Polyhedral Oligomeric Silsesquioxane as a Cross-linker for

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Anal. Chem. 2010, 82, 5447–5454

Polyhedral Oligomeric Silsesquioxane as a Cross-linker for Preparation of Inorganic-Organic Hybrid Monolithic Columns Minghuo Wu,†,‡ Ren’an Wu,*,† Ruibing Li,†,‡ Hongqiang Qin,†,‡ Jing Dong,† Zhenbin Zhang,†,‡ and Hanfa Zou*,† CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China, and Graduate School of Chinese Academy of Sciences, Beijing 100049, China An inorganic-organic hybrid monolithic capillary column was synthesized via thermal free radical copolymerization within the confines of a capillary using a polyhedral oligomeric silsesquioxane (POSS) reagent as the inorganic-organic hybrid cross-linker and a synthesized long carbon chain quaternary ammonium methacrylate of N-(2-(methacryloyloxy)ethyl)-dimethyloctadecylammonium bromide (MDOAB) as the organic monomer. The preparation process was as simple as pure organic polymer-based monolithic columns instead of using POSS as the nanosized inorganic-organic hybrid blocks (cross-linker) of the monolithic matrix. The pore properties and permeability could be tuned by the composition of the polymerization mixture. The characterization and evaluation results indicated that the synthesized MDOA-POSS hybrid monolith possessed the merits of organic polymer-based monoliths and silica-based monoliths with good mechanical and pH (pH 1-11) stabilities, which may be attributed to the incorporation of the rigid nanosized silica core of POSS. Column efficiencies of 223 000 and 50 000 N/m were observed in capillary electro-driven chromatography (CEC) and µ-HPLC, respectively. Peptides and standard proteins were baseline separated by this hybrid column in CEC and µ-HPLC, respectively, as well. The separation of bovine serum albumin (BSA) tryptic digest was also attempted to show its potential application in proteome analysis. Monolithic columns can be described as the integrated continuous porous separation media for separation sciences. In the past decade, monolithic columns, the novel state-of-the-art stationary phases, have been given comprehensive attention and applied widely in microscale chromatographic separations, such as capillary liquid chromatography (CLC), capillary electro-driven chromatography (CEC), and microfluidic devices. Microscale monolithic columns are usually prepared within the confines of * To whom correspondence should be addressed. Tel.: +86-411-84379610. Fax: +86-411-84379620. E-mail: [email protected] (H.Z.). Tel.: +86-41184379576. Fax: +86-411-84379620. E-mail: [email protected] (R.W.). † Chinese Academy of Sciences. ‡ Graduate School of Chinese Academy of Sciences. 10.1021/ac1003147  2010 American Chemical Society Published on Web 05/28/2010

capillaries, and with no need of supporting frits. Monolithic columns possess advantages that include easy preparation, versatility in surface modification, great permeability, and good peak capacity. These unique merits have made monolithic columns the attractive alternative to the packed and open-tubular columns in the analytical separation sciences.1-4 In recent years, with the rapid development of nanoscale chromatographic separation systems coupled to mass spectrometry, the use of capillary monolithic columns have emerged as a promising choice for the separation of complex biological samples to provide a lower backpressure drop, better column stability, and better resolution and sensitivity.5-7 On the basis of the chemical nature of monoliths, monolithic columns can be mainly classified into organic polymer-based and silica-based monolithic columns.8,9 However, the mechanical and solvent instability of polymer-based monoliths, and the pH sensitivity of silica-based monoliths are the inherent drawbacks for polymer-based and silica-based monoliths, respectively.10-12 Recently, the emerging organic-inorganic hybrid monolithic columns, incorporating organic moieties into inorganic (usually silica) monolithic matrices via the co-condensation of organofunctional trialkoxysilanes [(RO)3Si-R′: where R′ represents the organofunctional group] and conventional tetra-alkoxysilanes (i.e., TMOS or TEOS) by the sol-gel method, seem to be the promising choices for polymer- or silica-based monolithic columns.10-14 Organic-inorganic hybrid monolithic columns are supposed to combine the merits of the organic polymer and (1) Svec, F. J. Sep. Sci. 2004, 27, 1419–1430. (2) Zou, H. F.; Huang, X. D.; Ye, M. L.; Luo, Q. Z. J. Chromatogr. A 2002, 954, 5–32. (3) Tanaka, N.; Kobayashi, H.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Hosoya, K.; Ikegami, T. J. Chromatogr. A 2002, 965, 35–49. (4) Vlakh, E. G.; Tennikova, T. B. J. Sep. Sci. 2007, 30, 2801–2813. (5) Wu, R. A.; Hu, L. G.; Wang, F. J.; Ye, M. L.; Zou, H. J. Chromatogr. A 2008, 1184, 369–392. (6) Kasicka, V. Electrophoresis 2008, 29, 179–206. (7) Sandra, K.; Moshir, M.; D’Hondt, F.; Verleysen, K.; Kas, K.; Sandra, P. J. Chromatogr. B 2008, 866, 48–63. (8) Gusev, I.; Huang, X.; Horvath, C. J. Chromatogr. A 1999, 855, 273–290. (9) Guiochon, G. J. Chromatogr. A 2007, 1168, 101–168. (10) Hayes, J. D.; Malik, A. Anal. Chem. 2000, 72, 4090–4099. (11) Colon, H.; Zhang, X.; Murphy, J. K.; Rivera, J. G.; Colon, L. A. Chem. Commun. 2005, 2826–2828. (12) Yan, L. J.; Zhang, Q. H.; Zhang, H.; Zhang, L. Y.; Li, T.; Feng, Y. Q.; Zhang, L. H.; Zhang, W. B.; Zhang, Y. K. J. Chromatogr. A 2004, 1046, 255–261. (13) Xu, L.; Lee, H. K. J. Chromatogr. A 2008, 1195, 78–84.

