Anal. Chem. 2004, 76, 4911-4919
Accelerated Articles
Voltammetric Detection of Metal Nanoparticles Separated by Liquid Chromatography Yang Song, Michael LAV Heien, Victoria Jimenez, R. Mark Wightman, and Royce W. Murray*
Kenan Laboratories of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Fast-scan cyclic voltammetric (FSCV) detection of the separation of small (2.2 nm display a surface plasmon (SP) absorbance at ∼520 nm, while those with diameter of 103 nanoparticles. We concur with this point of view as it provides an emphasis to the need for alternative nanoparticle size methodology, based on the combination of a chemical separation and a physically based size determination. EXPERIMENTAL SECTION Chemicals. Tetraoctylammonium bromide (Oct4NBr, >98%), sodium borohydride (>98%), dichloromethane (>99%), silver nitrate (>99%), tetrabutylammonium perchlorate (Bu4NClO4, >99%), hexanethiol (HSC6OH, >95%), ferrocene (Fc, > 99%), and toluene (99%) were used as received from Aldrich. Ferrocenyloctanethiol (HSC8Fc)32 and HAuCl4‚3H2O33 were synthesized according to earlier reports. Hexanethiolate-stabilized Au140 clusters were prepared as reported previously.1,7,8 Briefly, AuCl4- was phase-transferred by Oct4NBr from water to toluene, where a 3:1 molar excess of hexanethiol (HSC6OH) was added. The toluene solution was then cooled to 0 °C, followed by addition of a 10-fold molar excess of BH4- reductant. The reducing reaction was allowed to proceed over a 24-h period. Solvent fractionation was used to produce clusters with comparatively better monodispersity.4 We have determined that this reaction produces MPCs34 with an average 1.6-nm core diameter and composition Au140[S(CH2)5CH3]53.8 A modified Brust synthesis was used to synthesize Au38 hexanethiolate MPCs. At the reduction step, the toluene solution was cooled in a dry ice/acetone bath to -78 °C. To this solution, a 10-fold molar excess of NaBH4 in Nanopure water was rapidly added with vigorous stirring. After ∼30 min., the product was isolated and cleaned by running through a silica gravity column.35 Mixed monolayer MPCs were prepared by a ligand exchange in which a 1:1 mole ratio of hexanethiolate-coated Au140 MPCs and HSC8Fc was stirred for 1 h in a THF solution. After removing the solvent under vacuum, the product was repeatedly rinsed with acetonitrile until no unbound ligands were observed by 1H NMR. The mixed monolayer composition was analyzed by 1H NMR following decomposition of the MPC monolayer to disulfides with I2 as described previously.1 Assuming that the total number of ligands (53) and cluster core size (Au140) remain the same,36 the number of ferrocenyl-octanethiolate ligands was determined to be an average of 9 per MPC, from the area ratio of ferrocene resonances at 4.38 and 4.81 ppm and methyl resonances at 0.9 ppm. The 1H NMR spectra were of methylene-d2 chloride (Cambridge Isotope Laboratories, Inc.) solutions containing ∼20 mg of cluster/mL (0.68 mM MPC and 0.038 M ligand) and were collected using a Bruker AC500 spectrometer. Reversed-Phase HPLC. A Waters 600 controller pump capable of gradient elution and a Waters 996 PDA detection system were used for high-performance liquid chromatography (HPLC). Injection was with a Rheodyne 7725 injection valve with (32) Chidsey, C. E.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. Soc. 1990, 112, 4301-4306. (33) 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. (34) Cleveland, C. L.; Landman, U.; Shafigullin, M. N.; Stephens, P. W.; Whetten, R. L. Z. Phys. D 1997, 40, 503-508. (35) Jimenez, V. J.; Georganopoulou, D.; White, R. J.; Harper, A. S.; Mills, A. J.; Lee, D.; Murray, R. W. Langmuir 2004, ASAP article. (36) Song, Y.; Murray, R. W. J. Am. Chem. Soc. 2002, 124, 7096-7102.
Figure 1. Cross section diagram (not to scale) of the electrochemical cell used in the fast-scan voltammetric detection of HPLC separations of gold MPCs. A glass-encased Pt cylinder microelectrode 25 µm in diameter and 50 µm in length was used as the working electrode. Ag/Ag+ (Ag wire/AgNO3 (1 mM)/Bu4NPF6 (0.1 M)/acetonitrile) was used as the reference electrode.
