Journal of Chromatography A, 1342 (2014) 70–77
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Preparation of polyhedral oligomeric silsesquioxane-based hybrid monolith by ring-opening polymerization and post-functionalization via thiol-ene click reaction夽 Zhongshan Liu a,b , Junjie Ou a,∗ , Hui Lin a,b , Hongwei Wang a , Jing Dong a , Hanfa Zou a,∗ a b
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Graduate School of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 6 January 2014 Received in revised form 17 March 2014 Accepted 20 March 2014 Available online 28 March 2014 Keywords: Hybrid monolith Thiol-ene reaction Disulfide bond Ring-opening polymerization Polyhedral oligomeric silsesquioxane
a b s t r a c t A polyhedral oligomeric silsesquioxane (POSS) hybrid monolith was simply prepared by using octaglycidyldimethylsilyl POSS (POSS-epoxy) and cystamine dihydrochloride as monomers via ring-opening polymerization. The effects of composition of prepolymerization solution and polycondensation temperature on the morphology and permeability of monolithic column were investigated in detail. The obtained POSS hybrid monolithic column showed 3D skeleton morphology and exhibited high column efficiency of ∼71,000 plates per meter in reversed-phase mechanism. Owing to this POSS hybrid monolith essentially possessing a great number of disulfide bonds, the monolith surface would expose thiol groups after reduction with dithiothreitol (DTT), which supplied active sites to functionalize with various alkene monomers via thiol-ene click reaction. The results indicated that the reduction with DTT could not destroy the 3D skeleton of hybrid monolith. Both stearyl methylacrylate (SMA) and benzyl methacrylate (BMA) were selected to functionalize the hybrid monolithic columns for reversed-phase liquid chromatography (RPLC), while [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (MSA) was used to modify the hybrid monolithic column in hydrophilic interaction chromatography (HILIC). These modified hybrid monolithic columns could be successfully applied for separation of small molecules with high efficiency. It is demonstrated that thiol-ene click reaction supplies a facile way to introduce various functional groups to the hybrid monolith possessing thiol groups. Furthermore, due to good permeability of the resulting hybrid monoliths, we also prepared long hybrid monolithic columns in narrow-bore capillaries. The highest column efficiency reached to ∼70,000 plates using a 1-m-long column of 75 m i.d. with a peak capacity of 147 for isocratic chromatography, indicating potential application in separation and analysis of complex biosamples. © 2014 Published by Elsevier B.V.
1. Introduction Up till now, monolithic column, as called continuous bed, has achieved much progress in preparation, characterization and application in separation of small molecules and biomacromolecules [1–14]. Generally, the monolithic columns are classified into organic polymer-based [15–17], inorganic silica-based [18] and
夽 Presented at the 40th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2013 Hobart), Hobart, Tasmania, Australia, 18–21 November 2013. ∗ Corresponding author at: Chinese Academy of Sciences, Dalian Institute of Chemical Physics, Key Laboratory of Separation Science for Analytical Chemistry, Dalian 116023, China. Tel.: +86 411 84379576/+86 411 84379610; fax: +86 411 84379620. E-mail addresses: [email protected] (J. Ou), [email protected] (H. Zou). http://dx.doi.org/10.1016/j.chroma.2014.03.058 0021-9673/© 2014 Published by Elsevier B.V.
