Preparation of Hybrid Monolithic Columns via âOne-Potâ Photoinitiated

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Analytical Chemistry

Fast preparation of hybrid monolithic columns via “one–pot” photo-initiated thiol-acrylate polymerization for retention-independent performance in capillary liquid chromatography

Haiyang Zhanga,b, Junjie Oua,*, Zhongshan Liua,c, Hongwei Wanga,c, Yinmao Weib,*, Hanfa Zoua,* a

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of

Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, 116023, China E-mail: [email protected], [email protected]; Fax: +86-411-84379620; Tel: +86-411-84379576, +86-411-84379610 b

Key Laboratory of Synthetic and Natural Function Molecule Chemistry of Ministry of

Education, College of Chemistry and Materials Science, Northwest University, Xi'an 710069, China E-mail: [email protected]; Fax: +86-29-81535026; Tel: +86-29-81535026 c

University of Chinese Academy of Sciences, Beijing 100049, China

1

ACS Paragon Plus Environment


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Abstract A novel “one–pot” approach was developed for ultra-rapid preparation of various hybrid monolithic columns in the UV-transparent fused-silica capillaries via photo-initiated thiol-acrylate polymerization of an acrylopropyl polyhedral oligomertic silsesquioxane (acryl-POSS) and a mono-thiol monomer (1-octadecanethiol or sodium 3-mercapto-1-propanesulfonate) within 5 minutes, in which the acrylate not only homopolymerizes, but also couples with the thiol. This unique combination of two types of free-radical reaction mechanisms offers a simple way to fabricate various acrylate-based hybrid monoliths. The physical characterization, including SEM, FT-IR and thermal gravimetric analysis was performed. The results indicated that the mono-thiol monomers were successfully incorporated into acryl-POSS based hybrid monoliths. The column efficiencies for alkylbenzenes on the C18-functionalized hybrid monolithic column reached to 60,000–73,500 plates per meter at the velocity of 0.33 mm/s in capillary liquid chromatography, which was far higher than that of previously reported POSS-based columns prepared via thermal-initiated free-radical polymerization without adding any thiol monomers. By plotting of plate height (H) of alkylbenzenes versus the linear velocity (u) of mobile phase, the results revealed a retention-independent efficient performance of small molecules in the isocratic elution. These results indicated that more homogeneous hybrid monoliths formed via photo-initiated thiol-acrylate polymerization, particularly, the use of multi-functional crosslinker possibly prevented the generation of gel-like micropores, reducing mass transfer resistance (C-term). Another sulfonate-containing hybrid monolithic column also exhibited hydrophobicity and ion-exchange mechanism, and the dynamic binding capacity was calculated as 71.1 ng/cm (75 µm i.d.).

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INTRODUCTION Macroporous organic monoliths emerged in 1990s possess a unique structure and exhibit many advantages, such as easy fabrication process, varieties of functionality, wide pH tolerance and good permeability1-3. Various organic monoliths including the polymethacrylates, polyacrylamides and polystyrenes have been successfully fabricated and applied for the separation science, especially for separation of large biomolecules4-10. Unfortunately, the swelling in organic solvents could lead to the change of pore structure and decrease of mechanical stability11,12. Additionally, compared to silica-based monoliths, organic monoliths usually exhibit low column efficiency for the separation of small molecules6,13,14. Great efforts have been made to improve the mechanical stability and separation efficiency of organic monoliths15-21. As a kind of organic–inorganic hybrid nanocomposite, polyhedral oligomeric silsesquixanes (POSS) developed at the end of last century are really nanostructural chemicals with sizes from 1 to 3 nm in diameter. It has been proved that the incorporation of POSS into polymeric networks can result in significant improvement in a variety of physical and mechanical properties due to the reinforcement at the molecular level and the inorganic framework’s ceramic-like properties22-25. Based on the excellent property of POSS, our group26-28 has adopted a multi-methacrylate POSS (POSS-MA) monomer to prepare several hybrid monolithic columns via thermal-initiated free radical polymerization. The preparation process was as simple as that of organic monoliths. As expected, these POSS-based hybrid monoliths exhibited better mechanical and pH stabilities by comparison with the common polymethacrylate monoliths. However, the relatively low column efficiencies (below 50,000 N/m) of those hybrid monoliths were obtained in capillary liquid chromatography (cLC)26. Fortunately, other POSS-based monoliths were successfully fabricated via epoxy-amine ring-opening polymerization29-32 and phosphine-catalyzed thiol-methacrylate click polymerization33. Surprisingly, the resulting monoliths exhibited the highest column efficiency of 195,000 N/m for butylbenzene in reversed-phase (RP) mode33. These results greatly inspired us to search for novel chemistry for fabrication of monoliths with satisfactory chromatographic efficiency. All above-mentioned approaches for preparation of POSS-based hybrid monoliths were 3



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performed at appropriate temperature, which would be finished in several hours. However, there are only a few reports on the preparation of POSS-based hybrid monoliths via photo-initiated polymerization, which could be almost completed in minutes rather than in hours and avoided considering the effect of polymerization temperature on monolith morphology. Alves and Nischang34 have synthesized hybrid porous materials by thermal/photo-initiated thiol-ene reaction of polyhedral oligomeric vinylsilsesquioxane

(vinylPOSS)

with

multi-thiol

monomers.

