Mass Spectrometric Identification of Silver Nanoparticles: The Case of

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Mass Spectrometric Identification of Silver Nanoparticles: The Case of Ag32(SG)19 Jingshu Guo, Santosh Kumar, Michael Bolan, Anil Desireddy, Terry P. Bigioni, and Wendell P. Griffith* Department of Chemistry, University of Toledo, Toledo, Ohio 43606, United States S Supporting Information *

ABSTRACT: Mass spectrometry has played a key role in identifying the members of a series of gold clusters, which has enabled the development of magic-number cluster theory. The successes of the gold cluster system have yet to be repeated in another metal cluster system, however. Silver clusters in particular have proven to be challenging due to their relative instability compared with gold clusters. Using the wellcharacterized gold nanocluster, Au25(SG)18, we present optimized electrospray ionization mass spectrometry (ESIMS) instrumental parameters for the maximal transmission of the intact cluster. Parameters shown to have the largest effect on intact cluster transmission/detection include trap and transfer collision energy, source temperature, and cone gas flow rate. Herein we describe a general strategy to acquire mass spectra of fragile metal clusters with reliable mass assignments. By also optimizing sample solution conditions, high-quality ESI mass spectra of a prototypical silver:glutathione (Ag:SG) cluster were obtained without significant fragmentation. By using gentle conditions and solution conditions designed to stabilize the clusters, fragmentation was dramatically reduced and mass spectra with isotopic resolution were measured. Using this strategy, we have made the first formula assignment for a ligand-protected Ag cluster of Ag32(SG)19.

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Precise mass assignments for larger Ag clusters have yet to appear in the literature.18−21 The lower stability of Ag clusters has proven to be a major limitation in mass spectrometric analyses, leading to assignments for only small Ag clusters,22−26 although some of these may be fragments of larger clusters. Clearly new mass analysis strategies are needed for rapid progress to be made in the identification and characterization of fragile metal cluster species. Here we describe a general strategy that enables the acquisition of high-quality mass spectra of fragile metal clusters that are needed to make reliable mass and formula assignments. By optimizing ESI-MS conditions and using solution conditions that stabilize the clusters, using a sliver:glutathione (Ag:SG) cluster as a model, fragmentation was dramatically reduced and mass spectra of the intact cluster with isotopic resolution were recorded. Using this strategy, we have made the first formula assignment for a large ligand-protected silver magic-number metal cluster: Ag32(SG)19.

agic-numbered clusters are being intensely studied as models for molecular precision in nanostructures.1−13 For example, a family of Au clusters has been identified wherein each family member has a precise number of metal atoms in their core and ligands in their protective outer shell.9−14 With a better understanding of such molecular precision and nanostructure stability, it may be possible to design materials with very specific properties for a variety of applications.15 Mass spectrometry has played a pivotal role in the characterization of magic-numbered Au clusters, giving the most valuable information short of complete structure determination by single-crystal X-ray diffraction (SC-XRD). Although laser desorption ionization and matrix-assisted laser desorption ionization (MALDI) have advanced the field, electrospray ionization (ESI) has been required to determine the molecular formulas for the family of Au clusters3−7,16,17 due to its gentler ionization. Thus far, the structures of only three members of the Au cluster family have fallen to total structure determination by SC-XRD,10−13 underscoring the value of mass spectrometric formula determination. Attention is now turning to different materials in an attempt to generalize the nanostructure stability theories that were based on magic-numbered Au clusters as model systems. Thiolate-protected silver metal clusters are being developed as the first interesting complementary system,18−26 since chemical, electronic, and optical differences between gold and silver provide useful contrasts while their similarities accelerate progress in their synthesis and characterization. While Au clusters have proven to be relatively stable, enabling successful mass determinations, Ag clusters have not. © 2012 American Chemical Society

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EXPERIMENTAL SECTION Materials. All chemicals used in this study were of analytical grade or better. The following reagents were purchased from Fisher Chemicals: methanol, ammonium acetate, and acetic acid. All purchased chemicals were used without further purification. HPLC-grade water was used for all mass Received: March 1, 2012 Accepted: May 17, 2012 Published: May 17, 2012 5304