hybrid organic–inorganic [6] monolithic columns. Polymer-based monolithic columns are prepared by in situ polymerization using organic monomers and crosslinkers in the presence of porogenic solvents to form organic polymers, such as polymethacrylates, polyacrylamides and polystyrenes [16]. Silica-based monolithic columns are commonly fabricated via sol–gel technique following a chemical modification on surface of matrix with silylation reagents. Compared with the former two, the hybrid monolith may somewhat combine the advantages of organic polymer-based and silica-based monoliths, such as mechanical stability, low shrinkage and controlling porous structure easily. Therefore, the hybrid monolith, mainly organic-silica monolith, has attracted more and more attentions. Since Hayes and Malik [3] incorporated organic functional moieties into inorganic silica monolithic matrices via sol–gel chemistry, demonstrating good separation efficiency in capillary electrochromatography (CEC), various types
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of organic-silica based monoliths have been reported by changing the siloxanes with different organic moieties, such as allyl, propyl, aminopropyl, vinyl, etc. [6,19–21]. However, the limited types of organic-trialkoxysilanes restricted the development of hybrid monolithic columns. To overcome this limitation, we have developed the “one-pot” approach for incorporation of organic moieties into the silica monolithic matrix in our previous works [7,22]. To some extent, it turns out that the “one-pot” approach is a facile method to prepare various functionalized hybrid monolithic columns. In our recent works, we have introduced polyhedral oligomeric silsesquioxanes (POSS) monomer, which can be regarded as the smallest possible particles of silica with sizes of from 1 to 3 nm in diameter, into monolithic column matrix via free radical polymerization [10,23–25]. This method has some merits comparing with the sol–gel method, such as without hydrolysis, condensation reactions of siloxane and good pH stability over a wide pH range. Furthermore, different from free radical polymerization, we selected octaglycidyldimethylsilyl POSS (POSS-epoxy) and diamines as monomers to prepare a series of hybrid monoliths with well-controlled 3D skeletons via ring-opening polymerization, gaining excellent chromatographic performance [11,26,27]. Herein, we prepared hybrid monolithic column via ring-opening polymerization using POSS-epoxy and cystamine dihydrochloride (Cys·2HCl) as monomers. Due to the introduction of cystamine, the skeletal surface of monolithic column possesses a lot of disulfide bonds. After simple reduction with dithiothreitol (DTT), thiol groups were generated as the active sites and subsequently reacted with methylacrylate monomers via thiol-ene click chemistry, which is famous for its merits of high selectivity and high conversion under a variety of mild conditions [28–32]. The resulting hybrid monoliths were successfully applied for capillary liquid chromatography (cLC). 2. Materials and methods 2.1. Chemicals and reagents (3-Aminopropyl)triethoxysilane (APTES), POSS-epoxy, Cys·2HCl, [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)ammonium hydroxide (MSA) and stearyl methylacrylate (SMA) were purchased from Aldrich (Milwaukee, WI, USA). DTT, cetyltrimethyl ammonium bromide (CTAB), benzyl methacrylate (BMA) and EPA610 were purchased from Sigma Chemical Co. (St Louis, Mo, USA). Dimethylphenylphosphine (DMPP) was obtained from J&K Scientific Ltd. (Beijing, China). The fused-silica capillaries with dimension of 50, 75 and 100 m i.d. were obtained from the Refine Chromatography Ltd. (Yongnian, Hebei, China). Benzene and butylbenzene were purchased from Beijing Chemical Works (Beijing, China). Ammonium bicarbonate, sodium hydroxide (NaOH), ethylbenzene, dimethyl formamide (DMF), hydroquinone and other standard compounds were obtained from Tianjin Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). HPLC-grade acetonitrile (ACN) was used for mobile phase and obtained from Yuwang Group (Shandong, China). The water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore Inc., Milford, MA, USA). Other chemical reagents were all of analytical grade. 2.2. Preparation of POSS-based hybrid monoliths Prior to use, the inner wall of fused-silica capillary was pretreated and modified with a layer of amino groups for anchoring monolith matrix according to the method described by Lin et al. [11]. Briefly, the capillary was rinsed by 1.0 mol/L NaOH, water,
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1.0 mol/L HCl and water, successively, which was later dried by nitrogen stream at room temperature. Then, the capillary was filled with APTES solution in methanol (50%, v/v), sealed with rubbers at both ends and submerged in water bath at 50 ◦ C for 12 h. Finally, the capillary was rinsed with methanol to flush out the residual reagent and dried under nitrogen flow. For preparation of POSS-based hybrid monolithic capillary column, the prepolymerization mixture with different composition as listed in Table 1 was introduced into the above-mentioned pretreated capillaries with a syringe. After sealing both ends with rubbers, the capillary was immersed in a water bath at different temperature for 12 h. The obtained POSS-based hybrid monolith column was then flushed with methanol/H2 O (60/40, v/v) to remove residuals. For preparing of bulk hybrid monoliths, to a centrifuge tube the prepolymerization mixture was added and reacted at 50 ◦ C for 12 h. Then the bulk hybrid monolith was cut into smaller pieces and extracted with methanol/H2 O (60/40, v/v) in a Soxhlet apparatus and dried in a vacuum. For the following reduction of disulfide bonds and modification, the monolith was immersed in 0.2 mol/L DTT in 0.1 mol/L aqueous ammonium