Capillary Electrophoresis, Mass Spectrometry, and UV-Visible

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Anal. Chem. 2008, 80, 2439-2446

Capillary Electrophoresis, Mass Spectrometry, and UV-Visible Absorption Studies on Electrolyte-Induced Fractionation of Gold Nanoclusters Chung Keung Lo,† Man Chin Paau,† Dan Xiao,*,‡ and Martin M. F. Choi*,†

Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, P.R. China, College of Chemistry and College of Chemical Engineering, Sichuan University, Chengdu 610065, P.R. China

We describe a novel and simple electrolyte-induced fractionation method to separate a polydisperse watersoluble gold nanocluster (Au NC) product. Different particle sizes of Au NC fractions can be easily centrifuged down as a function of the electrolyte concentration or lipophilicity of the solution. The changes in the absorption characteristic of the Au NC fractions under different electrolyte/ethanol conditions demonstrate the change in particle size distribution of the Au NC. Small gold nanoclusters, Au10, Au11, Au12, and Au15, were separated from the Au10-Au50 polydisperse Au NC product under various phosphate/ethanol conditions. The core size separation of Au NC was evaluated by their migration trends in capillary zone electrophoresis, UV-visible absorption, and mass spectra. The electrolyte-induced fractionation not only provides a convenient method to separate small Au NC mixture but also assists in the study of the photophysical properties of smaller Au NCs that are present with the larger Au NCs in a polydisperse Au NC product. The analytical separation of water-soluble gold monolayer protected cluster (Au MPC) is an important technique in the study and development of Au MPC because their properties are core size dependent,1 and they have high potential application for biosensing,2 catalysis,3 electronics,4 and nanotechnology.5 Furthermore, the synthesis of Au MPC with 1-5 nm often results in a mixture of core sizes, i.e., polydisperse MPC product and possibly also variations in the number of protecting ligands on each MPC.1,5 As such, many researchers have developed a variety of approaches to achieve particle separation and to narrow the size distribution of nanoparticles in polydisperse Au MPC products. Gel electrophoresis6-9 has been employed to separate the * To whom correspondence should be addressed. E-mail: mfchoi@hkbu. edu.hk (M.M.F.C.); [email protected] (D.X.). † Hong Kong Baptist University. ‡ Sichuan University. (1) Choi, M. M. F.; Douglas, A. D.; Murray, R. W. Anal. Chem. 2006, 78, 27792785. (2) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (3) Ghosh, S. K.; Kundu, S.; Mandal, M.; Pal, T. Langmuir 2002, 18, 87568760. (4) Sun, S. H.; Murray, C. B.; Weller, D.; Folks, L.; Moser, A. Science 2000, 287, 1989-1992. (5) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293-346. 10.1021/ac702135z CCC: $40.75 Published on Web 03/07/2008

© 2008 American Chemical Society

Au MPC products. However, the separation was time-consuming and the separation efficiency was relatively lower. Recently, ionpair chromatographic1 and capillary electrophoretic separation10 have been developed to separate the polydisperse Au MPC product efficiently. However, they were somewhat limited in throughput, and it was difficult to collect the separated MPC. Thus, new and simple methods are still in high demand to assist the development of this area of nanotechnology. Unlike molecular system, it is often difficult to employ crystallization for colloidal structures, and the methods that have been used successfully are performed primarily in organic media. In such media, fractionation is based on the greater solubility of the smaller MPCs in polar solvent mixtures.11 Recently, DNA-induced size selective assembly and separation of the binary and ternary mixture of Au nanoparticles have been described.12 However, their work only focused on the fractionation of Au nanoparticles in binary or ternary mixture rather than a genuine polydisperse Au MPC product. In this article, a novel approach of fractionating a polydisperse gold nanocluster (Au NC) product in aqueous medium is described. Different core sizes of water-soluble Au NC were precipitated out and centrifuged down by adding an electrolyte and an organic solvent in the aqueous medium. Smaller core Au NC could be easily separated from an as-prepared polydisperse NC product. The successful separation of smaller core Au NCs were verified by different analytical techniques including capillary electrophoresis, absorption, and mass spectra. The electrolyteinduced fractionation is a simple and time-saving method to separate the small Au NC for further studying of their photophysical properties. (6) Schaaff, T. G.; Knight, G.; Shafigullin, M. N.; Borkman, R. F.; Whetten, R. L. J. Phys. Chem. B 1998, 102, 10643-10646. (7) Schaaff, T. G.; Whetten, R. L. J. Phys. Chem. B 2000, 104, 2630-2641. (8) Negishi, Y.; Yakasugi, Y.; Sato, S.; Yao, H.; Kimura, K.; Tsukuda, T. J. Am. Chem. Soc. 2004, 126, 6518-6519. (9) Negishi, Y.; Nobusada, K.; Tsukuda, T. J. Am. Chem. Soc. 2005, 127, 52615270. (10) Lo, C. K.; Paau, M. C.; Xiao, D.; Choi, M. M. F. Electrophoresis, in press. (11) Whetten, R. L.; Khoury, J. T.; Alvarez, M. M.; Murthy, S.; Vezmar, I.; Wang, Z. L.; Stephens, P. W.; Cleveland, C. L.; Luedtke, W. D.; Landman, U. Adv. Mater. 1996, 8, 428-433. (12) Lee, J.-S.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2006, 128, 88998903.

