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Polymer Monoliths with Exchangeable Chemistries: Use of Gold Nanoparticles As Intermediate Ligands for Capillary Columns with Varying Surface Functionalities Qing Cao,†,‡ Yan Xu,† Feng Liu,‡ Frantisek Svec,§,† and Jean M. J. Fre´chet*,§,† College of Chemistry, University of California, Berkeley, California 94720-1460, The Molecular Foundry, E.O. Lawrence Berkeley National Laboratory, Berkeley, California 94720-8139, and Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry, Peking University, Beijing 100871, China Newly developed porous polymer monolithic capillary columns modified with gold nanoparticles coated with exchangeable functionalities allow easy switching of separation modes by a simple ligand exchange process. These columns are prepared from a poly(glycidyl methacrylateco-ethylene dimethacrylate) monolith through reaction of its epoxide moieties with cysteamine to afford a monolith rich in surface thiol groups. Gold nanoparticles prepared via in situ reduction of chloroauric acid within the column become attached to the surface of the pores of the monolith. Alternatively, a solution of colloidal gold nanoparticles can be pumped through the thiol modified column to achieve their attachment. While the first approach is faster, it affords a lower coverage of nanoparticles than the second method, while both methods preserve the excellent hydrodynamic properties that are typical of the monolithic columns. Functionalization of the surface of the bound gold nanoparticles is then carried out using low molecular weight thiol-containing surface ligands. The dynamic nature of the bond between gold and these surface ligands enables the replacement of one surface ligand by another through a simple solution exchange process. This novel approach expands the application range of monoliths as a single column can now be used in different separations modes. Applications of the columns with exchangeable chemistries are demonstrated with the capillary electrochromatographic separation of peptides and the nano-high-pressure liquid chromatography (HPLC) separation of proteins in both reversed phase and ion exchange modes. Monolithic columns consisting of rigid synthetic polymers have emerged in the early 1990s as an alternative to packed chromatographic columns.1,2 Because of their unusual porous structure, * To whom correspondence should be addressed. Phone: 510 643 3077. Fax: 510 643 3077. E-mail:
[email protected]. † University of California. ‡ Peking University. § The Molecular Foundry. (1) Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1992, 54, 820–822. (2) Wang, Q.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1993, 65, 2243–2248.
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monolithic columns provide for rapid convectional mass transport with very low resistance to flow. As a result, these stationary phases have been used in the very fast high-pressure liquid chromatography (HPLC) separations of large molecules such as proteins,2–4 nucleic acids,5,6 and synthetic polymers.7,8 Monolithic columns are especially attractive for use in capillary electrochromatography (CEC) as they are readily prepared in situ from liquid precursors, which alleviates most of the problems associated with packing of capillary columns with microparticles.9-13 The success of monolithic columns in CEC then led to the development of monolithic capillary columns for HPLC14,15 and microfluidics.16,17 These developments have been summarized in numerous reviews and books, e.g., ref 18. Two important variables that control the performance of polymer monoliths are their porous structure and their chemistry. The porous structure is controlled by factors such as the duration and temperature of the polymerization reaction, the composition of the pore-forming solvent mixture or porogen, and the percentage of cross-linking monomer in the polymerization mixture.18–21 Controlling these factors enables the fine-tuning of pore size within (3) Xie, S.; Allington, R. W.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1999, 865, 169–174. (4) Premstaller, A.; Oberacher, H.; Walcher, W.; Timperio, A. M.; Zolla, L.; Chervet, J. P.; Cavusoglu, N.; van Dorsselaer, A.; Huber, C. G. Anal. Chem. 2001, 73, 2390–2396. (5) Premstaller, A.; Oberacher, H.; Huber, C. G. Anal. Chem. 2000, 72, 4386– 4393. (6) Huber, C. G.; Premstaller, A.; Xiao, W.; Oberacher, H.; Bonn, G. K.; Oefner, P. J. J. Biochem. Biophys. Methods 2001, 47, 5–19. (7) Petro, M.; Svec, F.; Gitsov, I.; Fre´chet, J. M. J. Anal. Chem. 1996, 68, 315–321. (8) Petro, M.