Although

these

POSS-based monoliths were also prepared in the capillaries, their column efficiencies in cLC were not reported. Herein, a simple “one–pot” approach was successfully developed for fast preparation of hybrid monoliths with various functionalization via photo-initiated thiol-acrylate polymerization reaction, in which the acrylate not only homopolymerizes, but also couples with the thiol. An acrylopropyl-POSS (cage mixture) (acryl-POSS) monomer and a

mono-thiol

compound,

such

as

1-octadecanethiol

(ODT)

or

sodium

3-mercapto-1-propanesulfonate, were selected as the precursors, respectively, and two kinds of hybrid monoliths were quickly fabricated within 5 min. The physical characterization, including SEM, FT-IR and thermal gravimetric analysis, and chromatographic evaluation of two hybrid monoliths were carried out. EXPERIMENTAL SECTION Chemicals and Materials. The monomer of acryl-POSS is hybrid molecule with an inorganic silsequioxane at the core and organic acrylopropyl groups attached at the corners of the cage, which was obtained from Hybrid Plastics, Inc (Hattiesburg, MS, USA). ODT (C18H37SH), sodium 3-mercapto-1-propanesulfonate, methacryloxypropyl trimethoxysilane (γ-MAPS) (≥98%), EPA610, 2,5-dihydroxyl benzoic acid (DHB) and polystyrenes (Mw = 800, 4,000, 13,200, 35,000, 50,000, 90,000, 280,000 and 900,000) were purchased from Sigma (St Louis, MO, USA), and used directly without further purification. 1-Butanol and ethylene glycol were from J&K Scientific Ltd. (Beijing, China). 2,2-Dimethoxy-2-phenylacetopheone (DMPA, 99%) was purchased from Acros Organics (New Jersey, USA). Lysozyme (chicken egg white), bovine serum albumin (BSA), myoglobin (horse heart), ovalbumin,

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α-casein and RNase A were obtained from Sigma-Aldrich (St Louis, Mo, USA). Cytochrome C (bovine heart) was obtained from Aladdin (Shanghai, China). Dithiothreitol (DTT) and iodoacetamide (IAA) were products of Sino-American Biotechnology Corporation (Beijing, China). Thiourea, benzene, toluene, ethylbenzene, propylbenzene, butylbenzene, phloroglucinol, pyrocatechol, para-cresol, 2,6-dimethyl phenol, 2,4-dichlorophenol, caffeine, carbamazepine, 2,4-dinitroaniline, 4-aminobiphenyl, 2,6-dchloro-4-nitroaniline and other standard analytes were of analytical grade, and obtained from Tianjin Kermel Chemical Plant (Tianjin, China). The flexible fused-silica capillary (UV transparent coating) with inner dimension of 75 µm was purchased from Polymicro Technologies (Phoenix, AZ, USA). HPLC-grade acetonitrile (ACN) was from Yuwang Group (Shandong, China) and used for preparation of mobile phases. The water used in all experiments was doubly distilled and purified by Milli-Q system (Millipore Inc., Milford, MA, USA). “One–pot” Preparation of C18-functionalized Hybrid Monolithic Column (Monolith I). Prior to preparation of hybrid monolith, the capillary was pretreated with γ-MAPS to immobilize a layer of methacrylate groups into its inner wall. In brief, the capillary was rinsed sequentially with 0.1 mol/L NaOH (2 h), H2O (1 h), 0.1 mol/L HCl (2 h), H2O (1 h) and methanol (1 h). The washed capillary was filled with γ-MAPS/methanol (50%, v/v) for 0.5 h, and then sealed with rubber septa at both ends and submerged in a water bath at 50 oC for 16 h. Finally, the capillary was rinsed with methanol to flush out the residual reagent and dried under nitrogen flow. A multi-acrylate monomer (acryl-POSS) and a monothiol (ODT) were selected as the precursors to prepare hybrid monolith I as shown in Scheme 1. The detailed composition of polymerization solutions was given in Table 1. In brief, a polymerization solution comprised of acryl-POSS (32.3 mg, 0.024 mmol), ODT (14 mg, 0.048 mmol), 1-butanol, ethylene glycol and DMPA was homogenized by sonication for 5 min, and then introduced into the pretreated capillary. After sealing both ends with rubbers, the capillary was irradiated by UV light (λ = 365 nm, 120 mJ/cm2) for 5 min. The resulting monolithic column was then flushed with methanol to remove residuals. The rest of polymerization solution in the vial was also cured under UV light to form bulk monolithic material, 5



