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1 Supporting Information Heteroaggregation of Graphene Oxide with Nanometer- and Micrometer-Sized Hematite Colloids: Influence on Nanohybrid Aggregation and Microparticle Sedimentation Yiping Feng, 1, 2, 3, *, Xitong Liu, 1, Khanh An Huynh, 4 J. Michael McCaffery, 5 Liang Mao, 2 Shixiang Gao, 2 and Kai Loon Chen 1 1 Department of Environmental Health and Engineering, Johns Hopkins University, Baltimore, Maryland , United States 2 State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu , China 3 Institute of Environmental Health and Pollution Control, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou, Guangdong , China 4 Faculty of Environment and Natural Resources, Ho Chi Minh City University of Technology, Ho Chi Minh City, Vietnam 5 The Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, Maryland , United States Y. F. and X. L were equal contributors to this work. S1

2 Section S1: Additional Details on Materials and Methods Preparation of Graphene Oxide Stock Suspension. The used GO was produced by oxidizing crystal graphite with a mixture of sulfuric acid, sodium nitrate, and potassium permanganate (the Hummers method 1 ). The composition of GO is 79% carbon and 20% oxygen, the average flake size is micrometers with at least 80% of the sample being single layered. The GO stock suspension was prepared by dispersing 8 mg of GO powder in 200 ml deionized (DI) water (Millipore, MA) through ultrasonication (Branson B1510, 40 khz). The mixture was then passed through a 0.2 µm cellulose acetate filter and the filtrate was named GO stock suspension. The total organic carbon content of the GO stock suspension was determined through combustion catalytic oxidation at 680 C (TOC-L, Shimadzu, Japan). GO stock suspensions were stored in the dark at 4 C. Preparation of Nanometer- and Micrometer-Sized Hematite Colloids. Hematite colloids were synthesized through forced hydrolysis of FeCl 3. 2, 3 Briefly, FeCl 3 6H 2 O was dissolved in hot (100 C) aqueous HCl (0.002 mol L -1 ) to produce a 0.02 mol L -1 Fe 3 solution and prepared for the synthesis of HemNP, while 100 ml of NaOH (5.4 mol L -1 ) was slowly introduced into 110 ml of 2.0 mol L -1 FeCl 3 solution for the synthesis of HemMP. Then the solutions were respectively transferred into the high-density polyethylene bottles, and immediately placed in an oven (100 C) for 7 days to synthesis HemNP, and for 8 days to synthesis HemMP. After aging, the samples were cooled to room temperature. The HemNPs were collected through ultra-centrifugation (>5000g), while HemMPs were directly collected the particles settled on the bottom of the bottle. The obtained particles were thoroughly washed by DI water to completely remove Fe 3 and other impurities. The cleaned HemNPs solutions were then aged in an oven at 100 C for more than 24 h. Determination of Homoaggregation Kinetics. Homoaggregation kinetics of GO and S2

3 HemNPs were quantified by attachment efficiencies, α. This value is calculated from the initial aggregation rates as follows: 4, 5 α = 1 N0 (dd h (t) ) dt 1 (N0 ) fast (dd h (t) ) dt t 0,fast (1) where α is the attachment efficiency, N 0 is the initial particle concentration, D h is the hydrodynamic diameter of nanoparticles, and t is the time of aggregation. The linear regression of initial homoaggregation rate was performed over a time period in that D h,t reached 1.3 times or 2.0 times of the initial hydrodynamic diameter (D h,0 ) of HemNPs or GO. When the homoaggregation rate is very slow, such as D h,t failed to reach 1.3 D h,0, then the linear regression will be achieved over a time of 60 minutes. For all regressions, the y-intercepts of the fitted lines did not exceed 4 nm or 12 nm in excess of D h,0 for HemNPs or GO, respectively. The fast homoaggregation rate was obtained at the high salt concentrations. Transmission Electron Microscopy. 300 mesh carbon-coated TEM copper grids were used for TEM observation. For each ratio (0.004, 0.060, and 0.400), 3 grids were prepared in parallel, and at least 2 of them were observed. During TEM imaging, the pictures of several random areas were taken. The number of TEM images taken was 30, 108, and 76 for GO/HemNP mass concentration ratio of 0.004, 0.060, and 0.400, respectively. Adsorption of GO on HemMPs. Batch adsorption experiments were performed in 10- ml centrifuge tubes (Fisher Scientific). The suspensions had ph 5.2 and 0.1 mm NaCl. Initially, each tube contained 5 ml GO suspensions at different concentrations (0 6.0 mg TOC/L). HemMPs were then added to have a final concentration of 150 mg/l. All the tubes were shaken at 150 rpm, ca. 25 C for 0.25 h or 48 h. Afterward, the tubes were centrifuged (Beckman Coulter Avanti TM J-20 XPI) for 30 min at 3,000 rpm to separate hematite from GO suspension. After centrifugation, 4 ml of supernatant was carefully transferred to a clean borosilicate vial. S3