bicarbonate for 2 h, and then rinsed with methanol/H2 O (60/40, v/v). The monomer solutions (SMA, BMA or MSA, the precise proportion as seen below) were added to the above monolith for modification. The bulk hybrid monoliths were rinsed with methanol and dried for measuring in FT-IR. 2.3. Modification of hybrid columns via reduction of disulfide bonds and thiol-ene click reaction (Fig. 1) A 0.2 mol/L DTT in 0.1 mol/L aqueous ammonium bicarbonate was flushed through POSS-based hybrid monolith column by nitrogen pressure with 4 MPa for 2 h, and then rinsed with methanol/H2 O (60/40, v/v). Then the monomer solution of SMA/DMPP/ethanol (10/1/100, v/v/v) was flushed through hybrid monolith under nitrogen pressure for 2 h, and rinsed with methanol for chromatographic experiments. Similarly, the hybrid columns were also functionalized with BMA and MSA according to aforementioned procedures. The precise proportions of monomer solution were BMA/DMPP/ethanol = 10/1/100 (v/v/v) for BMA modification and MSA/DMPP/ethanol/H2 O = 20/2/400/100 (w/v/v/v) for MSA modification, respectively. 2.4. Instruments and methods The microscopic morphology of hybrid monoliths was obtained by scanning electron microscopy (SEM) (JEOL JSM-5600, Tokyo, Japan). Fourier-transformed infrared spectroscopy (FT-IR) characterization was carried out on Thermo Nicolet 380 spectrometer using KBr pellets (Nicolet, Wisconsin, USA). The cLC experiments were performed on LC system equipped with an Agilent 1100 micropump, a K-2501 UV detector (Knauer, Berlin, Germany) and a 7725i injector with a 20 L sample loop. A T-union connector was used as a splitter, with one end connected to a blank capillary (200 cm × 50 m i.d.) and the other connected to the monolithic column. The detection window was made by removing the polyimide coating of fused-silica capillary tubing. All chromatographic data were collected and evaluated using the software program HW-2000 from Qianpu Software (Shanghai, China). For illustrating the effects of all parameters more intuitively, permeability was calculated according to Darcy’s law [33] by the following, B0 = FL/(r2 P), where F (m3 /s) is the flow rate of mobile phase, is the viscosity of water (1.0 × 10−3 Pa s), L and r (m) are effective length and inner radius of the column, P (Pa) is the pressure drop of column. The data of P and F was measured
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Table 1 Detail composition of prepolymerization mixture for preparing POSS-based hybrid monolithic columns. Column
A B C D E #
POSS-epoxy (mg)
50 50 50 50 50
CTAB (mg)
14 16 18 16 16
Cys·2HCl/NaOH/H2 O (L)#
130 130 130 125 135
Ethanol (L)
125 125 125 125 125
Permeability/10−14 m2 45 ◦ C
50 ◦ C
55 ◦ C
13.7 12.2 0.001 0.15 142
3.1 3.0 – 0.07 55
2.4 2.0 – – 12
Cys·2HCl/NaOH/H2 O: 1080/383/7200, mg/mg/L. (Cys·2HCl, 12.47%; NaOH, 4.42%, wt%)
on an Eksigent one dimensional Plus Nano-HPLC system (Eksigent, Dublin). The mobile phase was water, and flow rate was set at 200–1500 nL/min.
3. Results and discussion 3.1. Preparation of POSS-based hybrid monolithic column The preparation of POSS hybrid monolith is outlined in Fig. 1. As expected, the morphology and permeability of hybrid monoliths were significantly affected by composition of prepolymerization solution and polycondensation temperature, which were investigated in detail. The mixture of propanol/1,4-butanediol and PEG10,000 was initially selected as porogenic solvents following the approach in our previous works [11,26,27]. However, it was found that the commercial reagent of Cys·2HCl could not react with POSS-epoxy because of low reaction activity and insolubility of Cys·2HCl in this porogenic system. We have attempted using anhydrous triethylamine and sodium hydroxide to generate free cystamine, and finally chose sodium hydroxide solution to neutralize hydrogen chloride in Cys·2HCl and form Cys·2HCl/NaOH/H2 O solution (Cys·2HCl/NaOH/H2 O, 1080/383/7200, mg/mg/L), and the ring-opening polymerization between the free cystamine (Cys) and POSS-epoxy could be carried out in the ethanolCTAB-Cys·2HCl/NaOH/H2 O solution. However, the content of
Cys·2HCl/NaOH/H2 O solution has a great influence on the morphology and permeability. A little change of Cys·2HCl/NaOH/H2 O content could result in a transformation of monoliths from dense to loose (Fig. 2d, b, e), which was also confirmed by column permeability changing from 1.5 × 10−15 to 1.42 × 10−12 m2 with increase content of Cys·2HCl/NaOH/H2 O solution at 45 ◦ C. Therefore, the Cys·2HCl/NaOH/H2 O solution was selected at 130 L for the following experiments. Owing to the hydrophobic property of POSS-epoxy, CTAB was needed to form a homogenous mixture. As discussed above, water was a poor solvent that led to earlier phase separation, supervening with large pores in monolithic material (Fig. 2e). CTAB may form micelles and enhance the uniformity and stability of prepolymerization mixture due to the interaction between water molecules and hydrophilic heads of CTAB. As a result, the phase separation may be postponed, and the average pore diameter of monolith is decreased with an increase of CTAB content. As shown in Table 1 (Columns A–C), increasing CTAB content from 14 to 18 mg, the permeability of monolithic columns was remarkably decreased from 1.37 × 10−13 to 1.0 × 10−17 m2 , which hardly allowed the mobile phase to pass through (Table 1, Column C). The SEM micrographs also verified that the pore size became smaller, especially when CTAB content was up to 18 mg in Fig. 2c. What’s worse, high CTAB content would lead to monolith matrix detaching from the inner wall of fused-silica capillary. This phenomenon was also observed as raising the content of ethanol in
Fig. 1. Preparation of POSS-based hybrid monolith via ring-opening polymerization and post-functionalization based on thiol-ene click reaction.