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which was rinsed with ethanol three times, cut into small pieces, grinded using mortar and pestle, and then dried in a vacuum at 50 oC for 24 h. “Two–step” Preparation of C18-functionalized Hybrid Monolithic Column (Monoliths II and III). A “two–step” approach was also adopted to prepare C18-funtionalized hybrid monolith as shown in Scheme 1. First, the hybrid monolith II was fabricated in the capillary by using acryl-POSS as the sole precursor via photo-initiated free radical polymerization. Briefly, as listed in Table 1, a polymerization solution containing acryl-POSS (32.3 mg, 0.024 mmol), 1-butanol and ethylene glycol and DMPA was homogenized by sonication for 5 min, and then introduced into the γ-MAPS-pretreated capillary. After sealing both ends with rubbers, the capillary was irradiated by UV light (λ = 365 nm, 120 mJ/cm2) for 5 min. The resulting monolithic column was then flushed with methanol to remove residuals. To modify the above–mentioned monolith, an ethanol solution containing 5.0% (w/v) ODT and 0.3% (w/v) DMPA was flushed through the hybrid monolith. Then the reaction was performed by UV light irradiation for 5 min. Finally, the hybrid monolith III was obtained by washing with methanol to remove the residuals. “One–pot” Preparation of Sulfonate-containing Hybrid Monolithic Column (Monolith IV). Similarly, another sulfonate-containing hybrid monolithic column (assigned as monolith IV) was also prepared with “one–pot” process by using a mono-thiol compound of sodium 3-mercapto-1-propanesulfonate to replace ODT. Ten microliters water was added into the porogenic solvents (7.1%, v/v) to dissolve the thiol compound. Instruments and Methods. The polymerization was irradiated in UV crosslinkers (XL-1500A, λ=365 nm, Spectronics Corporation, New York, USA). Fourier-transformed infrared spectroscopy (FT-IR) characterization was carried out on Thermo Nicolet 380 spectrometer using KBr pellets (Nicolet, Wisconsin, USA). The microscopic morphology of monolithic materials was obtained by scanning electron microscopy (SEM, JEOL JSM-5600, Tokyo, Japan). Pore size distribution was measured by MIP on a PoreMaster GT-60 (Quantachrome Instrument Corporation, USA). The specific surface area was calculated from nitrogen adsorption/desorption measurements of dry bulk monoliths 6


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using a Quadrasorb SI surface area analyzer (Quantachrome, Boynton Beach, USA). Thermogravimetric (TG) data were collected on Pyris 1 TGA (Perkin Elmer, USA). The permeability was calculated according to Darcy’s law by the equation, B0 = FηL/(πr2∆P), where F (m3/s) is the flow rate of mobile phase, η is the viscosity of mobile phase (0.90×10-3 Pa s for ACN/H2O = 40/60, v/v), L and r (m) are effective length and inner diameter of the column, respectively, ∆P (Pa) is the pressure drop of column. The data of ∆P and F were obtained on an ACQUITY UltraPerformance LC (Waters, USA). The flow rates of mobile phase were set at 0.1-4.0 µL/min. The cLC experiments were performed on LC system coupled with an Agilent 1100 micropump, a 7725i injector with a 20 µL sample loop and a K-2501 UV detector (Knauer, Berlin, Germany). A T-union connector was used as a splitter, with one end connected to a blank capillary (150 cm × 50 µm i.d.) and the other connected to the monolithic column. The detection window was made by removing the polyimide coating of fused-silica capillary tube with 50 µm i.d. in a position 5.0 cm from the separation monolithic column outlet. The real flow rate through monolithic column was measured by using a blank capillary (10 cm × 75 µm i.d.) connected to the monolithic column. It was calculated according to the equation, µ = πr2L/t, where L and r (m) are the length and inner diameter of the blank capillary, respectively, and t is the time that mobile phase (through monolithic column) flowed through the blank capillary. All chromatographic data were collected and analyzed using the software program HW-2000 from Qianpu Software (Shanghai, China). Preparation of the Tryptic Digest of Four Proteins and cLC-MS/MS Analysis. The tryptic digestion of four proteins (BSA, myoglobin, ovalbumin and α-casein) and cLC-MS/MS analysis were performed according to procedures previously reported by us with minor modification35. The sample trapping was achieved with a homemade C18-particle-packed trap column (4.0 cm length × 200 µm i.d.), and the subsequent separation was carried out on a hybrid monolith (monolith I or III) with an integrated emitter, which was prepared by directly tapering the tip from the outlet of capillary. (Detailed experimental procedures are provided in the Supporting Information, SI.)

