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Laser Desorption and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of 29-kDa Au:SR Cluster Compounds T. Gregory Schaaff*
Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6131
Positive and negative ions generated by laser-based ionization methods from three gold:thiolate cluster compounds are mass analyzed by time-of-flight mass spectrometry. The three compounds have similar inorganic core masses (∼29 kDa, ∼145 Au atoms) but different n-alkanethiolate ligands associated with each cluster compound (Au:SR, R ) butane, hexane, dodecane). Irradiation of neat films (laser desorption/ionization) and films generated by dilution of the cluster compounds in an organic acid matrix (matrix-assisted laser desorption/ ionization) with a nitrogen laser (337 nm) produced distinct ion abundances that are relevant to different structural aspects of the cluster compound. Laser desorption/ionization of neat Au:SR compound films produces ions consistent with the inorganic core mass (i.e., devoid of original hydrocarbon content). Matrix-assisted laser desorption/ionization produces either ions with m/z values consistent with the core mass of the cluster compounds or ions with m/z values consistent with the approximate molecular weight of the cluster compounds, depending on ionization conditions. The ion abundances, and ionization conditions under which they are detected, provide insight into desorption/ionization processes for these unique cluster compounds as well as other analytes typically studied by matrix-assisted laser desorption/ ionization. Gold:thiolate (Au:SR) cluster compounds (or monolayer protected cluster compounds) constitute a special subset of metallic nanostructures that have been the subject of numerous studies in recent years.1 Conceptually, these compounds consist of a dense metallic gold core surrounded by a shell of thiolate ligands (See Chart 1.). The measured optical properties are dominated by the electronic structure associated with metallic bonding within the inorganic core, while the gross chemical properties are derived with the organic (or biologic) ligands attached to that core. Similar metallic and semiconductor cluster compounds are finding applications in biologic imaging, such as strongly scattering centers * Phone: (865) 574-2297. Fax: (865) 574-9771. E-mail:
[email protected]. Current address: BWXT Y-12, P.O. Box 2009, MS 8189, Oak Ridge, TN 378218189. (1) Templeton, A. C.; Wuelfing, M. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. 10.1021/ac0353482 CCC: $27.50 Published on Web 10/06/2004
© 2004 American Chemical Society
for electron microscopy2,3 or bright fluorescent microscopy4-6 probes. Since the optical and electronic properties of cluster and nanocrystal compounds are inexorably linked to the size of the inorganic core, many future applications rely on the ability to synthesis and isolate compounds with either a narrow core size distribution or those that are molecularly pure (i.e., one single structure). Giant (nanometer-scale) cluster compounds have been isolated for selected systems recently (e.g., Pd145 metallic clusters7 and semiconductor Ag2S cluster compounds8). However, due to their inherent compositional and structural complexity, these types of compounds represent a significant challenge for routine analytical chemical techniques, even those developed for other macromolecular systems. Because chemical properties are derived from the organic ligands, one of the unique aspects of the Au:SR compounds is the ability to isolate and accumulate cluster compounds with distinct inorganic core sizes through fractionation,9-11 chromatography,11-13 supercritical extraction,14 and electrophoretic methods.15 As a result of such separations, it was possible to map the evolution of optical properties from bulklike (broad plasmon resonance excitation)10 to molecular-like (discrete electronic transitions)11,15 metallic electronic structure. In addition, other interesting properties have been discovered for these cluster (2) Hainfeld, J. F.; Furuya, F. R. J. Histochem. Cytochem. 1992, 40, 177-184. (3) Powell, R. D.; Halsey, C. M. R.; Hainfeld, J. F. Microsc. Res. Technol. 1998, 42, 2-12. (4) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759-1762. (5) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (6) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018. (7) Tran, N. T.; Powell, D. R.; Dahl, L. F. Angew. Chem., Int. Ed. 2000, 39, 4121-4125. (8) Wang, X. J.; Langetepe, T.; Persau, C.; Kang, B. S.; Sheldrick, G. M.; Fenske, D. Angew. Chem., Int. Ed. 2002, 41, 3818-3822. (9) 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. (10) Alvarez, M. M.; Khoury, J. T.; Schaaff, T. G.; Shafigullin, M. N.; Vezmar, I.; Whetten, R. L. J. Phys. Chem. B 1997, 101, 3706-3712. (11) Schaaff, T. G.; Shafigullin, M. N.; Khoury, J. T.; Vezmar, I.; Whetten, R. L.; Cullen, W. G.; First, P. N.; GutierrezWing, C.; Ascensio, J.; JoseYacaman, M. J. J. Phys. Chem. B 1997, 101, 7885-7891. (12) Song, Y.; Jimenez, V.; McKinney, C.; Donkers, R.; Murray, R. W. Anal. Chem. 2003, 75, 5088-5096. (13) Jimenez, V. L.; Leopold, M. C.; Mazzitelli, C.; Jorgenson, J. W.; Murray, R. W. Anal. Chem. 2003, 75, 199-206. (14) Clarke, N. Z.; Waters, C.; Johnson, K. A.; Satherley, J.; Schiffrin, D. J. Langmuir 2001, 17, 6048-6050. (15) Schaaff, T. G.; Whetten, R. L. J. Phys. Chem. B 2000, 104, 2630-2641.