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1996, 752, 59–66. (9) Ericson, C.; Liao, J. L.; Nakazato, K.; Hje´rten, S. J. Chromatogr., A 1997, 767, 33–41. (10) Palm, A.; Novotny, M. V. Anal. Chem. 1997, 69, 4499–4507. (11) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3646–3649. (12) Zhang, M. Q.; El Rassi, Z. Electrophoresis 2001, 22, 2593–2599. (13) Bedair, M.; El Rassi, Z. Electrophoresis 2002, 23, 2938–2948. (14) Moravcova, D.; Jandera, P.; Urban, J.; Planeta, J. J. Sep. Sci. 2003, 26, 1005–1016. (15) Lee, D.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 2004, 1051, 53–60. (16) Yu, C.; Xu, M.; Svec, F.; Fre´chet, J. M. J. J. Polymer Sci., Polym. Chem. 2002, 40, 755–769. (17) Stachowiak, T. B.; Rohr, T.; Hilder, E. F.; Peterson, D. S.; Yi, M.; Svec, F.; Fre´chet, J. M. J. Electrophoresis 2003, 24, 3689–3693. 10.1021/ac1015613 2010 American Chemical Society Published on Web 08/03/2010
a broad range spanning at least 2 orders of magnitude from tens to thousands of nanometers. Control of the surface chemistry of the monoliths is generally achieved using either the direct copolymerization of functional monomers,2,22,23 the chemical modification of preformed reactive monoliths,1,24-26 or the grafting of functional polymer chains onto the surface of pores.27-30 Although the single step approach involving direct copolymerization of functional monomers is powerful, it suffers from the fact that the nature of the functional monomer and composition of the polymerization mixture directly affect the porous structure of the resulting monolith. Thus, each time a new monomer is used, both the polymerization mixture and the reaction conditions must be adjusted to achieve the desired porous properties. Another fundamental limitation of this approach is the distribution of functional monomers throughout the monolith. The presence of retention sites buried within the dense polymer structure requires diffusional mass transport of analytes thus limiting the utility of the single step approach to the preparation of monoliths suitable for the separation of macromolecules which are too large to penetrate the polymer structure. In contrast, both the chemical modification and the grafting approaches enable the independent control of porous structure and surface chemistry. The newest approach leading to monoliths with specific chemical properties relies on functionalization of the pore surface with nanoparticles. Although functionalized nanoparticles have been used for the preparation of particulate column packings several decades ago,31 application of this process in the field of monoliths occurred only recently. For example, Hilder et al. have prepared a monolith containing sulfonic acid functionalities, to which they have subsequently attached monodisperse polystyrene latex nanoparticles with quaternary amine groups.32 These monoliths functionalized with reactive nanoparticles have shown excel(18) Svec, F.; Fre´chet, J. M. J. Science 1996, 273, 205–211. Deyl, Z.; Svec, F. Capillary Electrochromatography; Elsevier: Amsterdam, The Netherlands, 2001. Svec, F.; Tennikova, T. B.; Deyl, Z. Monolithic Materials: Preparation, Properties, and Applications; Elsevier: Amsterdam, The Netherlands, 2003. Svec, F.; Huber, C. G. Anal. Chem. 2006, 78, 2100–2107. Svec, F.; Stachowiak, T. B. Macroporous monoliths for chromatographic separations in microchannels. In Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques; Landers, J., Ed.; Taylor & Francis: Boca Raton, FL, 2007; pp 1297-1326. Guiochon, G. J. Chromatogr., A 2007, 1168, 101–168. Svec, F. J. Chromatogr., A 2010, 1217, 902–924. (19) Svec, F.; Fre´chet, J. M. J. Chem. Mater. 1995, 7, 707–715. (20) Viklund, C.; Svec, F.; Fre´chet, J. M. J.; Irgum, K. Chem. Mater. 1996, 8, 744–750. (21) Xie, S.; Svec, F.; Fre´chet, J. M. J. J. Polym. Sci., Polym. Chem. 1997, 35, 1013–1021. (22) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 2288–2295. (23) La¨mmerhofer, M.; Peters, E. C.; Yu, C.; Svec, F.; Fre´chet, J. M. J.; Lindner, W. Anal. Chem. 2000, 72, 4623–4628. (24) Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1995, 702, 89–95. (25) Xie, S.; Svec, F.; Fre´chet, J. M. J. Polym. Prepr. 1997, 38, 211–212. (26) Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3131–3139. (27) Viklund, C.; Svec, F.; Fre´chet, J. M. J.; Irgum, K. Biotechnol. Prog. 1997, 13, 597–600. (28) Viklund, C.; Irgum, K.; Svec, F.; Fre´chet, J. M. J. Macromolecules 2001, 34, 4361–4369. (29) Rohr, T.; Ogeltree, D. F.; Svec, F.; Fre´chet, J. M. J. Adv. Funct. Mater. 2003, 13, 265–270. (30) Rohr, T.; Hilder, E. F.; Donovan, J. J.; Svec, F.; Fre´chet, J. M. J. Macromolecules 2003, 36, 1677–1684. (31) Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, 1801– 1809. Stevens, T. S.; Langhorst, M. A. Anal. Chem. 1982, 54, 950–953. (32) Hilder, E. F.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 2004, 1053, 101–106.