RESULTS AND DISCUSSION 7



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Preparation of C18-functionalized hybrid monolithic columns. The “one–pot” preparation of C18-functionalized hybrid monolith (monolith I) was carried out according to the process shown in Scheme 1, and the composition of prepolymerization mixtures was listed in Table 1. It is well known that the porogenic system plays a vitally important role in the formation of monolithic materials. We finally selected a mixture of 1-butanol and ethylene glycol as the porogenic system after a series of experiments. As shown in Table 1, the volume ratio of 1-butanol to ethylene glycol had effect on the permeabilities of two kinds of hybrid monoliths (I and II). It could be seen that the permeability of monolith I was increased from 1.3 to 2.2×10-14 m2 with an increase of ethylene glycol in the porogenic system, which served as the macroporogenic solvent (poor solvent) in porogenic system. Meanwhile, the permeability of monolith II was also increased from 1.2 to 1.5×10-14 m2 with the increase of the volume of ethylene glycol. By comparison of two monoliths I-2 and II-2 prepared with the same porogenic system, the total precursor content in the prepolymerization solution for monolith I-2 was reached to 33.1% (w/v), which was remarkably higher than that for monolith II-2 (23.1%, w/v), however, the permeability of monolith I-2 (1.8×10-14 m2) was higher than that of monolith II-2 (1.4×10-14 m2). These results indicated that the thiol monomer, ODT, causes the chain transfer, affecting the degree of polymerization and the pore size of hybrid monolith. As expected, the acryl-POSS containing several acrylate groups homopolymerized for the formation of monolith II. It is easily understood that homopolymerization of acrylate is inevitable to form monolith I in the “one–pot” process. The molar ratio of thiol to acrylate groups in the prepolymerization solution for monolith I was about 1/4, so the thiol-ene addition reaction also proceeds. It seems that “one–pot” process proceeds largely through the homopolymerization at early stages of the reaction followed by a more like thiol-ene coupling reaction at the end. As a matter of fact, thiol-acrylate precursors undergo rapid polymerization processes because the acrylate couples with the thiol (thiyl addition to the acrylate bond followed by the hydrogen abstraction of thiol hydrogen) and homopolymerizes (acrylate group propagation)36,37. As shown in Scheme S1 (SI), two types of reaction mechanisms would simultaneously occur when using DMPA as photoinitiator38. Although the rate constant for the hydrogen abstraction of the 8


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thiol group by the carbon-centered radical on the acrylate radical chain end to the reaction is lower than that for acrylate homopolymerization, the mono-thiol ODT could be simultaneously integrated into the POSS-based monolith via “one–pot” thiol-acrylate reaction. Two reaction mechanisms could be convincingly proved by the formation of monolith II and following post modification to prepare monolith III via “two–step” approach. It was observed that the white solid emerged in the vial after 30 s UV irradiation for preparation of bulk hybrid monolith II, demonstrating the phase separation took place. However, phase separation for preparation of bulk hybrid monolith I occurred only after 10 s UV irradiation. We have tried to add more ODT in the prepolymerization solution to fabricate hybrid monoliths, but it was difficult to fill the prepolymerization solution into capillary, possibly due to the occurrence of thiol-acrylate polymerization reaction in the room even without any UV irradiation. This phenomenon could be attributed to an important advantage of relative insensitivity to oxygen inhibition in thiol-ene photo-initiated free-radical polymerization36,39. As shown in Scheme S1 (SI), the peroxy radicals are formed by the reaction of carbon-centered propagation radicals with molecular oxygen, facilitating the hydrogen abstraction of a thiol hydrogen to form the thiyl radicals, and then the main propagation steps continue. As a result, the polymerization occurs in air almost as rapidly as in an inert atmosphere. The major obstacle of oxygen inhibition in traditional free radical polymerization is essentially eliminated in thiol-ene (acrylate/methacrylate) polymerization.

Characterization of C18-functionalized hybrid monoliths prepared with “one–pot” and “two–step” approaches. Figure 1 presents the FT-IR spectra of acryl-POSS monomer, monoliths I, II and III, respectively. It could be observed from Figure 1a that the peak signals at 1728 and 1636 cm−1 implied the presence of α,β-unsaturated carbonyl (C=O) and the stretching vibration of C=C bond in the acryl-POSS monomer, while peak signal at 1728 cm−1 was changed to 1736 cm−1, and the intensity of peak at 1636 cm−1 was remarkably decreased in Figure 1b. This result indicated that free radical polymerization of the acrylate groups in acryl-POSS was carried out in the formation of monolith II, meanwhile, part of unreacted 9



