Supporting Information for: Little Adjustments Significantly Improve the Turkevich Synthesis of Gold Nanoparticles

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1 Supporting Information for: Little Adjustments Significantly Improve the Turkevich Synthesis of Gold Nanoparticles Florian Schulz,* Torge Homolka, Neus G. Bastús, Victor Puntes, Horst Weller and Tobias Vossmeyer*

2 Table of Contents Table of Contents... 2 Methods... 3 Characterization... 3 UV/vis spectroscopy DLS measurements Graphit furnace atom absorption spectrometry (GF-AAS)... 3 ph calculations... 3 Supporting Figures, Tables and Discussions... 4 Observations for the optimized protocol... 4 Statistical evaluation of TEM results... 5 DLS and UV-vis and yield of the reaction Influence of the ph on the AuNP-synthesis with the inverse method Protocol for optimized mixing with citrate solution instead of citrate buffer Effect of EDTA on the anisotropic growth of AuNPs Analysis of circularity ph-dependent stabilization of crystal facets and the role of EDTA Seeded growth of AuNPs at low ph References

3 Methods Characterization UV/vis spectroscopy. Absorbance spectra were recorded using a Varian Cary 50 spectrometer. UV-micro-cuvettes sealed with lids (Plastibrand, Carl Roth GmbH, Karlsruhe Germany) were used for all experiments. DLS measurements. DLS measurements were done with a Zetasizer Nano ZS (Malvern Instruments). The instrument uses a He-Ne- Laser (4.0 mw, 633nm). Data were analyzed using the software Dispersion Technology Software (Version 5.10). The number of runs per measurement was set to 30. UV-micro-cuvettes (Plastibrand, Carl Roth GmbH, Karlsruhe Germany) were used with sample volumes of 200 µl. Samples were filtered before measurements using PTFE (Poly(tetrafluoroethylene)) syringe filters with 0.2 µm pore size (Carl Roth GmbH, Karlsruhe Germany). All samples were measured three times and the graphical presentation of the results shows the average of these measurements as a percentage of volume. The standard deviation of the volume means of three measurements was below 3.0 %. The procentual volume distributions are presented because these were in best agreement with size distributions obtained by TEM analysis. Graphit furnace atom absorption spectrometry (GF-AAS) GF-AAS measurements were performed with a ContrAA-700 AAS-spectrometer (Analytik Jena, Germany) at 242,795 nm. The limit of detection (LOD) was 10 µg/l. Measurements were performed in triplicates and the relative standard deviation of the mean was 1-5 %. ph calculations ph-calculations were performed with the excel-spreadsheet CurTiPot which is available freely via the internet by courtesy of Gutz, I. R. G. ( accessed May 2014). 3

4 Supporting Figures, Tables and Discussions Observations for the optimized protocol Table S1 summarizes important parameters and observations for the syntheses with the protocol for optimized mixing and ph. The basic protocol is described in the methods section of the main text. The precursor concentrations c(haucl 4) are noted, the addition of EDTA and other substances, the preheating time t, the ph of the final AuNP-solutions after cooling, variations of the mixing and the inset of nucleation. The latter denotes the time after precursor addition when the first color-change from the clear solution was observed. Interestingly, the nucleation was faster when the synthesis was pursued in a baffled flask, the dispersity of these batches was not improved though (compare Table S4). Table S1. Variations of the optimized protocol and observed effects. other c(haucl 4) c(edta) t final Batch substances [µm] [mm] [min] ph added mixing inset of nucleation [s] rpm n.a rpm n.a rpm n.a rpm n.a rpm rpm rpm rpm rpm rpm rpm rpm rpm rpm µm CuCl rpm rpm rpm rpm rpm rpm rpm SC instead of buffer rpm rpm rpm rpm in baffled flask rpm in baffled flask rpm in baffled flask rpm in baffled flask rpm rpm 36 4

5 Statistical evaluation of TEM results The following tables show an exemplative TEM image of each batch and the binary image that was used for particle size analysis with the software ImageJ. The number of counted particles N is given and the mean diameter with the standard deviation. For most batches, N particles were counted from three or more TEM images, although just one example is shown for each batch. These data are summarized in the article in Figure 2. Variations of the standard protocols as described in the methods section are also reported. The EDTA concentrations are based on anhydrous tetrasodium EDTA (M = g/mol), e.g. 3.8 mg in the reaction mixture (V = 1000 ml) are assumed to yield. The EDTA was added to the boiling citric acid buffer solution before addition of the precursor. Table S2. Syntheses with the inverse method. 1 N = 867 = 9.8 ± 1.2 nm 2 N = 360 = 9.5 ± 1.4 nm 3 N = 232 = 14.7 ± 2.1 nm 4 N = 293 = 16.7 ± 2.9 nm 5