lent performance in the separation of sugars. In more recent work, Haddad and co-workers have used this concept for the preparation of columns for ion chromatography and CEC.33-40 Gold nanoparticles (GNP) described by Faraday more than 150 years ago41 have since been applied in many different fields and now constitute one of the most extensively studied nanomaterials, though few applications have been explored in separation science.42-47 They have been used to form a self-assembled layer in capillaries for open tubular electrochromatography48-50 and gas chromatography51,52 and as pseudostationary phase in capillary and microchip electrophoresis.53-57 To date, only two reports describe packings for capillary HPLC prepared via the coating of silica-gel and nonporous polystyrene particles with GNP.58,59 Recently, Connolly et al. have described the immobilization of gold nanoparticles on a poly(butyl methacrylate-co-ethylene dimethacrylate) monolith with photografted poly(4,4-dimethyl-2vinylazlactone) chains modified to bear amine groups.60 Application of their conjugate has yet to be demonstrated. We have developed monolithic columns containing GNP immobilized via thiol chemistry and demonstrated their use for the selective (33) Hutchinson, J. P.; Zakaria, P.; Bowie, A. R.; Macka, M.; Avdalovic, N.; Haddad, P. R. Anal. Chem. 2005, 77, 407–416. (34) Zakaria, P.; Hutchinson, J. P.; Avdalovic, N.; Liu, Y.; Haddad, P. R. Anal. Chem. 2005, 77, 417–423. (35) Hutchinson, J. P.; Hilder, E. F.; Macka, M.; Avdalovic, N.; Haddad, P. R. J. Chromatogr., A 2006, 1109, 10–18. (36) Hutchinson, J. P.; Hilder, E. F.; Shellie, R. A.; Smith, J. A.; Haddad, P. R. Analyst 2006, 131, 215–221. (37) Zhang, S.; Macka, M.; Haddad, P. R. Electrophoresis 2006, 27, 1069–1077. (38) Hutchinson, J. P.; Macka, M.; Avdalovic, N.; Haddad, P. R. J. Chromatogr., A 2006, 1106, 43–51. (39) Glenn, K. M.; Lucy, C. A.; Haddad, P. R. J. Chromatogr., A 2007, 1155, 8–14. (40) Haddad, P. R.; Hilder, E. F.; Evenhuis, C.; Schaller, D.; Pohl, C.; Flook, K. J. Abstracts of Papers, Spring ACS National Meeting, Salt Lake City, UT, March 22-26, 2009; ANYL-236. (41) Faraday, M. Philos. Trans. R. Soc. London 1857, 147, 145–181. (42) Zhang, Z.; Wang, Z.; Liao, Y.; Liu, H. J. Sep. Sci. 2006, 29, 1872–1878. (43) Nilsson, C.; Birnbaum, S.; Nilsson, S. J. Chromatogr., A 2007, 1168, 212– 224. (44) Guihen, E.; Glennon, J. D. Anal. Lett. 2003, 36, 3309–3336. (45) Nilsson, C.; Nilsson, S. Electrophoresis 2006, 27, 76–83. (46) Zhang, Z.; Yan, B.; Liao, Y.; Liu, H. Anal. Bioanal. Chem. 2008, 391, 925– 927. (47) Sykora, D.; Kasicka, V.; Miksik, I.; Rezanka, P.; Zaruba, K.; Matejka, P.; Kral, V. J. Sep. Sci. 2010, 33, 372–387. (48) O’Mahony, T.; Owens, V. P.; Murrihy, J. P.; Guihen, E.; Holmes, J. D.; Glennon, J. D. J. Chromatogr., A 2003, 1004, 181–193. (49) Yang, L.; Guihen, E.; Glennon, J. D. J. Sep. Sci. 2005, 28, 757–766. (50) Yang, L.; Guihen, E.; Holmes, J. D.; Loughran, M.; O’Sullivan, G. P.; Glennon, J. D. Anal. Chem. 2005, 77, 1840–1846. (51) Gross, G. M.; Nelson, D. A.; Grate, J. W.; Synovec, R. E. Anal. Chem. 2003, 75, 4558–4564. (52) Gross, G. M.; Grate, J. W.; Synovec, R. E. J. Chromatogr., A 2004, 1029, 185–192. (53) Neiman, B.; Grushka, E.; Lev, O. Anal. Chem. 2001, 73, 5220–5227. (54) Huang, M. F.; Kuo, Y. C.; Huang, C. C.; Chang, H. T. Anal. Chem. 2004, 76, 192–196. (55) Yu, C. J.; Su, C. L.; Tseng, W. L. Anal. Chem. 2006, 78, 8004–8010. (56) Pumera, M.; Wang, J.; Grushka, E.; Polsky, R. Anal. Chem. 2001, 73, 5625– 5628. (57) Lin, Y. W.; Huang, M. J.; Chang, H. T. J. Chromatogr., A 2003, 1014, 47– 55. (58) Ortiz, Y.; Cintron, J. M.; Colon, L. A. Abstracts of Papers, Spring ACS National Meeting, San Diego, CA, April 1-5, 2001; ANYL-074. (59) Kobayashi, K.; Kitagawa, S.; Ohtani, H. J. Chromatogr., A 2006, 1110, 95– 101. (60) Connolly, D.; Twamley, B.; Paull, B. Chem. Commun. 2010, 46, 2109– 2111. (61) Xu, Y.; Cao, Q.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2010, 82, 3352– 3358.