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acrylate groups still existed. The peak at 1636 cm−1 was slightly changed to 1634 cm−1, and its intensity was further decreased in Figure 1c, confirming the successful post-modification via thiol-acrylate coupling reaction between unreacted acrylate groups and ODT for the preparation of monolith III. It could be observed from Figure 1d that the peak at 1728 cm−1 was clearly changed to 1738 cm−1, and the peak signal at 1636 cm−1 was almost disappeared. These results forcefully indicated that both homopolymerization reaction and thiol-ene coupling reaction simultaneously occurred in the “one–pot” process. It could be found from the thermogravimetric analysis (Figure S1, SI) that no significant weight loss occured until the temperature reached to 350 oC, and the pyrolysis continued up to 650 oC for hybrid monoliths I, II and III, indicating better thermostability than those of hybrid monoliths reported previously. The residue for monolith I was only 36.19%, and lower than those for monoliths II (48.61%) and III (45.29%). This result also proved that more ODT was incorporated into the monolith I in the “one–pot” approach, while ODT could only react with acrylate sites located in the surface of monolith II in the “two–step” approach. SEM images showed that two kinds of C18-hybrid monolithic matrices were well attached to the inner walls of the γ-MAPS pretreated capillaries without any disconnection (Figure 2, S2 and S3, SI). Particularly, the monolith I contained much more macro through-pores around 1 µm, while the monolith II presented the cauliflower-like globular structure intertwined with typical micrometer-sized pores. Their specific surface areas were measured in the dry state and calculated as 178.9 (monolith I) and 38.2 (monolith II) m2 g-1 according to nitrogen adsorption/desorption isotherm, indicating that the mesopores possibly existed in monolith I, while lack of micropores and mesopores in monolith II. The size exclusion chromatography using THF as mobile phase also provided the porosity of two hybrid monoliths, as shown in Figure S4a and b (SI). The total porosity of monolith I was measured at 86.1%, while the total porosity of monolith II was only 62.3%, indicating better permeability for monolith I than that for monolith II. However, the pore size distribution for monolith I looked much wider than that for monolith II (Figure S4c and d, SI). The relationship between flow rate and back-pressure drop of hybrid monoliths I and II 10


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was also measured. As shown in Figure S5 (SI), the good linear relationship (R = 0.999) between back-pressure and flow rate was obtained. Two monoliths could undergo a high pressure over 45.0 MPa without any deterioration on the framework, indicating satisfactory mechanical strength. In addition, the permeabilities were calculated at 1.8 and 1.4 × 10-14 m2 for the columns I-2 and II-2 (Table 1), respectively, indicating good permeability. As a result, columns I-2 and II-2 were selected for following cLC evaluation, and the C18-modified column II-2 (assigned as monolith III-2) was also measured in cLC. In order to investigate the pH stability, the monolith I-2 was flushed by 60% ACN with different pH values for more than 100 h. The retention factor (k) was then monitored by cLC, and the result was shown in Figure S6 (SI). All the retention factor remained above 90% after flushing by the solution with different pH values (pH = 1, 2 and 10), indicating that the monolith had good pH stability. However, the monolithic matrice was flushed out from the capillary after flushing by the solution at pH 11 for 60 h. It may be due to the fact that the linkage between the matrice and capillary wall, the Si-O-Si-C bonds formed via γ-MAPS and silanol, was destroyed under such strong basic ambiance26. In addition, significant decrease of the retention factor and column efficiency of the monolith I-2 were not observed after keeping both end of the column in water for 3 months, which could remain 86% and 85%, respectively. This result indicated that the hybrid monolith had high stability. The repeatability and reproducibility of monolith I-2 were also characterized by measuring the relative standard deviations (RSDs) of the retention factor of toluene as test compound (thiourea as void time marker) under the mobile phase of 60% ACN. The RSDs of run-to-run, column-to-column and batch-to-batch were less than 2.5%, 2.7% and 3.3% (n=5), respectively. The results demonstrated that the repeatability and reproducibility of hybrid monolith were acceptable.

Chromatographic evaluation and application of two C18-functionalized hybrid monolithic columns prepared with “one–pot” and “two–step” approaches. The chromatographic assessment of three monolithic columns was carried out in reversed-phase

liquid

chromatography

(RPLC).

Five

alkylbenzenes

were

baseline-separated on monolith I with the mobile phase of ACN/H2O (60/40, v/v) within 11



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8 min, and the peaks were all symmetric (Figure 3a). The highest column efficiencies for 5 alkylbenzenes reached to 60,000–73,500 plates per meter (corresponding to 13.6–16.5 µm of plate height) at the velocity of 0.33 mm/s (Figure 3b). As shown in Figure 3c and S7 (SI), 5 alkylbenzenes were also baseline-separated on monoliths II and III under the same chromatographic conditions, indicating a typical RP retention mechanism in monolith II due to the hydrophobicity of acryl-POSS. Particularly, the highest efficiencies for 5 alkylbenzenes on monolith II reached to 84,600–102,700 plates per meter (corresponding to 9.7–11.8 µm of plate height) at the velocity of 0.84 mm/s (Figure 3d). The column efficiencies on two acryl-POSS-based columns prepared via photo-initiated thiol-acrylate polymerization with both “one–pot” and “two–step” modes were far higher than