50-µL sample loop. To enhance resolution in Figure 4,39 two columns were utilized in-linesboth containing 5-µm particles with 120-Å pore size; the first (250 × 4.6 mm i.d.) column had a silica bonded C8 stationary phase (Thermo Hypersil, Keystone Scientific Operations), and the second (150 × 4.6 mm i.d.) had a silica bonded phenyl stationary phase (Shandon HPLC Co.). In the experiments of Figures 3 and 6, only the first of the two columns was employed. The mobile-phase flow rate was set at 0.7 mL/ min, and the column temperature was 30 °C. The mobile phase consisted of dichloromethane, with 10 mM Bu4NClO4 added to provide the ionic conductivity required by electrochemical detectors. Electrodes and Electrochemical Detectors. Constant potential amperometry detection was done in a cross-flow LC-44 thinlayer electrochemical cell (Bioanalytical Systems, Inc.) connected in-line with the PDA detector; the delay time between the two detectors was ∼1.5 s when the flow rate was 0.7 mL/min. The electrodes were a 3-mm-diameter glassy carbon working electrode, a Ag/AgCl (aqueous, 3 M NaCl) reference electrode, and the stainless steel cell body served as the counter electrode. Signals were read by a locally constructed potentiostat (Department of Chemistry Electronics Shop, UNCsChapel Hill), converted through a Waters A/D converter and monitored by the Waters software along with the PDA-detected chromatograms. A battery-powered potential source was used to reduce ac noise. FSCV was done in a locally fabricated two-electrode electrochemical cell, connected in-line with the PDA detector. A delay of 2 s between the two detectors was seen at a flow rate of 0.7 mL/min. Figure 1 shows a cartoon of the cell arrangement. The working electrode was a glass-encased Pt microcylinder (25-µmdiameter Pt wire, from Sigma-Aldrich, St. Louis, MO), constructed starting with glass capillaries (A-M Systems, Carlsborg, WA) that had been tapered with a micropipet puller (Narashige, Tokyo, Japan). The platinum wire was inserted in a capillary, left protruding by ∼50 µm, and sealed with epoxy (Epon 828 with 14% m-phenylenediamine by weight, Miller-Stephenson Chemical Co., Danbury, CT). Excess epoxy was removed with acetone, and (37) Michael, D. J.; Joseph, J. D.; Kilpatrick, M. R.; Travis, E. R.; Wightman, R. M. Anal. Chem. 1999, 71, 3941-3947. (38) Heien, M.; Wightman, R. M., local user manual. (39) Jimenez, V. J.; Song, Y.; Harper, A. S., manuscript in preparation.
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Figure 2. Illustration of the data analysis procedure employed in all fast-scan microelectrode detector results presented in this report. In the illustration, Au38 hexanethiolate MPCs were detected. In the voltammogram plots, positive-going potential scans produce downward, anodic, current peaks.
the assembly was cured at 150 °C for 12 h (or at 100 °C for 2 days). The electrode was electrochemically cleaned before each run by repeatedly scanning in 0.05 M H2SO4 from -0.4 to 1.3 V for 5 min at a rate of 5 V/s. The reference electrode was Ag/Ag+ (Ag wire/AgNO3 (1 mM)/Bu4NPF6 (0.1 M)/acetonitrile). FSCV Data Acquisition and Analysis. Cyclic voltammograms were acquired under PC control using data acquisition hardware and previously described37 local software written in LabVIEW (National Instruments, Austin, TX). The potential waveform was generated and voltammetric currents were acquired with a National Instruments PC interface card (PCI-6052E); another interface card (PCI-6711E) synchronized waveform application and data acquisition. The waveform was input into a custombuilt potentiostat (Department of Chemistry Electronics Shop, UNCsChapel Hill). Background current subtraction, signal averaging, and digital filtering were all done under software control. Data analysis was performed with local software written in LabVIEW.38 The procedure is outlined in Figure 2. The collected three-dimension potential-current-time information is represented 4914 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
as a color plot (panel A), with x axis representing the triangular wave scan number (which can be translated into retention time based upon experimental settings), the y axis corresponding to electrode potential, and false color for the value of current detected. A “voltammetric chromatogram” can be extracted from these data by plotting current at some defined potential, such as the horizontal white dashed line in panel A, with the example result in panel B. A background-corrected voltammogram (panel C) is obtained by subtracting the current-potential profile at a sample-free elution time (blue circle in panel B) from the currentpotential profile at a time under an elution peak (red circle). In the example shown, a voltammogram of hexanethiolate-protected Au38 MPCs is produced as shown in panel C. RESULTS AND DISCUSSION The FSCV experiment was first evaluated with known substancessferrocene as a redox molecule and samples of reasonably monodisperse MPCs (hexanethiolate-coated Au140 and Au38). Curves 1A, 2A, and 3A in Figure 3 show their conventional
Figure 3. (A) Cyclic voltammograms of (1A) ferrocene (2 mM), (2A) hexanethiolate-coated Au38 MPC (1 mM), and (3A) hexanethiolatecoated Au140 MPC (0.2 mM), measured at 100 mV/s on a 3-mmdiameter Pt working electrode (E vs Ag/Ag+). Current scale S ) 20, 15, and 2 µA. (B) Background-corrected fast-scan CVs (scan rate 150 V/s) at a 25-µm-diameter, 50-µm-length Pt cylinder microelectrode, of the (1B) ferrocene redox molecule, (2B) hexanethiolatecoated Au38 MPCs, and hexanethiolate-coated Au140 MPC directly off the C8 HPLC column. Current scale S ) 15, 20, and 10 nA. ∆Epeak ) 90 and 193 mV for curves 1A and 1B. Average ∆V peak spacings 300, 435, 270, and 290 mV in curves 2A, 2B, 3A, and 3B, respectively. In the voltammogram plots, positive-going potential scans produce downward, anodic, current peaks. The chromatograms of these three species, detected optically with a PDA detector, showed only single eluted peaks. Flow rate was 0.7 mL/min, mobile phase was CH2Cl2 with 10 mM Bu4NClO4, and the injected samples were 50 µL of 0.05 mM solutions.