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retention of cysteine containing peptides and the simplification of a peptide mixture prior to its separation.63 In this report we exploit the dynamic nature of the bond between gold and thiol groups to introduce the concept of monoliths with exchangeable chemistries. This novel approach expands the application range of monoliths since a single column can be used in different separations modes as shown below with capillary electrochromatographic and nano-HPLC separations of peptides and proteins. EXPERIMENTAL SECTION Materials. Ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) were obtained from Sigma-Aldrich (St. Louis, MO) and purified by passing them through an inhibitor remover column. Cyclohexanol, 1-dodecanol, (3-methacryloyloxypropyl)trimethoxysilane, azobisisobutyronitrile (AIBN), chloroauric acid, sodium hydroxide, hydrochloric acid, cysteamine, sodium borohydride, trisodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, 1-octadecanethiol, sodium-2-mercaptoethanesulfonate, mercaptopropionic acid, 2-mercaptoethanol, formic acid, cytochrome c (bovine pancreas, pI 10.2, hydrophobicity index 92), ribonuclease A (bovine heart, pI 9.45, hydrophobicity index 75), myoglobin (horse skeletal muscle, pI 7.2, hydrophobicity index not available), peptides (Tyr-Gly, Tyr-Gly-Gly, and TyrGly-Gly-Phe-Leu), and HPLC-grade solvents (acetonitrile, methanol, acetone) were purchased from Sigma-Aldrich and used as received. Solutions of gold nanoparticles (GNP) with a size of 15 and 10 nm were obtained from BB International (Cardiff, U.K.). Peptides and proteins were dissolved in water at typical concentrations of 0.1 and 0.2 mg/mL, respectively. Teflon and polyimide coated 100 µm i.d. fused silica capillaries were purchased from Polymicro Technologies (Phoenix, AZ). Instrumentation. A syringe pump (Kd Scientific, New Hope, PA) was utilized for the washing steps after the modifications of monolithic columns with cysteamine. GNP colloids were pumped through the monolithic capillary columns using a high pressure 260D syringe pump (ISCO, Lincoln, NE) and a 7725 manual sixport sample injection valve (Rheodyne, Rohnert Park, CA) with a 2 mL loop. Capillary electrochromatography (CEC) experiments were carried out using an HP3D capillary electrophoresis system (Agilent Technologies, Santa Clara, CA) equipped with a diode array detector and an external pressurization system. The mobile phase consisted of 10 mmol/L sodium phosphate buffer solution pH 2.5 containing 50% (v/v) acetonitrile. The applied voltage was +10 kV for the column containing amine functionalities and -10 kV for the columns functionalized with carboxyl and hydroxyl groups. The UV detection wavelength was adjusted to 214 nm. Both ion-exchange and reversed phase nano-HPLC separations were carried out using an Agilent 1200 series nanoflow LC system consisting of a microvacuum degasser, a nanopump, a micro wellplate autosampler, and a multiple wavelength detector. A linear mobile phase gradient from 0.02 mol/L phosphate buffer, pH 7.9, to 0.6 mol/L sodium chloride in the phosphate buffer in 3.5 min at a flow rate of 4.0 µL/min was used for elution in ion-exchange chromatography and the separation detected at 210 nm. Reversed phase chromatography was carried out using a linear mobile phase 7418