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Chart 1
compounds, which also show strong size dependencies or quantum size effects (e.g., electrochemical charging of the cluster core,16,17 chiroptical effects in gold clusters with biologically derived ligands,15,18 and solid-state molecular crystal structure19). Laser desorption/ionization mass spectrometry has been highly efficient for analyzing the core mass of the cluster compounds to provide both rapid monitoring of size separation techniques and optimization of reaction parameters to produce specific cluster compounds in high yield and the qualitative observations regarding further reactions of the cluster compounds.20,21 Owing to the central role of laser desorption/ionization (LDI)-MS, many reports have differentiated the separated cluster compounds by their respective core masses. For example, 29kDa Au:SC4 refers to a gold cluster compound that, upon irradiation with UV irradiation, produces a group of ions centered at m/z 29 000 and has an associated ligand shell composed of butanethiolate. While LDI-MS mass spectrometry has served a central role in isolating Au:SR cluster compounds, implementation of low-fragmentation ionization methods (e.g., electrospray ionization and matrix-assisted laser desorption/ionization, MALDI) has only achieved sporadic success. While gold:thiolate cluster compounds have received increased attention in recent years due to the ease of preparation, the first analysis of gold cluster compounds by mass spectrometry was performed on gold:phosphane cluster compounds by McNeal and co-workers using 252Cf-plasma desorption mass spectrometry.22 (16) Chen, S. W.; 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. (17) Chen, S. W.; Murray, R. W. J. Phys. Chem. B 1999, 103, 9996-10000. (18) Schaaff, T. G.; Knight, G.; Shafigullin, M. N.; Borkman, R. F.; Whetten, R. L. J. Phys. Chem. B 1998, 102, 10643-10646. (19) Whetten, R. L.; Shafigullin, M. N.; Khoury, J. T.; Schaaff, T. G.; Vezmar, I.; Alvarez, M. M.; Wilkinson, A. Acc. Chem. Res. 1999, 32, 397-406. (20) Alvarez, M. M.; Khoury, J. T.; Schaaff, T. G.; Shafigullin, M.; Vezmar, I.; Whetten, R. L. Chem. Phys. Lett. 1997, 266, 91-98. (21) Schaaff, T. G.; Shafigullin, M. N.; Khoury, J. T.; Vezmar, I.; Whetten, R. L. J. Phys. Chem. B 2001, 105, 8785-8796. (22) McNeal, C. J.; Winpenny, R. E. P.; Hughes, J. M.; MacFarlane, R. D.; Pignolet, L. H.; Nelson, L. T. J.; Gardner, T. G.; Irgens, L. H.; Vigh, G.; J. P. Fackler, J. Inorg. Chem. 1993, 32, 5582-5590.
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From these studies, three to four molecular-like ions were observed for a class of gold-phosphane cluster compounds, which had previously been thought to contain a single component. Recently, one such compound has been separated in high purity by low-pressure chromatographic techniques and analyzed by matrix-assisted laser desorption/ionization mass spectrometry.23 In addition, other metallic-phosphane cluster compounds have recently been studied by electrospray ionization mass spectrometry.24 Despite many refinements in the methods for preparation and isolation (or separation) of the gold-thiolato metal cluster compound materials, they have never been conclusively demonstrated to exist as molecularly defined substances (e.g., singlecrystal structure determination). However, previous studies allude to a molecular system composed of cluster species with nearly identical composition and molecular weight. Clearly, an advanced mass spectrometry investigation, utilizing ultrasensitive and ultrasoft (gentle) ionization methods, should enable a direct, rapid determination of the distribution of assemblies present after synthesis and isolation. This report presents (among other things) progress toward this goal and illuminates some of the mechanisms and challenges inherent to the (MA)LDI processes, as applied to such materials. Results from both LDI and MALDI mass spectrometry are presented for three gold cluster compounds with the same Au core mass: 29-kDa Au:SC4, Au:SC6, and Au:SC12. The abundance of structurally relevant ions under both LDI and MALDI conditions was dependent on both the irradiance delivered (on a single-shot basis) and the total power delivered to the sample. Unlike many other macromolecular structures, structurally relevant high m/z ions were produced from Au:SR cluster compounds under many ionization conditions (i.e., desorption from neat films or from clusters diluted in organic matrixes). The ability to detect these ions under many ionization conditions provides insight into likely (23) Gutierrez, E.; Powell, R. D.; Furuya, F. R.; Hainfeld, J. F.; Schaaff, T. G.; Shafigullin, M. N.; Stephens, P. W.; Whetten, R. L. Eur. Phys. J. D 1999, 9, 647-651. (24) Kawano, M.; Bacon, J. W.; Campana, C. F.; Winger, B. E.; Dudek, J. D.; Sirchio, S. A.; Scruggs, S. L.; Geiser, U.; Dahl, L. F. Inorg. Chem. 2001, 40, 2554-2569.
desorption/ionization processes for the gold cluster compounds. In addition, this unique system may provide further detail concerning proposed mechanisms for MALDI of typical biologic analytes. EXPERIMENTAL SECTION The 29-kDa Au:SR cluster compounds used for these studies were synthesized and separated using procedures described in detail elsewhere.21 Briefly, the cluster compounds were prepared by a procedure based on methods described by Brust et al.,25 which are optimized to yield (in high abundance) the compound that produces ions under LDI conditions centered at m/z 29 000 for gold:butanethiolate, gold:hexanethiolate, and gold:dodecanethiolate clusters (Au:SC4, Au:SC6, and Au:SC12, respectively). The compounds were separated by fractional crystallization, which is effected by slow addition of acetone to a concentrated toluene solution. Neat films of the mixture and separated cluster compounds (for LDI-MS) were prepared by pipeting 1 µL of a concentrated solution (5 mg/mL in toluene) onto a sample plate and allowing the solution dry under ambient conditions. Separation of the compounds was monitored by LDI-MS, as shown below. As described by many reports dealing with matrix-assisted laser desorption/ionization, the ability to cocrystallize the analyte in a matrix crystal is highly advantageous, requiring a common solvent. For this reason, methylene chloride (HPLC grade, Baker Scientific) was used as a common solvent for the cluster compounds and the organic matrixes. Four matrixes (SigmaAldrich) were tested: 3,5-dihydroxybenzoic acid (DHB), 3,5dimethoxy-4-hydroxycinnamic acid (sinapinic acid), 1,8,9-anthracenetriol (dithranol), and trans-3-indoleacrylic acid. Films of the matrix-diluted cluster compounds were generated by diluting 20 µL of a 10 µg/mL methylene chloride solution of Au:SR cluster compounds 1:1 (v/v) with a saturated matrix solution in methylene chloride. Of the four matrix molecules, sinapinic and trans-3indoleacrylic acid produced comparable mass spectra, while DHB and dithranol did not seem to reduce fragmentation of the Au:SR cluster compounds as well (i.e., most spectra were similar mass spectra obtained by irradiating neat films). MALDI mass spectra shown in this report are derived from ions produced by irradiation of cluster compounds diluted in a sinapinic acid matrix. Laser desorption and matrix-assisted laser desorption/ionization mass spectra were obtained using Perseptive Biosystems Voyager DE linear time-of-flight mass spectrometer operating in delayed extraction mode with an accelerating voltage of 20 kV. The postdesorption delay time and space-focusing conditions were optimized using laser-desorbed cations and anions from the neat film by resolving the m/z 32 spacing in the ions detected at m/z values centered at 29 000. While these ions could not be baseline resolved, the periodic spacing of m/z 32 could still be used to ensure optimum resolution conditions and the calibration was correct under different ionization conditions (e.g., LDI vs MALDI) for this region of the mass spectrum. The Voyager DE mass spectrometer is equipped with a VSL-337 ND pulsed N2 laser (Laser Science, Newton, MA) with a 4-ns pulse width at 337 nm. To determine the magnitude of the laser irradiance delivered to the sample under different computer(25) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802.
