Incorporation of Single-Wall Carbon Nanotubesinto an Organic Polymer Monolithic StationaryPhase for µ-HPLC and CapillaryElectrochromatography

Yan Li, Yuan Chen, Rong Xiang, Dragos Ciuparu, Lisa D. Pfefferle, Csaba Horvath,† andJames A. Wilkins*

Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520-8286

Single-wall carbon nanotubes (SWNT) were incorporatedinto an organic polymer monolith containing vinylbenzylchloride (VBC) and ethylene dimethacrylate (EDMA) toform a novel monolithic stationary phase for high-performance liquid chromatography (HPLC) and capillaryelectrochromatography (CEC). The retention behavior ofneutral compounds on this poly(VBC-EDMA-SWNT) mono-lith was examined by separating a mixture of small organicmolecules using micro-HPLC. The result indicated thatincorporation of SWNT enhanced chromatographic reten-tion of small neutral molecules in reversed-phase HPLCpresumably because of their strongly hydrophobic char-acteristics. The stationary phase was formed inside afused-silica capillary whose lumen was coated with co-valently bound polyethyleneimine (PEI). The annularelectroosmotic flow (EOF) generated by the PEI coatingallowed peptide separation by CEC in the counterdirec-tional mode. Comparison of peptide separations on poly-(VBC-EDMA-SWNT) and on poly(VBC-EDMA) with an-nular EOF generation revealed that the incorporation ofSWNT into the monolithic stationary phase improved peakefficiency and influenced chromatographic retention. Thestructures of pretreated SWNT and poly(VBC-EDMA-SWNT) monolith were examined by high-resolution trans-mission electron microscopy, Raman spectroscopy, scan-ning electron microscopy, and multipoint BET nitrogenadsorption/desorption.

Carbon nanotubes (CNT) have been widely recognized asquintessential nanomaterials with high strength and uniquetopologically controlled electronic properties.1-5 Since their dis-covery in 1991, they have stimulated intensive research intopotential high-impact applications such as nanoelectronic devices,catalyst supports, biosensors, and hydrogen storage.

CNT have also shown potential in chromatographic applica-tions. Several studies focused on size separation and purificationof CNT themselves using chromatography.6-14 Different types ofchromatography were used to remove nanoparticles from CNTsamples and to separate functionalized CNT by length. Recently,electrochemical detectors with CNT modified electrodes wereused in liquid chromatography15-17 and capillary electrophore-sis18,19 achieving significantly lower operating potentials andyielding substantially enhanced signal-to-noise characteristics.

Because carbon materials have long been used as adsorbentsfor trapping or separation of volatile organic compounds, wedecided that, because of their unique physicochemical properties,CNT might offer interesting opportunities for the developmentof new stationary-phase materials for chromatography. Kartsova20

first discussed the sorption properties of CNT and therefore thepossibility of using CNT as a stationary-phase component inchromatography. However, it was noted that most CNT were

* Corresponding author. Tel: (203) 432-4373. Fax: (203) 432-4372. E-mail:james.wilkins@yale.edu.

† Author deceased.(1) Iijima, S. Nature 1991, 354, 56-58.(2) Haddon, R. C. Acc. Chem. Res. 2002, 35, 997.(3) Dai, H. Surf. Sci. 2002, 500, 218-241.(4) Yu, M.-F.; Files, B. S.; Arepalli, S.; Ruoff, R. S. Phys. Rev. Lett. 2000, 84,

5552-5555.(5) Hu, J.; Odom, T. W.; Lieber, C. M. Acc. Chem. Res. 1999, 32, 435-445.

(6) Duesberg, G. S.; Muster, J.; Krstic, V.; Burghard, M.; Roth, S. Appl. Phys.A: Mater. Sci. Process. 1998, A67, 117-119.

(7) Duesberg, G. S.; Burghard, M.; Muster, J.; Philipp, G.; Roth, S. Chem.Commun. (Cambridge) 1998, 435-436.

(8) Duesberg, G. S.; Blau, W.; Byrne, H. J.; Muster, J.; Burghard, M.; Roth, S.Synth. Met. 1999, 103, 2484-2485.

(9) Holzinger, M.; Hirsch, A.; Bernier, P.; Duesberg, G. S.; Burghard, M. Appl.Phys. A: Mater. Sci. Process. 2000, 70, 599-602.

(10) Chen, J.; Rao, A. M.; Lyuksyutov, S.; Itkis, M. E.; Hamon, M. A.; Hu, H.;Cohn, R. W.; Eklund, P. C.; Colbert, D. T.; Smalley, R. E.; Haddon, R. C. J.Phys. Chem. B 2001, 105, 2525-2528.

(11) Niyogi, S.; Hu, H.; Hamon, M. A.; Bhowmik, P.; Zhao, B.; Rozenzhak, S.M.; Chen, J.; Itkis, M. E.; Meier, M. S.; Haddon, R. C. J. Am. Chem. Soc.2001, 123, 733-734.

(12) Zhao, B.; Hu, H.; Niyogi, S.; Itkis, M. E.; Hamon, M. A.; Bhowmik, P.; Meier,M. S.; Haddon, R. C. J. Am. Chem. Soc. 2001, 123, 11673-11677.

(13) Chattopadhyay, D.; Lastella, S.; Kim, S.; Papadimitrakopoulos, F. J. Am.Chem. Soc. 2002, 124, 728-729.