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EXPERIMENTAL SECTION Chemicals. Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4‚ 3H2O, 99.9+ %) and sodium borohydride (NaBH4) (99%) were obtained from Aldrich (Milwaukee, WI). Mesityl oxide (MO > 98%) as the neutral electroosmotic flow (EOF) marker, 2,5dihydroxybenzoic acid (DHB, 98%), and N-acetyl-L-cysteine (NAC > 99%) were purchased from Sigma (St. Louis, MO). Methanol (MeOH), ethanol (EtOH) of HPLC-grade, and acetone of AR-grade were obtained from Labscan (Bangkok, Thailand). Disodium hydrogen phosphate dihydrate was from Fluka (Buchs, Switzerland). Glacial acetic acid and sodium hydroxide (NaOH) were purchased from Farco Chemical Supplies (Beijing, China). Purified water from a Milli-Q-RO4 water purification system (Millipore, Bedford, MA) with a resistivity higher than 18 MΩ cm was used to prepare all solutions. All reagents of analytical grade or above were used as received. Synthesis of N-Acetyl-L-cysteine-Protected Au NC. The N-acetyl-L-cysteine-protected Au NC was prepared according to a previous method.1 Briefly, 1.82 g (4.62 mmol) of HAuCl4‚3H2O was mixed with 2.27 g (13.9 mmol) of N-acetyl-L-cysteine in a solvent mixture of 200 mL of MeOH/glacial acetic acid (6:1 v/v) under stirring at 0 °C to form a white suspension of orange solution. The mixture was then reduced at once with 3.50 g (92.5 mmol) of NaBH4 in 45 mL of EtOH, forming a dark brown solution and stirred for 30 min. Then, the product was precipitated by adding 200 mL of acetone, and the precipitate was redissolved in 5 mL of H2O. The pH of the solution was adjusted to ∼1 by the dropwise addition of concentrated HCl. The Au NC was precipitated again by adding 200 mL of acetone. Further purification was done by dialysis of the product in a minimum amount of H2O loaded into 24 mm segments of Spectr/Por cellulose ester dialysis membrane tubes (MWCO ) 10 000, Spectrum Laboratories, Rancho Dominguez, CA), which were placed in a 5 L beaker of water, and stirred slowly, recharging with fresh water every ∼24 h over the course of 7 days. The dark brown, Au NC solutions were collected from the dialysis tubes and dried by a stream of N2 at room temperature. The spherical polydisperse NACprotected NC product with an average core size of 1.7 ( 0.5 nm, confirmed by transmission electron microscopy (TEM), was denoted as the CrudeNC product. Fractionation of Au NC. The fractionation of Au NC was performed in the following procedure: 2.0 mg of CrudeNC was dissolved in 50 µL of electrolyte (sodium phosphate buffer, pH 10). The final solution of the Au NC product was made up to 250 µL by adding known amounts of EtOH and electrolyte. The electrolyte concentrations were adjusted to 10, 20, 30, and 40 mM with 65% v/v of EtOH when studying the effect of electrolyte concentration. The concentration of the electrolyte was adjusted to 30 mM with various volume percentages (60, 65, and 75%) of EtOH when studying the effect of solvent lipophilicity. The resulting solution was centrifuged at 13 000 rpm for 15 min to precipitate out the solid Au NC. As such, two fractions were obtained: one was the dissolved fraction (DF) and the other was the precipitated fraction (PF) of the Au NC. Au NCs in the DF were further precipitated out by the addition of acetone. All Au NCs from the DFs and PFs were redissolved in 30 mM electrolyte solutions prior to most instrumental analysis. 2440