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inorganic silica-based monoliths, such as easy fabrication, wide pH range tolerance, good mechanical stability, and high permeability. Since Hayes and Malik10 prepared an organic-inorganic porous capillary monolithic column using N-octadecyldimethyl[3(trimethoxysilyl)propyl] ammonium chloride as the organofunctional alkoxysilane for CEC, a number of organic-inorganic hybrid columns have been reported by using different organofunctional alkoxysilanes including phenyltriethoxysilane, 3-aminopropyltriethoxysilane, C8-triethoxysilane, and methyltrimethoxysilane.11-19 Bridged silane monomers such as 1,2-bis(trimethoxysilyl)ethane and 1,2-bis(triethoxysilyl)ethane have also been used to prepare hybrid monoliths.20 Dulay et al.21 also prepared an organic-silica hybrid monolithic column, named the photopolymerized sol-gel (PSG) monolith, using methacryloxypropyl trimethoxysilane (MPTMS) as the monomer via polycondensation and photoinitiated polymerization. Different from the above-mentioned organic-inorganic hybrid monolithic columns, we have previously developed a “one-pot” approach to prepare the hydrophilic and hydrophobic organic-inorganic hybrid monolithic capillary columns via the in situ co-condensation and copolymerization between the organic polymerization precursors and the inorganic alkoxysilanes, which can be developed as a versatile method to synthesize the organic-silica hybrid monoliths by using a variety of organic monomers.22 Nevertheless, the use of the alkoxysilanes in the above-mentioned approaches would probably result in the residual silanol groups on monolith surface, which would possibly cause the peak tailing, broadening, or nonspecific adsorption in practice.23 Polyhedral oligomeric silsesquioxane (POSS) is a type of cagelike silsesquioxane, which embodies a truly inorganic-organic hybrid architecture containing an inner inorganic framework made up of silicon and oxygen.24-26 It refers to the structures with the empirical formula Rn(SiO1.5)n, where R represents a range of organofunctional groups, while n is an even integer g4. POSS chemical reagents are thought to be the smallest silica particles with sizes of 1-3 nm, which can be easily incorporated into common polymers via copolymerization, grafting, or blending. Using POSS reagents as the monomers in copolymerization processes is convenient with no dramatic change in reaction (14) Roux, R.; Puy, G.; Demesmay, C.; Rocca, J. L. J. Sep. Sci. 2007, 30, 3035– 3042. (15) Yan, L. J.; Zhang, Q. H.; Zhang, W. B.; Feng, Y. Q.; Zhang, L. H.; Li, T.; Zhang, Y. K. Electrophoresis 2005, 26, 2935–2941. (16) Yan, L. J.; Zhang, Q. H.; Feng, Y. Q.; Zhang, W. B.; Li, T.; Zhang, L. H.; Zhang, Y. K. J. Chromatogr. A 2006, 1121, 92–98. (17) Tian, Y.; Zhang, L. F.; Zeng, Z. R.; Li, H. B. Electrophoresis 2008, 29, 960– 970. (18) Constantin, S.; Freitag, R. J. Sol-Gel Sci. Technol. 2003, 28, 71–80. (19) Kanamori, K.; Yonezawa, H.; Nakanishi, K.; Hirao, K.; Jinnai, H. J. Sep. Sci. 2004, 27, 874–886. (20) Hutanu, D. Synthesis and characterization of novel stationary phases for small scale liquid chromatographic separations of proteins and nanoparticles. Ph.D. Thesis, Oregon State University, 2008. (21) Dulay, M. T.; Quirino, J. P.; Bennett, B. D.; Kato, M.; Zare, R. N. Anal. Chem. 2001, 73, 3921–3926. (22) Wu, M. H.; Wu, R. A.; Wang, F. J.; Ren, L. B.; Dong, J.; Liu, Z.; Zou, H. F. Anal. Chem. 2009, 81, 3529–3536. (23) Allen, D.; Rassi, Z. E. Analyst 2003, 128, 1249–1256. (24) Li, G. Z.; Wang, L. C.; Toghiani, H.; Daulton, T. L.; Koyama, K.; Pittman, C. U. Macromolecules 2001, 34, 8686–8693. (25) Li, G.; Wang, L.; Ni, H., C. U. P., Jr J. Inorg. Organomet. Polym Mater. 2001, 11, 123–154. (26) Pielichowski, K.; Njuguna, J.; Janowski, B.; Pielichowski, J. Adv. Polym. Sci. 2006, 201, 225–296.