those

of

previously

reported

POSS-MA-based

columns

prepared

via

thermal-initiated free radical polymerization26. The difference in column efficiency of monoliths I and II was mainly attributed to their distinction in morphology and pore-size distribution, which was vividly reflected in the van Deemter curves of alkylbenzenes on monoliths I and II (Table 2). The values of eddy diffusion term (A-term) for monolith I ranging from 3.77 to 6.48 were slightly smaller than those for monolith II ranging from 5.15 to 7.62, while the values of mass transfer resistance (C-term) for monolith I in the range of 13.1-15.9 were remarkably higher than those for monolith II in the range of 2.52-5.89. It is well known that A-term is the main contributor of the peak broadening, especially in the separation of small molecules. Herein, the relatively small values of A-term were much smaller than those of traditional organic monoliths40 and silica-based monoliths41 in LC, implying the limitation of eddy diffusion. Furthermore, the values of C-term were also lower (≤ 20.0 ms) than those of methacrylate-based monoliths42,43. Compared to monolith I, the smaller values of C-term for monolith II also indicated a better communication between the stationary phase and analytes (the mass transfer was faster as the molecules were transported almost solely by convection instead of diffusion). As a result, due to the combination of two effects, higher column efficiencies were achieved on monolith II in cLC separation of small molecules. It is deduced that the morphology and pore-size distribution properties of photo-initiated polyacrylate networks would be altered by the addition of thiol-containing monomers in the “one–pot” mode. 12


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As shown in Figure 3, it is also observed that the efficiency for the strong-retained compounds (such as butylbenzene) was slightly higher than those of weak-retained compounds (such as benzene). This result demonstrated that two hybrid monoliths revealed a retention-independent performance of small molecules in the isocratic mode. As shown in Table 2, it could be seen that the C-terms of butylbenzene were lower than those of benzene on two hybrid monoliths. This low mass transfer resistance is possibly attributed to the lack of micropores (the low specific surface areas indicated the lack of micropores in the dry state). It can be deduced that the employ of multi-functional crosslinker (acryl-POSS) prevents the hybrid monoliths from generation of gel-like micropores via free radical polymerization, which reduced the permeation of small molecules in the gel-like structure and the mass transfer resistance, and finally improved the efficiency of the strong-retained compounds 44,45. By comparison of the retention factors of alkylbenzenes on the hybrid monoliths II and III (Figure 3c and S7, SI), the latter exhibited more hydrophobic than the former, further indicating that the ODT was successfully post-modified on the surface of monolith II. Figure S8 (SI) presents the effect of ACN content in mobile phases on the retention factors of alkylbenzenes on three POSS-based monolithic columns, indicating the RP separation mechanism. The plots of logarithmic retention factors of alkylbenzenes versus the number of saturated carbon atoms in the alkyl chain of alkylbenzenes were all linear, and the methylene selectivities of three monolithic columns could be calculated, as given in Table S1 (SI). It is found that the hybrid monolith I exhibited the strongest hydrophobicity among three monoliths, whose methylene selectivity was determined to be 1.63 and 1.55 at 50% and 55% ACN in mobile phases, respectively. Although the post-modification could increase the hydrophobicity of monolith II, the hydrophobicity of monolith III, whose methylene selectivity was calculated to be 1.46 and 1.39 at 50% and 55% ACN in mobile phases, respectively, was still lower than that of monolith I. These results further proved that more ODT could be incorporated into the monolith I in the “one–pot” approach, while ODT could only react with acrylate groups located on the surface of monolith II during the “two–step” process. Due to better permeability and stronger hydrophobicity of the C18-functionalied hybrid monolith I prepared with “one–pot” process, it would be a good RP separation 13



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material. The separation of small molecules such as basic and phenolic compounds was performed, and satisfactory results were obtained (Figure S9, SI). Additionally, EPA610 and a mixture of proteins were also separated on hybrid monolith I with gradient mode in cLC (Figure S10, SI). Additionally, the separation of highly complicated samples was also performed on two hybrid monoliths. As illustrated in Figure 4, the tryptic digest of four proteins (BSA, myoglobin, ovalbumin and α-casein) was also used to evaluate the separation ability of hybrid monoliths. On the basis of the database search of the obtained chromatograms of tryptic digest, 91 unique peptides were positively identified with high protein sequence coverages (BSA 75%, myoglobin 68%, ovalbumin 65% and α-casein 39%) on the hybrid monolith I, while 80 unique peptides were identified with relative low protein sequence coverages (BSA 68%, myoglobin 63%, ovalbumin 61% and α-casein 39%) on hybrid monolith III. The former was also higher than that identified on previous POSS-based monolith prepared with thermal-initiated free radical polymerization26. All results demonstrated the potential of the hybrid monoliths in analysis of complicated environmental and biological samples. Preparation and characterization of sulfonate-containing hybrid monolithic column via “one–pot” approach For further demonstration of the success of this “one–pot” approach in the ultrafast preparation of POSS-based hybrid monolithic column via photo-initiated thiol-acrylate polymerization reaction, another sulfonate-containing hybrid monolith was also prepared by using sodium 3-mercapto-1-propanesulfonate to replace ODT. A ternary porogenic system (1-butanol/ethylene glycol/water) was adopted to dissolve the thiol monomer. After optimizing the preparation conditions, a sulfonate-containing hybrid monolith (monolith IV) was also fabricated within 5 min via “one–pot” approach. As shown in Figure S11 (SI), the hybrid monolith IV was well attached to the inner wall of the γ-MAPS pretreated capillary without any disconnection, and through-pores around 1 µm could be observed. Meanwhile, the eluent flushed out of the monolith IV was collected into a centrifuge tube and analyzed by a MALDI-TOF mass spectrometer. The oligomers with molecular weight ranging from 1350 to 2700 Da were detected. The repeating units with mass difference of 178 Da were observed (Figure S12, SI). This result illustrated that not all monomers could participate in the formation of monolith. 14


