Facile Preparation of Zwitterionic Organic-Silica Hybrid Monolithic

The chromatographic evaluation of hybrid monolithic columns was performed using an HPLC system including two LC-10AD VP pumps (Shimadzu, Kyoto, ...
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Facile Preparation of Zwitterionic Organic-Silica Hybrid Monolithic Capillary Column with an Improved “One-Pot” Approach for Hydrophilic-Interaction Liquid Chromatography (HILIC) Hui Lin,†,‡ Junjie Ou,*,† Zhenbin Zhang,†,‡ Jing Dong,† Minghuo Wu,†,§ and Hanfa Zou*,† †

CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China ‡ Graduate School of Chinese Academy of Sciences, Beijing 100049, China § College of Environmental and Chemical Engineering, Nanchang Hangkong University, Nanchang 330063, China S Supporting Information *

ABSTRACT: A simple single-step thermal-treatment “one-pot” approach for the preparation of organic-silica hybrid capillary monolithic columns is described. In this improved method, the cross-linker vinyltrimethoxysilane (VTMS) was replaced by 3methacryloxypropyltrimethoxysilane (γ-MAPS), which is more active in polymerization reactions, and only one thermal treatment step was required in the preparation of hybrid monoliths. Two zwitterionic organic-silica monolithic columns were successfully synthesized by using [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (MSA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) as the organic monomers. The effects of the tetramethoxysilane (TMOS)/γ-MAPS molar ratio, content of monomer, composition of porogenic solvent, and reaction temperature on the morphologies of the hybrid monoliths were investigated. The MSA-silica and MPC-silica hybrid monolithic columns exhibited good permeability and good mechanical stability. The monolithic columns were used for the separation of polar compounds by capillary hydrophilic-interaction chromatography (cHILIC). A typical HILIC retention mechanism was observed at higher organic solvent contents (>50% ACN). The MSA monoliths were further investigated in the separation of various neutral, basic, and acidic analytes, as well as small peptides, by capillary liquid chromatography (cLC), and high efficiency and satisfactory reproducibility were achieved. In addition, the analysis of a tryptic digest of bovine serum albumin (BSA) by cLC tandem mass spectrometry (cLC-MS/MS) with an MSA monolith further demonstrated its potential in the separation of biological samples.

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multidimensional high-performance liquid chromatography (HPLC) for the separation of complex samples.17−19 Additionally, HILIC is generally performed with mobile phases of high organic solvent content, which are quite compatible with mass spectrometry (MS) and provide sensitive detection for LC-MS analysis.19−21 There are several typical stationary phases for HILIC, including silica,22,23 amino,24,25 diol,26 polyhydroxyethyl aspartamide,4 and cyclodextrin-based packings.27,28 Zwitterionic material, which contains both positively and negatively charged sites,29 is one of the most popular HILIC stationary phases because of its hydrophilic character. Commercially available particulate-packed zwitterionic HILIC columns such as ZICHILIC and ZIC-pHILIC have been applied successfully in the separation of various hydrophilic molecules ranging from inorganic ions to proteins30 and exhibited good selectivity to isomeric N-glycans in glycol-proteomic analysis.31,32 However,

urrently, reversed-phase liquid chromatography (RPLC) is the most widely used mode in the separation of complex samples. However, it is not well-suited for the analysis of polar compounds because highly aqueous mobile phases are often required to achieve adequate retention.1 As an alternative approach, normal-phase liquid chromatography (NPLC) can provide effective retention for polar compounds; however, the poor solubility of polar molecules in nonaqueous mobile phases and a low reproducibility limit the use of NPLC for the separation of polar compounds such as carbohydrates and natural product extracts.2,3 Hydrophilic-interaction liquid chromatography (HILIC), as a variant of NPLC, which was developed by Alpert in 1990,4 has been successfully applied for the separation of carbohydrates,5,6 peptides,7,8 proteins,9,10 pharmaceuticals,11,12 and natural product extracts,13 as well as for the selective enrichment of glycopeptides and phosphopeptides in proteomics applications.14,15 In HILIC, a polar stationary phase and aqueous/organic mobile phases are utilized, and the direction of retention is opposite that of RPLC.16 As a result, HILIC has good separation orthogonality to RPLC, enabling their integration into two-dimensional or © 2012 American Chemical Society