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(Waters Corp.) and operated in the negative ionization mode using homemade continuous-flow fused silica emitters. Instrument parameters were maintained at the following optimal values for each experiment unless otherwise indicated: capillary voltage, 1.8−3.0 kV; sampling cone, 35 V; extraction cone, 4 V; cone gas, 45 L/h; trap collision energy, 0.5 V; transfer collision energy, 1.0 V; source temperature, 40 °C; desolvation temperature, 120 °C. Calibration was performed externally in the positive ionization mode in the range 400 ≤ m/z ≤ 4000 using a solution of sodium cesium iodide. Mass spectra were an average of approximately 1400 scans and processed using Masslynx 4.1 software (Waters Corp.). All mass spectra were collected in V-mode, except for Figure 3 (of main text), which was collected in W-mode for increased resolution and detection of the isotopic distribution. Isotopic distributions were simulated using the freeware, mMass, version 4.0 (Copyright 2005 by Martin Strohalm).27 All Au25(SG)18 and Ag:SG band 6 samples were diluted to a concentration of approximately 0.5 mg/mL in a solution of 50% methanol in water. Band 6 Ag:SG clusters were also diluted into solutions of 5 mM ammonium acetate containing 50% methanol at the pH indicated. Note that here pH corresponds to the pH of the 10 mM ammonium acetate solution before dilution in methanol. Best conditions (source temperature, cone gas flow rate, and trap/transfer collision energy) were first found for Au25(SG)18, due to its superior stability. Parameter values were varied to show the effect of either one or a combination of the ion source parameters as shown in Supplementary Table S1 in the Supporting Information. Mass spectra for these conditions are shown in Supplementary Figure S2 in the Supporting Information. The best conditions for Au25(SG)18 (trace H) were subsequently used for analysis of the Ag:SG band 6 clusters.

spectrometry analyses. For all other purposes, distilled deionized water (18.2 MΩ cm) was used. Synthesis and Purification of Nanoclusters. The synthesis and purification of Au:SG and Ag:SG clusters was carried out as previously described. In short, both Au:SG and Ag:SG clusters were synthesized by simple reduction of gold and silver salts with aqueous sodium borohydride solution in the presence of glutathione ligand. The metal salt compounds (0.25 mmol), either AgNO3 or HAuCl4, were dissolved in water followed by the addition of a 4-fold molar excess of glutathione (GSH). The resultant silver thiolate suspension was cooled in an ice bath for 30 min. A 10-fold molar excess of cooled NaBH4 was added dropwise to the ice-cold reaction mixture at a stirring rate of ∼1100 rpm. The reaction mixture was kept stirring for an additional 1 h after the addition of all of the NaBH4 and its volume subsequently reduced to ∼5 mL in a rotary evaporator without heating. Ag:SG and Au:SG clusters were precipitated by addition of ∼20 mL of methanol. The resultant precipitate was washed 3 times with methanol through ultrasonic dispersion-centrifugation to remove unreacted material.20 Purification of Ag:SG and Au:SG clusters was achieved using polyacrylamide gel electrophoresis (PAGE) with homemade gels: 30% acrylamide resolving gels with dimensions 20 cm × 20 cm × 1.5 mm and 4% acrylamide stacking gels. Gels were run on a Thermo Scientific vertical electrophoresis system (P10DS). A Tris/glycine electrolyte buffer was used. Particle loading was 1 mL of an approximately 40 mg/mL solution, which was run at constant 200 V with cooling (coolant temperature, 0 °C). Band 6 was excised from the PAGE gels, crushed, soaked in water, and refrigerated for 2−4 h while the clusters diffused out from the gel. The resultant colored solution was centrifuged to remove the remaining pieces of gel. The supernatant was filtered through a 0.22 μm syringe filter, concentrated with a 3 kDa cutoff filter, and the solution was evaporated to dryness in an Eppendorf Vacufuge Concentrator Speed-vac. UV−Visible Spectrophotometry. For UV−visible spectrophotometry measurements (depicted in Supplementary Figure S1A in the Supporting Information), Ag:SG clusters were extracted from the PAGE gel directly into a 100 mM ammonium acetate buffer solution. The solution was split into four equal parts, each of which was adjusted with acetic acid to pHs of 3, 4, 5, and 6. The solutions were left to age for 6 days under ambient conditions. Absorption spectra of the different pH Ag:SG solutions were recorded using a Nicolet Evolution 300 spectrophotometer (Thermo Electron Corp.). Measurements were made over the course of 6 days to monitor changes in concentration due to aging, as shown in Supplementary Figure S1 in the Supporting Information. While the absorbance of all solutions decreased during this time, the pH 5 solution showed the least degradation and was therefore chosen for mass spectrometric analysis. Additional UV−visible spectrophotometric measurements (Supplementary Figure 1B in the Supporting Information) were carried out on the Band 6 Ag:SG clusters solutions that were prepared in the same way as used in the ESI-MS data collection: at a concentration of approximately 0.5 mg/mL in 50% methanol in water, 50% methanol in 5 mM ammonium acetate pH 7, and 50% methanol in 5 mM ammonium acetate pH 5. Mass Spectrometry. All mass spectrometry data were collected on a Synapt HDMS quadrupole-time-of-flight ion mobility mass spectrometer equipped with a nanospray source