Figure 1. Low m/z (a) and high m/z (b) regions of negative ion laser desorption ionization mass spectrum obtained by irradiating a neat film of a mixture of gold:thiolate cluster compounds with N2 laser at 2.4 MW/cm2.
controlled settings, the beam was diverted outside the vacuum chamber with a 90% transmission prism into a J3-09 pyroelectric/ silicon Joulemeter (Molectron). The laser power settings in the instrument control software were changed, and the output from the Joulemeter (fluence) was monitored with a digital storage oscilloscope. The irradiance values listed below were calculated for a focus of 200 µm (diameter) on the sample plate and corrected for additional transmission losses due to the focusing lens and quartz window installed on the vacuum chamber. Reported values were obtained by averaging the measured fluence from 20 individual laser shots. RESULTS The Au:SR cluster compounds (where R ) butane, C4; hexane, C6; and dodecane, C12), produced different types of high m/z ions upon irradiation with a pulsed UV laser (N2, 337 nm) under various ionization conditions. To differentiate these two conditions in the results to follow, LDI refers to irradiation of neat (thick) films generated by depositing 1-2 µL of a concentrated (10-20 mg/mL) solution of Au:SR cluster compounds and MALDI refers to irradiation of cluster compounds dispersed within a sinapinic acid matrix. LDI mass spectra of gold:thiolate were obtained by irradiating neat films of the Au:SR cluster compounds, typically at an irradiance of 2-3 MW/cm2. Figure 1 shows different regions of the negative ion LDI mass spectrum generated from a mixture of Au:SR cluster compounds (R ) C12). Two distinct types of anions are produced when neat Au:SR films are irradiated. Ions in the low m/z region (Figure 1a) are extremely intense compared to those at higher m/z values (on the order of 10-100 times signal intensity) and can produce large background signals (due to Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
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detector relaxation) that extends to m/z ∼5000. In addition to the m/z 197 [Au]- anion, groups of anions are detected with nominal differences in m/z of 229. The lowest m/z ions in each group corresponds to the “bare” [AuNSM]- anion (see labels in Figure 1a). Higher m/z ions in each group are detected at m/z values that are separated by either m/z 12 or 13, which presumably corresponds to addition of elemental carbon or the combination of carbon and hydrogen. The relative intensity of these ions increases with increasing laser irradiance and are invariant with respect to ligand composition. Therefore, the remainder of this report concentrates on the higher m/z ions found at lower relative abundance that have a direct relation to the structure of the cluster compound as it exists in the condensed phase. Similar to previous reports,9,11,20 the higher m/z region of LDI mass spectra shown in Figure 1b is composed of peaks corresponding to negative ions in distinct regions centered about m/z 8000, 15 000, 22 000, and 29 000. The spectrum represents the average of 512 individual mass spectra obtained at an irradiance of 4.1 MW/cm2. To lower the contribution of noise arising from the low m/z ions, a low-mass cutoff was used to blank the mass spectrum below m/z 4000. The mass spectrometer resolution (e.g., space focus and delayed extraction) was optimized for the mean m/z shown in the Figure (m/z ∼15 000) to obtain the best resolution for the entire window. The effect of delayed extraction on mass resolution and measured ion abundances is well documented26 and has been found to produce LDI mass spectra from mixtures of Au:SR’s that do not always reflect the true abundances of components in the mixture being analyzed.27 Thus, when monitoring separations or optimizing reaction conditions, LDI mass spectra are acquired with static acceleration and fixed space focusing to obtain a qualitative, but more accurate representation of component abundance. When mixtures of Au:SR cluster compounds are irradiated, the negative ions (and positive ions, not shown) detected have periodic spacing from m/z ∼6000 until it is impossible to resolve the spacing due to instrumental resolution limitations. The ions detected can be described as having major and minor m/z spacing consistent with the composition of the inorganic cluster core. The minor spacing in each group of ions corresponds to an m/z difference of 32 (S). The major m/z spacing between the most abundant ion in each adjacent group corresponds to a difference of either 197 (Au) or 229 (AuS). The N, M labels in the inset correspond to ions having the general formula [AuNSM]. Ions of the same general AuNSM- composition were observed by Arnold and Reilly for the separated compound that produces negative ions centered at m/z 15 000.28 Abundant ions follow this general progression from approximately m/z 6000 until the m/z 32 spacing cannot be resolved. While Au is monoisotopic, S has four natural isotopes: 32S (95.02), 33S (0.75), 34S (4.21), and 36S (0.02). The resolution of the mass spectrometer is not sufficient to resolve the isotopic abundances; thus assignments and calibration were made using the exact mass of 197Au (196.966 55) and the average mass of S (32.065). (26) Reilly, J. P.; Colby, S. M. Anal. Chem. 1996, 68, 1419-1428. (27) Schaaff, T. G.Preparation and Characterization of Thioaurite Cluster Compounds. Ph.D. Dissertation, School of Chemistry and Biochemistry, Georgia Institute of Technology, 1998. (28) Arnold, R. J.; Reilly, J. P. J. Am. Chem. Soc. 1998, 120, 1528-1532.