Received: November 14, 2011 Accepted: February 22, 2012 Published: February 22, 2012 2721

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capillaries with dimensions of 75 or 200 μm i.d. and 365 μm o.d. were obtained from Refine Chromatography Ltd. (Yongnian, Hebei, China). Trypsin was purchased from Promega (Madison, WI). Bovine serum albumin (BSA), toluene, dimethylformamide (DMF), formamide, thiourea, benzoic acid, and other standard compounds were all obtained from Sigma (St. Louis, MO). Dithiothreitol (DTT) and iodoacetamide (IAA) were purchased from Sino-American Biotechnology Corporation (Beijing, China). HPLC-grade acetonitrile (ACN) was used for the preparation of mobile phases. The water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore Inc., Milford, MA). Phe-Phe, Tyr-Phe, Tyr-Val, Gly-Tyr-Gly, and Gly-Gly-Glu-Ala were obtained from Serva GmbH (Heidelberg, Germany). C18 particles (5 μm, 120 Å pores) were purchased from DAISO (Osaka, Japan). Other chemical reagents were of analytical grade. Preparation of MSA Monoliths. The MSA monoliths were prepared according to the “one-pot” process reported previously44,46 with minor modification. Prior to use, to anchor the monoliths to the capillary wall, the fused-silica capillary was pretreated and rinsed, successively, by 1.0 M NaOH for 4 h, water for 30 min, 1.0 M HCl for 14 h, and water for another 30 min and then dried in a nitrogen stream at room temperature for further use. A prepolymerization mixture containing acetic acid (0.01 M, 5.0 mL), PEG (10000 MW, 540 mg), urea (800 mg), TMOS (1.8 mL), and γ-MAPS (500 μL) was stirred for 2.5 h at 0 °C and hydrolyzed to form a homogeneous solution. Then, MSA (40 mg) and AIBN (2 mg) were added to 0.5 mL of the hydrolyzed mixture with 10 min of sonication for mixing and degassing. After that, the mixture was manually introduced into the pretreated capillary to an appropriate length by a syringe. After both ends of the capillary had been sealed with two pieces of rubber, the mixture was incubated at 40 °C for 12 h for simultaneous condensation and polymerization. The obtained hybrid monolithic capillary column was completely flushed with water and methanol successively to remove the PEG and other residuals. As a comparison, an MSA monolith with the previous “onepot” approach using VTMS as the cross-linker was also prepared. The prepolymerization mixture was composed of acetic acid (0.01 M, 5.0 mL), PEG (10000 MW, 540 mg), urea (675 mg), TMOS (1.8 mL), and VTMS (600 μL) and was stirred for 1.0 h at 0 °C and hydrolyzed to form a homogeneous solution. After sonication for 10 min, the mixture was manually introduced into the pretreated capillary to an appropriate length by a syringe. After both ends of the capillary had been sealed with two pieces of rubber, the mixture was incubated at 40 °C for 12 or 24 h or at both 40 and 60 °C for 12 h for condensation and polymerization. The obtained hybrid monolithic capillary columns were completely flushed with water and methanol successively to remove the PEG and other residuals. Preparation of MPC Monoliths. Preparation of the MPC monoliths was similar to that of the MSA monoliths except for the use of MPC instead of MSA and the amount of the urea (300 mg) in the prepolymerization mixture. Capillary Liquid Chromatography and Scanning Electron Microscopy. The chromatographic evaluation of hybrid monolithic columns was performed using an HPLC system including two LC-10AD VP pumps (Shimadzu, Kyoto, Japan) and a UV detector (K-2501, Knauer, Berlin, Germany). Data were collected at 214 or 254 nm and processed by a

