Anal. Chem. 2003, 75, 199-206
HPLC of Monolayer-Protected Gold Nanoclusters Victoria L. Jimenez, Michael C. Leopold,† Carolyn Mazzitelli, James W. Jorgenson, and Royce W. Murray*
Kenan Laboratories of Chemistry CB-3290, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Polydisperse samples of Au nanoparticles protected with monolayers of hexanethiolate ligands (C6 MPCs) and with mixed monolayers of hexanethiolate and mercaptoundecanoic acid (C6/MUA MPCs) have been chromatographically separated using C8 120-Å columns and acetone/ toluene mobile phase. The spectral details of eluted peaks and of quantized double-layer charging features in the differential pulse voltammetry of collected fractions were used to show that the elution orders of C6 MPC mixtures and of C6/MUA MPC mixtures were different. For C6 MPCs, the smallest MPCs were eluted first, whereas the smallest C6/MUA MPCs were eluted last. The reversal of order of elution was rationalized in terms of intermolecular interactions with the stationary phase, dominant for the C6 MPC, being suppressed by the heightened polarity of the monolayer surface of the C6/MUA MPCs, making a size exclusion mechanism dominant. The range of apparent core diameters of the separated nanoparticles was 1.3-2 nm. Metal nanoparticles are of considerable interest in the world of nanoscience and technology owing to, for example, their connotations in electronic1, optical,2,3 magnetic,4 catalytic,5 and chemical sensing4 applications. Like the better known area of quantum dot semiconductor nanoparticles,6,7 monolayer-protected clusters (MPCs, the dimensionally smallest known category of isolatable metal nanoparticles, in the present work ∼1-3-nm diameter) also exhibit size-dependent electronic absorbance and fluorescence spectra8-11 and electronic core charging in electrolyte * Corresponding author. E-mail:
[email protected]. † Present address: Department of Chemistry, University of Richmond, Richmond, VA 23173. (1) Schon, G.; Simon, U. Colloid Polym. Sci. 1995, 273, 202. (2) Encyclopedia of Chemical Technology, 4th ed.; Wiley: New York, 1994; Vol. 12, pp 569-571. (3) Freeman, R. G.; Grabar, K. C.; Allison, K. J.; Bright, R. M.; Davis, J. A.; Guthrie, A. P.; Hommer, M. B.; Jackson, M. A.; Smith, P. C.; Walter, D. G.; Natan, M. J. Science 1995, 267, 1629. (4) Thomas, J. M. Pure Appl. Chem. 1988, 60, 1517. (5) Henglein, A. J. Phys. Chem. 1993, 97, 5457. (6) Glazman, L. I. J. Low Temp. Phys. 2000, 118 (5/6), 247. (7) Grieve, K.; Mulvaney, P.; Grieser, F. Curr. Opin. Colloid Interface Sci. 2000, 5, 168. (8) 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. (9) Schaaff, T. G.; Shafigullin, M. N.; Khoury, J. T.; Vezmar, I.; Whetten, R. L.; Cullen, W. G.; First, P. N. J. Phys Chem. B 1997, 101, 7885-7891. (10) Landes, C. F.; Braun, M.; El-Sayed, M. A. J. Phys. Chem. B 2001, 105, 10554-10558. (11) Huang, T.; Murray, R. W. J. Phys. Chem. B 2001, 105, 12498-12502. 10.1021/ac0260589 CCC: $25.00 Published on Web 12/06/2002
© 2003 American Chemical Society
media.12 For ∼1.6-nm core diameters, we refer to core charging as13,14 quantized double-layer (QDL) charging. MPCs consist of a nanosized central metal core (usually Au) surrounded by a monolayer of protective ligands (usually thiolates). The protective thiolate ligands prevent aggregation of Au metal cores when they are isolated in the dried state. This is an enormously enabling property because it allows manipulation of Au MPCs as molecular entities, including both metal15 and ligand exchange,16 chemical derivatization12 of the monolayer, and deployment of a variety of analytical measurements.17 That the properties of metal and semiconductor nanoparticles are size dependent makes it of fundamental interest to isolate them according to their size, to understand better the origins of property-size relations. This can mean devising synthetic routes that produce nanoparticles of uniform size (monodisperse) or deploying postsynthetic procedures capable of narrowing the size dispersion of an already prepared nanoparticle sample. Synthetic methods that have been employed to control the size or the polydispersity of metal nanoparticles include use of reverse18 and normal19 micelles, Langmuir-Blodgett films,20 zeolites,21 two-phase liquid-liquid systems,22 and organometallic techniques.23 While the average nanoparticle size can be manipulated by such synthetic approaches, they rarely directly produce nanoparticles with strictly uniform size, at least if one views core radius variability in relation to the change in nanoparticle radius corresponding to the increment of a single closed shell of metal atoms, which for Au is ∼0.25 nm. The synthetic tribulations in achieving this goal are especially severe for the smallest nanoparticles (like (12) Hostetler, M. J.; Green, S. J.; Stokes, J. J.; Murray, R. W. J. Am. Chem. Soc. 1996, 118, 4212-4213. (13) Hicks, J. F.; Templeton, A. C.; Chen, S.; Sheran, K. M.; Jasti, R.; Murray, R. W.; Debord, J.; Schaaff, T. G.; Whetten, R. L. Anal. Chem. 1999, 71, 37033711. (14) Chen, S.; Murray, R. W.; Feldberg, S. W. J. Phys. Chem. B 1998, 102, 98989907. (15) Shon, Y. S.; Dawson, G. B.; Porter, M.; Murray, R. W. Langmuir 2002, 18, 3880-3885. (16) Hostetler, M. J.; Templeton, A. C.; Murray, R. W. Langmuir 1999, 15, 37823789. (17) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (18) Pileni, M. P. J. Phys. Chem. 1993, 97, 6961. (19) Lisieki, I.; Billoudet, F.; Pileni, M. P. J. Phys. Chem. 1996, 100, 4160. (20) Fendler, J. H.; Meldrum, F. C. Adv. Mater. 1995, 7, 607. (21) Herron, N.; Wang, Y.; Eddy, M.; Stucky, G. D.; Cox, D. E.; Moller, K.; Bein, T. J. Am. Chem. Soc. 1989, 111, 530. (22) Brust, M.; Walker, D.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801. (23) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706.