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Figure 2. Positive (a) and negative (b) ion laser desorption/ ionization mass spectra (at 2.4 MW/cm2) from a Au:SC4 cluster compound after separation from smaller and larger size cluster compounds, as seen in Figure 1. The insets in (a) and (b) show the narrower m/z range centered at m/z 29 000. The peaks detected at higher m/z values correspond to dimer, trimer, etc., ions of the base ion at m/z 29 000.
After fractionation and isolation, the 29-kDa (core mass) Au: SC4 cluster compound was separated from its other compounds having disparate core masses. Figure 2 shows the negative and positive ion LDI mass spectra for this compound obtained with an irradiance of 4.1 MW/cm2. The spectra shown are the average of mass spectra obtained from 32 laser shots. The acquisition of 32 shots has been observed to aid in the ability to resolve the m/z 32 spacing. When a larger set of spectra are averaged, the m/z 197 and 229 spacing is resolved well (as seen in Figure 1b), but the m/z 32 spacing cannot be resolved in the m/z 30 000 region of the mass spectrum. This is likely due to slight changes in laser power, electronic jitter, or both, which can become more pronounced when averaging larger sets of spectra. Comparison of the positive and negative ion mass spectra indicates that similar groups of ions are produced at what seems to be approximately the same intensity in both positive and negative ion modes. As can be seen in the insets of Figure 2, the same general m/z 197 and 229 spacing is prevalent. The distribution of ion abundances starts as an abrupt rise at m/z ∼26 500, and ions at lower m/z values with the characteristic m/z 32 and 197 spacing are not detected below that cutoff. The distribution of ions reaches a maximum at m/z ∼29 000 and decreases until ions with the characteristic m/z 32 and 197 spacing are not detected (m/z 32 000). Peaks detected at higher m/z values correspond to multimers of the m/z 29 000 ions (i.e., 2 × 29 000, 3 × 29 000, etc.). These multimer ions have been observed up to 10 × 29 000 under negative ion operation and up to 8 × 29 000 under positive ion operation. Threshold irradiance for detection of positive ions from the Au:SC4 cluster compound was measured at 1.5 MW/cm2, which
Figure 3. Positive ion laser desorption/ionization mass spectra obtained from the separated cluster compound shown in Figure 2 at increasing irradiance (from top to bottom). The numbers above each mass spectrum correspond to irradiance in MW/cm2.
produces a broad, featureless peak (similar to the top spectrum in Figure 3) at a signal-to-noise level of 2. Figure 3 illustrates the changes in positive ion abundance as a function of irradiance. At slightly above threshold for production of positive ions from neat films, no m/z 32 or 197 spacing is observed in the ion abundances (1.7 MW/cm2, top spectrum). In addition, at this lower laser power, the peak apex is centered at m/z ∼30 000. As irradiance is increased, not only do the ion abundances start to show the characteristic m/z 32 and 197 spacing, but the apex of the ion abundances shifts to lower m/z values (∼29 000). At high irradiance (>5 MW/cm2), the m/z 32 spacing cannot be resolved and the peak apex has shifted still lower, to m/z ∼28 500. A similar dependence of ion abundances on irradiance was observed negative ions generated from neat films of the 29-kDa Au:SC4 cluster compounds. Similar to high-fragmentation conditions for MALDI, shown below, it should be noted that the abundance of ions detected at m/z ∼29 000 were relatively invariant with respect to chainlength of the thiolate ligand. For example, similar ion abundances were detected for both LDI-generated positive and negative ions from Au:SC4, Au:SC6, and Au:SC12 cluster compounds with core masses of 29 kDa. MALDI has been used to mitigate fragmentation of the Au: SR cluster compounds during ionization repeatedly with a number of typical organic matrix molecules. To date, the best matrix has been determined to be sinapinic acid. However, the use of trans3-indoleacrylic acid (typically used for MALDI of organic polymers) was found to have performance comparable to that of sinapinic acid. Unlike many other MALDI analytes, it appears that two desorption/ionization regimes exist for MALDI of Au:SR cluster compounds (high and low fragmentation), which are dependent on the incident irradiance and total irradiance delivered to the matrix/cluster mixture. Figure 4 shows the dependence of
Figure 4. Positive ion matrix-assisted laser desorption/ionization mass spectra obtained from the separated Au:SC4 cluster compound shown in Figure 2 at increasing irradiance (from top to bottom). The numbers above each mass spectrum correspond to irradiance in MW/ cm2. The * above spectra at 2.1-3.0 MW/cm2 corresponds to m/z 28 400.