the packing of microparticles in a capillary for microscale analysis requires tremendous skill, otherwise leading to poor reproducibility. Monolithic columns have been attracting increasing attention and have been extensively studied and widely applied in microHPLC/capillary HPLC (μLC/cLC) because of their easy preparation, good permeability, and fast mass transfer.33−37 Among these columns, organic-silica hybrid monolithic columns have been attracting increasing attention compared with traditional polymer-based or silica-based monolithic columns.38,39 However, because of a lack of commercially available hydrophilic silane monomers, most organic-silica hybrid monolithic columns are functionalized with hydrophobic moieties such as octyl,40 phenyl,41 allyl,42 and propyl43 groups, so the study of such columns in HILIC mode is very limited.2 The “one-pot” approach recently developed could effectively overcome the problem. Many organic monomers can be incorporated directly into the silica matrix through the sol−gel process with tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS) with subsequent free-radical polymerization.44−46 In this approach, the sol−gel formation usually takes place at low temperature to form a hybrid monolith skeleton with high porosity and good permeability, whereas the subsequent free-radical polymerization generally occurs at an adequate rate at a relatively high temperature. As a result, a twostep thermal treatment is required to ensure that the organic monomers are fully incorporated into the silica matrix.44−46 This preparation procedure is tedious, which likely further leads to a lowering of the reproducibility of hybrid monolithic columns. Increasing either the temperature of the sol−gel process or the reaction rate of free-radical polymerization at the same temperature would overcome this problem. Actually, the former is related to the composition of the sol−gel prepolymerization mixture47 and significantly affects the monolith morphology. An option to address the problem is to increase the rate of free-radical polymerization. In this study, the “one-pot” approach was further improved by replacing the widely used VTMS by 3-methacryloxypropyltrimethoxysilane (γ-MAPS), which could increase the reaction rate of free-radical polymerization with organic methacrylate monomers, and only a single thermal treatment step was employed in the preparation process. By using two commercially available organic monomers, [2(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (MSA) and 2-methacryloyloxyethyl phosphorylcholine (MPC), two novel zwitterionic organic-silica hybrid monolithic capillary columns were successfully fabricated. The obtained hybrid monolithic columns were successfully applied to the separation of neutral, basic, and acid analytes, as well as small peptides and protein tryptic digest under HILIC mode. Compared to the previous “one-pot” approach, this single-step thermal-treatment “one-pot” approach would represent a simpler and more convenient way to prepare organic-silica hybrid monolithic columns using a variety of organic monomers.

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EXPERIMENTAL SECTION Materials. MSA, MPC, poly(ethylene glycol) (PEG, Mn = 10000), and γ-MAPS were purchased from Aldrich (Milwaukee, WI). Azobisisobutyronitrile (AIBN) was obtained from Shanghai Chemical Plant (Shanghai, China) and recrystallized in ethanol before use. TMOS was purchased from Chemical Factory of Wuhan University (Wuhan, China). Fused-silica 2722

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Figure 1. Schematic incorporation of zwitterionic monomers MSA and MPC into silica-based monolithic matrixes through a single-step thermaltreatment “one-pot” process.

Table 1. Effects of Synthesis Parameters on the Formation of MSA Monoliths with Observations by Optical Microscopy

**

Study considered the effects of (a) PEG content, (b) γ-MAPS content, (c) MSA content, (d) temperature.

us with minor modifications.48 The sample trapping was achieved with a homemade C18-particle-packed trap column (4 cm length × 200 μm i.d.), and the subsequent separation was carried out on either an MSA monolith (15 cm length × 75 μm i.d.) or a C18-particle-packed column (12 cm length × 75 μm i.d.) with an integrated emitter, which was prepared by directly tapering the tip from the outlet of the capillary.49 (Detailed experimental procedures are provided in the Supporting Information.)

chromatography workstation (Beijing Cailu Scientific Instrument Ltd., Beijing, China). A 7725i injector with a 20 μL sample loop was used. A T-union connector served as a splitter, with one end connected to the capillary monolithic column and the other end to a blank capillary (95 cm length, 50 μm i.d., and 365 μm o.d.). The split ratio was controlled at about 1/400. The outlet of the hybrid monolithic column was connected by a Teflon tube to an empty fused-silica capillary (75 μm i.d. and 365 μm o.d.), where a detection window was made by removing a 2 mm length of the polyimide coating in a position 5.5 cm from the separation monolithic column outlet. Mobile phases were prepared by mixing appropriate volumes of ACN and H2O (or salt solutions) to reach desired organic levels (and salt concentrations). Throughout this article, the mobile-phase pH values and salt concentrations refer to the aqueous portion only, except for the study of the effect of the salt concentration. In that case, the salt concentrations refer to the final concentration in the mobile phase. In the pH-effect study, a stock solution of 33.3 mM formic acid and 33.3 mM acetic acid was first prepared, and then the pH of the stock solution was adjusted to the desired pH value (pH 3.0, 4.0, 5.0, 6.0, and 7.2) by addition of ammonium hydroxide. Scanning electron microscopy (SEM) images were obtained using a JEOL JSM-5600 scanning electron microscope (JEOL, Tokyo, Japan). Preparation of BSA Tryptic Digest and cHPLC-MS Analysis. The tryptic digest of BSA and cHPLC-MS analysis were performed according to procedures previously reported by