cyclic voltammetry, while curves 1B, 2B, and 2C show fast potential scan voltammograms taken of their elution peaks from an HPLC column (silica bonded C8 stationary phase). The corresponding PDA-detected chromatograms (not shown) exhibit only a single eluted peak. The voltammograms in curves 1A and 1B display the signature ferrocene electrochemistry. The 90-mV ∆EPEAK in the macroelectrode voltammetry and the 193-mV ∆EPEAK in the FSCV result are not surprising considering the fast scan rate in the latter and that no particular measures were taken to limit iRUNC (uncompensated iR drop) effects. The cyclic voltammetry of hexanethiolate-coated Au38 MPCs (Figure 3, curve 2A) shows two one-electron oxidations spaced by ∆V ) 300 mV (with ∆EPEAK ) 160 mV) and by other oxidation steps ∼0.6V more positive. We have previously described the Au38 MPC voltammetry;10 the doublet of oxidation steps is associated with a twoelectron occupancy of a HOMO level (highest occupied molecular
orbital), and the further oxidations with a somewhat lower-lying molecular orbital. The doublet of oxidation steps can be clearly seen in the FSCV result (curve 2B). There is some distortion in that their ∆EPEAK values are larger (like curve 1B) and also that for the Au38+1/0 wave (400 mV) is larger than that of the Au38+2/+1 wave (250 mV). ∆EPEAK is the same for the two waves in curve 2A, so the distortion in curve 2B is probably not an electrontransfer kinetic effect but rather an artifact associated with the background current correction. Figure 3, curve 3A shows the well-studied9,41 series of oneelectron transfers of Au140 hexanethiolate MPCs, each corresponding to change of the cluster core charge by a single oxidative or reductive electron transfer. This pattern represents a quantized double layer charging property. The fast-scan CV of the Au140 sample (curve 3B) also shows the series of roughly even-spaced voltammetric peaks (average spacing ∆V ) 290 mV). In summary, the results in Figure 3 show that (a) the general features of conventional voltammetry of the chosen substances are reproduced in the FSCV of their eluted HPLC peaks, (b) iRUNC effects are present attendant to fast scanning and a nonoptimum flow cell design, and (c) there is some distortion of the voltammetry at the limits of the potential scan window due to hysteresis from the cell time constant turnover in which the charging current background correction does not exactly match that when the sample is present. Nonetheless, the fast-scan technique successfully detected the nanoparticle elutions and thereby offers potential information on patterns of electron-transfer steps of as-yet undetected electrochemically chargeable nanoparticles. Next discussed are comparisons of three different HPLC detection modes, in a separation of a hexanethiolate-coated MPC sample that is polydisperse in core size. Figure 4 shows results for detection by optical absorbance (top, PDA detector), constant potential amperometry (middle), and FSCV (bottom, the current chromatogram presented is selected at 250 mV versus Ag/Ag+ from the current-potential record of 150 V/s fast-scan cyclic voltammetry). It is immediately evident that the three chromatograms display nearly identical patterns of eluting peaks and at similar retention times; i.e., the three detection methods all respond to the nanoparticles and show similar chromatographic resolution. We will discuss the detector responses as obtained for the group of peaks A and B as labeled in Figure 4A. The PDA electronic UV-visible spectra of clusters eluting under peak A and peak B, are shown in Figure 5, top. The two spectra are distinctively different; that from peak A has a steplike fine structure suggestive of small MPC core sizes (