mass spectra on the irradiance when the 29-kDa (core mass) Au:SC4 cluster compound is diluted in a matrix of sinapinic acid. Because it is well known that MALDI matrixes can produce socalled “hot spots” (where the signal is much stronger in specific areas of the sample), the mass spectra shown in Figure 4 were obtained by continuously repositioning the laser spot over the entire sample surface while acquiring data. At lower irradiance (below 3 MW/cm2), the abundances of ions centered at m/z 29 000 wwere similar to those measured from LDI-MS of neat films (top 3 spectra, Figure 4), though the ions detected correspond to slightly lower m/z values. At nearthreshold irradiance, the ions detected correspond to a broad, featureless peak with an apex at m/z 29 200, similar to the nearthreshold LDI from neat films but at lower m/z values. At slightly higher irradiance (2-3 MW/cm2), the m/z spacing is resolved, also consistent with resolution of these characteristic ions under LDI conditions at higher irradiance. While the m/z 32 spacing is not resolved to the degree found in LDI from neat films, the m/z 197 spacing is partially resolved for approximately three or four groups of ions. The first group of ions detected (i.e., the first peak in the progresses of m/z 197 spacing, denoted by * in Figure 4) corresponds to m/z 28 400. At 3 MW/cm2, the ions corresponding to m/z values between 28 000 and 30 000 remain relatively unchanged, while a distribution of ions corresponding to the broad featureless peak at m/z 32 000 increases in relative abundance. Above 3.0 MW/cm2, the low m/z peaks again appear featureless and the ions corresponding to m/z 32 000 are detected at much higher relative abundance. The absolute intensity of the ions detected does not change appreciably, but the relative signal-to-noise ratio increases slightly. Reproducible features are partially resolved, superimposed on the Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
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Figure 5. Positive ion matrix-assisted laser desorptionionization mass spectra obtained at 2.1 MW/cm2 from the Au:SC4, Au:SC6, and Au:SC12 cluster compounds (from top to bottom, respectively). Again, the * above the spectra corresponds to m/z 28 400, common among all three compounds.
broad peak centered at m/z 32 000, but resolution at this m/z range is not sufficient to make qualified assignments of ion composition, nor do the m/z values contain recognizable spacing (e.g., consistent with loss of whole ligand molecules from the ion). Ion abundances detected for the 29-kDa Au:SC6 and Au:SC12 cluster compounds showed similar irradiance-induced effects. Figure 5 shows the average of 32 mass spectra obtained at low irradiance (2.4 MW/cm2) from the three 29-kDa cluster compounds (top to bottom): Au:SC4, Au:SC6, and Au:SC12. With only slight differences, the three compounds produce remarkably similar groups of ions with m/z 197 spacing, but the m/z 32 spacing was not resolved for these compounds. While mass resolution limitations preclude unequivocal assignment of the ions detected, it is clear that the position of the first abundant group of ions (with the characteristic m/z 197 spacing) is measured at approximately the same m/z (28 400) for all three cluster compounds (denoted by * in Figures 4 and 5). The distribution of ions in this region is narrower than those detected with LDI. As seen in Figure 5, the rise in abundance under this ionization condition occurs at m/z 27 500, compared to m/z 27 000 for LDI (inset, Figure 1a), and the ion abundance returns to near baseline at m/z ∼31 000 (compared to m/z ∼32 000 for LDI). Upon repeated experiments to evaluate changes in ion abundances as a function of irradiance for the three compounds, it was apparent that the relative ion abundances were also affected by the total power delivered to the sample. Mass spectra obtained from repeated pulses delivered to dense sample areas (i.e., areas with large crystalline matrix structures) changed with subsequent laser shots, while those obtained from sparse or thin areas on the sample were consistently similar to those seen in Figure 5. Focusing the laser on an area in the matrix/cluster sample region, 6192 Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
Figure 6. Evolution of positive ion matrix-assisted laser desorption/ ionization mass spectra obtained from the Au:SC4 cluster compound at irradiance of 4.0 MW/cm2. The numbers above each mass spectrum correspond to the number of laser shots delivered to the matrix/analyte sample.
which appeared optically dense, and triggering the laser (off and on) manually generated mass spectra such as those shown in Figure 6. The irradiance used for the spectra in Figure 6 was 4.0 MW/cm2. The mass spectrum obtained in the first few laser shots is similar to both low-irradiance MALDI (Figures 4 and 5) and LDI from neat films. During these first few shots, the distribution of ions has an apex at m/z ∼28 000, but the characteristic m/z 197 spacing is only partially resolved across the distribution of ion abundances detected. After the first 10-20 laser pulses, the ion abundances centered at m/z 29 000 decreases and the higher m/z ions are detected at an increased relative abundance centered at m/z 32 000. At ∼30 laser shots, ions centered at m/z 29 000 and 32 000 are approximately the same abundance. Finally, at ∼75 laser shots, the ions at m/z 29 000 are completely suppressed and the ions detected are centered at m/z 32 000. In addition, initial mass spectra (first 10-20 shots) had ion abundances centered at m/z 58 000, 87 000, etc., corresponding to the multimers of the ions centered at m/z 29 000 as seen in LDI mass spectra obtained from irradiating neat films. The relative abundance of multimers remained constant with respect to the abundance of the ions at m/z 29 000. However, no ion abundances were detected at the m/z values corresponding to multimers of the ions centered at m/z 32 000 (see discussion below). After determining the changes in the abundance of desorbed ions under different laser conditions for the 29-kDa Au:SC4 compound, the other two 29-kDa cluster compounds were investigated using similar ionization conditions. Figure 7 shows the low-fragmentation MALDI mass spectra from three different 29-kDa cluster compounds (from top to bottom): Au:SC4, Au: SC6, and Au:SC12. The position of the peak apex and tailing edge changes consistent with the longer chain length ligands of the different cluster compounds. It is also interesting to note that the
Figure 7. Positive ion matrix-assisted laser desorption/ionization mass spectra obtained by irradiating the matrix/sample area at an irradiance of 4.0 MW/cm2 for 60-70 laser pulses and then acquiring mass spectra for 32 additional laser pulses. The mass spectra (from top to bottom) correspond to ions generated from the Au:SC4, Au: SC6, and Au:SC12 cluster compounds, which produced m/z ∼28 400 ions shown in Figure 5. The arrows in each mass spectrum correspond to the expected m/z for a molecular-type ion from an intact cluster compound with an Au:SR ratio of 2.57:1, as determined by elemental analysis.