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UV-Visible Absorption and Photoluminescence Spectroscopy. The UV-visible absorption spectra of the Au NC solutions were acquired with a Cary 100 Scan UV-visible spectrophotometer (Varian, Palo Alto, CA) over the wavelength range from 250 to 800 nm. The photoluminescence properties of the fractions were recorded by a QM4 spectrofluorometer (Photon Technology International, Lawrenceville, NJ). Capillary Zone Electrophoresis. Electrophoretic separations were performed on a P/ACE MDQ CE system (Beckman Instruments, Fullerton, CA) in conjunction with a diode-array detector (DAD) monitoring at 214 nm or a laser-induced fluorescence (LIF) detector monitoring at excitation/emission wavelengths of 488/550 nm. Bare fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) with 50 µm i.d., 375 µm o.d., total capillary length of 40.2 cm, and effective length of 30 cm were used for Au NC separations. New capillaries were initialized by flushing with water (5 min), 1.0 M NaOH (30 min), 0.10 M NaOH (30 min), water (50 min), and run buffer (30 min) before use. Between analyses the capillary was rinsed with water (2 min), 0.10 M NaOH (2 min), water (2 min), and run buffer (5 min). Injection was performed at a pressure of 100 mbar for 3 s. Normal polarity conditions, i.e., EOF toward the cathode, were used for all the work, and most separations were performed at 25 °C. The run buffer was prepared by dissolving an appropriate amount of sodium phosphate with water. The pH of the phosphate buffer solutions was adjusted to 10 by adding NaOH. It was then mixed with EtOH to form the run buffer of 30 mM phosphate (pH 10) in 20% v/v EtOH. The pH measurements were taken on an Orion combined pH glass electrode (Chicago, IL). The run buffer pH was specified as that of the aqueous buffer before mixing with EtOH. In most of the sample injections, 10 mM of MO was mixed with the sample solution. All sample solutions and run buffers for capillary zone electrophoretic (CZE) analysis were filtered through the 0.45 µm cellulose acetate syringe filters (Alltech, Deerfield, IL). Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry. The fractions were analyzed by a matrix-assisted laser desorption ionization-mass spectrometer (MALDI-MS) (Autoflex, Bruker, Germany) similar to the reported method.7 The Au NC fractions were mixed (1:1 v:v) with a 1 M solution of DHB in MeOH/H2O (1:1 v/v). Then 4 µL of this solution was deposited on a MALDI target plate and air-dried. The sample was inserted into the instrument and irradiated by a pulsed N2 laser working at 337 nm. In general, 30 laser shots were averaged for each spectrum. RESULTS AND DISCUSSION Fractionation under Different Electrolyte Concentrations. The dissolved fraction (DF) and precipitated fraction (PF) of the CrudeNC product under different concentrations (10-40 mM) of electrolyte (sodium phosphate, pH 10) in 65% v/v EtOH were separated and collected. Their UV-visible absorption spectra are displayed in Figure 1. These spectra are normalized at 250 nm to remove the effect of concentration differences so as to allow focus on comparison of spectral shape and band position. Negishi et al.9 and our previous work1 observed that smaller core Au NCs possess a sharper decrease in absorbance than that of larger core Au NCs from shorter to longer wavelengths. In essence, large core Au NC produces an absorption spectrum with relatively

Figure 1. UV-visible absorption spectra of different Au NC fractions collected under various concentrations (10-40 mM) of electrolyte: (a) PF and (b) DF.