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conditions, as long as the POSS monomers are soluble in the monomer mixture.25 The POSS reagents also offer a unique opportunity to prepare truly molecularly dispersed nanocomposites, which can be used as rigid hard building blocks in polymer for various hybrid materials.27 The interest in POSS materials is based on the facts that the rigid silicon and oxygen framework could greatly enhance the mechanical and thermal stability of the resulted POSS-containing nanohybrid polymers.24,28 In this work, we applied a POSS reagent of POSS-methacryl substituted (POSS-MA) as the cross-linker to prepare an inorganicorganic hybrid monolithic column, for the first time to our best knowledge, via the copolymerization with a functional monomer of N-(2-(methacryloyloxy)ethyl)-dimethyloctadecylammonium bromide (MDOAB) in a toluene-dodecanol porogen system. The resulting inorganic-organic hybrid monolithic capillary column (MDOA-POSS) was systematically investigated, which exhibited good mechanical stability and good pH stability. This approach would represent a versatile method to prepare the inorganicorganic hybrid monolithic column by using a variety of organic functional monomers for copolymerization. EXPERIMENTAL DETAILS Materials. POSS-methacryl substituted (cage mixture, N ) 8,10,12, POSS-MA) was purchased from Acros (NJ, USA). 2,2(Dimethylamino)ethyl methacrylate (DEMA) was purchased from Nanjing Chemlin Chemical Industry Co., Ltd. (Nanjin, China). γ-Methacryloxypropyltrimethoxysilane (γ-MAPS) and trifluoroacetic acid (TFA) were purchased from Sigma (St Louis, MO, USA). Ribonuclease B from bovine pancreas, bovine serum albumin (BSA), cytochrome C from bovine heart, enolase from yeast, insulin, and ovalbumin were all purchased from Sigma (St Louis, MO, USA). Lysozyme from chicken egg white was obtained from Sino-American Biotechnology Co. (Beijing, China). Azobisisobutyronitrile (AIBN) was purchased from Shanghai Chemical Plant (Shanghai, China) and recrystallized in ethanol before use. A fused-silica capillary with 75 µm i.d. and 375 µm o.d. was purchased from Reafine Chromatography Ltd. (Hebei, China). Dithiothreitol (DTT), iodoacetamide, and the protease inhibitors cocktail were all purchased from Sino-American Biotechnology Co. (Beijing, China). Daisogel ODS-AQ (5 µm, 120 Å pore) was purchased from Daiso (Osaka, Japan). HPLC-grade acetonitrile (ACN) from Merck (Darmstadt, Germany) was used for the preparation of mobile phases. Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore Inc., MA, USA). Other chemical reagents were of analytical grade. Synthesis of N-(2-(Methacryloyloxy)ethyl)dimethyloctadecylammonium Bromide (MDOAB). The synthesis of MDOAB was similar to that of allyldimethyldodecylammonium bromide (ADDAB) as in a previous work.22 DEMA (4 mL, 23.8 mmol) was added dropwise to a solution of 1-bromooctadecane (9 mL, 26.5 mmol) in ethanol (20 mL), which was stirred for 10 min at room temperature. Then, the obtained solution was heated to 55 °C and stirred for 36 h. After removal of the solvent by a rotary evaporator under vacuum, the resulting yellow liquid was (27) Livage, J. Curr. Opin. Solid State Mater. Sci. 1997, 2, 132–138. (28) Tanaka, K.; Inafuku, K.; Adach, S.; Chujo, Y. Macromolecules 2009, 42, 3489–3492.

Figure 1. Schematic preparation of MDOA-POSS hybrid monolithic column using POSS-MA as cross-linker and MDOAB as monomer.

recrystallized in 40 mL ethyl acetate/hexane (20/80, v/v). After incubated at 4 °C for 1 h, followed by filtration and vacuum drying, 9.2 g white solid product (MDOAB) was obtained with 95% yield. The product was characterized by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), m/z at 410.33 [M+]. Preparation of the MDOA-POSS Hybrid Monolithic Column. Before the preparation of the MDOA-POSS hybrid monolithic column, the fused-silica capillary was respectively rinsed by 1.0 M NaOH for 6 h, water for 30 min, 1.0 M HCl for 6 h, and water for 30 min, and was dried by nitrogen stream at room temperature. A 50% γ-MAPS methanol (v/v) solution was used to introduce the CdC bonds onto the inner surface of capillary to anchor monolithic matrix as previously reported.29 First, the solution was injected into the capillary, and then the capillary was submerged in a water bath at 50 °C overnight with the both ends sealed with silicone rubbers. After that, the capillary was rinsed by methanol to flush out the residuals and dried by nitrogen stream again for further use. The polymerization mixture consisting of monomer MDOAB, cross-linker POSS-MA, initiator AIBN, and binary porogen of toluene-dodecanol were ultrasonicated in a bath sonicator (90 W) for 15 min to degas. The obtained homogeneous solution was manually introduced into the γ-MAPS pretreated capillary to an appropriate length with a syringe. By sealing the both ends of the capillary with silicone rubbers, the capillary was incubated at 55 °C for 12 h. Finally, the prepared monolith capillary column was washed by methanol to remove unreacted monomers and porogens. The structures of POSS-MA and MDOAB and the schematic synthesis of the MDOA-POSS hybrid monolith are illustrated in Figure 1. Preparation of BSA Tryptic Digest. The tryptic digest procedure of BSA was according to that previously reported by us with minor modifications.22 BSA (2 mg) was dissolved in 1 mL of denaturing buffer containing 8 M urea. After the addition of 10 µL DTT solution, the mixture was incubated at 60 °C for 1 h to reduce the disulfide bonds of the protein. After that, 3.7 mg IAA was added, and the mixture was incubated at room temperature in the dark for 40 min. Finally, the mixture was diluted 8-fold with 100 mM ammonium bicarbonate buffer (pH 8.2) and digested at 37 °C for 20 h with trypsin at the enzymeto-substrate ratio of of 1:40 (w/w). After digestion, the pH value of the obtained BSA tryptic digest solution was adjusted to 2-3 by 10% TFA aqueous solution. Followed by a solid-phase extraction (SPE) of the BSA tryptic digest with a homemade C18 cartridge, (29) Dong, X. L.; Dong, J.; Ou, J. J.; Zhu, Y.; Zou, H. F. Electrophoresis 2006, 27, 2518–2525.

