Anal. Chem. 2006, 78, 2779-2785
Ion-Pair Chromatographic Separation of Water-Soluble Gold Monolayer-Protected Clusters Martin M. F. Choi,† Alicia D. Douglas, and Royce W. Murray*
Kenan Laboratories of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
We demonstrate the efficacy of ion-pair chromatography for separations of samples of charged, polydisperse, water-soluble gold nanoparticles protected by monolayers of N-acetyl-L-cysteine and of tiopronin ligands. These nanoparticle mixtures have 1-2-nm-diameter Au core sizes as estimated from UV-visible spectra of the separated components. This size range encompasses the transition from bulk metal to molecular properties. The nanoparticle mixtures were resolved, the smallest nanoparticles eluting first, on an octadecylsilyl (C18) column using isocratic elution with a methanol/water mobile phase containing tetrabutylammonium fluoride (Bu4N+F-) and phosphate buffer. The column retention increases with Bu4N+F- concentration, lowered pH, and decreasing methanol volume fraction. The retention mechanism is dominated by ion-pairing in either the mobile phase or at the stationary/mobile-phase interface. Size exclusion effects, used in many previous nanoparticle separations, are insignificant. Gold colloids have fascinated science from the time of alchemists. In the past two decades, Au nanoparticles have attracted considerable research attention owing to their unique chemical and physical properties.1-6 Nanoscience has also become an exciting pioneer field in analytical chemistry. Au nanoparticles protected from metal-metal aggregation by monolayers of thiolate ligands (monolayer-protected clusters, MPCs)2,5 have special interest because of the ease of manipulating the monolayer * Corresponding author. E-mail:
[email protected]. † Visiting scientist on sabbatical leave from: Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, P.R. China. (1) (a) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293-346. (b) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (2) Whetten, R. L.; Khoury, J. T.; Alvarez, M. M.; Murthy, S.; Vezmar, I.; Wang, Z. L.; Stephen, P. W.; Cleveland, C. L.; Luedtke, W. D.; Landman, U. Adv. Mater. 1996, 5, 428-433. (3) (a) Chen, S.; Ingram, R. S.; Hostetler, M. J.; Pietron, J. J.; Murray, R. W.; Schaaff, T. G.; Khoury, J. T.; Alvarez, M. M.; Whetten, R. L. Science 1998, 280, 2098-2101. (b) Hicks, J. F.; Templeton, A. C.; Chen, S.; Sheran, K. M.; Jasti, R.; Murray, R. W. Anal. Chem. 1999, 71, 3703-3711. (4) Schmid, G.; Baumle, M.; Geerkens, M.; Oseman, C.; Sawitowski, T. Chem. Soc. Rev. 1999, 28, 179-185. (5) (a) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802. (b) Terrill, R. H.; Postlethwaite, T. A.; Chen, C.-H.; Poon, C.-D.; Terzis, A.; Chen, A.; Hutchison, J. E.; Clark, M. R.; Wignall, G.; Londono, J. D.; Superfine, R.; Falvo, M.; Johnson, C. S., Jr.; Samulski, E. T.; Murray, R. W. J. Am. Chem. Soc. 1995, 117, 1253712547. (6) Weare, W. W.; Reed, S. M.; Warner, M. G.; Hutchison, J. E. J. Am. Chem. Soc. 2000, 122, 12890-12891. 10.1021/ac052167m CCC: $33.50 Published on Web 02/28/2006
© 2006 American Chemical Society
chemistry. Our laboratory has been concerned with the analytical chemistry of MPCs and particularly with Au nanoparticles with 1-3-nm core diameters. This is an important size range, since within it, bulk Au metal properties are giving way to moleculelike properties, and properties become size-dependent. The availability of well-defined, purified nanoscopic materials is essential in investigating relationships between the size of a nanoparticle and its chemical, optical, and physical properties. Our understanding of such relationships remains very incomplete, including nanoparticles based on metallic elements. An important and challenging fact is that syntheses of small-size Au nanoparticles (1-5-nm core diameters) generally result in mixtures of core sizes (polydispersity)7 and possibly also variations in the number of protecting ligands on each MPC. Much effort has consequently been directed at improving MPC core uniformity, at ways to measure it, and at developing advanced separation techniques. Chromatographic techniques offer powerful new approaches. The size dependency of optical spectra and voltammetry of separated MPCs can be visualized by chromatographic systems coupled with photodiode array (PDA)8-10 and electrochemical detectors,9 respectively. Chromatographic and electrophoretic methods that have been explored include gel electrophoresis,11,12 capillary electrophoresis,13-15 size exclusion chromatography (SEC),8,16-18 reversed-phase HPLC,9,10,19 and ion (7) Hostetler, M. J.; Wingate, J. E.; Zhong, C. J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Murray, R. W. Langmuir 1998, 14, 17-30. (8) Wei, G.