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In order to prove the incorporation of sulfonate group in the hybrid monolith IV, a breakthrough curve was measured. A solution of dipeptide Gly-Tyr (238.2 Da) (0.5 mg/mL), which bears a 2-charge at pH 2.3, was used to determine the dynamic binding capacity of monoliths IV and II-2 (13 cm × 75 µm i.d.). As shown in Figure 5c, the dynamic binding capacity of the monolith II-2 was calculated as 22.9 ng/cm. Meanwhile a sharp increase in the baseline could be observed when the column was saturated (Figure 5b), and the dynamic binding capacity of monolith IV was calculated as 71.1 ng/cm, which was about 3 times as many as that of monolith II-2. These results indicated that the sulfonate group was integrated into the hybrid monolith IV, which exhibited ion-exchange mechanism. As shown in Figure S13 (SI), 5 alkylbenzenes could be baseline-separated on monolith IV by using the mobile phase of ACN/H2O (50/50, v/v). It is due to the hydrophobicity of POSS unit in hybrid monolith IV, indicating a typical RP retention mechanism. However, the methylene selectivity of monolith IV was determined to be 1.37 and 1.32 at 50% and 55% ACN in the mobile phase, respectively (Table S1, SI), and lower than those of monolith II, whose methylene selectivity was 1.39 and 1.34 at 50% and 55% ACN in mobile phases, respectively. These results further demonstrated the successful incorporation of hydrophilic thiol of 3-mercapto-1-propanesulfonate into acryl-POSS-based monolith via “one–pot” approach, and this monolith actually exhibited a reversed-phase and ion-exchange mixed-mode retention mechanism.

CONCLUSION We have developed a novel “one–pot” approach for rapid preparation of various POSS-containing monolithic columns in a UV-transparent fused-silica capillary via photo-initiated thiol-acrylate polymerization. The resulting hybrid monoliths exhibited good permeability and separation ability for small molecules and proteins in RPLC. In the “one–pot” process, the acrylate monomer not only homopolymerizes, but also couples with the thiol-containing monomer, indicating several advantages over the “two–step” approach. First, reducing the procedures would facilitate to enhance preparation reproducibility of monolithic columns. Second, the polymerization rate in “one–pot” approach was much higher than that of the formation of polyacrylate monoliths in traditional photo-initiated free radical polymerization. It is attributed to the addition of 15



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Page 16 of 27

thiol into the “one–pot” thiol-acrylate polymerization, and the major obstacle of oxygen inhibition

is

essentially

eliminated

in

the

thiol-ene

(acrylate/methacrylate)

polymerization. As a result, ultra-rapid photo-initiated polymerization is almost completed in minutes rather than in hours, and avoids the tedious works to optimize the preparation conditions, particularly, it is not necessary to consider the effect of temperature on the morphology of monoliths compared with thermal-initiated polymerization. In a word, this unique combination of two types of free-radical polymerization mechanisms offers a novel way to directly fabricate various acrylate-based monolithic materials. It is anticipated that thiol-ene (acrylate/methacrylate) polymerization will be further applied as a useful tool for widespread applications in the area of material science, especially for the synthesis of novel stationary phases in chromatographic research.

AUTHOR INFORMATION Corresponding Author *E-mail:

[email protected]

(J.J.

Ou);

[email protected]

(H.F.

Zou);

[email protected] (Y.M. Wei)

ACKNOWLEDGEMENTS Financial support is gratefully acknowledged from the China State Key Basic Research Program Grant (2013CB-911203, 2012CB910601), the National Natural Sciences Foundation of China (21235006), the Creative Research Group Project of NSFC (21321064), and the Knowledge Innovation program of DICP to H. Zou as well as the National Natural Sciences Foundation of China (No. 21175133) to J. Ou.