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MPCs), where the total number of metal atoms and the number of atom layers (e.g., 3-6) in a closed-shell core structure are small. Postsynthetic techniques for narrowing the dispersity of asprepared metal nanoparticle size include heating,24 vapor treatments,25,26 etching procedures,27,28 annealing,29 and solubility fractionation.30 Heating and vapor treatments are somewhat effective for larger nanoparticle dimensions but are ineffective for very small nanoparticles. Etching procedures are most successful in decreasing MPC core sizes. Our laboratory has used13 solubility fractionation, originally introduced by the Whetten group,30 which rests on the greater solubility in polar solvents of alkanethiolate MPCs having smaller cores. This method can be effective but is both time-consuming and less effective for nanoparticles with longchain alkanethiolate monolayers. Annealing chemistry has in recent work29 been shown to be effective in improving monodispersity in one specific case, namely, of Au140 clusters bearing hexanethiolate monolayers. In the above context, it is of equal interest to learn how to analytically characterize nanoparticle dispersity. Methods that respond to nanoparticle dispersity include mass spectrometry,30 voltammetry of quantized double-layer charging,13,14 and electronic spectra.31 The Whetten group has used30 mass spectrometry to great effect; nonetheless, the resolution of all of these approaches has limits. Differential migration methodology is obviously needed in nanoparticle science. Several chromatographic methods have been employed, including size exclusion chromatography (SEC),32-35 capillary electrophoresis (CE),35,36 and ion exchange chromatography (IEC).37 Each of these has virtues and disadvantages, the latter including irreversible adsorption by SEC column packing material and the inapplicability of IEC to neutral nanoparticles. Of reports on very small nanoparticles, only CE has resolved36 a complex mixture and then only partially. Additionally, as far as we are aware, there has been no successful resolution of nanoparticle mixtures below the ∼2-nm average core diameter range. In light of the above, and considering the relevance of monodisperse MPC materials to QDL studies, we launched an HPLC investigation into separating MPCs using silica bonded C8 (24) Devenish, R. W.; Goulding, T.; Heaton, B. T.; Whyman, R. J. Chem. Soc., Dalton Trans. 1996, 673-679. (25) Maye, M. M.; Zheng, W.; Leibowitz, F. L.; Ly, N. K.; Zhong, C. J. Langmuir 2000, 16, 490-497. (26) Zhong, C. J.; Zhang, W. X.; Leibowitz, F. L.; Eichelberger, H. H. Chem. Commun. 1999, 1211-1212. (27) Schaaff, T. G.; Whetten, R. L. J. Phys. Chem. B 1999, 103, 9394-9396. (28) Wilcoxon, J. P.; Martin, J. E.; Parsapour, F.; Wiedenman, B., Kelley, D. F. J. Chem. Phys. 1998, 108, 9137. (29) Hicks, J. F.; Miles, D. T.; Murray, R. W. J. Am. Chem. Soc. 2002, 124, 13322-13328. (30) 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. (31) Hostetler, M. J.; Wingate, J. E.; Zhong, C.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Kwignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir 1998, 14, 17-30. (32) Wei, G. T.; Liu, F. J. Chromatogr., A 1999, 253-260. (33) Wei, G.; Liu, F.; Wang, C. R. C. Anal. Chem. 1999, 71, 2085-2091. (34) Wilcoxon, J. P.; Martin, J. E.; Provencio, P. Langmuir 2000, 16, 99129920. (35) Schnabel, U.; Fischer, C.; Kenndler, E. J. Microcolumn Sep. 1997, 9, 529534. (36) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121, 7081-7089. (37) Bos, W.; Steggerda, J. J.; Yan, S.; Casalnuovo, J. A.; Mueting, A. M.; Pignolet, L. H. Inorg. Chem. 1988, 27, 948-951.