-T.; Liu, F.-K.; Wang, C. C. R. Anal. Chem. 1999, 71, 2085-2091. (9) Song, Y.; Jimenez, V.; McKinney, C.; Donkers, R.; Murray, R. W. Anal. Chem. 2003, 75, 5088-5096. (10) Jimenez, V. L.; Leopold, M. C.; Mazzitelli, C.; Jorgenson, J. W.; Murray, R. W. Anal. Chem. 2003, 75, 199-206. (11) (a) Schaaff, T. G.; Knight, G.; Shafigullin, M. N.; Borkman, R. F.; Whetten, R. L. J. Phys. Chem. B 1998, 102, 10643-10646. (b) Schaaff, T. G.; Whetten, R. L. J. Phys. Chem. B 2000, 104, 2630-2641. (12) Negishi, U.; Nobusada, K.; Tsukuda, T. J. Am. Chem. Soc. 2005, 127, 52615270. (13) Schnabel, U.; Fischer, C.: Kenndler, E. J. Microcolumn Sep. 1997, 9, 529534. (14) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121, 7081-7089. (15) (a) Rodriguez, M. A.; Armstrong, D. W. J. Chromatogr., B 2004, 800, 7-25. (b) Liu, F.-K.; Lin, Y.-Y.; Wu, C.-H. Anal. Chim. Acta 2005, 528, 249-254. (16) (a) Wei, G.-T.; Liu, F.-K. J. Chromatogr., A 1999, 836, 253-260. (b) Fischer, Ch.-H.; Lilie, J.; Weller, H.; Katsikas, L.; Henglein, A. Ber. Bunsen-Ges. 1989, 93, 61-64. (c) Fischer, Ch.-H.; Weller, H.; Katsikas, L.; Henglein, A. Langmuir 1989, 5, 429-432. (17) (a) Wilcoxon, J. P.; Martin, J. E.; Provencio, P. Langmuir 2000, 16, 99129920. (b) Wilcoxon, J. P.; Martin, J. E.; Provencio, P. J. Chem. Phys. 2001, 115, 998-1008. (18) Siebrands, T.; Giersig, M.; Mulvaney, P.; Fischer, C.-H. Langmuir 1993, 9, 2297-2300.
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exchange chromatography.20 Ultracentrifugation and molecular imprinting as separation tools have also been recently reported.21 While TEM has been widely employed to measure nanoparticle size and shape, its results are increasingly uncertain for cores smaller than ∼2 nm, as has been discussed in some detail.17 A large fraction of the separations research has dealt with (water-insoluble) nanoparticles having nonpolar protecting layers, such as alkanethiolate-protected MPCs. Considering the potential bioanalytical and biomedical applications of MPCs, aqueous stability and solubility are also important, and a number of watersoluble nanoparticles have been reported,11,12,14,22-25 including glutathione-protected Au MPCs fractionated by gel electrophoresis11,12 and tiopronin-protected Au MPCs separated by capillary electrophoresis.22 The above contributions notwithstanding, the efficiency of nanoparticle separations remains quite primitive in comparison to the better-developed fields of separations of small molecules by gas or liquid chromatography. Nanoparticles are large entities, often with poorly understood chemical composition. As part of an effort to expand the methodology applicable to nanoparticle separations, we describe here the first use of ion-pair chromatography26 for separations of samples of water-soluble, polydisperse Au core MPCs coated with N-acetyl-L-cysteine or with tiopronin ligands. The former are newly synthesized MPCs. N-Acetyl-L-cysteine is a therapeutic drug27 used as a mucolytic reagent,28 and in the treatment of acetaminophen hepatotoxicity, and is of interest for antioxidant/radical-scavenging activity.29 We have described tiopronin-coated Au MPCs previously.14,22 The estimated average core sizes of both MPCs lie in the interesting metal-to-molecule dimension range. They are negatively charged owing to dissociation of some of the numerous carboxylic acid functionalities present in the thiolate monolayer shell. Ion-pair chromatography, while well known26 for small ions and charged molecules, has not been employed previously in nanoparticle separations. All separations of charged nanoparticles cited above have employed either SEC or electrophoretic methods, and none has relied on ion-pair interactions. Ion-pair (ion-interaction) chromatographic separations are conceptually straightforward, involving interaction of a (necessarily ionic) sample with a hydrophobic modifier counterion at a (19) Song, Y.; Heien, M. LAV; Jimenez, V.; Wightman, R. M.; Murray, R. W. Anal. Chem. 2004, 76, 4911-4919. (20) Bos, W.; Steggerda, J. J.; Yan, S.; Casalnuovo, J. A.; Mueting, A. M.; Pignolet, L. H. Inorg. Chem. 1988, 27, 948-954. (21) (a) Calabrette, M.; Jamison, J. A.; Falkner, J. C.; Liu, Y.; Yuhas, B. D.; Matthews, K. S.; Colvin, V. L. Nano Lett. 2005, 5, 963-967. (b) Koenig, S.; Chechik, V. Chem. Commun. 