6 5 N = 323 = 12.2 ± 1.8 nm 6 N = 306 = 12.1 ± 1.7 nm t = 10 min 7 N = 359 = 12.0 ± 1.5 nm t = 20 min 8 N = 208 = 16.4 ± 2.3 nm Table S3. Syntheses with the ph optimized method. 1 N = 1152 = 15.7 ± 1.8 nm ph control by addition of 100 µl HCl (2 M) 6

7 2 N = 936 = 13.9 ± 1.5 nm 3 N = 497 = 15.3 ± 1.7 nm 4 N = 481 = 16.6 ± 1.6 nm 0.1 mm EDTA 5 N = 728 = 14.1 ± 1.5 nm Table S4. Syntheses with optimized ph and mixing. 1 N = 1164 = 11.7 ± 0.9 nm 7

8 2 N = 457 = 11.5 ± 1.0 nm 3 N = 704 = 11.7 ± 0.9 nm 4 N = 688 = 11.9 ± 0.9 nm 5 N = 1436 = 11.7 ± 0.9 nm 6 N = 1419 = 12.6 ± 0.8 nm 8

9 7 N = 1073 = 12.4 ± 0.8 nm 8 N = 952 = 12.2 ± 0.8 nm + 15 % Precursor 9 N = 1033 = 12.4 ± 0.9 nm 10 N = 894 = 12.3 ± 0.8 nm 11 N = 1127 = 12.0 ± 0.8 nm 9

10 12 N = 917 = 12.1 ± 0.8 nm 13 N = 1106 = 11.9 ± 0.7 nm 14 N = 1097 = 12.3 ± 0.8 nm 16 N = 1000 = 12.4 ± 0.8 nm 0.03 mm EDTA 17 N = 700 = 12.1 ± 0.8 nm 10

11 18 N = 969 = 11.9 ± 0.7 nm 19 N = 1028 = 11.7 ± 0.7 nm 20 N = 1120 = 11.8 ± 0.7 nm 21 N = 1062 = 12.1 ± 0.6 nm 23 N = 961 = 11.4 ± 0.8 nm 11

12 24 N = 922 = 11.4 ± 0.6 nm 25 N = 1279 = 11.4 ± 0.8 nm synthesis in baffled flask 26 N = 746 = 11.8 ± 0.7 nm synthesis in baffled flask 27 N = 1000 = 11.7 ± 0.8 nm - 23 % Precursor synthesis in baffled flask 28 N = 929 = 11.9 ± 0.8 nm + 23 % Precursor synthesis in baffled flask 12

13 29 N = 1112 = 12.2 ± 0.7 nm - 23 % Precursor 30 N = 1087 = 11.7 ± 0.7 nm + 23 % Precursor 13

14 DLS and UV-vis and yield of the reaction The AuNP batches synthesized with the different methods were routinely characterized by UV-vis spectroscopy and DLS. Representative results are shown in Figure S1. Figure S1. Exemplative absorbance spectra (left) and hydrodynamic diameters d H determined by DLS (right) for citratestabilized AuNPs synthesized with the different methods presented in this communication. Red lines: inverse method (batch 2 in Table S2); Green lines: ph optimized method (batch 2 in Table S3); Blue lines: ph and mixing optimized method (batch 1 in Table S4); Grey dashed lines: ph and mixing optimized method with addition of EDTA (batch 19 in Table S4). For the DLS measurements the volume means of d H with standard deviation (width of the volume distribution as defined by the software Dispersion Technology Software 5.10, Malvern Instruments Ltd.) are indicated in the respective colors. For comparison, the diameters obtained by TEM analysis for the according samples are also given in parentheses. The absorbance spectra show the characteristic peak at nm indicative of the localized surface plasmon resonance of the AuNPs. The samples for DLS measurements usually have to be purified by syringe filtration (PTFE, 0.2 µm) to remove aggregated organic byproducts of the synthesis. Because these typically cause broad signals which can vary from d H ~ nm, they are possibly due to the formation of polymolecular complexes proposed for the mechanism of the Turkevich synthesis, and studied in detail by Mikhlin et al. 1 Because the intensity of scattered light scales with ~d 6, even minute amounts of such contaminants affect the results of DLS measurements. The presence of AuNP-aggregates causing these signals can be ruled out based on UV-vis- and TEM-analysis. The strong plasmonic interparticle coupling in AuNP-aggregates would lead to an increase in absorbance in the range nm, 2 which was not observed and no aggregates were observed on the TEMsamples. The mean d H of the volume distributions are in good agreement with the TEM results when the samples are purified. The CVs, however, cannot be determined as accurately as with TEM-analysis. A sample of batch 20 (Table S4) was centrifuged for 20 minutes at 20,000 g to separate AuNPs and gold complexes. The supernatant was then analyzed by graphite furnace atom absorption spectrometry (GF-AAS). No gold was detected in the supernatant and considering the limit of detection (LOD) of 10 µg/l, the yield in this reaction was > 99.9 %. 14