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columns with 120-Å pores. Like solubility fractionation,30 differential partition (or partition-like interactions) in reversed-phase HPLC should, ideally, yield separations paralleling the solubility properties of the nanoparticles. Additionally, the small pore size of the silica particles was selected with an eye to the potential of a size exclusion sensitivity in the nanoparticle separation. An attractive feature of HPLC is its potentially large resolving power and, when used in conjunction with a photodiode array (PDA) spectrophotometer, information on the MPC core dimension from the UV-visible electronic spectral fine structure that accompanies very small core size.8,9 This paper presents our initial HPLC investigation of polydisperse samples of two kinds of MPCs, those with protective monolayers composed solely of hexanethiolate ligands (C6 MPCs) and those (C6/MUA MPCs) with mixed monolayers of C6 thiolate ligands and (10%) mercaptoundecanoic acid (MUA) thiolate ligands. The synthetic procedure used to prepare the C6 MPCs produces31 a dispersity of nanoparticle sizes with an average composition of Au140(C6)53. These MPCs are used to prepare the mixed monolayer C6/MUA MPCs. The separations are detected using a PDA spectrophotometer and, for collected fractions, with differential pulse voltammetry (DPV), which allows an additional core size evaluation. EXPERIMENTAL SECTION Chemicals. HAuCl4‚xH2O was synthesized according to established procedures.38 Tetrabutylammonium perchlorate (Bu4NClO4), hexanethiol (C6SH), sodium borohydrate, and MUA were purchased from Aldrich. HPLC grade toluene, acetonitrile, dichloromethane, and acetone (Aldrich) were used as received. House distilled water was purified with a Barnstead NANOpure system (18.2 MΩ). MPC Synthesis. A modified Brust procedure22 was used to synthesize MPCs on a 2-g scale. To a vigorously stirred solution of 5-6 g of Oct4NBr in 200 mL of toluene was added 3.1 g of HAuCl4‚xH2O in 110 mL of Nanopure water. After stirring for ∼30 min, 3.76 mL of C6SH was added and the resultant mixture was stirred for another ∼30 min. The solution was then cooled in an ice/water bath and 3.8 g of NaBH4 in 20 mL of Nanopure water quickly added to the vigorously stirred solution, which was allowed to react for ∼30 min. Collecting the organic phase, the solvent was removed on a rotary evaporator, and the product resuspended in acetonitrile (which dissolves excess thiol but not nanoparticle), collected on a glass frit (medium porosity), and washed copiously with acetonitrile. Place Exchange (Ligand Exchange) Reaction. A 1-g sample of C6 MPCs and 170 mg of MUA were codissolved in 300 mL of THF in a covered round-bottom flask, which was allowed to stir for ∼3 days, after which the solvent was removed by a rotary evaporator. The black product was suspended in acetonitrile and collected on a glass filtration frit after repeated washings with acetonitrile. NMR analysis of the MPC revealed a 10% loading of MUA on the C6 MPCs. Detailed descriptions of place exchange reactions can be found elsewhere.12,39,40 (38) (a) Handbook of Preparative Inorganic Chemistry; Brauer, G., Ed.; Academic: New York. 1965; pp 1054-1059. (b) Block, B. P. Inorg. Synth. 1953, 4, 14-17. (39) Ingram, R. S.; Hostetler, M. J.; Murray, R. W. J. Am. Chem. Soc. 1997, 119, 9175.
Measurements. One of the two HPLC chromatographs employed was a Waters 600 controller pump capable of gradient elution and equipped with a Waters 996 PDA detection system and a Waters fraction collector II. Injection was by a Rheodyne 7725 injection valve with 50-µL sample loop. The other chromatograph, employed during the first phase of this investigation, was an older Waters system (Waters 510 isocratic pump, Waters 484 single-wavelength UV-visible detector set to 400 nm, and Rheodyne 7010 injection valve with a 50-µL loop). The chromatograms shown in Figures 1 and 8 were obtained on the older instrument. A stainless steel silica (pore size 120 Å) bonded C8 (250 × 4.6 mm i.d., ThermoHypersil) column was used on both instruments; the purchased columns, however, exhibited somewhat different behaviors. The column temperature was set at 30 °C. The mobile phase (flow rate, 1 mL/min) consisted of toluene/acetone mixtures proportioned by trials to yield efficient separations. It was found that some of the nanoparticles tended to “stick” on the column, and on the newer instrument, at the end of each chromatogram, the mobile phase was switched to the solvent component (usually toluene) that best solubilized these MPCs. (It was nonetheless difficult to entirely free the column from strongly adherent nanoparticles, and column life was not long.) After flushing the column, for the next chromatogram, the mobile phase was switched back to the solvent mixture of choice. Fractions for electrochemical and TEM analysis were collected over time intervals targeting specific peaks on the chromatogram. Absorbance spectra (300-900 nm) of the MPC sample solution (same solvent as mobile phase), before HPLC separation, were taken with an ATI-Unicam UV4 dual-beam spectrophotometer in 0.1-cm-path length quartz cuvettes. 