(14) Farkas, E.; Anderson, E. M.; Chen, Z.; Rinzler, A. G. Chem. Phys. Lett. 2002,363, 111-116.

(15) Xu, J.; Wang, Y.; Xian, Y.; Jin, L.; Tanaka, K. Talanta 2003, 60, 1123-1130.

(16) Zhang, W.; Xie, Y.; Ai, S.; Wan, F.; Wang, J.; Jin, L.; Jin, J. J. Chromatogr.,B 2003, 791, 217-225.

(17) Zhang, W.; Wan, F.; Xie, Y.; Gu, J.; Wang, J.; Yamamoto, K.; Jin, L. Anal.Chim. Acta 2004, 512, 207-214.

(18) Wang, J.; Chen, G.; Wang, M.; Chatrathi, M. P. Analyst (Cambridge, U. K.)2004, 129, 512-515.

(19) Wang, J.; Chen, G.; Chatrathi, M. P.; Musameh, M. Anal. Chem. 2004, 76,298-302.

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insoluble in the known solvents, which restricted their applicationin chromatography. In 2003, Li21 studied CNT as a gas chromato-graphic column packing and concluded that CNT showed strongerretention of compounds but lower efficiency. The structure of CNTresembles one or more graphite sheets rolled up into a cylinderthat consists of hexagon-rich sp2 carbon at the wall and a fewpentagons at the caps, getting more sp3-like as tube diametersbecome smaller (<0.7 nm). Because of their curved surface, CNTare expected to show a stronger binding affinity for hydrophobicmolecules compared to a planar carbon surface.21 On the basis oftheir unique characteristics, we predicted that CNT present inthe chromatographic stationary phase might enhance the retentionof compounds with hydrophobic character and that the columnsmight also display unique retention characteristics.

CNT can be single-wall or multiwall structures. Single-wallcarbon nanotubes (SWNT) consist of a graphene sheet rolled intoa cylinder with a typical diameter on the order of 1 nm. Multiwallcarbon nanotubes (MWNT) consist of concentric cylinders withan interlayer spacing of 3.4 Å and a diameter typically on the orderof 10-20 nm.3 Although MWNT have been applied in gaschromatographic column packing, to our knowledge, applicationof CNT in liquid chromatography column stationary-phase syn-thesis has not previously been explored. CNT solubility must beretained while maintaining their unique structure during incor-poration into the stationary phase. It was observed that MWNTwere insoluble in common solvents while SWNT were found toform suspensions.22 Therefore, SWNT used in the current studywere acid treated to enhance their solubility. The smaller diameterSWNT with unique electronic properties are expected to havestronger interactions with biomolecules because carbon-carbonbonds become strained and convert from sp2 to more sp3-likebonding. SWNT used here (mean diameter of 0.84 nm) were onthe smaller end of the SWNT commercially produced.

The popularity of monolithic columns in high-performanceliquid chromatography (HPLC) and capillary electrochromatog-raphy (CEC) results from the ease of their in situ preparation,good permeability due to their porous properties, and readilyavailable surface chemistries, both organic and inorganic.23,24 Allof the above features make them an attractive alternative togranular packed columns.25-27 Monolithic stationary phases de-signed for peptide separations typically employ functionalitiessimilar or identical to those used in conventional reversed-phasegranular stationary-phase materials.28,29 To take advantage ofmonolithic column architecture and the specific features of CNT,we report here our initial efforts to incorporate SWNT into anorganic polymer monolith to form a new generation stationaryphase for micro-HPLC (µ-HPLC) and CEC. The goal of this study

was to investigate the influence of SWNT on the chromatographicproperties of a polymeric stationary phase that had been previouslystudied30,31 and to determine the feasibility of using SWNT tomodify the adsorption characteristics of monolithic stationaryphases.

MATERIALS AND METHODSMaterials. Ethylene glycol dimethacrylate (EDMA) and poly-

ethyleneimine (PEI, MW 10 000, 30% aqueous) were purchasedfrom Polysciences (Warrington, PA). Vinylbenzyl chloride (VBC)was from Dow (Midland, MI), and azobisisobutyronitrile (AIBN)(98%) was from Pfaltz & Bauer (Waterbury, CT). (3-Glycidoxy-propyl)trimethoxysilane (GPTMS), 3-(trimethoxysilyl)propyl meth-acrylate, 2,2-diphenyl-1-picryhydrazyl hydrate (DPPH), trifluoro-acetic acid (TFA), succinic acid, toluene, 1-propanol, and forma-mide were from Aldrich (Milwaukee, WI). HPLC reagent gradesulfuric acid, dimethylformamide (DMF) (99%), and analytical-grade hydrochloric acid, monobasic, dibasic, and tribasic sodiumphosphates, and sodium hydroxide (98.8%) were from J. T. Baker(Phillipsburg, NJ). Dimethyl sulfoxide (DMSO) was purchasedfrom Burdick and Jackson (Muskegon, MI). HPLC-grade metha-nol, acetone, acetonitrile, methylene chloride, and triethylaminewere purchased from Fisher (Fair Lawn, NJ). Water was purifiedand deionized with a NANOpure system (Barnstead, Boston, MA).A reversed-phase test mixture containg uracil, phenol, N,N-diethyl-m-toluamide, and toluene in acetonitrile/water (58:42) and apeptide mixture including methionine enkephalin, leucine en-kephalin, angiotensin II, Val-Tyr-Val, and Gly-Tyr were bothpurchased from Sigma (St. Louis, MO). The peptide mixture wasdissolved in pure water. Fused-silica capillary tubing of 75-µm i.d.and 375-µm o.d. with a polyimide outer coating was purchasedfrom Quadrex Scientific (New Haven, CT). Materials related tothe SWNT synthesis can be found in a previous report.32