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gradient of 0-60% acetonitrile in 0.1% aqueous formic acid in 3.5 min at a flow rate of 4.0 µL/min and the separation detected at 200 nm. Scanning electron micrographs of capillary cross section and energy dispersive spectra were obtained using a Hitachi S-4300 SE/N Schottky emission scanning electron microscope (SEM) integrated with a backscattered electron (BSE) detector (Hitachi, Pleasanton, CA) and an energy dispersive X-ray spectrometer (EDS, Thermo Electron). The monoliths were coated with a thinlayer of conductive carbon to avoid charging and heating of the sample irradiated with the electron beam during both imaging and compositional analysis. Preparation of Generic Monoliths. The inner wall of the 100 µm i.d. fused-silica capillary was vinylized to enable covalent attachment of the monolith.62 A mixture consisting of 24% GMA (functional monomer), 16% EDMA (cross-linker), 30% cyclohexanol and 30% 1-dodecanol (porogens), and 1% AIBN, (with respect to monomers) (all in weight %) was homogenized by sonication for 15 min and deaerated by purging with nitrogen for 15 min. The vinylized capillary was filled with the polymerization mixture using a syringe and sealed with a rubber septum at both ends. The polymerization was carried out in a water bath at 60 °C for 24 h. Functionalization with Thiol Groups. A modified method described by Burfield et al. was used to obtain monoliths with free thiol functionalities.63 Briefly, a 2.5 mol/L cysteamine solution in water was pumped through the GMA-EDMA monolith at room temperature at a flow rate of 1 µL/min for 30 min. The column was then rinsed with water until the pH of the eluent was neutral. The monolith contained 1.05 mmol/g thiol groups determined using the reaction with sodium-hydrogen sulfide.64 In Situ Preparation of GNP. The apparatus for the in situ method consisted of a T-piece with two inlets connected to two syringe pumps for delivery of aqueous solutions of HAuCl4 and sodium citrate, respectively. The column with thiol groups was attached to the outlet. With the use of this simple approach, mixing of both reagents was achieved before entering the column. The optimized conditions are described in detail elsewhere61 and include 50 mmol/L HAuCl4, 200 mmol/L trisodium citrate pumped for 30 min at a flow rate of 1.0 µL/ min, each while keeping temperature of the column at 100 °C. The column was rinsed with water to remove excess reagents and stored in the refrigerator at 4 °C. Modification with GNP Solution. Columns for CEC were prepared by pumping GNP through the monolith using a simple syringe pump. Since this approach is slow, we have also used a high pressure 260D ISCO pump for this process. Gold colloid solution was filled in the 2 mL loop attached to a six-port injection valve. The valve was switched, and the contents of the loop were pumped using the high pressure 260D ISCO pump in the thiol modified monolithic columns at a flow rate of 5 µL/min until the whole column turned deep red and pink solution was observed (62) Eeltink, S.; Geiser, L.; Svec, F.; Fre´chet, J. M. J. J. Sep. Sci. 2007, 30, 2814–2820. (63) Burfield, D. R.; Gan, S. N.; Smithers, R. H. J. Chem. Soc. Perkin Trans. 1 1977, 666–671. (64) Preinerstorfer, B.; Bicker, W.; Lindner, W.; La¨mmerhofer, M. J. Chromatogr., A 2004, 1044, 187–199.