the collected peptides elution was dried under vacuum and dissolved into a 0.1% formic acid aqueous solution (1 mL), which was stored in a -4 °C freezer before µ-HPLC-MS/MS analysis. Capillary Electrochromatography. All CEC experiments were carried out on a CE instrument-P/ACE MDQ System (Beckman, Fullerton, CA, USA) equipped a UV detector with temperature at 25 °C and detection wavelength at 214 nm. A detection window was made by removing the polyimide coating of a fused-silica capillary with a razor blade in the empty section of the capillary at the edge of the hybrid monolithic continuous bed. The total length of the prepared capillary column was 31 cm with an effective length of 21 cm. The monolithic column was first preconditioned by running buffer for at least 30 min with a manual syringe pump, and then, equilibrated on an instrument by applying a low voltage (10 kV, ramping time for 10 min) until a stable current was obtained. All data obtained were based on three runs. The retention factor (k′) was defined as (tr - t0)/t0, where tr and t0 represent the retention times of an analyte and an unretained compound (thiourea was used as the void time marker here), respectively. Micro-HPLC System. A µ-HPLC system consists of an agilent pump, a UV detector (K-2501, Knauer), a chromatography workstation (Cailu, Beijing), and a injection valve (model 7125, Rheodyne) fitted with a T-joint using a fused-silica capillary (50 µm i.d. and 375 µm o.d., length of 95 cm) as a splitter. The split ratio was controlled at ca. 1/400. The outlet of the hybrid monolithic column was connected to a fused-silica capillary (50 µm i.d. and 375 µm o.d.,) with a Teflon tube. The detection window was made by burning off a 2 mm polyimide coating at a position 6 cm from the separation monolithic column outlet. A µ-HPLC system (ThermoFinnigan, San Jose, CA) for protein tryptic digest separation consists of a degasser, a quaternary Surveyor MS pump, and a six-port/two-position valve. A capillary of 50 µm i.d. was used for splitting, and the flow rate after splitting was adjusted to ca. 200 nL/min. Mass Spectrometry Detection. The LTQ linear ion trap mass spectrometer was equipped with a nanospray ion source. The temperature of the ion transfer capillary was set at 200 °C. The spray voltage was set at 1.8 kV, and the normalized collision energy was set at 35.0%. One microscan was set for each MS and MS/MS scan. All MS and MS/MS spectra were acquired in the data dependent mode. The mass spectrometer was set that one full MS scan was followed by six MS/MS scans on the six most intense ions. The dynamic exclusion function was set as follows: repeat count 2, repeat duration 30 s, and exclusion duration 90 s. System control and data collection were done by Xcalibur software Analytical Chemistry, Vol. 82, No. 13, July 1, 2010

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Table 1. Composition of Polymerization Mixture for the Preparation of MDOA-POSS Hybrid Monolithic Columnsa col

toluene (µL)

dodecanol (µL)

MDOAB (mg)

permeability

homogeneity

A B C E F

80 90 100 90 90

260 250 240 250 250

5 5 5 8 12

good good poor good good

poor good good good good

a

EOF (cm2/(V s) × 10-4)

k′ benzene

k′ butylbenzene

1.71

0.383

1.017

1.51 0.93

0.329 0.344

0.881 0.908

Other preparation conditions: polymerization temperature, 55 °C; POSS, 50 mg; AIBN, 2 mg.