Instrumentation. µ-HPLC experiments were performed usingan LC system from Micro-Tech Scientific, Inc. (Vista, CA),equipped with a 5-µL six-port microbore valve from Valco(Houston, TX) for small sample injections and a Ultra-plus IIsolvent delivery system, which provides a flow rate range of 0.1-300 µL/min with software flow calibration and solvent compress-ibility compensation. The binary solvent system was formed from(A) water + 0.1% TFA and (B) acetonitrile + 0.1% TFA. For sampledetection, a model 2000 (Thermal Separations, San Jose, CA)variable-wavelength, manually operated UV/visible detector wasused. Data acquisition was controlled by a Chrom Perfect System(Justice Laboratory Software, Denville, NJ). All samples weredetected at a wavelength of 214 nm.

Capillary electrochromatography and capillary electrophoresisexperiments were carried out on a HP3DCE capillary electrophore-sis unit (Agilent Technologies, Wilmington, DE) equipped with adiode array UV detection system and controlled by a P150personal computer (Hewlett-Packard, Palo Alto, CA). Windows95 (Microsoft, Redwood, WA) and Chemstation V.4.01 (Agilent)were installed to control the instrument functions and to process

(21) Li, Q.; Yuan, D. J. Chromatogr., A 2003, 1003, 203-209.(22) Chen, J.; Hamon, M. A.; Hu, H.; Chen, Y.; Rao, A. M.; Eklund, P. C.; Haddon,

R. C. Science (Washington, D. C.) 1998, 282, 95-98.(23) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Hirao, K.; Tanaka, N. Colloids

Surf, A 2001, 187, 273-279.(24) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Nagayama, H.; Hosoya,

K.; Tanaka, N. Anal. Chem. 2000, 72, 1275-1280.(25) Svec, F. J. Sep. Sci. 2004, 27, 747-766.(26) Li, Y.; Xiang, R.; Wilkins, J. A.; Horvath, C. Electrophoresis 2004, 25, 2242-

2256.(27) Hilder, E. F.; Svec, F.; Frechet, J. M. J. Electrophoresis 2002, 23, 3934-

3953.(28) Gusev, I.; Huang, X.; Horvath, C. J. Chromatogr., A 1999, 855, 273-290.(29) Zhang, S. H.; Huang, X.; Zhang, J.; Horvath, C. J. Chromatogr., A 2000,

887, 465-477.

(30) Li, Y.; Xiang, R.; Horvath, C.; Wilkins, J. A. Electrophoresis 2004, 25, 545-553.

(31) Zhang, S. H.; Zhang, J.; Horvath, C. J. Chromatogr., A 2001, 914, 189-200.

(32) Lim, S.; Ciuparu, D.; Pak, C.; Dobek, F.; Chen, Y.; Harding, D.; Pfefferle,L.; Haller, G. J. Phys. Chem. B 2003, 107, 11048-11056.

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the data. Both inlet and outlet vials were pressurized with nitrogenup to 12.0 bar. The temperature in the column cartridge was 25°C, and the wavelength of the UV detector was set at 214 nm.

High-resolution transmission electron microscopy (HR-TEM)images of SWNT were obtained using a Tecnai F20 200 kVmicroscope. The solid sample was dispersed in pure ethanol bysonication, and 0.05 mL of this suspension was placed on a coppermesh coated with an amorphous holey carbon film. The ethanolwas evaporated prior to the TEM analysis.

Raman spectra of SWNT samples were recorded using anexcitation wavelength of 532 nm on a LabRam instrument fromJobin Yvon Horiba equipped with an Olympus confocal micro-scope.

SEM images of purified nanotubes and capillary cross sectionon the micrometer scale were obtained using energy-dispersivespectrometry (EDS). EDS images and data were collected withthe EDAX Phoenix Pro EDS/imaging system (EDAX, Mahwah,NJ) on the JEOL JXA-8600 electron microprobe (JEOL, Peabody,MA) in the Department of Geology and Geophysics at YaleUniversity. Images acquired with this system are secondaryelectron images. EDS data were used for both qualitative elementidentification and semiquantitative compositional estimates. Capil-lary columns were cut into 2-mm pieces and coated with conduc-tive carbon to diminish charge buildup and heating of the samplefrom the electron beam, a requisite for both imaging andcompositional analysis under high vacuum. All images and EDSdata were collected at an accelerating voltage of 10 kV. Beamcurrents ranged from ∼275 pA for higher resolution images to∼5 nA for EDS acquisitions.

To obtain information about the surface area and mesoporesize distribution of the polymer monolithic microspheres, adsorp-tion-desorption isotherms of liquid nitrogen were measured usinga model Autosorb-1C (Quantachrome, Boynton Beach, FL) staticvolumetric instrument at -196 °C. Prior to the measurements,the monolithic polymer structure was ground and degassed at110 °C to a residual pressure lower than 10-2 Pa. A Baratron (0.1-1000 Pa) pressure transducer was used for low-pressure measure-ments.