higher absorbance than that of small core NC in the visible light region. The difference in the normalized absorption spectra of the Au NC precipitated fractions PFs (Figure 1a) and dissolved fraction DFs (Figure 1b) well demonstrate the change in particle size distribution of the Au NC under electrolyte-induced fractionation. The spectral characteristics of our small and large core size Au NCs are in complete agreement with the previous reported Au NC spectra.9 The subscripts in DF or PF represent the use of different experimental conditions in fractionation, i.e., DF10mM and PF10mM denote that 10 mM electrolyte was used during fractionation of the dissolved and precipitated Au NCs. The absorption spectra of PFs (except PF10mM) show higher absorbances while DFs (except DF10mM) produce lower absorbances than that of the CrudeNC product. Besides, the absorbances of PF increase (absorbance of PF10mM < PF20mM < PF30mM < PF40mM) and DF decrease (absorbance of DF10mM > DF20mM > DF30mM > DF40mM) with the increase in electrolyte concentrations. This infers that higher proportions of large core Au NCs in the PFs and lower proportions in the DFs can be obtained by increasing the electrolyte concentration during fractionation. In order to further interpret the change in size distribution of the CrudeNC product during electrolyte-induced fractionation, a CZE method was also applied to analyze the DFs and PFs. Our previous work demonstrated that CZE is a powerful tool to evaluate the size distribution of Au NC products synthesized under different experimental conditions.10 Figure 2 depicts the electropherograms of various PFs (Figure 2a) and DFs (Figure 2b) of the collected Au NCs using different electrolyte concentrations during fractionation. The first peak in each electropherogram is the neutral marker MO, and the migration peaks after MO are the Au NCs, i.e., the peaks are separated according to the chargeto-size ratio. The smaller the Au NC core size, the longer the migration time.10 The CrudeNC trace at the bottom of the electropherograms is also displayed, and all the Au NC peaks are normalized to the peak height of Au NC peak 1 for ease of comparison and interpretation. The separated Au NC peaks are

labeled in ascending order according to their relative core sizes, i.e., a larger number represents a larger core size. In general, the proportions of peaks 4-11 (larger core NCs) increase and peaks 1-3 (smaller core NCs) are more or less the same in PFs (Figure 2a) with increasing electrolyte concentration. By contrast, the proportions of peaks 7-11 (larger core NCs) decrease and peaks 1-6 (smaller core NCs) are almost constant in DFs (Figure 2b). The increase in larger core Au NC proportions explains the increase in absorbance of PFs as shown in Figure 1a. The decrease in the larger core Au NC proportion vindicates the decrease in absorbance of the DFs (Figure 1b) with the increase in electrolyte concentration. When only the smallest core Au NCs (peaks 1-3 and 6) are present in PF10mM, it produces an UV-visible absorption spectrum of lower absorbance than that of the CrudeNC product (Figure 1a) while its complementary DF10mM gives a spectrum with slightly higher absorbance (Figure 1b). Here, we found that no precipitate was induced when no electrolyte was added under the same % v/v of EtOH. Because most of the surface-attached ligands of Au NC were deprotonated13 (pKa of free N-acetyl-L-cysteine is ∼4.1 in pure water1) in alkaline condition (pH 10), Au colloids and latex spheres typically containing charged surface functional groups aggregate in high salt conditions.14-16 Aggregation is attributed to the reduction of Coulombic repulsion between same-charged particles upon electrolyte addition. Once the repulsion between particles are reduced, van der Waals interactions bring the particles together and lead to aggregations.17 Thus, on the initial addition of electrolyte, i.e., (13) Hostetler, M. J.; Wingate, J. E.; Zhong, C.-J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir 1998, 14, 1730. (14) Colloidal Gold: Principles, Methods, and Applications; Hayat, M. A., Ed.; Academic Press: San Diego, CA, 1991. (15) Baschong, W.; Lucocq, J. M.; Both, J. Histochemistry 1985, 83, 409-411. (16) Working with FluoSpheres Fluorescent Microspheres; Molecular Probes, Inc. Product information, http://www.probes.com, 1999. (17) Introduction to Soft Matter; Hamley, I. W., Ed; Wiley: Chichester, England, 2000.

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Figure 2. Electropherograms of different Au NC fractions collected under various concentrations (10-40 mM) of electrolyte: (a) PF and (b) DF. The detection wavelength was 214 nm. All the peaks were normalized relative to peak 1 for each electropherogram for ease of comparison.