version 1.4 (Thermo). The scan range was set from m/z 400 to m/z 1600. Data Analysis. The acquired MS/MS spectra was searched on the database using the MASCOT (version 2.2.04) protein identification platform (Matrix Science, London, UK) and the MS/ MS spectra of pull-down were searched against IPI_bovine BOVIN_3.32 (32 946 sequences; 16 109 453 residues). Cysteine residues were searched as fixed modification of 57.0215 Da, and methionine residues as variable modification of 15.9949 Da. Peptides were searched using fully tryptic cleavage constraints and up to two internal cleavages sites were allowed for tryptic digestion. The mass tolerances were 2 Da for parent masses and 1 Da for fragment masses. RESULTS AND DISCUSSION Preparation and Characterization of the MDOA-POSS Hybrid Monolithic Column. The preparation conditions of the MDOA-POSS hybrid monolithic column have been investigated. Considering the cross-linker of POSS-MA can be well-dissolved in toluene, a binary porogenic solvent of toluene-dodecanol, which is a broadly used porogen in the preparation of organic polymer-based monolithic columns,30-33 was utilized for this hybrid monolithic column. The amounts of toluene and dodecanol used in the formation of the MDOA-POSS hybrid monolithic column were listed in Table 1. As can be seen from columns A-C in Table 1, the permeability of the formed monoliths decreased as the amount of dodecanol (toluene) in the porogenic solvent decreased (increased), while the homogeneity of the generated monoliths increased conversely. This phenomenon is similar to that in the preparation of polymer-based monolithic columns by using toluene-dodecanol as the porogenic solvent,30,31 where the toluene was used as the good solvent for hydrophobic monomers and dodecanol used as the solvent for polar monomers. The ratio change of toluene to dodecanol affected the solubility of the monomer of MDOAB and the cross-linkers of POSS-MA, and consequently influenced the phase separation in the formation of the hybrid inorganic-organic monolith. The ratio of toluene to dodecanol used for the preparation of the hybrid monolith was 90/250. As shown in Table 1, the MDOA-POSS hybrid monolithic columns (columns E and F) could be synthesized with this rational ratio, though the amount of the organic monomer of MDOAB was increased from 5 to 12 mg. Interestingly, with the increase (30) Li, Y.; Zhang, J.; Xiang, R.; Yang, Y. H.; Horvath, C. J. Sep. Sci. 2004, 27, 1467–1474. (31) Ou, J. J.; Dong, J.; Tian, T. J.; Hu, J. W.; Ye, M. L.; Zou, H. F. J. Biochem. Biophys. Meth. 2007, 70, 71–76. (32) Li, Y.; Gu, B. H.; Tolley, H. D.; Lee, M. L. J. Chromatogr. A 2009, 1216, 5525–5532. (33) Lubbad, S. H.; Buchmeiser, M. R. J. Sep. Sci. 2009, 32, 2521–2529.

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of the amount of MDOAB in the polymerization mixture, the electroosmotic flow (EOF) of the synthesized MDOA-POSS hybrid monolithic column was decreased from 1.71 to 1.51 and 0.93 for 5, 8, and 12 mg of MDOAB, respectively. This is actually in conflict with the general thought that increasing the functional monomer in copolymerization would increase the incorporated monomer and consequently would generate the increased EOF for CEC. In the comparison of retention factors (k′) among columns B, E, and F, it was also observed that the k′ for benzene and butylbenzene of column E and F were much less than that of column B. These results indicated that the amount of the MDOAB copolymerized into the hybrid monolith decreased when the MDOAB content high than 5 mg in the prepolymerization mixture. The factors which affects of the polymerization mixture of cationic monomers were complicated including the distance between the quaternary ammonium groups and the radical position, charge density, ionic strength, hydrophobic interactions, etc.34 In this experiment, we observed that the polymerization time increased with the increase of the MDOAB content in the polymerization mixture. The cationic group of MDOAB results in a repulsion effect between each other which would prevent the formation of higher molecular weight polymer. Additionally, due to the hydrophobic interaction, the long chain of the MDOAB monomer tends to approach to the POSS-MA, which would hinder the copolymerization between MDOAB and POSS-MA. Additionally, the incompatibility of the reactivity ratios of MDOAB to POSSMA might also impact the copolymerization process. On the basis of these observations, a polymerization mixture consisting of 90 µL toluene, 250 µL dodecanol, 5 mg MDOAB, 50 mg POSS-MA, and 2 mg AIBN was chosen and polymerized at 55 °C to obtain the homogeneous and permeable MDOA-POSS hybrid monolithic column and column B was used for further experiment. The cross-section morphology of the prepared MDOA-POSS hybrid monolithic column (column B) was characterized by SEM, and the obtained SEM images were shown in Figure 2 with two different magnifications. As shown in Figure 2A, a uniform MDOA-POSS monolithic matrix with large through-pores was obtained within the capillary. In Figure 2B, it can be seen that the formed organic-POSS monolithic matrix was attached to the inner capillary wall well. This was because of not only the successful pretreatment of capillary by γ-MAPS but also the copolymerization happened among γ-MAPS, MDOAB, and POSS-MA. If looking at the aggregated polymer clusters of the synthesized MDOA-POSS hybrid monolith, one can tell that the obtained MDOA-POSS hybrid monolith was a kind of organic monolith. (34) Losada, R.; Wandrey, C. Macromolecules 2009, 42, 3285–3293.

Figure 2. SEM images of a MDOA-POSS hybrid monolithic column. Magnification: 2000× for A and 5000× for B.