Samples and Solutions for CEC. A stock solution ofphosphate buffer (80 mM) was made and then diluted andadjusted to various values of pH and concentration. The runningbuffer was prepared by mixing phosphate buffer and the appropri-ate volume of acetonitrile. Before CEC experiments, the runningbuffer was degassed with helium for ∼20 min. The peptides andelectroosmotic flow (EOF) marker dimethyl sulfoxide (DMSO)used in this study were dissolved in deionized water and dilutedto the appropriate concentrations. Between runs, the capillarycolumn was rinsed with acetonitrile for 10 min, followed withrunning buffer for 15 min at 1 × 106 Pa inlet pressure. The columnwas equilibrated electrokinetically at the operating voltage untilthe baseline was stable.

Preparation of Soluble SWNT. The SWNT used in this studywere grown using a cobalt catalyst incorporated into the pore wallsof mesoporous silica templates. The Co-MCM-41 catalysts wereprepared with 1 wt % Co loading following the procedure describedelsewhere.33-35 Co-MCM-41 were prereduced in hydrogen to 500

°C for 0.5 h, and then SWNT were grown for 1 h under 6 atm COpressure at 800 °C. The silica template was removed by refluxingin 2.5 wt % NaOH for 1 h. The protocol we chose for thepreparation of soluble SWNT in 2-propanol was an acid oxidativemethod36 using H2SO4/H2O2 treatment, because it is minimallydestructive toward the SWNT structure as confirmed by Ramanand TEM experiments. About 10 mg of SWNT was added into 20mL of a 9:1 mixture of 98% H2SO4 and 30% H2O2, both in aqueoussolution. The mixture was stirred for 30 min. After the reaction,the mixture was placed in an ultrasonic bath and sonicated for 10min. The resulting SWNT suspension was diluted using 250 mLof distilled water and filtered through a 0.45-µm Millipore poly-carbonate membrane. The resulting SWNT mat was subsequentlywashed using 10 mM NaOH and distilled water until the pH ofthe filtrate was 7. The wet SWNT mat was then separated fromthe filter by dispersion into 2-propanol. The SWNT sampledissolved in 2-propanol gave a suspension of SWNT at a concen-tration of 0.1 mg/mL, which was used as one of the porogens inthe preparation of the monolithic stationary phase. The SWNTsuspension in 2-propanol was sonicated again for 15 min im-mediately before use. The suspension must be used soon afterpreparation because SWNT bundles formed after several daysremain in suspension and cannot be debundled by furthersonication.

Preparation of the Monolithic Column. (1) Pretreatmentof Fused-Silica Capillaries. A 0.5-m length of fused-silicacapillary tubing was washed with water and filled with 1.0 MNaOH. With both ends sealed, the tubing was heated at 100 °Cfor 2 h in the oven of a Sigma 2000 gas chromatograph (Perkin-Elmer, Norwalk, CT). Thereafter, it was washed with deionizedwater for 10 min, 1.0 M HCl for 5 min, deionized water for 30min, and acetone for 10 min. Subsequently, the capillary tubingwas placed again in the oven at 120 °C and purged with nitrogenfor 1 h to remove residual water. Siloxane groups at the innersurface of raw fused-silica capillaries were hydrolyzed by pre-treatment to increase the density of silanol groups serving asanchors for the subsequent silanization.37,38

(2) Preparation of the Porous Monolithic StationaryPhase for µ-HPLC. Fused-silica capillaries were silanized afterpretreatment to covalently anchor the monolithic column bed tothe capillary inner wall.38 A solution containing 30% (v/v) 3-(tri-methoxysilyl)propyl methacrylate and 0.1% (w/v) inhibitor DPPHwas prepared in dry DMF, deaerated with helium for 10 min, andfilled into the pretreated capillary. After both ends were sealed,the column was placed in the oven at 120 °C for 6 h. The capillarywas then taken out and washed extensively with DMF, methanol,and methylene chloride at room temperature and was blown drywith nitrogen for 1 h. Thereafter, a solution containing 20% (v/v)VBC (monomer), 20% (v/v) EDMA (cross-linker), 40% (v/v)2-propanol containing pretreated SWNT and 20% (v/v) formamideas the porogens, and 0.3% AIBN (w/v) as the polymerizationinitiator was prepared and mixed ultrasonically into a homoge-

(33) Ciuparu, D.; Chen, Y.; Lim, S.; Haller, G. L.; Pfefferle, L. J. Phys. Chem. B2004, 108, 503-507.

(34) Chen, Y.; Ciuparu, D.; Lim, S.; Yang, Y.; Haller, G. L.; Pfefferle, L. J. Catal.2004, 225, 453-465.

(35) Chen, Y.; Ciuparu, D.; Lim, S.; Yang, Y.; Haller, G. L.; Pfefferle, L. J. Catal.2004, 226, 351-362.

(36) Zhao, W.; Song, C.; Pehrsson, P. E. J. Am. Chem. Soc. 2002, 124, 12418-12419.

(37) Huang, X.; Zhang, J.; Horvath, C. J. Chromatogr., A 1999, 858, 91-101.(38) Zhang, J.; Zhang, S.; Horvath, C. J. Chromatogr., A 2002, 953, 239-249.