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RESULTS AND DISCUSSION An ESI mass spectrum for the gold-glutathione cluster Au25(SG)18 acquired under typical ESI conditions is shown in Figure 1A. A clear charge state distribution was observed from

Figure 1. (A) ESI-MS of Au25(SG)18 clusters acquired using typical ESI instrumental parameters, showing four charge states for the intact clusters. (B) ESI-MS of band 6 Ag:SG clusters acquired under the same instrumental conditions as in part A, showing only fragments with no clear charge state distribution. See Table 1 for the parameter values. 5305

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[M − 4H+]4− through [M − 8H+]8−. Deconvolution of charge states 4− through 7− provided a mass of 10 438 Da for the intact Au cluster, consistent with the calculated mass of Au25(SG)18. Although a significant fragmentation background was found at high mass, the most abundant species were fragments at m/z 501.8 and 1004.6, corresponding to Au(SG) and Au2(SG)2. The same typical conditions were applied to the analysis of Ag:SG magic-number clusters. The sixth band from the gel electrophoresis of this family (band 6) was similar in size to Au25(SG)18 and was therefore used in this study.20 For ESI analysis of purified band 6 in a solution of 50% methanol in water (typical solvent used in ESI MS analyses for glutathione conjugates), no intact clusters were detected. Large Ag:SG clusters were identified in the same size range as Au25(SG)18 but no distinct charge state distribution could be observed nor mass assignment made (Figure 1B). The most abundant ion intensities in the mass spectrum were assigned to large fragments ranging from Ag24(SG)15 through Ag31(SG)19 with charge states ranging from 3− to 5−, indicating that Ag:SG clusters are significantly more fragile than Au:SG clusters and require more delicate handling. In the past decade, significant progress has been made to improve MS detection of fragile noncovalent complexes in aqueous media.28,29 It was demonstrated that the ESI interface conditions used for labile species must be as gentle as possible to maintain the intact complex.30 Under typical ESI conditions, there is a delicate balance between desolvation and the dissociation of these complexes. It has been shown that using nanospray instead of conventional electrospray ionization results in a much gentler desolvation process and more reliable detection of labile complexes, since the smaller initial droplet size requires fewer and less energetic collisions for complete ion desolvation.31 Likewise, the addition of more volatile organic solvents, such as methanol, to aqueous analyte solutions also helps promote gentle desolvation. While nanospray sources and organic solvents can greatly facilitate desolvation, perhaps of the most important variables that affect the transmission and consequent detection of these gas phase complexes are source temperature, ion activation, and collisional cooling of ions.32 In our initial experiments, these parameters were tuned for gentle detection of Au25(SG)18 and were then used in the analysis of the band 6 Ag:SG clusters. First, the ion source temperature was decreased from 80 to 40 °C, which increased the intensities of peaks corresponding to intact clusters by approximately 2-fold relative to fragment ion peaks (Supplementary Figure S2B in the Supporting Information). Next, collisional cooling was improved by increasing the cone gas flow rate from 0 to 45 L/h, since it is known to reduce fragmentation.33 This resulted in an even more marked increase in intact cluster peak intensities, making them the principal species in the mass spectrum (Supplementary Figure S2C in the Supporting Information). Reducing the trap/transfer collision energies (CE) from 6.0/4.0 V to 0.5/ 1.0 V had the largest effect on reducing fragmentation (Supplementary Figure S2D in the Supporting Information). Decreasing the trap CE relative to the transfer CE reduces collision-induced dissociation and allows for only low-energy collisions, which are necessary to remove weakly bound species such as solvent or adducts. The data shown here emphasize the importance of careful tuning of these parameters for reliable mass spectrometric analyses of fragile metal clusters.