To examine the mechanical stability of the obtained MDOAPOSS hybrid monolithic column, a column (length, 25.5 cm) was connected to a HPLC pump (Agilent) with the flow rate ranged from 0.1 to 2.2 µL/min (in splitter mode) using the water and ACN as the mobile phase, respectively. The measured backpressure was linearly increased as the flow rate was increased (See the Supporting Information, Figure S1), which indicated that the MDOA-POSS hybrid monolith possessed good mechanical stability. Using Darcy’s Law35 of permeability B0 = FηL/(πr2∆P), where F is the flow rate of the mobile phase (m3/s), η is the viscosity of the mobile phase (Pa · s), L is the effective length of column (m), r is the inner radius of the column (m), and ∆P is the pressure drop of the column (Pa), the permeability of the MDOA-POSS hybrid monolithic column was calculated as 7.19 × 10-14 m2 and 5.35 × 10-14 m2 for water (η = 0.89 cP) and ACN (η = 0.38 cP), respectively, which indicated the good permeability of the prepared monolithic column. As the mobile phase changed from water to ACN, the change of column permeability was ca. 25.6% which was less than the estimated changes of ca. 36.1% for poly(butyl methacrylate-co-ethylene dimethacrylate) monolith.36 However, the change of the permibility was large than that of poly[hydroxyethyl acrylate-copoly(ethylene glycol) diacrylate] monoliths which showed nearly no swelling or shrinking in different polarity solvents.37 This result indicates the acceptable solvent swelling of the prepared monolithic column by using the POSS as the crosslinker. By changing the mobile phase back to the previous one, the column permeability could be recovered within 30 min, which means that the swelling and the shrinking of this hybrid monolith was reversible. To investigate the pH stability, the obtained MDOA-POSS hybrid monolithic column was flushed for more than 100 h by 50% ACN with different extreme pH values at the flow rate of 0.2-0.3 µL/min. The chromatographic performances including the theoretical plate number N and the retention factor k′ of the flushed column were monitored by CEC at several flush intervals. The obtained results were illustrated in Figure S2 (of the Supporting Information), which describes the performance trace of columns flushed by 50% ACN solution at extreme low and high (35) Stanelle, R. D.; Sander, L. C.; Marcus, R. K. J. Chromatogr. A 2005, 1100, 68–75. (36) Gu, C.; Lin, L.; Chen, X.; Jia, J.; Wu, D.; Fang, N. J. Sep. Sci. 2007, 30, 2866–2873. (37) Li, Y. Y.; Tolley, H. D.; Lee, M. L. Anal. Chem. 2009, 81, 9416–9424.

pH values. As seen in Figure S2A, both theoretical plate number N and retention factors (k′) were remained above 90% after flushing by 0.1 M HCl containing 50% ACN solution (pH ) 1.1) for over 100 h, which indicats that the MDOA-POSS hybrid monolithic column has good pH stability at a low pH value. In contrast, the obtained hybrid monolithic column was also flushed by a basic solution at a high pH value to test its pH stability at basic conditions. Figure S2B shows the similar performance trace of N and k′ under high pH conditions. The result shows that the retention factors (k′) of hydrophobic compounds remained about 75% after flushing by a 10 mM phosphate solution containing 50% ACN at pH 11 for 35 h and, then, stayed at nearly the same level for over 100 h. At this high pH value, N was stable at the initial period of 35 h. After that, N increased first and then decreased as the flush time increased to ca. 120 h. Even higher pH values of 12 and 13 have also been used for the pH stability test. It was found that N could remain constant for 24 h under such extreme pH conditions. After flushing for 24 h at pH 13, k′ was decreased remarkably, while the slight detachment of the monolith from the inner wall of the capillary was also observed by an optical microscope. This may be due to the fact that the linkage between the hybrid monolith and capillary, the Si-O-Si-C bonds formed between γ-MAPS and the capillary wall, was destroyed under such strong basic ambience. CEC and µ-HPLC Performance. In CEC, EOF is the basic requirement for driving mobile phases through a capillary column. To prepare the hybrid monolithic column for CEC with a sufficient EOF, a synthesized quaternary ammonium bromide monomer of MDOAB, which acted as the main source to generate the EOF from cathode to anode, was used for the hybrid monolithic matrix fabrication. The examined relationship between the EOF and the pH value of the running buffer is presented in Figure S3 (of the Supporting Information). It can be seen that the MDOA-POSS hybrid monolithic column could provide strong EOF (greater than 1.98 × 10-4 cm2/(V s), from cathode to anode) in CEC in a wide range of pH values (from 2 to 10) of running buffer. The strong EOF maintained at high pH condition was due to the strong ionization of the quaternary ammonium group of MDOAB, which indicates the successful in situ copolymerization of MDOAB and POSS-MA. The obtained strong EOF of the MDOA-POSS hybrid monolithic column would be a potential advantage for the separation of positively charged analytes in CEC by using a high pH value of the running buffer. Analytical Chemistry, Vol. 82, No. 13, July 1, 2010

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Figure 3. Separation of alkylbenzens on MDOA-POSS hybrid monolithic column. Experimental conditions for CEC: mobile phase, 10 mM Na2HPO4-citric acid buffer containing 80% ACN at pH 3; separation voltage, -20 kV; injection, -5 kV for 1 s; column effective length, 21 cm; total length, 31 cm; detection wavelength, 214 nm. Experimental conditions for µ-HPLC: mobile phase, 0.1% TFA buffer containing 70% ACN; column effective length, 34 cm; outlet to detection window, 6 cm; injection volume, 4 µL in split mode; detection wavelength, 214 nm. Analytes: (0) thiourea; (1) benzene; (2) toluene; (3) ethylbenzene; (4) propylbenzene; (5) butylbenzene.