1400 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005

neous solution. It was then filled into the column with nitrogen.The capillary column was then sealed and heated for 16 h at 75°C31 in the oven of a model Sigma 2000 gas chromatograph(Perkin-Elmer). The column was then washed with methanol anddeionized water extensively. A 10 mM solution of aqueous sodiumhydroxide was pumped through the column containing the porouspoly(VBC-EDMA-SWNT) monolith packing to hydrolyze theresidual benzyl chloride groups at the surface of monolith. Afterthe column was washed with deionized water and methanol, itwas purged overnight with nitrogen at room temperature. Themonolithic column without SWNT incorporated was preparedusing an identical procedure substituting pure 2-propanol insteadof 2-propanol containing SWNT as the porogen. The length ofboth columns was 40 cm.

(3) Preparation of the Porous Monolithic StationaryPhase with Annular EOF Generation for CEC. The monolithiccolumn for CEC separation was prepared according to a previouslypublished method.30 The fused silica capillary was first silanizedwith 10% (v/v) GPTMS with 1% (v/v) triethylamine in dry toluenefor 3 h at room temperature. Afterward, the capillary was flushedwith a solution of 7.5% (v/v) PEI in 0.05 M succinate buffer (pH6.0) for 5 h at room temperature. Primary amino groups on PEIwere covalently linked to the GPTMS coating to form an annularpositively charged polymer layer in the column for the generationof EOF. A neutral monolithic stationary phase with SWNT wasthen prepared by in situ copolymerization of VBC and EDMA inthe presence of 2-propanol with SWNT and formamide as theporogen as described above. The benzyl chloride functionalitieson the monolith were subsequently hydrolyzed to benzyl alcoholgroups as previously described.30 Finally, a 1-2-mm-wide segmentof the polyimide outer coating at a distance of 8.5 cm from theoutlet end was heated with an Archer model B microtorch (RadioShack, New Haven, CT) while the capillary column was purgedwith oxygen at 8.3 × 105 Pa. This treatment removed thepolymeric packing inside the segment, creating a detectionwindow. Subsequently, the capillary column was washed withmethanol and deionized water. The column length for both thecontrol and the SWNT column was 40 cm from the inlet to thedetection window.

RESULTS AND DISCUSSIONIncreasing the Solubility of SWNT. To incorporate SWNT

into the monolithic stationary phase while maintaining a uniformpolymer matrix in the capillary column, it was necessary to evenlydisperse SWNT in the polymer solution. Preparation of well-dispersed SWNT has proved to be a nontrivial task, and severalacid oxidative methods have been developed to solve thisproblem.10,36,39 Most of those methods, however, are destructive.For our application, SWNT should not be cut too short, and theformation of defects during the solubilization procedure shouldbe minimized. To study the impact of SWNT on the chromato-graphic performance of the column, a mild solubilization method36

is necessary so the effect of defect level can be independentlyexamined. SWNT were therefore treated with H2SO4/H2O2 toincrease their solubility through the introduction of hydroxylmoieties on their surface. The treated SWNT were then dispersed

by sonication in 2-propanol. By applying this acid treatment, SWNTwere well dispersed and quite stable in 2-propanol. No precipitationwas observed from the 2-propanol suspension after two weeks(Figure 1). Before polymerization of the stationary phase, 2-pro-panol with SWNT was added directly into the monomer mixtureas a porogen to incorporate SWNT into the monolithic stationaryphase.

SWNT and Column Characterization. As-synthesized SWNTsamples and purified (after silica template removal) SWNTsamples were studied by HR-TEM (Figure 2). Micrograph A inFigure 2 shows SWNT bundles evolving from the pores of theCo-MCM-41 catalyst, micrograph B in Figure 2 shows tubebundles with cobalt clusters after silica template removal, andmicrograph C shows a more detailed look of those SWNT bundles.These results confirmed that the SWNT structure was notdamaged after silica template removal. Raman spectroscopy wasalso used to characterize the SWNT before and after acidtreatment as shown in Figure 3. The dotted line in Figure 3 showsthe spectrum of the as-synthesized SWNT with Co-MCM-41catalysts, while the dashed line was collected for the solubleSWNT mat on membrane filter after H2SO4/H2O2 treatment. Thesolid line shows the Raman spectrum of poly(VBC-EDMA-SWNT)monolith polymer. There are two features of interest in the

(39) Barber, A. H.; Cohen, S. R.; Wagner, H. D. Phys. Rev. Lett 2004, 92, 186103-186101-186103-186104.

Figure 1. SWNT suspension in 2-propanol after H2SO4/H2O2

pretreatment. No precipitation of SWNT was observed during the firsttwo weeks.