1H NMR spectra of CD2Cl2 Au MPC solutions were collected at 200 MHz on a Brueker AC200 spectrophotometer. Differential pulse voltammetry was performed with a BAS 100B electrochemical analyzer. The voltammetry of separated fractions of C6SH MPCs (from which the original mobile phase was allowed to evaporate) was performed in a 0.1 M Bu4NClO4/CH2Cl2 solution with a Pt working electrode (macro), Pt auxiliary electrode, and a Ag wire quasi-reference electrode. Voltammetry of collected fractions of C6/MUA MPCs in the same electrolyte solution was done on films of the nanoparticles immobilized on a gold working electrode using network nanoparticle polymer chemistry previously described.41 RESULTS AND DISCUSSION Chromatography of C6SH MPCs. Figure 1 shows a chromatogram of a sample of C6 MPCs on a silica bonded C8 column with 120-Å pores. A dominant set of peaks is seen starting at 6 min that was common in C6 MPC samples under these conditions. Our experience has been that the detailed appearance of both the dominant and minor peaks varies in the chromatography of C6 MPC samples prepared using nominally identical conditions, meaning that the dispersity of the C6 MPC is sensitive to the precise details of the synthesis. Also, the appearance of a chromatogram of C6 MPCs, for any given sample, reflects both the progress of prior treatments (such as annealing or fractional (40) Green, S. J.; Stokes, J. J.; Hostetler, M. J.; Pietron, J. J.; Murray R. W. J. Phys. Chem. B 1997, 101, 2663-2668. (41) Zamborini, F. P.; Hicks, J. F.; Murray, R. W. J. Am. Chem. Soc. 2000, 122, 4514-4515.
Figure 1. Chromatogram (absorbance detected at 400 nm) of asprepared C6 MPC. The mobile phase was 2:1 acetone/toluene ratio at a flow rate of 1 mL/min, and the column was a RP C8, 120-Å pore size column connected to a Waters 510 isocratic pump. Fractions were collected and electrochemically analyzed as in Figure 2.
Figure 2. Differential pulse voltammetry of indicated C6 MPC samples. The 2- and 6-min fractions from Figure 1 were collected over time windows of 1 min each; that at 8 min was collected over a 3-min window.
precipitation30 undertaken to reduce the polydispersity of the MPC sample) and any column degradation caused by irreversible nanoparticle adsorption. Figure 1 is thus presented as just an illustrative demonstration of the effectiveness of reversed-phase HPLC in separation of a polydisperse nanoparticle sample, which we will next show has a quite narrow range of dimensions. Figure 1 was obtained after taking trial chromatograms to establish a suitable mobile-phase composition for isocratic separation. Pure toluene produces low-capacity factors and little separation of alkanethiolate MPCs in general. The MPCs are incompletely soluble in pure acetone. The 2:1 acetone/toluene mixture used in Figure 1 was reasonably optimum for the experiment, although fully resolved Gaussian-shaped peaks were never observed and the column must be flushed with toluene between experiments as noted in the Experimental Section. The next question, of course, is what do the peaks represent? The simplest version of this question is whether the separation represents some order of nanoparticle elution related to core size. The question of elution order was addressed by taking DPVs of collected chromatographic fractions (Figure 2). This figure shows current peaks that represent sequences of single-electron charging of the MPC cores, as they diffuse to the electrode from the MPC solution. The discretization of the charging is called quantized double-layer charging.14 We know from prior work that nanoparticles with smaller core sizes exhibit larger peak-to-peak voltage Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
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Table 1. C6 MPC Quantized Double-Layer Peak Potential Spacing and Capacitances (CCLU) Taken from Plots in Figure 3, and Apparent Core Diameters Calculated from Eq 2
Figure 3. Charge state and peak potential from DPV shown in Figure 2 for (A) as-prepared C6 MPC and (B) fractions collected at 2 (dots), 6 (solid line), and 8 min (dashes).