full width half-maximum (fwhm) of the distribution of ions does not change appreciably with different chain lengths. A previously isolated 29-kDa Au:SC12 cluster compound was ionized by MALDI with 3,5-dihydroxybenzoic acid as a matrix.21 However, in that case, the ion distribution extended from m/z 28 000 to 40 000. In summary, the three 29-kDa Au:SR compounds (R ) C4, C6, and C12) produced nearly identical mass spectra both at low irradiance and during the initial irradiation of the matrix/cluster sample. The ions produced under this particular ionization condition were centered at m/z 29 000, with the first detected (resolved) ion being m/z 28 400 for all three compounds. For the Au:SC4, Au:SC6, and Au:SC12, higher irradiance or additional laser pulses produced higher m/z ions that were centered at m/z 32 000, 33 200, and 37 400, respectively. In previous reports, the Au:S ratio for the 29-kDa (core mass) Au:SR compounds was determined by elemental analysis to be 2.57:1.21 The arrows in Figure 7 denote the m/z value that would correspond to an intact ion assuming the peak at m/z 28 400 corresponds to the number of gold atoms in the cluster core. DISCUSSION While matrix-assisted laser desorption/ionization has proven extremely useful for the analysis of thermally labile macromolecules, the implementation of this “soft” ionization method to inorganic cluster compounds still faces significant challenges. Considering the structure of the cluster compounds (i.e., inorganic core surrounded by a hydrophobic monolayer), there are similarities to typical MALDI analytes (similar in size to proteins, chemical
properties similar to hydrophobic polymers). As would be expected for a complex molecular structure, many other subtle properties must be considered for application of MALDI to metallic cluster compounds. For example, the near-IR, visible, and ultraviolet optical properties are quite different from that of typical MALDI analytes. The extinction coefficient (at 337 nm) for the 29-kDa cluster compounds is on the order of that for the sinapinic matrix molecules in which they are dispersed. In addition, the hydrophobicity of the cluster compounds may preclude their dispersal into various matrixes. Overcoming these differences and understanding specific ionization processes in these complex compounds are required before soft ionization techniques can be applied to this class of cluster compounds, or more importantly, used to develop similar techniques for other types of cluster compounds (e.g., semiconductor and other metallic cluster compounds). The lack of mass spectrometry-based studies of cluster compounds is likely due to a number of factors, with one of the most important being sample purity of available cluster compounds. To draw on the biologic MALDI analogy, understanding and applying MALDI to metallic cluster compounds before isolation by cluster size would be similar to developing MALDI for proteins by starting with an extract of all proteins from a whole cell without any prior chemical separations. As shown in Figure S1 (Supporting Information), the changing abundances of ions as a function of laser irradiance and number of laser shots produce mass spectra with peaks superimposed from different size nanocrystal cores or, more detrimental, at m/z values that normally correspond to ions generated from LDI of neat films. Thus, without separations, it is impossible to determine which ions are produced under which conditions. Of the gold:thiolate cluster compounds, the compound that produces ions centered at m/z 29 000 by LDI is the best understood compound (structurally and electronically),21 which is why this compound (with different ligand molecules) was chosen for these studies. The measurement of ion abundances corresponding to the approximate core mass of the cluster compound is consistent with other analytical techniques more commonly used to analyze cluster sizes, e.g., X-ray diffraction,19 electron microscopy,11 and scanning probe microscopy.29,30 Under LDI conditions, the fwhm of the distribution of ions centered at m/z 29 000 is ∼2500. This in turn translates into a core diameter dispersion (typically determined by high-resolution electron microscopic analysis) of 1.67 ( 0.02 nm using the formula
Deq )
(
NAu
(π/6)d
)
(1/3)
(1)
which is based on the density (d ) 59 atoms/nm3) of gold in its native fcc structure. While the relative error associated with the mass spectrally derived diameter (0.02 nm) clearly cannot translate into a physical parameter, it does illustrate the precision associated with the measurement, which cannot be achieved by either electron microscopy or powder X-ray diffraction measurements. (29) Harrell, L. E.; Bigioni, T. P.; Cullen, W. G.; Whetten, R. L.; First, P. N. J. Vac. Sci. Technol. B 1999, 17, 2411-2416. (30) Bigioni, T. P.; Harrell, L. E.; Cullen, W. G.; Guthrie, D. E.; Whetten, R. L.; First, P. N. Eur. Phys. J., D 1999, 6, 355-364.