Analytical Chemistry, Vol. 77, No. 5, March 1, 2005 1401

spectra: First, the peaks in the Raman breathing mode (RBM)region characteristic for SWNT indicate that H2SO4/H2O2 treat-ment preserves the SWNT structure. Importantly, the RBM peakon the VBC-EDMA-SWNT polymer confirms that SWNT weresuccessfully incorporated into the polymer structure. The relativelyweak intensity may arise from the low concentration of SWNT inthe polymer and/or the perturbation of the SWNT breathingvibration because of its attachment to the polymer. Second, theD band in the treated SWNT spectrum (dashed line), assignedto disordered or defective carbon species, increases somewhatindicating some damage from carbon-carbon bond breaks andfrom the introduction of hydroxyl moieties by the H2SO4/H2O2

treatment. The stronger D band on the VBC-EDMA-SWNTpolymer (solid line) also indicated incorporation of some damagedSWNT into the monolith polymer. EDS can be used for phaseidentification and recognition of compositional variability. Theelemental analysis of SWNT pretreated with H2SO4/H2O2 is shownas an inset in panel A of Figure 4. EDS indicated that the SWNTsample was composed of 73% (w/w) carbon and ∼5% (w/w)oxygen, with cobalt accounting for most of the rest suggesting

the sample was mainly composed of SWNT with hydroxyl moietiesintroduced onto the surface by oxidation. The structure of themonolithic stationary phase in the capillary was also characterizedby SEM as shown in Figure 4B-D, which indicated that thepolymer matrix with SWNT incorporated had a uniform structure.

Separation of a Reversed-Phase Test Mixture in µ-HPLC.A reversed-phase test mixture containing uracil, phenol, N,N-diethyl-m-toluamide and toluene was used to examine the chro-matographic behavior of the poly(VBC-EDMA-SWNT) monolithiccolumn in µ-HPLC. To study the influence of SWNT on columnretention, the four compounds were separated using both a poly-(VBC-EDMA-SWNT) monolithic column and a poly(VBC-EDMA)(“control”) monolithic column under isocratic conditions with 50%(v/v) acetonitrile as the mobile phase, shown in Figure 5.

Table 1 shows the effect of incorporating SWNT into themonolithic stationary phase on the chromatographic retentionfactor k′, which is calculated as follows:

where tr and t0 are the elution times of the retained analytes andunretained marker (we used acetone in this study), respectively.We found that the permeability of the SWNT column wassignificantly lower than that of the control column (0.26 vs 0.32darcy, respectively), perhaps because of an effect of SWNT onthe polymerization reaction. This led to an increase in theunretained marker time (4.36 min for the SWNT column vs 3.23min for the control column). The chromatographic retentionfactors of these four small molecules on the monolithic columnwith SWNT incorporated were significantly higher than those onthe control column. This result suggested that CNT present inthe stationary phase enhanced chromatographic retention of thesemolecules.

This reversed-phase test mixture was also separated on apacked octadecyl silica column under isocratic conditions inµ-HPLC with the aqueous acetonitrile concentration fixed at 35%;shown in Figure 6A. It is also noted that the four compounds hadmuch longer retention times in µ-HPLC on the poly(VBC-EDMA-SWNT) monolith. Toluene, the most hydrophobic compound ofthe four neutral compounds, could not be eluted from the lattercolumn when the acetonitrile concentration in the mobile phase

Figure 3. Raman spectra recorded for as-synthesized SWNT,soluble SWNT mat on a membrane filter after H2SO4/H2O2 treatment,and the VBC-EDMA-SWNT polymer. Clear bands are seen for SWNTincorporated into the polymer.

Figure 2. HR-TEM images showing the structure of SWNT. (A) SWNT bundles evolving from the pores of the Co-MCM-41 catalyst onas-synthesized samples; (B) SWNT bundles with cobalt clusters after silica removal; (C) SWNT with cobalt clusters after silica removal.

k′ ) (tr - t0)/t0 (1)

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was less than 50% (v/v). However, uracil (U), phenol (P), N,N-diethyl-m-toluamide (D), and toluene (T) were almost coelutedon the silica packed column, when the acetonitrile concentrationwas 50% (v/v); shown in Figure 6B. The higher acetonitrileconcentration needed for elution of the compounds from theSWNT column suggested stronger retention brought about bySWNT incorporation.

It was important to examine further the mechanisms by whichincorporated SWNT influence column adsorbsion characteristics.It is well known, for example, that pore size plays an importantrole in determining stationary-phase characteristics. We reasoned

that incorporation of SWNT might simply alter the porousproperties of the monolithic stationary phase, resulting in thedifferent retention properties observed. However, the porousproperties of both columns were found to be quite similar (seebelow). Since analyte retention after incorporation of SWNT wassignificantly enhanced without corresponding changes in columnporosity, we propose that the specific structure, size, and chargecharacteristics of CNT all may play a role in these phenomena.CNT can be thought of as a graphite plane rolled up into a cylinderwith the surface consisting of regular hexagons. Analytes maybe drawn onto the nanotube surface or channels between

Figure 4. SEM images of H2SO4/H2O2 pretreated SWNT (A) and poly(VBC-EDMA-SWNT) monolith (B-D).

Figure 5. Chromatograms of a reversed-phase test mixture separated on (A) control monolith and (B) poly(VBC-EDMA-SWNT) monolithunder isocratic elution conditions (50% aqueous acetonitrile + 0.1% (v/v) TFA). Flow rates: (A) 0.5 µL/min (permeability, 0.32 darcy); (B) 0.4µL/min (permeability, 0.26 darcy). Both columns, 0.075 mm (diameter) × 400 mm (length). Temperature, room temperature; UV detection, 214nm. Peaks: U, uracil; P, phenol; D, N,N-diethyl-m-toluamide; T, toluene.

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nanotube bundles due to their surface tension and capillary effectsand, therefore, exhibit longer retention times.