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RESULTS AND DISCUSSION Optimization of Synthesis Conditions for MSA Monoliths. MSA monoliths were prepared by the single-step thermal-treatment “one-pot” approach. The scheme of this approach is shown in Figure 1. The process involves two major reactions: the polycondensation of hydrolyzed precursors of γMAPS and TMOS and the free-radical copolymerization of the precondensed siloxane and MSA. It is known that the morphology of monolithic columns can be tuned by changing conditions such as the composition of the polymerization mixture and the reaction temperature.47 Herein, several synthesis parameters affecting the formation of MSA monoliths were evaluated, and the results are reported in Table 1. As the porogen has a significant effect on the obtained porosity of hybrid monoliths, we kept the amounts of urea and TMOS at 800 mg and 1.8 mL, respectively, and optimized the amounts of PEG and γ-MAPS. The amount of PEG was optimized first by comparing columns A−C listed in Table 1. It 2723

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Figure 2. SEM images of an MSA monolith prepared with the improved “one-pot” approach. Magnification: (A) 3000× and (B) 5000×.

Figure 3. (A) Separation of standard neutral polar solutes and (B) dependence of their plate heights on the linear velocity of the mobile phase by the MSA monolith in cLC. Experimental conditions: column dimension, 47.5 cm × 75 μm i.d.; mobile phase, ACN/water (90/10, v/v); flow rate for A, 100 μL/min (before split); injection volume, 2.5 μL in split mode; detection wavelength, 214 nm.

monolith. Thus, the column synthesized at 40 °C (column B) was selected as having acceptable column permeability. As we developed the “one-pot” method, the organic monomer was simultaneously mixed with the silane mixture. The presence of MSA would affect monolith formation, so the amount of organic monomer was examined. The precondensation solution was prepared with different amounts of MSA (30, 40, and 50 mg). No significant difference between the three monolithic columns could be observed by optical microscope. Comparison of their cross-sectional morphologies (as shown in Figure 2 and Supplementary Figure 1, Supporting Information) showed that uniform organic-silica hybrid monolithic matrixes were obtained in all cases. In addition, compared to the columns prepared with 30 and 50 mg os MSA in the precondensation mixture (columns F and G), the column prepared with 40 mg of MSA in the precondensation mixture (column B) showed the highest column efficiency by cLC evaluation. As a result, the optimal preparation conditions for column B in Table 1 were employed in the following experiments. Characterization of the Optimized MSA Monolith. The mechanical stability of the obtained hybrid monolithic column was examined by connecting a 49.0-cm-long column to a μUPLC pump (Waters) using 40% ACN as the mobile phase. The measured back-pressure increased linearly (R = 0.9998) from 0.72 to 18.9 MPa as the flow rate was increased from 0.02 to 0.50 μL/min. This indicates that the MSA monolith had good mechanical stability. According to Darcy’s law50 of

was found that column B, for which 540 mg of PEG was used, exhibited the most homogeneous morphology and the best permeability, whereas use of lower or higher amounts of PEG (for columns A and C, respectively) decreased the permeability (data not shown). The effect of the ratio of TMOS to γ-MAPS on the hybrid monoliths was also investigated. As seen from the morphologies of columns B, D, and E, corresponding to TMOS/γ-MAPS ratios of 9:2, 18:5, and 18:7 (v/v), respectively, a lower content of γ-MAPS (column D) produced a transparent monolithic matrix, whereas a higher content of γMAPS (column E) led to a nonrigid monolith. When the ratio of TMOS to γ-MAPS was 18:5 (v/v), a monolith of good morphology could be obtained, consistent with the observation of the lowest back-pressure during flushing with methanol at the same flow rate. Accordingly, a TMOS/γ-MAPS ratio of 18:5 (v/v) was employed for further experiments. The temperature of co-condensation and polymerization plays a vital role in the skeleton and pore formation of hybrid monoliths, so the preparation temperature was also carefully optimized. It was observed that monoliths could not be well formed at 37 °C (column H). Large through-pores were observed in column H under an optical microscope, and the monolith matrix was detached from the inner capillary wall, which can be attributed to the incomplete co-condensation of the silane monomers. A higher temperature would improve the homogeneity of the monolith with tight anchoring to the inner surface of the capillary. However, temperatures higher than (or equal to) 45 °C (column I) resulted in poor permeability of the 2724