Combining the lower ion source temperature with either the decreased trap/transfer collision energies (Supplementary Figure S2F in the Supporting Information) or the higher cone gas flow rate (Supplementary Figure S2G in the Supporting Information) showed the expected improvement in intact cluster peak intensity. The largest improvement was the combination of higher cone gas flow rate and lower trap/ transfer collision energies (Supplementary Figure S2E in the Supporting Information). The ion source temperature was found to have the smallest effect on intact cluster detection. A comparison of best conditions and typical instrumental parameters is provided in Table 1. Table 1. Comparison of Best Conditions and Typical Parameters instrumental parameter

best conditions

typical value

trap collision energy (V) transfer collision energy (V) source temperature (°C) cone gas flow rate (L/h)

0.5 1.0 40 50

6.0 4.0 80−120 0

While improved ESI conditions resulted in a significant reduction in fragmentation and an overall improvement of the mass spectrum for Au25(SG)18 (compare Figure 1A and Supplementary Figure S2H in the Supporting Information), applying the same strategy to the band 6 Ag:SG clusters resulted in a dramatic transformation of the mass spectrum (compare Figures 1B and 2A). Fragmentation was significantly

Figure 2. Negative ESI mass spectra of band 6 Ag:SG clusters electrosprayed from (A) 50% methanol in water, (B) 50% methanol in 5 mM ammonium acetate pH 7, and (C) 50% methanol in 5 mM ammonium acetate pH 5. Fragments are singly deprotonated ions: Ag2(SG), Ag2(SG)2, and Ag3(SG)2 refer to [Ag2(SG) − H+]−, [Au2(SG)2 − H+]−, and [Au3(SG)2 − H+]−, respectively.

suppressed and a charge state distribution from [M − 3H+]3− through [M − 6H+]6− became immediately apparent, making mass assignment possible. The majority of the ions were found in the [M − 4H+]4− state, which appears to be the preferred charge state of these Ag:SG clusters. The remaining fragmentation produced a set of peaks for each charge state 5306

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as well as small fragments at m/z 517.82, 824.95, and 931.86, which corresponded to Ag2(SG), Ag2(SG)2, and Ag3(SG)2. Solution conditions were also considered for Ag:SG cluster mass analysis. Solution phase spectrophotometric studies found that the stability of band 6 clusters in ammonium acetate varied with pH (Supplementary Figure S1 in the Supporting Information). Although cluster concentrations decayed at all pHs, ammonium acetate solutions at pH 5 were found to be significantly more stable than other pHs. This suggested that ammonium acetate solutions could benefit mass analysis. Indeed, ESI mass spectra of methanolic solutions of Ag:SG clusters with aqueous ammonium acetate at pH 7 (Figure 2B) showed improved stability over methanolic solutions in water. This was manifested in the marked suppression of smaller fragments at m/z 520 and 827, which corresponded to Ag2(SG) and Ag2(SG)2. A concomitant suppression of larger cluster fragments was also observed, which narrowed the ion distribution for the −4 charge state but also increased the mass for the most abundant species by one Ag atom. Decreasing the pH from 7 to 5 led to a further reduction in fragmentation, narrowing the ion distributions for both the 4− and 5− charge states (Figure 2C). This increased the mass for the most abundant species by one Ag atom for each charge state, but crucially, this also resulted in one mass peak emerging as a compelling candidate for the parent ion mass. The [M − 4H+]4− peak at m/z 2320 and the [M − 5H+]5− peak at m/z 1856 both increased in intensity to become the dominant ion species while the corresponding fragment peaks one Ag atom lower in mass were suppressed. However, ion species occurring at higher mass did not increase in intensity with the improvement in conditions, indicating that they were not related to the parent ion. The deconvoluted mass for the most abundant species in Figure 2C was 9279 Da (Supplementary Figure S4 in the Supporting Information). On the basis of this mass, a formula assignment of Ag32(SG)19 has been made. To verify this formula assignment, the flight path was doubled (W-mode) to obtain isotopic resolution. The measured mass distribution was compared with the simulated isotopic distributions for the 4− charge state of Ag32(SG)19. The isotopic distributions were found to be in good agreement (Figure 3), thus confirming the molecular formula assignment. One interesting point to note is that in comparison to the Au25(SG)18 nanoparticle, in Ag32(SG)19 an increase in 7 metal atoms in the core results in an increase of only a single ligand.