10 mM, the smaller core Au NCs tend to aggregate first because their charge-to-size ratios are larger;10 thus, they are more efficiently screened by the added electrolyte and precipitated from the CrudeNC product as the PF. When the electrolyte concentration continues to increase, larger core Au NCs are then successively precipitated out. As a result, the population of the large NCs in the DF decreases and the average size of the NCs in the DF is smaller on increasing the electrolyte concentration. It can be observed that larger core Au NCs (peaks 8-11 of PF30mM and PF40mM in Figure 2a) have a more profound effect on absorption in the UV-visible spectrum (PF30mM and PF40mM in Figure 1a) than the smaller core Au NCs (PF10mM in Figure 1a). The existence of a small proportion of larger core Au NC in the Au NC mixture has a greater effect on the shape of the spectrum. Large core Au NCs usually have higher absorbances and featureless characteristics in both the UV and visible regions whereas small core Au NCs provide fine steplike structure in the UV region.1,9 When a Au NC product comprises both small and large Au NCs such as our CrudeNC product, it will only produce one resultant UV-visible absorption spectrum without any steplike feature. Our fractionation method provides a simple way to remove the larger core Au NCs from the polydisperse Au NC mixture to facilitate the study of the spectral fine structure of the smaller core Au NCs. The absorption characteristics of the smaller core Au NCs at ∼350-400 nm (PF10mM in Figure 1a) are more prominent because they were first precipitated. Fractionation under Various Volume % of EtOH. Furthermore, the effect of various volume % of EtOH on the electrolyteinduced fractionation was also investigated. Figure 3 depicts the UV-visible absorption spectra of Au NC fractions collected using different volume % of EtOH with 30 mM electrolyte. The increase in volume % of EtOH decreases the absorbance of both DF and PF. The initial addition of EtOH induces only the precipitation of those larger core Au NCs. On further increase of EtOH, the medium and smaller NCs are successively precipitated. It is wellknown that Au NCs with smaller cores have greater solubility in 2442

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polar solvents;18 thus, they will be separated from the solvent mixture only when the polarity of the solvent system is low. Our results support that the solubility of Au NC decreases as the lipophilicity of the solution increases. As such, the absorption spectrum of PF70%, which was prepared from the highest lipophilic solution, resembles closely that of the CrudeNC product because most Au NCs (mainly the larger ones) were precipitated. By contrast, the absorption spectrum of the complementary fraction (DF70%) shows the largest difference from that of CrudeNC, attributing to the relatively larger amounts of small Au NC still present in the DF fraction. Similarly, CZE was employed to study the change of particle size distribution in each Au NC fraction. Figure 4 displays the electropherograms of Au NC fractions prepared from different volume % of EtOH. The Au NC peaks are again normalized to the peak height of Au NC peak 1. Increasing the lipophilicity of the solution increases the proportion of Au NC peaks 1-11 in PFs (Figures 4a) but decreases the proportion in DFs (Figure 4b). It is also observed that smaller core Au NC peaks (e.g., peaks 2-5 and 8) in PFs increases progressively with EtOH concentration. As a result, PF tends to be more similar to the CrudeNC product in terms of the Au NC peak distribution in the electropherograms and absorption spectral characteristics. Conversely, the subsequent removal of smaller core Au NCs by increasing the lipophilicity of the solution resulted in the progressive diminishing of the smaller core Au NC peaks (peaks 2-4) in the electropherograms (Figure 4b). In conclusion, larger core Au NCs are precipitated first under 60% v/v EtOH and 30 mM electrolyte, followed by the smaller core Au NCs as the lipophilicity of the solution increases. Photoluminescence of Au Nanoclusters. It has been reported that NCs possess size-dependent photoluminescence behavior.8,19-21 As such, we first study our Au NCs by CZE coupled with laser-induced fluorescence (LIF) detection. Figure 5 depicts (18) Donkers, R. L.; Lee, D.; Murray, R. W. Langmuir 2004, 20, 1945-1952. (19) Yang, Y.; Chen, S. Nano Lett. 2003, 3, 75-79. (20) Negishi, Y.; Tsukuda, T. Chem. Phys. Lett. 2004, 383, 161-165.

Figure 3. UV-visible absorption spectra of different Au NC fractions collected under various volume percentages (60-70%) of ethanol with 30 mM electrolyte: (a) PF and (b) DF.