leaving the capillary. The columns were then rinsed thoroughly with water. Columns for CEC. A procedure comprising pumping the solutions of reagents at a flow rate of 1 µL/min for 60 min followed by washing with water for 30 min at the same flow rate was used in these experiments. First, aqueous 3-mercaptopropionic acid solution (1 mol/L) was pumped using a syringe pump through the monolith modified with in situ formed GNP to obtain a capillary column with carboxylic acid functionalities. This column was then flushed with 1 mol/L aqueous 2-mercaptoethanol solution to afford monolith containing hydroxyl functionalities. Finally, pumping 1 mol/L aqueous cysteamine solution through this monolith resulted in a column with amine functionalities. Column for Reversed Phase MicroHPLC. An ethanol solution of 1-octadecanethiol (20 mmol/L) was pumped using an ISCO pump (vide supra) at a flow rate of 30 µL/h for 10 h through the monolith modified with GNP colloid solution followed by washing with water and acetonitrile. Column for Ion-Exchange NanoHPLC. An aqueous solution of sodium-2-mercaptoethanesulfonate (50 mmol/L) was pumped at a flow rate of 30 µL/h for 10 h through the monolith modified with GNP colloid solution followed with water and acetonitrile. RESULTS AND DISCUSSION In Situ Modification of Preformed Monolith with Gold Nanoparticles. Gold nanoparticles prepared by in situ reduction of HAuCl4 using sodium citrate65 are readily assembled onto the surface of the pores of a thiol-functionalized monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) column. Typically, this process leads to a monolithic column deep-red in color and containing 15.37 ± 1.61 (n ) 3) atom % of gold with GNP 40-50 nm in size. Figure 1 shows SEM micrographs of the monolith before and after modification with the GNP. The GNPs are held firmly onto the surface of the monolith by robust multivalent linkages that prevent them from being released and eluted from the column even when the eluent contains competing thiol containing compounds.61 Therefore, the reactive surface of the GNPs attached to the monolith can be adjusted through the binding of a variety of thiol-containing moieties. Because such linkages are dynamic in nature, the exposed surface of the GNPs may be modified at will by exchanging the surface thiols with a solution containing another functional thiol.66,67 A ready application of this process involves the separation of thiolcontaining peptides since they will be retained by the GNP modified column but can later be released by elution with an aqueous solution of 2-mercaptoethanol. This unique feature can also be used to exchange the “separation chemistry” of the monolithic column through the use of thiols that, in addition to the SH group, contain another exposed functionality. This process affords monoliths with chemistries suitable for chromatography while the strong multivalent binding of the gold nanoparticles to the monolith still ensures the stability of this stationary phase. The surface functionalization is performed under mild conditions at room temperature just by pumping the solution of the new (65) Liu, W.; Yang, X.; Xie, L. J. Colloid Interface Sci. 2007, 313, 494–502. (66) Heister, K.; Allara, D. L.; Bahnck, K.; Frey, S.; Zharnikov, M.; Grunze, M. Langmuir 1999, 15, 5440–5443. (67) Xu, H.; Hong, R.; Wang, X. Y.; Arvizo, R.; You, C. C.; Samanta, B.; Patra, D.; Tuominen, M. T. Adv. Mater. 2007, 19, 1383–1386.
Figure 1. SEM micrograph of the internal structure of original poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith (a) and its counterpart modified with gold nanoparticles prepared in situ (b). The white spots in part b are the GNP.
compound through the monolith. The high coverage of thiols on the gold surface provides for a high quantity of functional groups and helps to minimize the undesirable interactions between the supporting polymer and the target compounds. In addition, the attached molecules can be exchanged just by flushing with excess of another thiol-containing compound,67 thus enabling the use of the same GNP-containing monolith in various separation modes. Figure 2 outlines the process involved in switching column functionalities from an initial GNP column with carboxylic acid initial functionalities to one with hydroxyl moieties and finally an amine-functionalized column. Needless to say, each of these functional columns can be prepared in a single step from the original GNP monolith as indicated with the dotted arrows or the switching order can be changed as desired. Figure 3 demonstrates the effect of these changes in surface chemistry on the separation of three peptides in the CEC mode. It is clear that changing the surface chemistry has a drastic effect on the separation process with the fastest separation (