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From the seminal work by Tanaka and co-workers31 and others to follow,32,33 it is well documented that metallic colloids can effect liberation of large molecular biologic ions from a glycerol matrix to produce “fragmentation-free” mass spectra similar to those obtained routinely with organic matrixes. Bulk gold and silver also share similar photochemically driven reactions that occur under intense UV irradiationsselective cleavage of the S-C bond.34,35 The propensity to selectively cleave the S-C bond under UV irradiation provides a mechanism similar to those proposed for the irradiation of organic matrix crystals (i.e., violent disruption of a specific crystal structure into an expanding plume) to liberate molecular ions from biopolymers. Presumably, this violent disruption of the Au:SR cluster compounds can allow liberation of “intact” cluster cores. Thus, the detection of high m/z ions such as those shown in Figure 1 may be the consequence of the Au:SR clusters acting both as a poor matrix and an analyte. The mechanism for desorption and ionization of these large structurally relevant ions from neat films of Au:SR cluster compounds is likely similar to those proposed for MALDI. Presumably, the high m/z ions are generated in a dense, expanding plume populated by both high m/z ions and low m/z ions. Both fragmentation of these high m/z ions and aggregation with themselves (e.g., multimers in Figure 1b) and with low m/z ions (as shown in Figure 1a) likely occurs within this dense plume to add to the distribution of ions detected in the final LDI mass spectrum. Reactions within a dense plume (under LDI conditions) would be consistent with the narrower distribution of ions detected under certain MALDI conditions (plume is likely not as dense with MALDI prepared samples). The gold:thiolate cluster compounds are unique (compared to other thermally labile macromolecules) in that they produce structurally relevant high m/z ions under all ionization conditions. Many have reported on a phenomenon in MALDI in which it takes a few laser pulses to observe ions being produced from the matrix/analyte sample.36-38 This has been previously attributed to a so-called “cleaning off” effect, where protein molecules not incorporated in the matrix crystal or amorphous material is ablated from the face of well-cocrystallized matrix/analyte samples and protein ions are not formed. While this effect is difficult to observe directly in the analysis of proteins (because they form either metastable ions or no ions at all during this process), this effect is observed more clearly in MALDI of the cluster compounds. Considering the changes in mass spectra under different ionization conditions, it is likely that gold cluster compounds are present both as a surface (or amorphous) layer and cocrystallized within the matrix crystals. Even though the cluster compounds (31) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-156. (32) Lai, E. P. C.; Owega, S.; Kulczycki, R. J. Mass Spectrom. 1998, 33, 554564. (33) Schurenberg, M.; Dreisewerd, K.; Hillenkamp, F. Anal. Chem. 1999, 71, 221-229. (34) Lewis, M.; Tarlov, M.; Carron, K. J. Am. Chem. Soc. 1995, 117, 95749575. (35) Rieley, H.; Price, N. J.; Smith, T. L.; Yang, S. H. J. Chem. Soc., Faraday Trans. 1996, 92, 3629-3634. (36) Bolbach, G.; Riahi, K.; Spiro, M.; Brunot, A.; Breton, F.; Blais, J. C. Analusis 1993, 21, 383-387. (37) Perera, I. K.; Kantartzoglou, S.; Dyer, P. E. Int. J. Mass Spectrom. Ion Processes 1996, 156, 151-172. (38) Pittenauer, E.; Schmid, E. R.; Allmaier, G.; Puchinger, L.; Kienzl, E. Eur. Mass Spectrom. 1996, 2, 247-262.
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Figure 8. Illustration of ion formation of Au:SR cluster compounds dispersed in an organic matrix under high- and low-fragmentation conditions (left and right images, respectively). The left image corresponds to the processes that occur at low irradiance or initial irradiation at high-irradiance levels to produce spectra similar to those generated from neat films due to either a neat film on the matrix/ analyte crystal or an amorphous layer, which allows for extensive fragmentation. The right illustrates a situation in which the amorphous (or neat) layer has been ablated and the cluster compounds are ejected (or liberated) from the matrix/analyte crystal similar to other macromolecules.
and the matrix molecules are both soluble in methylene chloride, the differences in hydrophobicity can likely still cause a high degree of segregation during the evaporation of the methylene chloride solvent. For example, the Au:SR cluster compounds with R ) C4, C6, and C12 are only soluble in nonpolar solvents or slightly polar solvents. On a microscopic scale, cocrystallization with a highly polar molecule (e.g., an organic acid) would presumably produce either a highly amorphous solid or a neat film of the cluster compounds on the matrix crystal (See Figure 8.). At low irradiance, erosion of the amorphous or neat layer would presumably require many shots to finally reach a portion of the matrix/analyte crystal in which the clusters are adequately dispersed within the crystal structure. This cleaning-off period would be substantially less at higher irradiance, which is consis-
Figure 9. Positive ion matrix-assisted laser desorption/ionization mass spectra for the Au:SC4 cluster compound obtained by irradiating the matrix/sample area at an irradiance of 4.0 MW/cm2 for the initial 64 laser pulses (a) and the average of mass spectra from 32 subsequent laser pulses (b). The dimer ion of the ions centered at m/z 29 000 is detected at m/z 58 000 in the first 64 laser pulses. Upon suppression of the m/z 29 000 ions, a distribution of ions at m/z 16 000 is detected in the mass spectrum corresponding to final 32 laser pulses, presumably the doubly charged species of the group of ions producing the distribution centered at m/z 32 000.
tent with the observation that, under low irradiance (and the first few laser shots under high irradiance), the mass spectra obtained from the matrix/analyte sample is similar to that obtained from neat films. After the ablation of the neat film or amorphous material, the ions are instead generated from a well-cocrystallized matrix/analyte structure in which the cluster compounds are highly diluted to produce molecular-like ions. Because ions are formed under many ionization conditions, it is possible to probe the transition from neat film (or amorphous matrix/cluster crystallinity) with studies such as those shown in Figure 6. Several aspects of the complete mass spectra obtained in this intermediate stage provide additional information in support of ablation of a neat or amorphous film. Figure 9 shows an extended mass spectrum obtained at 4.0 MW/cm2 during the changeover from high- to low-fragmentation regimes. In addition to the two distributions of ions centered at m/z 29 000 (high fragmentation) and 32 000 (low fragmentation), two additional distributions of ions are detected centered at m/z 16 000 and 58 000. The ion at m/z 58 000 is the characteristic dimer ion produced under LDI conditions, but the m/z 32 000 ion does not produce a corresponding dimer at m/z 64 000. The peak at m/z 16 000 presumably corresponds to the doubly charged species of the ions that contribute to the peak detected at m/z 32 000. The detection of the two additional peaks centered at m/z 58 000 and 16 000 are consistent with the ablation of an amorphous or neat layer (Figure 8a) followed by desorption and ionization of