Figure 4. Electropherograms of different Au NC fractions collected under various volume percentages (60-70%) of ethanol with 30 mM electrolyte. (a) PF and (b) DF. The detection wavelength was 214 nm. All the peaks were normalized relative to peak 1 for each electropherogram for ease of comparison.

the electropherograms of the CrudeNC product captured by (a) a LIF detector (excitation/emission at 488/550 nm) and (b) DAD at 214 nm. Au NC peak 8 shows the strongest luminescence whereas the neighborhood peaks 7, 9, 10, and 11 have weak emissions. Au NC peaks 1-5 are not observed under the LIF detection. Small Au NCs display only the UV excited blue emission (vide infra). Thus, our CE-LIF seems to be useful only for monitoring the larger core Au NCs (peaks 7-11) in the visible light region. Figure 6 displays the electropherograms of the Au NC fractions detected by LIF under various volume % of EtOH. The Au NC fractions detected by LIF under different electrolyte concentrations are shown in Figure S1 (Supporting Information). (21) Lee, D.; Donkers, R. L.; Wang, G.; Harper, A. S.; Murray, R. W. J. Am. Chem. Soc. 2004, 126, 6193-6199.

Increasing the lipophilicity of the solvent or the electrolyte concentration is favorable to the precipitation of the strongest emission Au NC (peak 8) which is tentatively assigned to Au22Au25 by mass spectrometry (vide infra). As our Au NCs show photoluminescence property, it is also worthwhile to study the photoluminescence spectroscopy of PFs and DFs. The corrected fluorescence excitation (Ex) and emission (Em) spectra of PF70% (red lines), DF70% (blue lines), CrudeNC (black lines), PF10mM (green lines) fractions are shown in Figure 7. All Em spectra were obtained at an excitation wavelength of 518 nm whereas the Em spectrum for PF10mM was recorded at excitation 350 nm. Ex spectra were captured at their corresponding Em maxima. All spectra were normalized at their Em/Ex maxima (λem/λex) for ease of comparison. The Em spectrum for Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

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Figure 5. Capillary electrophoretic separation of a polydisperse Au NC product detected by laser-induced fluorescence (excitation/emission at 488/550 nm) and diode-array (absorption at 214 nm) detectors. Traces a and b are the fluorescence and absorption signals, respectively.

Figure 6. Electropherograms of different Au NC fractions collected under various volume percentages (60-70%) of ethanol with 30 mM electrolyte: (a) PF and (b) DF. The signals were captured by the laser-induced fluorescence (excitation/emission at 488/550 nm) detector.

PF70% is bathochromically shifted compared to that of DF70%, indicating a higher proportion of larger core Au NCs in PF70%. The Em spectrum of the CrudeNC product has an Em band between PF70% and DF70% as it comprises both small and large core Au NCs. PF10mM produces the shortest emission band (λem 435 nm) since it contains mostly the smallest core Au NCs (vide supra). This size-dependent emission is consistent with the reported size-dependent HOMO-LUMO gap of Au NC.20,22-24 Negishi et al.9 reported that Au10 to Au12 have absorption bands from 300 to 400 nm and λem at ∼435 nm, Au22 has a ∼520 nm absorption peak and a λem ∼700 nm whereas Au29, Au33, Au35, Au38, and Au39 have broad absorption bands from 700 to 800 nm. (22) Mohamed, M. B.; Volkov, V.; Link, S.; El-Sayed, M. A. Chem. Phys. Lett. 2000, 317, 517-523. (23) Huang, T.; Murray, R. W. J. Phys. Chem. B 2001, 105, 12498-12502. (24) Chen, S.; Ingram, R. S.; Hostetler, M. J.; Pietron, J. J.; Murray, R. W.; Schaaff, T. G.; Khoury, J. T.; Alvarez, M. M.; Whetten, R. L. Science 1998, 280, 2098-2101.

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Moreover, when the electropherograms and absorption spectra of PF10mM, DF70%, PF70%, and CrudeNC are plotted and compared in Figure S2 (Supporting Information), they clearly show that Au NC peaks 1-6, 7, 8, and 9-11 in the electropherograms are responsible for the absorption features at 300-400 nm with fine steplike structure, ∼520 nm, and 700-800 nm absorption bands, respectively. Thus, the Au NC peaks in the electropherogram (Figure 2) can be assigned to Au10-Au 15 for peaks 1-6, Au22 for peaks 7, 8, and Au29-Au39 for peaks 9-11 according to their photophysical properties similar to that of Negishi et al.9 This deduction is corroborated by the mass spectrometric analysis of the Au NC fractions (vide infra). MALDI-MS Analysis of Au NC Fractions. MALDI-MS is a useful analytical technique to probe the inorganic core size of NC fractions. We obtained the mass spectra of (a) DF70% and (b) PF70% as shown in Figure 8, confirming the findings of UV-visible absorption spectra and CZE analysis. At the lower m/z range

Figure 7. Corrected fluorescence excitation (Ex) and emission (Em) spectra of different electrolyte-induced Au NC fractions. All the spectra are normalized at their respective excitation (λex) and emission (λem) maxima. The dotted and solid lines represent the emission and excitation spectra of the Au NC fractions, respectively.