spacing (∆V) in the DPV patterns of QDL charging.42 The average ∆V spacing between the * peaks in Figure 2 for the 2-, 6-, and 8-min fractions is 313 ( 67, 300 ( 38, and 278 ( 32 mV, respectively. Quantized double-layer charging is seen in electrolyte solutions of nanoparticles that have extremely small double-layer capacitances (CCLU). Single-electron changes on the state of nanoparticle core charge cause shifts ∆V () e/CCLU, where e is the electronic charge, 1.6 × 10-19 C) in the Fermi level energy of the nanoparticle. For MPCs of the size dimension discussed here, ∆V exceeds kBT (which expressed in electrochemical voltage, at room temperature, is ∼26 mV). The single-electron changes are readily observed in experiments such as DPV,14,42 cyclic voltammetry,39 microelectrode voltammetry,14 and ac impedance.43 Assuming that the capacitance of the nanoparticle in the electrolyte solution is constant, which seems to be the case nearest EPZC (the potential of zero charge), one can define14 apparent “formal potentials” for the successive one-electron changes in electronic charge (z) on the nanoparticle,
EZ,Z-1 ) EPZC + (z - 1/2)e/CCLU
(1)
where EPZC is about -0.1 to -0.2 V in CH2Cl2.14 This relation predicts linear plots of formal potential against nanoparticle charge state, as shown in Figure 3 for the chromatographic fractions and for the original C6 MPC sample. Table 1 gives results for the average voltage spacing ∆V and CCLU. The values of CCLU can be translated into MPC core dimensions based14 on a concentric sphere capacitor model, in which the metal core is the inner conducting sphere, the monolayer represents the dielectric, and the outer sphere is circumscribed by the monolayer/electrolyte solution boundary. The appropriate relation is
∈∈0 r + d r CCLU ) ACLU ) 4π∈∈0 (r + d) r d d
(2)
where ∈0 is the permittivity of free space, ∈ is the monolayer static (42) Chen, S.; Ingram, R. S.; Hostetler, M. J.; Pietron, J. J.; Murray, R. W.; Schaff, T. G.; Khoury, J. T.; Alvarez, M. M.; Whetten, R. L. Science 1998, 280, 2098-2101. (43) Chen, S.; Murray, R. W. J. Phys. Chem. B 1999, 103, 9996-10000.
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cluster fraction
∆V spacing (mV)
CCLU (aF)b
apparent core diameterc (nm)
as-prepared C6 MPCs peak at 2 mina peak at 6 mina peak at 8 mina
277 ( 20 313 ( 67 300 ( 38 278 ( 32
0.57 0.50 0.53 0.58
1.65 1.51 1.57 1.67
a Figure 1. b Figure 3. c Equation 2: dielectric constant ∈ ) 3, permittivity of free space ∈0 ) 8.85 × 10-12, thickness of the protecting dielectric monolayer d ) 0.77 nm. The value of ∈ ) 3 is taken from capacitance measurements on C6 self-assembled monolayers.47 Transmission electron microscopy measurements are unable to distinguish differences in core diameters of the magnitudes shown and, although attempted, were inconclusive.
dielectric constant, r and ACLU are the radius and surface area of the MPC core, respectively, and d is the monolayer dielectric thickness.14 Table 1 gives the values of core radii calculated using eq 2. (Assumptions and values of the other parameters used in the calculation are the same as previously13,14 used in applying this model and are given in the table footnotes.) For comparison, cores with truncated octahedral shapes and containing 116 and 140 Au atoms, approximate31 spheres of diameters 1.42 and 1.62 nm, respectively. From this analysis, and from the preponderance of an analogous peak in similar MPC separations, the large peak(s) eluting in the 3-min time interval starting at 8 min in Figure 1 are assigned to MPCs with approximately Au140 cores. The Au140 core composition is thought, from prior work,13,31 to be the majority MPC produced using the synthetic conditions cited in the Experimental Section. While there is some obvious statistical uncertainty, the peaks that elute at earlier times exhibit larger ∆V voltage spacing and correspond to MPCs having smaller cores or at least smaller double-layer capacitance. This conclusion is supported by spectral results, presented next. In further experiments, C6 MPC separations were carried out using a different instrument, equipped with a Waters 996 PDA detection system. The UV-visible spectra thus obtained provide an additional criterion for assessing the order of chromatographic elution, in terms of MPC core size. It is known that spectra of smaller MPCs exhibit fine structure related to core size. Whetten and co-workers8,9 have shown that the optical absorbance spectra of MPCs that have been well-fractionated according to core size include the following: (i) an onset of strong absorption located near 1.7 eV that becomes more distinct with decreasing core size, (ii) a broad absorption band centered at 2.4 eV that is diminished with decreasing core size, and (iii) a weak steplike fine structure in the spectra of smaller core size MPCs that starts from the absorption onset at 1.7 eV. An example C6 MPC chromatogram is shown in Figure 4 along with absorbance spectra of the indicated eluted peaks. The spectral results (inset) show that the peak eluting at the shorter time (2.2 min) exhibits a distinct steplike spectral structure and that latereluted peaks display much less spectral detail. According to the previous results,8,9 the steplike spectral fine structure is indicative of a smaller core size cluster (8-14 kDa by mass spectrometry). The peak eluting at longer time (21 min) exhibits a spectrum with
Figure 4. Chromatogram (absorbance detected at 400 nm) of asprepared C6 MPC. The MPC sample used was not the same as that employed in Figure 1 but was prepared in nominally a similar way. The difference between Figures 1 and 4 represents in part the sampleto-sample synthesis variation and in part the variation in the column material (different column lot specification). The mobile phase was 66% acetone/34% toluene at a flow rate of 1 mL/min. Separation was performed on an RP C8, 120-Å pore size column connected to Waters 600 controller pump. Detection was with a photodiode array which produced the spectra shown in the inset for materials eluted at the indicated times. Inset also shows spectrum of original, as-prepared C6 MPC. Ticks on spectra correspond to energies of (2.2 min) 1.8, 2.8, and 3.1 eV; (7.8 min) 2.2 and 3.3 eV; and (21.1 min) 2.4 and 3.3 eV.