Separation of Peptides in CEC. Because of our laboratory’sinterest in the analysis of proteins and peptides, we decided toinvestigate peptide separations using the SWNT monolithiccolumns. The unique interplay of chromatographic retention,electrophoretic migration, and hydrodynamic properties offeredby the CEC format26 provides a good platform on which to testmonolithic columns containing SWNT in this application. To avoidirreversible adsorption and electrostatic interaction between thestationary phase and the peptides in CEC, a porous neutral poly-(VBC-EDMA-SWNT) monolith with annular EOF generation wasprepared according to the method we have established.30

According to the current measurements, the conductivity andelectrokinetic porosity in the monolithic columns were calculatedusing the following equations:40

where i is the current flowing through the column, L is the lengthof the column, V is the applied voltage, σmonolith and σopen are theconductivities of the monolithic column and open tube, Φ is the

conductivity ratio, εT is the electrokinetic porosity, and theempirical constant m )1.5 in this case. The total surface area andthe size of mesopores within the monolithic globules weredetermined using liquid nitrogen physisorption. Valuable informa-tion on the presence and shape of the mesopores can be deducedfrom the shape of the nitrogen adsorption and desorption isothermaccording to the BET method. The electrokinetic and porousproperties of control and SWNT modified monolithic columns arelisted in Table 2. As shown in Table 2, there were appreciableincreases in EOF mobility and conductivity on the poly(VBC-EDMA-SWNT) monolith. Electrokinetic porosity and mesoporesize measurements of control and poly(VBC-EDMA-SWNT)monoliths indicated that the monolith with SWNT was only slightlymore porous than the control column. We speculate that SWNTmay influence the polymerization process itself and thereforesubtly change the porous properties of the monolith. Althoughsmall increases in mesopore size and electrokinetic porosity wereobserved (∼3 and ∼11% respectively), the surface area wassignificantly increased (28%) by incorporation of SWNT (Table2). Thus, a large increase in surface area along with a modestincrease in pore size apparently account for the enhanced retentionof the analytes in µ-HPLC. Since the incorporation of SWNT hadrelatively little effect on the porous properties of the column, wepropose that increases seen in EOF, conductivity, and retentionderive primarily from the unique properties of incorporated CNT.The SWNT used in these experiments were reported to show thenormal 1:2 distribution between metallic and semiconductingspecies,41 which may transport electric current ballistically withoutheat dissipation. Considering that liquid flow through the columnis driven by the electric field in CEC, we suspected that theelectronic characteristics of SWNT might affect CEC separationin view of the changes in EOF and conductivity seen in the SWNTcolumn.

A peptide mixture containing Gly-Tyr (G), Val-Tyr-Val (V),methionine enkephalin (M), leucine enkephalin (L), and angio-tensin II (A) was successfully separated isocratically on both poly-(VBC-EDMA-SWNT) and control monolithic columns with annularEOF generation by counterdirectional CEC using a hydroorganicmobile phase containing acetonitrile and phosphate buffer at pH2.5, shown in Figure 7. Table 3 shows the comparison of retentionfactors and separation efficiency of peptides on the monolithic

Table 1. Comparison of Measured Retention Factor (k′)of Compounds in a Reversed-Phase Test Mixture onControl and SWNT Monolithic Columnsa

poly(VBC-EDMA)

poly(VBC-EDMA-SWNT)

compounds t (min) k′ t (min) k′

marker 3.23 0 4.36 0uracil 3.23 0 8.90 1.04phenol 9.12 1.82 15.70 2.60N,N-diethyl-m-toluamide 11.18 2.46 17.73 3.07toluene 22.99 6.12 37.52 7.61

a The retention of an unretained marker compound (acetone) wasmeasured. These values were used along with retention times of thetest compounds to calculate retention factors (k′). The permeabilitiesof the two columns were 0.32 darcy for the control column and 0.26darcy for the SWNT column.

Figure 6. Chromatograms of a reversed-phase test mixture separated on 0.075 mm (d) × 100 mm (l) octadecyl silica column packed withSupelco Discovery wide-pore octadecyl silica (300 Å, 5-µm particle size) under isocratic elution conditions: (A) 35% aqueous acetonitrile +0.1% (v/v) TFA; (B) 50% aqueous acetonitrile + 0.1% (v/v) TFA. Flow rate, 0.5 µL/min; Temperature, room temperature; UV detection, 214 nm.Peaks: U, uracil; P, phenol; D, N,N-diethyl-m-toluamide; T, toluene.

σ ) iL/VA (2)

Φ ) σmonolith/σopen ) εTm

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columns (without incorporated SWNT) in CEC. It was found thatboth the retention factors and the separation efficiency on thepoly(VBC-EDMA-SWNT) monolithic column were superior tothose obtained on the control column. Although the EOF mobilityfor the poly(VBC-EDMA-SWNT) stationary phase was higher thanthat on the control column, the retention factors for all fivepeptides on the monolithic column with SWNT were higher thanthose on the control column (Table 3). These results agreed withdata from µ-HPLC separations of small molecules shown above.We conclude that both retention and separation efficiency wereenhanced by incorporation of CNT into the stationary phase. Itshould also be noted that peak symmetry on the poly(VBC-EDMA- SWNT) monolith was enhanced relative to the column made

without SWNT, as shown in Table 2. Hence, it is clear that SWNTincorporation improved column performance with respect toseveral aspects of its adsorption characteristics.