Figure 8. MALDI-mass spectra of (a) DF70% and (b) PF70%. The insets display the higher m/z range from 5500 to 20 000 Da.

2200-5500 Da, both fractions show the presence of Au10, Au11, Au12, Au15, and Au22. It is worth noting that some of the MS peaks probably originated from the same Au cores but with different numbers of attached ligand (NAC), i.e., a portion of ligands dissociated from the same parent cluster. As shown in Figure S2, Au NC peaks 1-11 are present in PF70% whereas DF70% contains only peaks 1-8 but is without peaks 9-11. Combination of the MS results, electropherograms, and absorption spectra, Au NC peaks 1-8 can be tentatively assigned to Au10, Au11, Au12, Au15, and Au22. It should be noted that the total number of MS peaks may not match exactly with the total number of peaks in the electropherogram as CZE is able to separate NCs differing by

only one or more protected ligands of the same Au core size. Second, larger core NCs may be fragmented to the smaller NCs in the mass spectra. At the higher m/z range 5500-20 000 Da, NCs larger than Au25 are not observed in DF70%, indicating that they have been centrifuged down as PF70%. By contrast, Au NCs larger than 9000 Da are observed in PF70% which can be tentatively assigned to Au29, Au33, Au35, Au38, Au39, and Au50 corresponding to the NC peaks 9-11 in the electropherogram (Figure S2). Similarly, mass spectra of PF10mM and PF70% are shown in Figure S3 (Supporting Information). It shows that Au10-Au15 are observed in both fractions, but no Au22-Au25 are observed in PF10mM. Combining the MS results (Figure S3) and electropherograms Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

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(Figure S2), Au NC peaks 1-6 and 7, 8 are assigned to Au10Au15 and Au22-Au25, respectively. Although it is possible that small Au clusters are fragmented from the large Au clusters in the MALDI-mass spectra, the close similarity of the photophysical properties (absorption and emission) between our Au NC fractions and the glutathione-protected Au NC supports that our as-prepared Au NC product contains Au10, Au11, Au12, Au15, Au22, Au25, Au29, Au33, Au35, Au38, Au39, and Au50. In addition, Au10-Au15 NCs can be separated from the Au10-Au50 polydisperse Au NC product using our electrolyte-induced fractionation method. Finally, most MS spectra produced an intense peak at ∼1436 Da (not shown), corresponding to Au4(NAC)4 fragmented from the larger core Au NCs, which are consistent with the results obtained by Gies et al.25 Au NCs are more likely to be fragmented in the form of Au tetramer during MS analysis. CONCLUSION In summary, a simple method for fractionating water-soluble Au NCs from a polydisperse Au NC product has been proposed. The electrolyte-induced fractionation depends on the electrolyte concentrations and the lipophilicity of the fractionated solution. (25) Gies, A. P.; Hercules, D. M.; Gerdon, A. E., Cliffel, D. E. J. Am. Chem. Soc. 2007, 129, 1095-1104.

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Nanoclusters of Au10, Au11, Au12, and Au15 can be separated from the polydisperse Au10-Au50 product and analyzed by CZE-DAD and LIF. The cluster sizes of the fractions were further confirmed by the mass spectrometric method. The hypsochromic shift of the photoemission spectrum and finelike absorption feature have been revealed after the removal of large core Au NCs by fractionation. The electrolyte-induced fractionation is general and can provide a simple and efficient method to fractionate watersoluble Au NCs so that their size-dependent phenomena and properties can be studied. ACKNOWLEDGMENT The work described in this paper was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project HKBU 200606). C. K. Lo would like to acknowledge Hong Kong Baptist University for a postgraduate studentship. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review October 17, 2007. Accepted February 6, 2008. AC702135Z