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Figure 4. Separation of (A) benzoic acids derivatives, (B) phenols, (C) purines and pyrimidines, and (D) nucleosides in cLC. Analytes: (A) (1) 2HB, (2) benzoic acid, (3) 2,5-DHB, (4) 4-HB; (B) (1) phenol, (2) catechol, (3) resorcinol, (4) hydroquinone, (5) pyrogallol; (C) (1) thymine, (2) uracil, (3) adenine, (4) cytosine; (D) (1) adenosine, (2) cytidine, (3) guanosine. Experimental conditions: column dimensions (A) 46.5 cm × 75 μm i.d., (B) 47.5 cm × 75 μm i.d., (C,D) 48.6 cm × 75 μm i.d.; mobile phase (A) ACN/water (containing 40 mM NH4Ac) (80/20, v/v), (B) ACN/ water (90/10, v/v), (C,D) ACN/water (containing 10 mM NH4Ac) (80/20, v/v); flow rate, 100 μL/min (before split); injection volume, 4 μL in split mode; detection wavelength (A,B) 214 nm, (C,D) 254 nm.

permeability, B0 = FηL/(πr2ΔP), where F is the linear velocity of the eluent, η is the dynamic viscosity of the mobile phase (0.801 cP51), L is the effective length of the column, r is the inner radius of the column, and ΔP is the pressure drop across the column. The permeability of the monolithic column was calculated as 3.80 × 10−14 m2, indicating good permeability. The reproducibility of the MSA monolith was assessed through the percent relative standard deviation (RSD) for the retention factor of thiourea as the test compound (toluene as the void time marker). The run-to-run (n = 3) and day-to-day (n = 4) reproducibilities were 0.76% and 1.63%, respectively. Seven monolithic columns were prepared with the same conditions, three from one batch and four from different batches. The RSD values of the retention factor of thiourea for the column-to-column (n = 3) and batch-to-batch (n = 4) tests were 3.17% and 3.42%, respectively. In addition, no clear deterioration of the columns was observed after hundreds of injections (in continuous use for about 3 months), which indicates the high robustness of these MSA monoliths. Chromatographic Evaluation of the Optimized MSA Monolith. A test mixture containing toluene, DMF, formamide, and thiourea was used to investigate the HILIC properties of the optimized MSA monolith. As shown in Figure 3A, these compounds were completely separated using a mobile phase of ACN/water (90/10, v/v), and the peaks were eluted

according to their polarity in order from low to high (toluene < DMF < formamide < thiourea). A typical HILIC retention mechanism was exhibited with these neutral polar compounds on the MSA monolith. In our previous reports,44−46 the two-step thermal treatment “one-pot” approach was employed to prepare VTMS-based hybrid monolithic columns. Although that method is relatively mature, its process is not very convenient. Herein, a single-step thermal-treatment “one-pot” approach was developed to simplify the preparation procedures. To confirm the effectiveness of this approach, several VTMS-based MSA monoliths were similarly prepared at different temperatures with different reaction times, and their separation efficiencies for the four test compounds were also investigated (detailed results are shown in Supplementary Figure 3 in the Supporting Information). It can be seen that DMF and formamide were not baselineseparated on the VTMS-based columns made through a singlestep thermal treatment at 40 °C for 12 and 24 h (Supplementary Figure 3A,B, Supporting Information). However, they were baseline-separated on the VTMS-based MSA monolith prepared with the previous two-step thermaltreatment “one-pot” approach (Supplementary Figure 3C, Supporting Information). This indicates that a two-step thermal-treatment process was necessary when VTMS was selected as the cross-linker. Comparison of the separation 2725