4 As shown in Figure S1a, GO used in this study showed a maximum absorbance at 200 nm. The concentrations of GO in the supernatants were determined at this wavelength using an UV Vis spectrophotometer (UV-1800, Shimadzu, Japan) based on the calibration curve shown in Figure S1b. The adsorption capacity (q, mg g 1 ) of the GO adsorbed on HemMPs after incubation was calculated using the following equation: 6 q = (C 0 C e )V m (2) where C 0 and C e are the initial and equilibrium concentrations of GO in the suspensions (mg L 1 ), V is the volume of the suspension (L), and m is the weight of the adsorbent (HemMPs) (g). EPMs of the residual HemMPs and GO suspensions at the bottom of the centrifuge tube (after centrifugation) were then measured to further investigate the interaction between GO and HemMPs. S4

5 Abs ( =200 nm) Absorbance (a.u.) Section S2: Additional Figures 1.6 (a) Wavelength (nm) 1.6 (b) R 2 = GO Concentration (mg/l) Figure S1. (a) UV-Vis absorption spectra of graphene oxide and (b) standard curve of graphene oxide at an absorbance of 200 nm. All the solutions were prepared in 0.1 mm NaCl at ph 5.2. S5

6 Hydrodynamic Diameter (nm) Hydrodynamic Diameter (nm) (a) 0 mm 6 mm 10 mm 20 mm 60 mm 100 mm 500 mm Time (min) 1200 (b) 60 mm mm 160 mm 400 mm 500 mm 600 mm Time (min) Figure S2. Representative homoaggregation profiles of (a) HemNPs at a concentration of 0.44 mg/l and (b) GO at a concentration of 0.66 mg TOC/L at different NaCl concentrations. S6

7 Hydrodynamic Diameter (nm) 200 GO/HemNPs= mg(toc)/l GO 0.44 mg/l HemNPs Time (s) Figure S3. Homoaggregation profiles of GO and HemNPs, together with a heteroaggregation profile of GO and HemNPs at GO/HemNP mass concentration ratio of All the experiments were conducted at 0.1 mm NaCl and ph 5.2. S7

8 Figure S4. Proposed heteroaggregation mechanisms at different GO/HemNP mass concentration ratios. S8

9 Figure S5. Representative TEM images of GO HemNP nanohybrids. S9

10 Figure S6. TEM image of micrometer-sized hematite particles (HemMPs). S10

11 Absorbance GO/HemMP Ratio Time (min) Figure S7. Change in the absorbance at 760 nm during the sedimentation of HemMPs in the absence and presence of GO at 0.1 mm NaCl and ph 5.2. Note that the initial drop of absorbance of HemMP suspensions in the presence of GO was attributed to the change in the optical properties of the suspensions stemming from the formation of heteroaggregates. S11

12 Figure S8. Representative TEM images of the heteroaggregation of GO with 2-µm sized hematite colloids. Scale bar is 1 µm in the figure. S12

13 (a) (b) Repulsion (c) Strong - attraction GO Concentration Increasing Weak attraction GO HemMP Figure S9. Proposed GO-HemMP heteroaggregation mechanisms at different GO concentrations. S13

14 Absorbance Hydrodynamic Diameter (nm) (a) GO/HemNP Ratio (b) Time (min) GO/HemNP Ratio Time (min) Figure S10. (a) Representative heteroaggregation profiles of GO and HemNPs in absence and presence of GO at 0.1 mm NaCl and ph 5.2. (b) Change in the absorbance at 416 nm of 30 mg/l HemNPs in absence and presence of GO at 0.1 mm NaCl and ph 5.2. S14

15 References 1. Hummers, W. S.; Offeman, R. E., Preparation of Graphitic Oxide. J Am Chem Soc 1958, 80, (6), Chen, K. L.; Mylon, S. E.; Elimelech, M., Enhanced aggregation of alginatecoated iron oxide (hematite) nanoparticles in the presence of calcium, strontium, and barium cations. Langmuir 2007, 23, (11), Matijevic, E.; Scheiner, P., Ferric Hydrous Oxide Sols.3. Preparation of Uniform Particles by Hydrolysis of Fe(Iii)-Chloride, Fe(Iii)-Nitrate, and Fe(Iii)- Perchlorate Solutions. Journal of colloid and interface science 1978, 63, (3), Chen, K. L.; Mylon, S. E.; Elimelech, M., Aggregation kinetics of alginate-coated hematite nanoparticles in monovalent and divalent electrolytes. Environ Sci Technol 2006, 40, (5), Chen, K. L.; Elimelech, M., Aggregation and deposition kinetics of fullerene (C- 60) nanoparticles. Langmuir 2006, 22, (26), Madadrang, C. J.; Kim, H. Y.; Gao, G. H.; Wang, N.; Zhu, J.; Feng, H.; Gorring, M.; Kasner, M. L.; Hou, S. F., Adsorption Behavior of EDTA-Graphene Oxide for Pb (II) Removal. Acs Applied Materials & Interfaces 2012, 4, (3), S15

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