Figure 5. Absorbance spectra comparison of Au clusters: (A) spectrum of 10.4-kDa Au/glutathione MPC,44 (Au28(SG)16, 5.6-kDa core); (B) spectrum from Figure 4 for peak at 2.21 min; (C) spectrum A magnified by 10. (Curves A and C adapted from Figure 4 of ref 44, by permission.)
a surface plasmon peak at 2.4 eV (520 nm) that is indicative of a larger core size cluster. To further the size comparison, Figure 5 compares the spectrum of the early (2.2 min) eluted peak in Figure 4, to a published spectrum of a 5.6 kDa core (Au28) gold-glutathione monolayer-protected cluster.44 These spectra are remarkably similar, showing features with, first, a strong absorbance at ∼670 nm, followed by a minimum at ∼600 nm, and then a steplike structure at higher energies/lower wavelengths. Whetten has (44) Schaaff, T. G.; Knight, G.; Shafigullin, M. N.; Borkman, R. F.; Whetten, R. L. J. Phys. Chem. B 1998, 102, 10643-10646.
Figure 6. Chromatograms (absorbance detected at 400 nm) of two different preparations of ethanol-soluble fractions of C6 MPCs. Mobile phase was 50% acetone/50% toluene at a flow rate of 0.6 mL/min; chromatography was performed on an RP C8, 120-Å pore size column connected to a Waters 600 controller pump. Insets are spectra taken with the photodiode array detector of the prominent peak eluting at ∼13 min.
stated46 that spectra of such small-size MPCs do not significantly change when the thiolate ligand is an alkanethiolate as opposed to the relatively polar glutathione ligand. We have observed on the other hand that details of the spectrum of this early-eluted peak vary from sample to sample, including the exact wavelength maximum of the absorption peak located at ∼670 nm, of the adjacent minimum at higher energy, and of the relative absorbances of the higher energy steplike spectral features. The source(s) of these variations are unknown, but may involve small differences in the number of core atoms or in the number of thiolate ligands attached to the gold core. These issues aside, the spectral comparison of Figure 5 strongly supports the Figures 2 and 3 conclusion that the earlier eluting peak in Figure 4 corresponds to an MPC having a smaller Au core size. As stated previously, the size dispersity of C6 MPC samples is quite sensitive to the details of their synthesis and to the success of postsynthesis attempts at improving monodispersity by, for example, fractional precipitation30 or annealing29 procedures. An ethanol-soluble fraction of the MPC synthesis is thought13 to consist of a higher fraction of MPCs that have a Au140 core size. Chromatographic results are shown in Figure 6 for two different (45) Schaaff, T. G.; Shafigullin, M. N.; Khoury, J. T.; Vezmar, I.; Whetten, R. L. J. Phys. Chem. B 2001, 105, 8785-8796. (46) Schaaff, T. G.; Whetten R. L. J. Phys. Chem. B 2000, 104, 2630-2641. (47) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559.
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Figure 7. Absorbance spectra comparison of Au clusters. (A) spectrum from Figure 6 of C6 MPCs eluting at 12.9 min; (B) spectrum of 29-kDa Au/glutathione MPC,45 (Au144-146(SG)50-60). (Curve B adapted from Figure 7a of ref 45, by permission.) Ticks on spectra correspond to energies of 1.8, 2.4, 2.6, and 3.2 eV.
C6 ethanol-soluble MPC samples synthesized, using approximately the same procedure, by one of the authors and by another student in our laboratory. The ethanol-soluble C6 MPC samples were separated using the same HPLC parameters, for which optimum separation conditions were obtained with a mobile-phase ratio of 1:1 toluene/acetone. We believe that the dominant peak at ∼13 min in both separations is the ubiquitous45 Au140 nanoparticle. Spectra obtained from each chromatogram showed similar spectral features. Figure 7 compares the spectrum of this dominant chromatographic peak to that of the 29-kDa Au-SR cluster compound (Au144-146 core) described by Whetten.45 The nanoparticle spectra are reasonably similar, with the HPLC result showing more detail, presumably from its improved chromatographically derived purity. A few additional isocratic elution (50% toluene and 50% acetone) experiments (not shown) were performed on MPCs with dodecanethiolate ligand monolayers (C12 MPCs) and on C6 MPCs using a linear gradient elution (from 100% acetone to 100% toluene). In both cases, the spectra of the eluted peaks (by the spectra steplike structure) indicate an order of elution from smaller to larger clusters. The above measurements show that, under the chromatographic conditions described, the separations of hexanethiolatecoated Au nanoparticles result in the smaller MPCs being eluted first. The key observations are the variations in DPV peak voltage spacing (∆V, and thus CCLU) of collected fractions and the spectral fine structure of early-eluted peaks. Both indicate that small core clusters are eluted first and larger ones later. Previous chromatographic investigations32-35 have pointed to size exclusion separations, in which larger nanoparticles are eluted first. Our observations show that the mechanism operating in the HPLC separation of C6 MPCs on a silica bonded C8 column with 120-Å column is not a size exclusion process but is instead a reverse-phase chromatographic separation. The classical explanation of reversedphase HPLC (nonpolar stationary phase, more polar mobile phase) is that sample components that are (relatively) less soluble in (or have weaker intermolecular interactions with) the stationary phase are eluted first. This observation is consistent with fractionation 204 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
Figure 8. Chromatogram (absorbance detected at 400 nm) of mixed-monolayer C6/MUA MPC. Ten percent of the monolayer was MUA ligand. Mobile phase was 6:1 acetone/toluene at a flow rate of 1 mL/min. Separation was performed on an RP C8, 120-Å pore size column connected to Waters 510 isocratic pump. Fractions were collected and electrochemically analyzed as in Figure 9.