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pH values and salt concentrations refer to the aqueous portion only). It can be seen that four benzoic acid derivatives were baseline-separated, and good peak shapes were obtained. Furthermore, the effects of the mobile phase pH and the salt concentration on retention of benzoic acids derivatives were also investigated. The mobile phase pH strongly affects the retention factors, but the salt concentration has only a slight effect. These results further confirmed that both hydrophilic interaction and electrostatic interaction would contribute to the retention of the charged analytes on the MSA monolith.31,52−56 (Detailed results and discussion are provided in the Supporting Information.) To further validate the potential of MSA monoliths, a series of phenols were selected as the analytes for cLC separation. As shown in Figure 4B, the weakly polar phenol, with only one hydroxyl group, was eluted first, followed by the more polar catechol, resorcinol, and hydroquinone, with two phenolic hydroxyl groups, and the most polar pyrogallol, with three phenolic hydroxyl groups, eluted last. Additionally, two positional isomers, catechol and resorcinol (or hydroquinone), were well separated. It can be concluded that the separation of phenols on the MSA monolith was dependent on both the number and the positions of the hydroxyl groups. Mixtures containing thymine, adenine, uracil, and cytosine or adenosine, cytidine, and guanosine were also used for the evaluation of the MSA monolith. As shown in Figure 4C,D, the four purines and pyrimidines and three nucleosides were baseline-separated with the optimized chromatographic conditions. No peak tailing was observed in the separation of these basic compounds. Because of the time-consuming derivatization,30 the separation of small hydrophilic peptides by RPLC is still a problem.30,57 HILIC has proved to be a very useful solution to this problem.30,31,54,58 Here, five peptides (Tyr-Phe, Tyr-Val, Phe-Phe, Gly-Tyr-Gly, and Gly-Gly-Glu-Ala) were selected as test compounds to investigate the selectivity of the MSA monolith. The HILIC separation was optimized, and a typical chromatogram is given in Supplementary Figure 5 (Supporting Information). The peptides were separated successfully with good peak shapes with the mobile phase ACN/water (80/20, v/v) (containing 40 mM NH4Ac), except for Gly-Tyr-Gly, whose poor peak shape might be due to the impurity of the sample or some unknown secondary interaction between the analyte and the stationary phase. The optimized monolithic column was also evaluated in the cLC-MS/MS analysis of a BSA tryptic digest. The BSA tryptic digest (1.2 μL) was loaded onto the monolithic column in HILIC mode, and the chromatogram obtained is shown in Figure 5. Based on a database search of the chromatogram, 37 unique peptides were positively identified with protein sequence coverage of 53.60% (RSD = 2.23%, n = 3), which was comparable to the results obtained on a C18-particlepacked column in RP mode, where 38 unique peptides were positively identified with protein sequence coverage of 54.26% (RSD = 2.73%, n = 3) under RP mode (as shown in Supplementary Figure 7, Supporting Information). The peaks of the peptide K.LKPDPNTLCDEFK.A were further extracted from the two chromatograms of HILIC and RP separation and compared (as shown in Supplementary Figure 8, Supporting Information). The peak widths at 0.613 height were 0.455 and 0.428 min for the MSA monolith (15 cm length × 75 μm i.d.) and C18-particle-packed column (12 cm length × 75 μm i.d., 5 μm, 120 Å pore), respectively. Although the separation performance on the monolithic column was not as good as