As shown in Figure 1, the synthesized monomer of MDOAB has a methacrylate group and a quaternary ammonium cation and a long aliphatic carbon chain (C18). After the in situ copolymerization with POSS-MA, the long aliphatic carbon chain of MDOAB played as the hydrophobic functional group for chromatographic separation. In this work, the alkylbenzenes were used to investigate the chromatographic performance of the obtained MDOA-POSS hybrid monolithic column in both CEC and µ-HPLC. Figure 3 shows the resultant chromatograms of these neutral aromatic compounds with good peak shapes on a MDOA-POSS hybrid monolithic column with the running buffer of 80% ACN at pH 3 for CEC and 0.1% TFA solution containing 70% ACN for µ-HPLC. The analytes were all eluted in the order of thiourea < benzene < toluene < ethylbenzene < propylbenzene < butylbenzene, which is corresponding to the hydrophobicities of these analytes from low to high. This result indicated the reversed-phase separation of these compounds on the hybrid monolithic column, which was further examined by investigating the change of retention factors (k′) over the ACN content in the mobile phase in CEC. As shown in Figure S4 (of the Supporting Information), the logarithms of the retention factors of alkylbenzenes decreased linearly with the increase of the ACN content in mobile phase, which confirmed the typical reversed-phase chromatographic property of the MDOA-POSS hybrid monolithic stationary phase toward the hydrophobic solutes.10 The column efficiencies of the obtained MDOA-POSS hybrid monolithic column in CEC and µ-HPLC were also evaluated. In CEC, the applied voltage was changed from 5 to 29 kV, and the relationship between the flow velocity and the plate height for thiourea, benzene and toluene were demonstrated in Figure 4. The column efficiency in the range of 166 000-187 000 N/m for alkylbenzenes and 223 000 N/m for thiourea was observed. The column remained at high efficiency with the linear velocity ranging from 1.0 to 1.6 mm/s in CEC. The relationship between the flow velocity and the plate height evaluated in µ-HPLC mode was also 5452

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Figure 4. Relationship of the plate height on the linear velocity of the mobile phase on the MDOA-POSS hybrid monolithic column. Experimental conditions for CEC and µ-HPLC are the same as those in Figure 3 except the flow velocity.

shown in Figure 4, and the lowest plate height of ca. 20 µm was obtained. The run-to-run reproducibility was evaluated on a single capillary monolithic column in CEC. The relative standard deviations (RSDs) for EOF and retention time of analytes (thiourea, benzene, and toluene) on the capillary monolithic column were less than 4.2% for 5 runs in CEC. Both column-to-column and batch-to-batch reproducibilities were also evaluated in term of the RSDs of EOF and retention times of analytes, which were less than 6.7% (n ) 3) and 9.2% (n ) 3), respectively. These results indicated that the reproducibility of these prepared MDOA-POSS hybrid monolithic columns was acceptable. Application of MDOA-POSS Hybrid Monolithic Column. The MDOA-POSS hybrid monolithic column was used for the separation of peptides in CEC. The separation mechanism of negatively charged compounds on this MDOA-POSS hybrid monolithic column is the combination of electrophoretic mobility, strong anion exchange (SAX) interaction, and hydrophobic interaction. As shown in Figure 5, five peptides were separated and four of them were eluted before the void time which was mainly due to the electrophoretic mobility. The peptides separated here should be negatively charged at pH 6.5, so the direction of electrophoretic migration was same to EOF which would lead to short separation time especially for the acidic peptides such as Gly-Gly-Asp-Ala (peak 1). It also indicated that the hydrophobic interaction were relatively weak since they were highly polar solutes and showed weak retention on hydrophobic reversed phase stationary phase. The SAX interaction was due to the existence of quaternary ammonium groups on the matrix surface. The effect of ionic strength in the mobile phase was also investigated. Figure 5 shows that the higher salt concentration (Figure 5A) could improve the peak shape since the SAX interaction could be suppressed by higher salt concentration to some content. The suppression of anion exchange interaction was favored for quicker electrophoretic migration which could lead to shorter separation time. This hybrid monolithic column was also used for the separation of standard proteins in µ-HPLC mode. Figure 6 (The drift of the baseline was due to the gradient elution) shows that seven standard proteins including ribonuclease B, cytochrome C, insulin, lysozyme, BSA, enolase, and ovalbumin were successfully sepa-

Figure 5. Separation of peptide mixture on MDOA-POSS hybrid monolithic column by CEC. CEC conditions: mobile phase, 20 mM Na2HPO4-citric acid buffer for A and 10 mM Na2HPO4-citric acid buffer for B containing 35% ACN at pH 6.5; separation voltage, -20 kV; injection, -5 for 5 s. Other CEC conditions are the same as those in Figure 3. Analytes: (1) Gly-Gly-Asp-Ala; (2) Gly-Ala-Ala; (3) SerSer-Glu-Ala-Asn-Leu-Arg; (4) Ala-Thr-Val-Leu-Asn-Tyr-Leu-Pro; (5) Leu-Tyr-Leu. Other peaks are unknown impurities.