experiments in which, qualitatively, smaller MPCs tend to be more soluble in polar solvents, as is seen in precipitation30 and ethanol extraction13 behavior. The HPLC order elution is thus explicable on a core size basis, in that smaller core MPCs exhibit differentially weaker interactions with the nonpolar stationary phase, relative to the more polar mobile phase. The central question of exactly why the smaller core MPCs are more soluble in polar solvents than larger core ones remains unsettled. Additionally, what MPC compositional features produce the numerous other chromatographic peaks is unknown; do they represent a dispersion of MPC core size or of thiolate ligand content on the nanoparticle? Further studies were performed on C6 clusters that have mixed monolayers, to introduce a decidedly polar constituent into the monolayer. Mixed-Monolayer C6/MUA MPC Separations. Chromatographic experiments were carried out on MPCs with mixed monolayers of hexanethiolate and MUA ligands. The latter ligands, introduced by place exchange (see Experimental Section) at a 10% loading (percentage of the original C6 ligands), introduce a strongly polar component into the MPC monolayer (with solubilities in polar solvents enhanced and depressed in nonpolar ones). Optimal chromatographic resolution using the C8 120-Å pore size column was now obtained using a more polar 6:1 acetone/toluene solvent mixture, in which the cluster was not completely soluble (meaning that some solubility fractionation probably occurred before the chromatography). Performing solvent fractionation before injection of the sample onto the HPLC column could drastically change the size distribution of the nanocluster core in the sample. The resulting chromatogram (Figure 8) exhibits three distinct peaks and is quite different in appearance from Figure 1 (obtained using the same column). The difference can be attributed to a combination of the possible solvent fractionation that occurred
Figure 9. Differential pulse voltammetry of indicated mixed-monolayer C6/MUA MPC samples. “As-prepared” refers to as-prepared C6 MPC following place exchange with MUA ligands. The fractions labeled peaks 1-3 from Figure 8 were collected over time windows of 1 min each. Table 2. Mixed Monolayer C6/MUAMPC Quantized Double-Layer Peak Potential Spacing and Capacitances (CCLU) Taken from Plots in Figure 10, and Apparent Core Diameters Calculated from Eq 2
Figure 10. Charge state and peak potential from DPV shown in Figure 9 for (A) as-prepared C6 MPC and (B) peak 1 (dashes), peak 2 (dots), and peak 3 (solid line).
before injection (see above) and, more importantly, the fact that the separation mechanism is apparently changed (see below) by the more polar character of the C6/MUA MPC. The three peaks were fraction-collected and subjected to DPV analysis (as was the as-prepared mixed-monolayer material), with results shown in Figure 9. Because the chromatographic fractions were too dilute to give useful voltammetry, the MPCs were preconcentrated by binding them into a network polymer film41 on the electrode using MUA-based carboxylate/Cu2+/carboxylate bridges. The resulting voltammetric signal to background was substantially increased in this way, albeit still not outstanding. The MPC capacitances taken from potential-core charge-state plots (eq 1, Figure 10) are presented in Table 2. The MPC capacitances in such network polymer films have in previous work41 been similar to their dissolved monomer MPC counterparts, and we assume that to be the case here. Therefore, the same parameters are used with eq 2 to calculate apparent diameters of the eluted cluster cores (Table 2). The electrochemical results in Figure 9 and Table 2 show that the voltage spacing between the quantized double-layer charging
cluster fraction
∆V spacing (mV)
CCLU (aF)
apparent diameter (nm)
as prepared peak 1 peak 2 peak 3
342 ( 80 200 ( 96 275 ( 48 400 ( 57
0.55 0.85 0.56 0.39
1.61 2.12 1.63 1.27
peaks of the fraction-collected nanoparticles is smaller for the early-eluted peaks. The early peak thus corresponds to clusters with larger apparent core sizes. Based on previous studies,31 and assuming closed-shell core structures, the average diameters in Table 2 correspond to MPC cores, in the three eluted peaks, containing approximately 1289 Au atoms (2.12-nm diameter), 140 Au atoms (1.63-nm diameter), and 79 Au atoms (1.27-nm diameter). This elution order result is the reverse of that revealed in the C6 MPC experiments (Table 1). This was confirmed in further experiments using spectral detection of the eluted peaks (Waters 996 detection system, Figure 11). The chromatogram displays the same general elution trend as Figure 8. Spectral fine structure is absent in the early-eluted peaks but is present in the later ones. The spectral results are consistent with the later-eluting peaks corresponding to MPCs with smaller core sizes. The results of Figures 8-11 were initially surprising. The order of elution of the nanoparticles is as if the chromatographic separation behaves as a size exclusion process, meaning that partition-like intermolecular interactions between the MPC and the C8 stationary phase have become less important than the relationship between MPC size and stationary-phase pore size. This is, however, explicable based on the profound effect of polar monolayer functional groups on MPC properties such as solubility. Our general experience with ligand exchanges of MPCs has been that introduction of relatively modest numbers of polar functional Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