efficiencies of the test compounds on VTMS-based columns with those on γ-MAPS-based columns indicates that the latter exhibited much better resolution and higher column efficiency. This might be because the conditions for preparation of the VTMS-based hybrid column are not optimal, and more effort is necessary to further optimize the preparation conditions with the two-step thermal-treatment “one-pot” approach. When γ-MAPS-based MSA monoliths were prepared by incubating at 40 °C for 24, 36, and 48 h, there was no significant change in efficiency (data not shown). Additionally, a γ-MAPS-based hybrid column was also fabricated at 40 °C for 12 h and 60 °C for another 12 h through a two-step thermaltreatment “one-pot” approach, and the separation ability was not dramatically improved. These results demonstrate that the organic monomer was effectively introduced into the γ-MAPSbased hybrid monolithic matrix through the single-step thermal-treatment process. The influence of the ACN content in the mobile phase on the retention factors (k′) of the four test compounds was investigated (as shown in Supplementary Figure 4, Supporting Information). The results indicated that the k′ values of DMF, formamide, and thiourea all increased with increasing content of ACN in the range from 50% to 90% (v/v). Thiourea showed the strongest retention on the polar monolith because it has the highest hydrophilicity of the four test compounds, and both DMF and formamide behaved similarly to thiourea but with weaker retention. Moreover, the column exhibited good retention and separation ability for polar compounds, as these four compounds were baseline-separated even in a mobile phase of 50% (v/v) ACN/water. These results indicate a typical hydrophilic-interaction mechanism for separation of these four compounds. They also suggest that the hydrophilic monomer MSA was successfully introduced into the silica monolithic matrix through the single-step thermal-treatment “one-pot” approach. The column efficiency of the resulting MSA monolith was evaluated by changing the flow rate of the mobile phase in cLC. Figure 3B shows the relationship between the flow rate and the plate height for toluene, DMF, formamide, and thiourea. A column efficiency varying from 50000 to 110000 N/m was achieved for different test compounds at the optimal flow rate. No significant loss in column efficiency was observed with linear velocities ranging from 0.4 to 1.5 mm/s, which means that the MSA monolith is promising for rapid separations. The prepared MPC monolith was also evaluated in the separation of various samples. The influence of the ACN concentration on retention factors and the dependence of the plate height of analytes on the linear velocity of the mobile phase were investigated. Similar chromatographic performances were observed for the MPC and MSA monoliths. (Detailed separation data for neutral, acidic, and basic compounds and small peptides are shown in Supplementary Figures 10−12 in the Supporting Information.) Applications of the MSA Monolith for HILIC Separation. The MSA monolith has a zwitterionic surface, which raises the possibility of weak electrostatic interaction with charged analytes.30 A mixture of benzoic acid (B) and its derivatives, salicylic acid (2-HB), p-hydroxybenzoic acid (4HB) and 2,5-dihydroxybenzoic acid (2,5-DHB) was selected as the test analytes to verify the possibility. Figure 4A exhibits the separation of four benzoic acid derivatives in cLC with the mobile phase of ACN/water (containing 40 mM NH4Ac) (80/ 20, v/v) (note that unless otherwise stated, the mobile phase 2726

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ACKNOWLEDGMENTS

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REFERENCES

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Financial support of H.Z. by the National Natural Sciences Foundation of China (No. 20975101) and the Creative Research Group Project by NSFC (No. 21021004) is gratefully acknowledged, as is support of J.O. by the National Natural Sciences Foundation of China (No. 21175133) and the Hundred Talents Program of the Dalian Institute of Chemical Physics of Chinese Academy of Sciences.

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Figure 5. Base-peak chromatogram of cLC-MS/MS analysis of a BSA tryptic digest on MSA monolith. Experimental conditions: column dimensions, 15 cm × 75 μm i.d.; mobile phase: buffer A, 100% water (containing 0.1% formic acid), buffer B, 100% ACN (containing 0.1% formic acid); separation gradient, buffer B from 95% to 20% in 65 min; flow rate, 200 nL/min (after split).

that on the C18-particle-packed column, the good permeability and lower back-pressure of the monolithic column could enable the use of longer columns, thereby achieving higher column efficiency and separation performance.

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CONCLUSIONS A single-step thermal-treatment “one-pot” approach was successfully developed for the preparation of hybrid monoliths by replacing the cross-linker silane reagent of VTMS with γMAPS. Compared to the previous two-step thermal-treatment “one-pot” approach, the single-step approach is simpler and more convenient. Capillary columns with two types of novel zwitterionic organic-silica hybrid monoliths were successfully fabricated using MSA and MPC as the organic monomers through this approach, and the obtained monolithic columns were successfully used for the HILIC separation of neutral, acidic, and basic polar compounds, demonstrating that this improved “one-pot” approach can be used as a general method for preparing different types of monolithic columns. In addition, the optimized MSA monolith not only is capable of hydrophilic interaction with polar and charged analytes but also offers the possibility of weak electrostatic interactions with charged analytes. In addition, the cLC-MS/MS analysis of a protein tryptic digest was carried out on the optimized MSA monolith, demonstrating that MSA monoliths are potentially useful tools for the efficient separation of some complex samples. It is necessary to further assess the breadth of this improved “one-pot” approach by preparing hybrid monoliths with different types of hydrophilic and hydrophobic monomers.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-411-84379610 (H.Z.), +86-411-84379576 (J.O.). Fax: +86-411-84379620 (H.Z.), +86-411-84379620 (J.O.). Email: [email protected] (H.Z.), [email protected] (J.O.). Notes

The authors declare no competing financial interest. 2727

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