This highlights the possibility that unlike the spherical structure of the Au25(SG)18 species,12 the Ag32(SG)19 nanoparticle may be anisotropic. While the solution composition was critical in obtaining high-quality ESI mass spectra of the parent ion, the precise nature of the interactions is unclear. The improved stability at pH 5 was consistent with the solutions studies, which may indicate that charge stabilization was important. The presence of ammonium acetate also appears to play a role, however, since solutions with pH 7 ammonium acetate produced better ESI mass spectra than solutions without. This could have been due to increased ionic strength of the solution. It is important to note that we were not able to collect ESI mass spectra at pH > 7 or pH < 5 due to precipitation of the nanoparticles, indicating a relatively narrow pH range for stability. UV−visible spectra of these solutions (Supplementary Figure S1B in the Supporting Information) show a marked increase in the intensity of the absorption peak and hence stabilization of the Ag:SG nanoparticles in pH 5 ammonium acetate, which is in good agreement with the ESI-MS results. Curiously, overnight aging of the Ag:SG cluster solutions resulted in a cleaner mass spectrum with improved signal-tonoise ratio. Clusters were aged at room temperature in 50% methanolic solutions of 5 mM ammonium acetate at pH 5. While the exact reasons for the observed improvements are not clear, it is possible that aging allowed time for less stable species, present as impurities, to decay. This would have enriched the solution in the most stable component, namely, the band 6 magic number cluster.

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CONCLUSION

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ASSOCIATED CONTENT

We demonstrate a potentially general strategy to overcome the inherent challenges of mass analysis of aqueous ligandprotected metal clusters, in particular for those that are fragile relative to the well-characterized Au25(SG)18 clusters. Gentle conditions for detection of metal clusters involved decreased source temperature, decreased trap/transfer collision energies, and increased cone gas flow rate for improved collisional cooling. The nature of the solvent used in the analysis was also shown to have a significant effect on the stability of Ag:SG clusters and on the predominant species detected by ESI-MS. On the basis of these observations, we have made the first assignment of Ag32(SG)19 for the molecular formula of the band 6 Ag:SG cluster. While this work is a key step to enable the full characterization of the family of Ag magic-number clusters, the general strategy could also be applied to a wide range of cluster compounds. This manuscript lays the foundation for successful ESI-MS analyses of a range of less stable metal cluster systems.

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

Figure 3. Comparison of the experimental (black trace) and simulated (bars) isotopic distributions for the 4− charge state of Ag32(SG)19. The flight path was doubled (W-mode). The simulated distribution was scaled for clarity and shifted −0.15 m/z to correct for a −65 ppm difference in mass measurement due to external calibration.

*Address: Wendell P. Griffith, Department of Chemistry, MS602, University of Toledo, 2801 W. Bancroft Street, Toledo, OH 43606. E-mail: Wendell.Griffi[email protected]. Phone: (419) 530-7964. Fax: (419) 530-4033. 5307

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Notes

(31) Benesch, J. L.; Ruotolo, B. T.; Simmons, D. A.; Robinson, C. V. Chem. Rev. 2007, 107, 3544. (32) Potier, N.; Rogniaux, H.; Chevreux, G.; Van Dorsselaer, A. Biol. Mass Spectrom. 2005, 402, 361. (33) Harkness, K. M.; Hixson, B. C.; Fenn, L. S.; Turner, B. N.; Rape, A. C.; Simpson, C. A.; Huffman, B. J.; Okoli, T. C.; McLean, J. A.; Cliffel, D. E. Anal. Chem. 2010, 82, 9268.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by start-up funds to Wendell P. Griffith from the University of Toledo and partially supported by NSF Grant CBET-0955148 to T.P.B.. The authors would also like to thank Dr. Dragan Isailovic for useful discussions.

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