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Figure 11. Chromatogram (absorbance detected at 400 nm) of mixed-monolayer C6/MUA MPC. Mobile phase was 85% acetone/ 15% toluene at a flow rate of 1 mL/min; chromatography was performed on an RP C8, 120-Å pore size column connected to a Waters 600 controller pump. Insets are spectra taken with the photodiode array detector of the peaks eluting at 3.3, 3.8, 4.4, and 6.2 min. Spectra are offset for clarity. Ticks at left of spectra represent zero absorbance. Ticks on spectra correspond to energies of (3.8 min) 3.2 eV; (4.4 min) 1.8, 2.8, and 3.1 eV; and (6.2 min) 1.8, 2.8, and 3.3 eV.
groupings into the MPC monolayer can produce quite substantial increases in the solubility of the MPCs in polar solvents. One would anticipate, against this background, that partition-like intermolecular interactions between the C6/MUA MPCs and the C8 stationary phase should be substantially weakened (relative to those of C6 MPCs). The retention time separation window would dramatically decrease, as well, if it were separating based on a size exclusion mechanism. The chromatographic differences between Figures 1 and 8 indicate that this indeed occurs, leaving pore size permeation as the dominant chromatographic separation effect. CONCLUSIONS While obviously not outstanding in relation to contemporary HPLC of small molecules, the chromatographic separations illustrated in Figures 1, 4, 5, 8, and 11 are outstanding in relation to previously reported nanoparticle chromatography. The voltammetric analyses of core size by quantized double-layer charging properties show that nanoparticles differing in core size by quite small amounts (Table 1) can be separated. HPLC has to be regarded as an extremely promising tool for use with small nanoparticles, provided attention is paid to factors influencing elution order
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For the selected column materials, the mechanism of nanoparticle separation is revealed to be a rather delicate balance between partition-like intermolecular interactions and size exclusion effects. Our interpretation is that the separation of alkanethiolate-coated Au clusters (C6 MPCs) is dominated by the relatively weaker partition-like intermolecular interactions between the smaller core size C6 MPCs and the nonpolar C8 stationary phase, resulting in the smaller core size C6 MPCs eluting first. Smaller core size alkanethiolate-coated MPCs are known to be relatively more soluble in polar solvents. Thus, although size exclusion effects are probably present, the reversed-phase separation mechanism dominates. Introducing carboxylic acid polarity into the MPC monolayers suppresses the partition-like interactions and allows the size exclusion effect to become dominant. Lacking, at present, is evidence for the influence of and balance between these two separation mechanisms over a more extended range of MPC monolayer chemistries and core sizes. That is a topic for further research. Finally, a caveat must be added with regard to the nature of the separated nanoparticles. We have analyzed the different peaks in the HPLC separations according to their differences in quantized double-layer charging properties and have expressed those properties in terms of MPC core size, using eq 2. Equation 2 is, however, just a model of the nanoparticle as a tiny capacitor and a very simple one at that. Certainly the MPC core size and the thickness and chemical nature of the monolayer dielectric (the hexanethiolate ligands) will jointly influence the value of nanoparticle capacitance CCLU, However, there is no analytical information as yet on whether all MPCs with a given Au core size actually bear identical numbers of thiolate ligands. We should, therefore, point to the possibility that some of the minor peaks in the chromatography, and perhaps some of the broadening of the chromatographic bands, arise not from a dispersity in core size but from a dispersity in the number of ligands on the MPC cores. The chromatographic partitioning process should be much more sensitive to the ligand count than the double-layer charging. HPLC should be a fruitful tool for exploring this chemistry. ACKNOWLEDGMENT This research was supported in part by research grants from the National Science Foundation and the Office of Naval Research. The authors thank Dr. Jocelyn Hicks of UNC (now at Dupont Central Research Laboratories, Wilmington, DE) for providing ethanol soluble C6 MPC, and Dr. Kamlesh Patel of UNC (now at Sandia National Laboratories) for initial assistance in equipment setup.
Received for review August 15, 2002. Accepted November 5, 2002. AC0260589