15 Influence of the ph on the AuNP-synthesis with the inverse method Figure S2. Influence of the ph on the AuNP-synthesis. A: ph of 100 ml 2.2 mm citric acid buffers (mixtures of sodium citrate and citric acid, c total = 2.2 mm) with different ratios of sodium citrate (SC) at room temperature and the ph of these buffers after addition of 666 µl 25 mm HAuCl 4. The resulting concentration c(haucl 4) = mm corresponds to that used in the inverse method. In the synthesis at T ~100 C the ph is additionally affected by the involved reactions. B: Mean diameters with standard deviation of AuNPs synthesized with the inverse method using different citric acid buffers. C, D and E: TEM-analyses of the batches shown in B and exemplative TEM-images. Sphericity of the AuNPs is assumed for analyses. Although the sphericity of AuNPs synthesized with 50 % SC (E) is low, their analysis is shown for comparison. The lowest coefficient of variation CV was obtained in 75 % SC, corresponding to a ph 5.9, which decreases to ~5.5 during the reaction (compare Table S1). 15

16 Protocol for optimized mixing with citrate solution instead of citrate buffer Figure S3. TEM images of AuNPs synthesized with the protocol for optimized mixing and a 2.2 mm citrate solution instead of citrate buffer. TEM images of AuNPs synthesized under the same conditions but in citrate buffer are presented in Figure 5 (no EDTA) in the main text and in Table S4, batches 1-3, 5 and 23. Effect of EDTA on the anisotropic growth of AuNPs Analysis of circularity The circularity of the samples was analyzed as described in the method section in the main text. Figure S4 illustrates the smoothing that is necessary to obtain meaningful results from TEM-images. Figure S4. Effect of smoothing on the circularity. From left to right: Cutout of an TEM image showing one AuNP, the same cutout in binarized form and after 10-fold smoothing and re-binarization. Without smoothing the circularity is 0.77, after smoothing it is The low circularity without smoothing is due to irregular boundaries after binarization. The extent of these irregularities depends on the contrast of the original TEM-image and is not controllable. Thus, analysis of the circularities without smoothing is not meaningful. 16

17 The mean circularity of batches with triangular prismatic AuNPs changes just slightly because just a few percent of non-spherical AuNPs are formed at maximum (Figure 5 and Figure S5B). The standard deviations of the circularities, SD(circ.), however, change significantly with the number of non-spherical AuNPs because the latter represent subpopulations with very low circularity (Figure 5 and Figure S5C and D). Thus, SD(circ.) is suitable to describe the uniformity of AuNPs. The analysis shows that both, CV (Figure S5A) and uniformity (Figure S5C), can be optimized by addition of. Figure S5. Characterization of AuNP-batches synthesized with the protocol for optimized ph and mixing in the presence of different amounts of EDTA. The preheating time t was 15 min for all these batches. A: Mean CVs with standard deviation of several AuNP-batches synthesized in the presence of 0 to 0.03 mm EDTA. The numbers n of analyzed batches are indicated at the data points. B: Mean circularities of the same batches as in A. Red error bars indicate the standard deviation of the mean circularities, for 0.03 mm EDTA no error bar is shown since n = 1. Grey error bars indicate the mean standard deviations of the circularities, SD(circ.). C: Plotting the mean relative SD(circ.) with standard deviation versus c(edta) displays significant differences of AuNPs uniformity. The error bar at is too small to be observed, for 0.03 mm EDTA no error bar is shown since n = 1. The subpopulations of non-spherical particles significantly affect SD(circ.), the mean circularity is less affected. Thus, SD(circ.) is more suitable to compare the uniformity of the particles. D: Exemplative TEM-images and circularitydistributions for AuNP-batches synthesized in the presence of 0.01 and 0.03 mm EDTA as indicated. Additional data for 0.00 and are presented in the main text, Figure 5. 17