2005, 4110-4112. (22) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir 1999, 15, 66-76. (23) Huang, T.; Murray, R. W. J. Phys. Chem. B 2001, 105, 12498-12502. (24) Le´vy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076-10084. (25) Woehrle, G. H.; Brown, L. O.; Hutchison, J. E. J. Am. Chem. Soc. 2005, 127, 2172-2183. (26) (a) Hearn, M. T. W., Ed. Ion-Pair Chromatography: Theory and Biological and Pharmaceutical Applications; Marcel Dekker: New York, 1985. (b) Fritz, J. S. J. Chromatogr., A 2005, 1085, 8-17. (c) Stahlberg, J. J. Chromatogr., A 1999, 855, 3-55. (27) Prescott, L. F.; Illingworth, R. N.; Critchley, J. A. J. H.; Stewart, M. J.; Adam, R. D.; Proudfoot, A. T. Br. Med. J. 1979, 2, 1097-1100. (28) Boman, G.; Ba¨cker, U.; Larsson, S.; Melander, B.; Wa¨hlinder, L. Eur. J. Respir. Dis. 1983, 64, 405-415. (29) Sa¨rnstrand, B.; Tunek, A.; Sjo¨din, K.; Hallberg, A. Chem.-Biol. Interact. 1995, 94, 157-164.
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hydrophobic stationary-phase interface. Detailed models26 for the process remain debated and include the formation of ion pairs in the stationary phase followed by partition, transformation of the stationary phase into an ion exchange/ion pairing surface through partition of the hydrophobic modifier (dynamic ion exchange model), and a nonstoichiometric, double layer model of thusly charged stationary-phase interfaces. More than one of these phenomena may in fact be concurrently involved. Our objective in the nanoparticle separations is not to address the issue of mechanistic models, although we note that the fact that the nanoparticles are a different kind of analyte may open some new routes to inquiry about the mechanism. The known solubilization into toluene, of water-soluble carboxylated gold nanoparticles30 by tetraoctylammonium bromide, suggests that separations of polydisperse N-acetyl-L-cysteine and tiopronin-protected MPCs (both with carboxylic acid-bearing monolayers) might be possible using a quaternary ammonium modifier and a nonpolar stationary phase. Indeed, tetrabutylammonium ions have been shown to partition at a reversed-phase C18 column.26 To explore this idea, we chose a C18 column, tetrabutylammonium fluoride (Bu4N+F-) as modifier, and a buffered, mixed methanol/water mobile phase. The separations proved to be rather efficient, but require careful selection of mobile-phase composition (concentration of Bu4N+F-, methanol (MeOH) content, and pH). The separations were detected using a PDA detector, so that UV-visible spectra were recorded of separated nanoparticles. Au MPCs coated with water-solubilizing thiolate ligands display optical absorbance spectra that are qualitatively diagnostic of MPC core size. Notably, a broad surface plasmon absorbance31 at ∼520 nm is seen for with larger cores (>2.2 nm), while smaller core (98%) were obtained from Aldrich (Milwaukee, WI), and N-acetyl-Lcysteine (>99%) and N-(2-mercaptopropionyl)glycine (tiopronin, 99%) were from Sigma (St. Louis, MO). HAuCl4‚3H2O was synthesized according to the literature.32 Acetone, ethanol, glacial acetic acid, MeOH (HPLC-grade), sodium hydrogen phosphate, and tetrahydrofuran (HPLC grade) were purchased from Fisher Chemicals (Fair Lawn, NJ), and sodium dihydrogen phosphate monohydrate (NaH2PO4‚H2O) were from Mallinckrodt Chemical (Phillipsburg, NJ). Water was purified by passing house-distilled water through a Barnstead (Dubuque, IA) NANOpure system (g18 MΩ). All reagents were of analytical reagent grade or above and were used as received. (30) Yao, H.; Momozawa, O.; Hamatani, T.; Kimura, K. Chem. Mater. 2001, 13, 4692-4697. (31) 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. (32) 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.
Synthesis of N-Acetyl-L-cysteine-Protected MPC. The synthesis is similar to that of tiopronin-protected MPCs.22 NaBH4 (3.57 g, 94.3 mmol) in 45 mL of ethanol was added with rapid stirring to a 0 °C solution of 1.82 g (4.63 mmol) HAuCl4‚3H2O and 2.28 g (13.9 mmol) of N-acetyl-L-cysteine codissolved in 200 mL of 6:1 (v/v) MeOH/glacial acetic acid. The dark-brown solution formed was stirred for 30 min and the solvent removed under vacuum and at
