almost constant after 20 min. Hence, an extraction time of
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20 min was chosen for the subsequent experiments.
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constant values. As is shown in Fig. S3,
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Fig. S3. Effect of the extraction time on the adsorption of three flavonoids in three matrices.
Operation in the batch mode. (a) green tea;(b)red wine;(c)urine.
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Effect of solution pH
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The pH of the sample solution plays an important role for the adsorption
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the sorbents. The pH not only changes the formation of the analytes, but also alters the
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interaction between the sorbents and the analytes. The solution pH will also change the charge
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property of the surface of GO/Fe3O4, which is a primary factor that affects the adsorption
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property towards the analytes. In this study, the addition of different PH of phosphate
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buffered saline into the sample solutions by adjusting the pH in the range of 2-9 was studied.
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As seen from Fig. S4, the highest extraction efficiencies for the three flavonoids were
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obtained at pH 5.8 in both green tea and red wine, 7.0 in urine. The enhanced extraction
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efficiency can be explained by the electrostatic force between the sorbent and analytes. The
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pKa1 values of the three flavonoids are reported to be 7.04, 7.36, 8.09 respectively [31]. When
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pH value increased above 7.0, the most flavonoids molecules were transformed to neutral
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state and a few were in protonated form, which resulted in gradually weakened electrostatic
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force and slightly lower extraction efficiency. To obtain highest extraction efficiency, the
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urine sample was adjusted to pH 7.0 and the green tea and red wine was 5.8 for the following
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experiments.
of the analytes to
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Fig. S4. Effect of the PH on the adsorption of three flavonoids in three matrices. Operation in
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the batch mode. (a) green tea;(b)red wine;(c)urine.
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Desorption conditions
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The type and volume of desorption solvent are vital for the desorption efficiency. So the
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choice of desorption solvent and its optimum volume should be carefully taken into account.
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In this work, different kinds of organic solvents (methanol, methanol containing 1 % or 2 %
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HAc, acetonitrile, acetonitrile containing 1 % or 2 % HAc, actone, actone containing 1 % or
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2 % HAc) were investigated. As a result, the eluting power of acetone containing 2 % HAc
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was much stronger than methanol and other desorption solvent. Thus acetone containing 2 %
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HAc was selected as the desorption solvent. The effect of desorption solution volume was
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also investigated. Results revealed that the best recoveries were obtained by using 1 mL
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actone containing 2 % HAc (0.5 mL every time and washed two times).The desorption time
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was optimized by increasing the vortex time from 0.5 to 10 min. The result indicated that 2.0
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min was enough to elute the flavonoids from magnetic graphene oxides.
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