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

References 16


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17



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Figure captions Scheme 1. “one–pot” and “two–step” approaches for preparation of C18-functionalized hybrid monolithic columns via photo-initiated thiol-acrylate polymerization. Figure 1. FT-IR spectra of (a) acryl-POSS monomer, (b) monolith II, (c) monolith III and (d) monolith I. Figure 2. SEM images of hybrid monoliths (a, b) I-2 and (c, d) II-2. Magnification: (a, c) 5000×; (b, d) 10,000×. Figure 3. (a, c) Separation of alkylbenzenes and (b, d) dependence of the plate height of alkylbenzenes on the linear velocity of mobile phase on hybrid monoliths (a, b) I-2 and (c, d) II-2 in cLC, respectively. The analytes: (1) thiourea, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene and (6) butylbenzene. Experimental conditions: column dimensions, (a, b) 21 cm × 75 µm i.d., (c, d) 22 cm × 75 µm i.d.; mobile phase, ACN/H2O (60/40, v/v); flow rates for (a), 170 µL/min (before split), 64 nL/min (after split), for (c), 170 µL/min (before split), 41 nL/min (after split); detection wavelength, 214 nm. Figure 4. Chromatograms of tryptic digest of four proteins (BSA, myoglobin, ovalbumin and α-casein) on hybrid monoliths I-2 (a) and III-2 (b) in cLC-MS/MS analysis. Experimental conditions: column dimensions, (a) 43 cm × 100 µm i.d., (b) 32 cm × 100 µm i.d.; mobile phase A, 0.1% formic acid in water, mobile phase B, 0.1% formic acid in ACN; the gradient 5-35% B in 85 min, 35-90% in 3 min, retained 90% B for 10 min; flow rate, 60 µL/min. Protein sequence coverage: (a) BSA (75%), myoglobin (68%), ovalbumin (65%), α-casein (39%); (b) BSA (68%), myoglobin (63%), ovalbumin (61%), α-casein (39%). Figure 5. Dynamic adsorption curves of frontal analysis on (a) void capillary, (b) monolith IV and (c) monolith II. Experimental conditions: column dimension, 13 cm × 75 µm i.d.; mobile phase, ACN/H2O (10/90, v/v) solution containing Gly-Tyr (0.5 mg/mL); flow rate, 40 µL/min (before split) (15 nL/min for monolith IV and 25 nL/min for monolith II, after spilt), detection wavelength, 214 nm.

18


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R O O Si R O Si R= O R Si O Si O O RO (C6H9O2)n(SiO1.5)n R O Si O O Si O R n=8,10,12 Si O Si O Acryl-POSS R R

"one-pot"

"two-step" Monolith II

hv

CHCH2 m

o

C18H37SH

o R

hv

R

Monolith I

hv C18H37SH

Monolith III o CHCH2 m o o SC18H37 o o SC18H37 o

Scheme 1.

19



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Figure 1.

20


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Figure 2.

21



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Figure 3.

22


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Figure 4.

23



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Figure 5.

24


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Table 1. Detailed composition of prepolymerization mixtures and the permeability of hybrid monoliths. Hybrid monolitha I-1 I-2 I-3 II-1 II-2 II-3 a

ODT (mg) 14 14 14 0 0 0

Porogenic solvents (µL/µL) b 115/25 112/28 110/30 115/25 112/28 110/30

Total monomer content (w/v, %) 33.1 33.1 33.1 23.1 23.1 23.1

Permeability (10-14 m2) 1.3 1.8 2.2 1.2 1.4 1.5

Specific surface area (m2/g) — 178.9 — — 38.2 —

The prepolymerization solution also contained the monomer of 32.3 mg acryl-POSS and the

photoinitiator of 1.5 µL 10% DMPA (w/v, DMPA/1-butanol). The capillary was irradiated in UV light (wavelength 365 nm, 120 mJ/cm2) for 5 min. b The porogenic solvents were the mixture of 1-butanol and ethylene glycol (v/v).

25



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Page 26 of 27

Table 2. Van Deemter coefficients measured with alkylbenzenes for monoliths I-2 and II-2 in cLC. Analytes benzene toluene ethylbenzene propylbenzene butylbenzene

A/µm 4.49 5.09 6.48 3.77 4.81

I-2 B/(µm2 s-1) 1870 1860 1660 2040 1930

C/ms 15.9 15.0 13.1 14.7 13.5

A/µm 5.29 5.15 6.67 6.40 7.62

II-2 B/(µm2 s-1) 1980 1850 1310 1350 1120

26


C/ms 5.89 5.77 3.81 3.58 2.52

Page 27 of 27

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for TOC only

"one-pot" R O Si R O Si R Si O Si O O RO R O O O Si O Si R Si O Si O R R O R= O n=8,10,12 Acryl-POSS

(Monolith I)

C18H37SH hv

o CHCH2 m o

Thiol-acrylate polymerization CHCH2 m

o

hv

hv

o R

C18H37SH

(Monolith III)

R

(Monolith II)

"two-step"

27


o SC18H37 o o SC18H37 o

Preparation of Hybrid Monolithic Columns via âOne-Potâ Photoinitiated

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