(40) Rathore, A. S.; Wen, E.; Horvath, C. Anal. Chem. 1999, 71, 2633-2641.(41) Odom, T. W.; Huang, J.-l.; Lieber, C. M. Ann. N. Y. Acad. Sci. 2002, 960,

203-215.

Table 4. Migration Order of Peptides Separated onDifferent Columnsa

peptide migration order 1 2 3 4 5

HPLC C18 reversed phase column G V M L Apoly(VBC-EDMA-SWNT) annular column M L V A Gpoly(VBC-EDMA) annular column M L A V Gopen tube with GPTMS-PEI coating A M L V G

a M, methionine enkephalin; L, leucine enkephalin; V, Val-Tyr-Val;A, angiotensin II; G, Gly-Tyr.

Table 2. Comparison of Electrokinetic and Porous Properties of the Monolithic Columns in CEC

columnsEOF mobilitya

(× 10-8 m2 V-1 s-1)conductivity

(×10-2 Ω-1 m-1)peak

asymmetryaelectrokinetic

porosityamesoporesize (nm)

surface area(m2/g)

poly(VBC-EDMA-SWNT)with annular EOF generation

2.53 11.20 1.10 0.58 3.35 11.01

poly(VBC-EDMA) withannular EOF generation

1.95 9.62 1.39 0.52 3.26 7.90

open tube with GPTMS-PEIcoating

2.67 23.54 1.04 n/ab n/a n/a

a Measurement were made with DMSO as the inert and neutral marker. b n/a, not applicable.

Figure 7. Electrochromatograms of a peptide mixture on poly(VBC-EDMA-SWNT) and poly(VBC-EDMA) monolithic columns with annularEOF generation. (A) Column, 0.075 mm (d) × 225 mm (l), fused-silica capillary with porous poly(VBC-EDMA-SWNT) monolith and GPMTS-PEI coating on the inner wall; (B) column, 0.075 mm (d) × 225 mm (l), fused-silica capillary with porous poly(VBC-EDMA) monolith and GPMTS-PEI coating on the inner wall. Mobile phase, 20 mM aqueous sodium phosphate buffer, pH 2.5 containing 25% (v/v) acetonitrile; applied voltage,-20 kV; temperature, 25 °C; UV detection, 214 nm. Peaks: (M) methionine enkephalin, (L) leucine enkephalin, (V) Val-Tyr-Val, (A) angiotensinII, and (G) Gly-Tyr.

Table 3. Comparison of Migration Factor and Separation Efficiency of Peptides on Monolithic Columns in CECa

poly(VBC-EDMA-SWNT)with annular EOF generation

poly(VBC-EDMA)with annular EOF generation

peptidet

(min) k′cec NH

(µm)peak

asymmt

(min) k′cec NH

(µm)peak

asymm

methionine enkephalin (M) 4.08 0.32 20574 11.9 0.88 4.42 0.12 11482 19.6 0.77leucine enkephalin (L) 4.19 0.36 38923 6.3 0.90 4.75 0.21 10918 20.6 0.80Val-Tyr-Val (V) 4.71 0.53 18708 13.1 0.87 5.57 0.41 7750 17.7 0.90angiotensin II (A) 5.06 0.64 42082 5.8 0.96 5.08 0.29 12734 29.0 0.75Gly-Tyr (G) 6.54 1.12 20690 11.8 0.95 7.33 0.86 10053 22.4 0.92

a N, number of theoretical plates; H plate height.

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To explore the separation mechanism in CEC, the migrationbehavior of the five peptides was studied. The order of peptideretention on poly(VBC-EDMA-SWNT) monolith was different fromthat observed in the control column under identical conditions,as shown in Table 4. The four peptides G, V, M, and L exhibitedsimilar behavior in CEC and in CZE with a GPTMS-PEI-coatedopen tube under the same separation conditions. However, asreported earlier,30 the migration behavior of peptide A, which wasapparently differentially affected by the hydrophobicity of thestationary phase, clearly showed that chromatographic retentionplayed a central role during the separation on monolithic columns.Table 4 shows the change in the order of retention with respectto peptide A, which migrated third between L and V on the controlcolumn, fourth between V and G on poly(VBC-EDMA-SWNT)column, and last in conventional reversed-phase HPLC. Thechange of the migration order of peptide A in the SWNTseparation further demonstrated the influence of CNT on elec-trochromatographic retention. Specific interactions between CNTand proteins and peptides have been reported previously.42,43

Experiments to gain a more detailed understanding of theinteraction between CNT and the analytes in this study along witha broader range of molecules are underway. Future experimentswill also focus on control of the concentration, diameter, andlength of the SWNT incorporated and the design of specificallymodified SWNT for targeted chromatographic separations.

ACKNOWLEDGMENTWe are grateful to Dr. Sangyun Lim, Yanhui Yang, and Dr.

Gary L. Haller at Yale University for performing BET measure-ments and for providing Co-MCM-41 catalysts for SWNTsynthesis. This work was supported by Grant GM 20993 from theNational Institute of Health, U.S. Department of Health andHuman Services, and DOE, office of Basic Energy Sciences. Wededicate this article to our friend and mentor Professor CsabaHorvath.

Received for review November 17, 2004. AcceptedNovember 30, 2004.

AC048299H(42) Katz, E.; Willner, I. Chemphyschem 2004, 5, 1085-1104.(43) Banerjee, I. A.; Yu, L. T.; Matsui, H. Nano Lett. 2003, 3, 283-287.

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