Figure 6. Separation of standard proteins on MDOA-POSS hybrid monolithic columns in µ-HPLC. Conditions: column length, 34/40 cm; mobile phase, (A) 0.1% TFA in water, (B) 0.1% TFA in ACN; gradient, 15 to 45% B in 10 min; inject volume, 10 µL in split mode; injected sample amount of these analytes after split were in the range of 4-8 ng; flow rate 300 nL/min; detection wavelength, 220 nm. Analytes: (1) ribonuclease B, (2) cytochrome C, (3) insulin, (4) lysozyme, (5) BSA, (6) enolase (7) ovalbumin. Other peaks are unknown impurities.

rated in 17 min with good peak shapes. Since the separation mechanism is a mixed mode containing SAX and hydrophobic interaction, the separation efficiency of proteins as well as peptides would not be comparable to that of pure reversed phased separation in µ-HPLC, so the fabrication of a pure reversed phase hybrid monolithic column with nonionic monomers such as octylor dodecyl-methacrylate was our following work. However, these separation results indicated that this hybrid monolith column was capable of separating both small molecules and big biomolecules. Besides the separation of alkylbenzenes, peptide mixtures, and standard proteins, the separation of peptide mixtures of BSA tryptic digest was also attempted on this obtained MDOA-POSS hybrid monolithic column by µLC-MS/MS for further investigating the potential use in the analysis of complex samples. One

Figure 7. Base peak chromatogram of µ-HPLC-MS/MS analysis of BSA tryptic digest. Experimental conditions: MDOA-POSS hybrid monolithic column, 40 cm × 75 µm i.d.; flow rate, 200 nL/min; sample amount, 1 pmol; mobile phase (A) 0.1% formic acid in water, (B) 0.1% formic acid in ACN; the separation gradient was buffer B from 2 to 35% in 40 min and from 35 to 80% in 5 min. After flushing with 80% buffer B for 5 min, the separation system was equilibrated with 2% buffer B for 25 min.

picomolar BSA tryptic digest was manually loaded onto a capillary monolithic column with a capillary (21 cm × 75 µm i.d.) for the µHPLC-MS/MS in RP mode with a gradient elution from 2 to 35% ACN within 40 min, and the chromatogram was shown in Figure 7. On the basis of the database search of the obtained chromatogram of BSA tryptic digest, 53 unique peptides were positively identified and the protein coverage was 71% (RSD ) 6.4%, n ) 3). These results were comparable to that obtained from a particulatepacked commercial column (5 µm, 120 Å pore, 12 cm × 75 µm i.d.), where 59 unique peptides (RSD ) 4.1%, n ) 3) and 76% protein coverage (RSD ) 3.1%, n ) 3) for 1 pmol BSA tryptic digest using the same µ-HPLC MS/MS analysis conditions (a chromatogram of the particle packed column was shown in the Supporting Information, Figure S5). The extract peaks (K.TCVADESHAGCEK.S, ions score 34) between the particle packed column and MDOA-POSS hybrid monolithic column were compared (Supporting Information, Figure S6). The peak widths at the 0.613 height were 0.184 and 0.188 min for the MDOA-POSS hybrid monolithic column and particle packed column, respectively. These results also indicated that this MDOA-POSS hybrid monolithic column was capable of separating some complex samples. CONCLUSIONS The monolithic column is being considered as the new generation of column for the chromatographic separation sciences, and the difficulties in the preparation of the desired monolithic column, either organic polymer-based or silica-based monolithic columns, has motivated the development of new methods for column preparation. Here, we prepared a novel inorganic-organic hybrid monolithic capillary column by the in situ thermal initiated free radical copolymerization within the confine of a capillary using a POSS regent (POSS-MA) as the inorganic-organic hybrid cross-linker and a synthesized aliphatic long chain methacrylate quaternary ammonium salt as monomer. To the best of our knowledge, this is the first time to use a POSS reagent as the Analytical Chemistry, Vol. 82, No. 13, July 1, 2010

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inorganic-organic hybrid cross-linker in the preparation of hybrid monolithic column. The whole preparation process was simple and very similar to that of organic polymer-based monolithic columns instead of the use of the inorganic-organic hybrid POSS reagent as cross-linker. The pore structure and the permeability of the synthesized inorganic-organic hybrid monolithic column are tunable by changing the polymerization conditions. After the characterization of the resultant MDOA-POSS hybrid monolithic columns, it was found that the hybrid monolithic column exhibited good mechanical stability and good pH stability. The monomer of MDOAB incorporated in the hybrid monolithic matrix could provide not only a strong EOF in a wide pH range (pH 2-10), but also the high hydrophobicity for separation of small neutral analytes in RP mode both in CEC and µ-HPLC. The column efficiencies of 223 000 and 50 000 N/m for thiourea were observed in CEC and µ-HPLC, respectively. The separation of peptides, standard proteins, and protein tryptic digest showed its potential in analysis of big biomolecules and complex samples. The success of using the inorganic-organic hybrid POSS reagent as the cross-linker does provide an interesting clue for the preparation of hybrid monolithic column by using other multifunctional hybrid materials or reagents as the basic nanoblocks for monoliths. In fact, POSS functionalized with various

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reactive organic groups can be incorporated into any existing polymer system through either grafting or copolymerization.25 Additionally, a variety of POSS reagents have been commercial available, which would be a great advantage to prepare various inorganic-organic hybrid monoliths by copolymerization with unrestricted organic monomers for different application purposes. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 20735004) and the State Key Basic Research Development Program of China (No. 2005CB522701) to H.Z.; the National Natural Sciences Foundation of China (No. 20875089), the National High Technology Research and Development Program of China (No. 2008AA02Z211), and the Hundred Talent Program of the Chinese Academy of Sciences to R.W. SUPPORTING INFORMATION AVAILABLE Figures S1-S6. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review October 27, 2009. Accepted May 11, 2010. AC1003147