18 ph-dependent stabilization of crystal facets and the role of EDTA Calculations indicate that in a solution of the citrate-citric acid system the dominant species is dihydrogen citrate at ph 4, whereas at ph 5.5 hydrogen citrate is the dominant species (Figure S6). At ph 7 the concentration of dihydrogen citrate should be ~0. Park and Shumaker-Parry very recently presented a detailed study of the adsorption of citrate and dihydrogen citrate on gold surfaces and one important conclusion in this context is that the adsorption strength of dihydrogen citrate on Au(100) and Au(110) facets should be significantly lower than that of citrate ions. 3 The most stable and dominant facet in AuNPs is Au(111), followed by Au(100) and Au(110), and large differences in stabilization of these facets promote anisotropic growth. E.g. Xia et al. hypothesized that underpotential deposition of silver(i)-ions stabilizes (100) and (110) facets in AuNP-synthesis, thereby promoting uniformity of the final particles. 4 We found that catalytic amounts of Cu also improve the uniformity of the AuNPs and lead to a faster nucleation. The CV of the obtained AuNPs is higher, though. In the optimized protocol the ph is controlled at ~5.5, so significant amounts of dihydrogen citrate should be present in the mixture (~12 %) besides the main species hydrogen citrate (~75 %, Figure S6). In consequence, the (100) and (110) facets of some AuNPs might be weakly stabilized, thus promoting anisotropic growth. The pk a-values of the EDTA carboxylic functions are much lower than those of citrate and these carboxylic groups are not protonated at ph 5.5 (Figure S7). Stabilization of the (100) and (110) facets by EDTA might be a reason for its effect of suppressing anisotropic growth. This hypothesis, however, has to be backed up by additional experiments which are beyond the scope of this study and are part of ongoing and future studies. The advantage of using EDTA to suppress anisotropic growth is, that the CV is not as sensitively affected as by the use of Ag- or Cu-ions. If the concentrations of these ions are not exactly adjusted to an optimal value, high CVs of the quite shape uniform AuNPs are the result (Figure S8 and S9). High EDTA concentrations (0.5 mm, data not shown) also lead to polydisperse AuNPs, but in a range of low concentrations, mm, EDTA improves the dispersity and uniformity of the AuNPs (Figure S5). Figure S6. Alpha plots for citric acid. These plots show the molar fraction of each ionic species as a function of the solutions ph. The plots were calculated for a total concentration of 2.2 mm. 18

19 Figure S7. Alpha plots for EDTA calculated for a total concentration of 0.02 mm. In the relevant ph range ~5.5 for the presented synthesis all carboxylic groups of EDTA are deprotonated. In the presence of Ag- as well as Cu-ions fast nucleation after ~5-10 s was observed and no or nearly no triangles were found (Table S1, Figures S8 and S9). The CVs of the AuNPs, however, were high or very high. Figure S8. Effect of Ag-ions. AuNPs synthesized with the inverse method in the presence of 5 µm AgNO 3. Figure S9. Effect of Cu-ions. AuNPs synthesized with the protocol for optimized ph and mixing in the presence of 25 µm CuCl 2. 19

20 Seeded growth of AuNPs at low ph Figure S10. AuNPs synthesized with the seeded-growth protocol as described previously, 5 but using citric acid buffer (SC/CA: 75/25, c = 2.2 mm) instead of citrate solution in every step. The AuNPs are the fourth generation of the seeded growth. The high CV of the AuNPs suggests that the presented buffer strategy cannot be transferred to this seeded growth synthesis. 20

21 References (1) Mikhlin, Y.; Karacharov, A.; Likhatski, M.; Podlipskaya, T.; Zubavichus, Y.; Veligzhanin, A.; Zaikovski, V. Submicrometer intermediates in the citrate synthesis of gold nanoparticles: New insights into the nucleation and crystal growth mechanisms. J. Colloid Interface Sci. 2011, 362, (2) Ghosh, S. K.; Pal, T. Interparticle Coupling Effect on the Surface Plasmon Resonance of Gold Nanoparticles: From Theory to Applications. Chem. Rev. 2007, 107, (3) Park, J.-W.; Shumaker-Parry, J. S. Structural Study of Citrate Layers on Gold Nanoparticles: Role of Intermolecular Interactions in Stabilizing Nanoparticles. J. Am. Chem. Soc. 2014, 136, (4) Xia, H.; Bai, S.; Hartmann, J.; Wang, D. Synthesis of Monodisperse Quasi-Spherical Gold Nanoparticles in Water via Silver(I)-Assisted Citrate Reduction. Langmuir 2010, 26, (5) Bastus, N. G.; Comenge, J.; Puntes, V. Kinetically Controlled Seeded Growth Synthesis of Citrate- Stabilized Gold Nanoparticles of up to 200 nm: Size Focusing versus Ostwald Ripening. Langmuir 2011, 27,

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