desorbed molecules ions from a diluted state within the organic matrix (Figure 8b). Complete mitigation of fragmentation during ionization still seems elusive for this class of macromolecular compounds, but the combination of different ionization conditions provides a consistent description of the molecular structure of the Au:SR cluster compounds. Considering the plume density is likely lowest for the high-fragmentation MALDI conditions (less propensity for broadening the distribution of ions due to aggregation with low m/z ions), this method would provide the more accurate determination for the core mass of the cluster compound at m/z 28 400 (though this number likely includes a small contribution from remaining sulfur). In addition to sharing a remarkably similar core mass, the number of ligands is also similar. Assuming the m/z 28 400 represents the lowest fragment of the inorganic core, the core size is estimated at 144 gold atoms. With this assumption, the number of ligands associated with all three compounds (i.e., mass difference between 28 400 and arrows shown in Figure 7) suggests all three compounds have ∼53-56 ligand molecules associated with the condensed-phase structure. While it is still not possible to unequivocally assign a true “molecular weight”, the approximate molecular weights of the Au:SC4, Au:SC6 and Au:SC12 cluster compounds is determined to be ∼33 500, 35 000, and 39 000, respectively. Of course, an alternative interpretation considers the mass distribution of ions produced by MALDI under both high and low fragmentations. The fwhm of ions produced under all MALDI conditions is approximately the same for all three compounds. That is, a similar distribution of ions is detected regardless of ligand or ionization conditions. Considering the distribution of ions in this manner would lead to the assumption that an “island of stability” exists for gold cluster compounds, which is centered at ∼145 atoms. This is also a plausible explanation, considering different theoretical models that have predicted a number of gold cluster structures of exceptional stability between 140 and 150 atoms.39,40 From this interpretation, the peak positions and widths can be treated statistically to determine level of “purity” or size/ molecular weight dispersion, somewhat analogous to a polymer system. Fitting the distribution of ion abundances under different ionization to a typical Gaussian distribution function can provide statistical information regarding constraints on the assembly composition. For this set of compounds, the core mass is 29 100 ( 800 Da, corresponding to a Au core number of 147 ( 4. Considering some sulfur content is still present, the apex estimate is likely skewed toward slightly higher core mass, and the distribution is likely larger than that in the intact cluster compound. The molecular weights and associated distributions of the Au:SC4, Au:SC6, and Au:SC6 compounds obtained by the same type of fitting are determined to be 32 000 ( 700, 33 000 ( 1100, and 37 000 ( 1000, respectively. Regardless of the interpretation (e.g., based on assumption of single core and fragmentation or a statistical treatment of the distribution of ion abundances), it is clear the overall molecular assembly is highly uniform in structure when the masses of the respective components are considered. (i.e., gold 197 Da and (39) Cleveland, C. L.; Luedtke, W. D.; Landman, U. Phys. Rev. Lett. 1998, 81, 2036-2039. (40) Cleveland, C. L.; Landman, U.; Schaaff, T. G.; Shafigullin, M. N.; Stephens, P. W.; Whetten, R. L. Phys. Rev. Lett. 1997, 79, 1873-1876.
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thiolate ligands C4 89 Da, C6 117 Da, and C12 201 Da). The similarity of the MW distributions to the core mass distribution though, brings into question this type of treatment. For example, if MALDI is producing intact ions from compounds with a distribution of core sizes, there is no (or only slight) statistical deviation in the number of ligand molecules associated with the different core sizes. It should also be noted that the approximate compositions extracted from the statistical approach (Au143-151SC433, Au143-151SC632-34, Au143-151SC1238-40) are both inconsistent with elemental analysis and counterintuitive (e.g., more SC12 ligands associated with assembly than SC6 or SC4). The single core and fragmentation discussed in the previous paragraph are both consistent with elemental analysis and intuitively attractive, considering the isolation of the Pd145 cluster compound by Tran and co-workers.7 CONCLUSIONS Cluster compounds are an increasingly important class of macromolecular structures. In addition to providing excellent model systems for understanding fundamental quantum effects in nanostructures, many applications could be potentially impacted by their further development: catalysis, sensor development, biologic tags, and molecular-scale electronics. Because the optical and electronic properties of clusters and nanocrystal compounds are inexorably linked to the core size of the compound, realization of these proposed applications requires that methods and techniques be developed to accurately synthesize and isolate the compounds with distinct inorganic core sizes. Accompanying this need for isolation of molecular-like structures on the nanometer scale, high-throughput analytical methods need to address separations and determination of purity, in much the same way as they are used for other macromolecular structures across chemistry and biology. The ability to quickly “size” the gold:thiolate cluster through monitoring ions generated by LDI-MS of neat films allowed for efficient optimization of synthetic parameters, as well as a convenient technique for monitoring size separations. There are two advantages to the mass spectrometry approach over traditional inorganic materials analytical methods (e.g., X-ray diffraction and TEM) for analysis of these nanostructured materials: speed and statistics. The typical time required to analyze the cluster compound by MALDI (or LDI) mass spectrometry is on the order of 2-5 min from sample preparation to data collection and analysis.
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The data obtained by mass spectrometry represent the statistical average of the complete ensemble of cluster compounds. While X-ray diffraction represents the statistical average of the compounds, it cannot provide information regarding the relative abundance of different cluster sizes in mixtures or offer qualitative information regarding the sample purity. For high-resolution electron microscopy, typical electron micrographs (and histograms generated from them) represent only a few hundred clusters, which could be considered a statistically valid representation of the entire ensemble on cluster compounds. However, the time in which the structurally relevant information is extracted from these two methods is usually on the order of hours, not minutes. With the increasing interest in nanostructured materials, there is a distinct need for efficient and reliable analytical tools for their analysis. Mass spectrometry, combined with ionization techniques developed for other macromolecules, is promising because of its high sensitivity and ever-increasing mass range and mass resolution. While it is still problematic to obtain completely fragmentation-free MALDI mass spectra for the gold:thiolate and gold: phosphine cluster compounds, these compounds still constitute the bulk of any studies involving mass spectrometry and metallic clusters. Advances in both ionization of the cluster compounds and application of these advances to other inorganic cluster compounds will rely on further studies of ionization processes and optimization of ionization conditions for these selected types of cluster compounds and other related nanocrystals. The author thanks Robert L. Whetten and Robert L. Hettich for their advice and suggestions regarding the drafting of the manuscript and Gregory B. Hurst for use of the time-of-flight instrumentation. Research was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences, U.S. Department of Energy at Oak Ridge National Laboratory, managed and operated by UT-Battelle, LLC under Contract DE-AC05-00OR22725. 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 November 14, 2003. Accepted April 23, 2004. AC0353482