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Fig. 2. SEM micrographs of POSS-based hybrid monolithic columns with (a) Column A, (b) Column B, (c) Column C, (d) Column D and (e) Column E prepared at 45 ◦ C, (f) Column E prepared at 50 ◦ C, (g) Column E prepared at 55 ◦ C. The composition of prepolymerization mixture as shown in Table 1.
prepolymerization mixture (Fig. S1). Therefore, the CTAB content of 16 mg was controlled. Another important effect on formation of monolithic column was polycondensation temperature. In fact, prepolymerization mixture would transform to opaque monolithic materials in 2 h at temperatures above 45 ◦ C, which implied ring-opening reaction basically finished. As indicated in Table 1, the permeability was decreased with an increase of polycondensation temperature for all prepolymerization mixtures with different ratios. For example, as for column A, the permeability was decreased from 1.37 × 10−13 to 2.4 × 10−14 m2 as polycondensation temperature increasing from 45 to 50 ◦ C. Additionally, they all exhibited more uniform and smaller pore size at high temperature (Fig. 2e–g). This trend may result from high temperature accelerating the ringopening polymerization, which lead to little diffusion of monomers
and phase separation in situ rapidly. Concretely, low polycondensation temperature made the column good permeability but low column efficiency, while too high temperature resulted in the monolith hard to flush through and lowered the reproducibility. As for Column B (Table 1) being prepared at 55 ◦ C, the values of reproducibility were 9.4% (column-to-column) and 12.1% (batch-to-batch), respectively. So we finally controlled the polycondensation temperature at 50 ◦ C, which also significantly improved column reproducibility. The reproducibility of hybrid monolith was evaluated through the relative standard deviation (RSD) for the retention factor (k) of toluene as model analyte (thiourea as the void time marker). The run-to-run (n = 4), column-to-column (n = 4) and batch-to-batch (n = 4) were 0.17%, 0.33% and 0.50%, respectively. All results above indicated the good reproducibility of POSS-based hybrid monolith.
Fig. 3. The retention factor (k) of toluene on unmodified and SMA-modified hybrid monolithic columns with different DTT reduction time.
Fig. 4. FT-IR spectra of (a) unmodified monolith, (b) SMA-modified monolith and (c) MSA-modified monolith.
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Fig. 5. (a) and (b): separation of alkylbenzenes on the hybrid monolith by cLC. Analytes: (1) thiourea, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene and (6) butylbenzene. (c) and (d): Dependence of the plate height of analytes on the linear velocity of mobile phase by the hybrid monolith capillary column. (e) and (f): The effect of ACN content in mobile phase on retention factor of alkylbenzenes. Experimental conditions: effective length of 40.7 cm × 100 m i.d.; off column detection, 6 cm × 50 m i.d.; mobile phase, ACN/H2 O (55/45, v/v, for a, b, c, d); flow rate, 160 L/min (before split, for a, b, e, f); detection wavelength, 214 nm. (Note: a, c, e for unmodified and b, d, f for SMA-modified monolithic columns, respectively.)
3.2. Modification and characterization of hybrid monoliths In recent reports, Svec et al. has prepared polymeric monolith using glycidyl methacrylate and ethylene dimethacrylate, to which thiol groups were introduced via post-modification and disulfide bond reduction of cystamine dihydrochloride [30,34]. In present work, POSS-based hybrid monolithic column, which intrinsically
contained disulfide bonds, was facilely prepared by “one-step” method. Thiol groups were produced on surface of hybrid monolith after reduction with DTT [35]. Considering that the amount of thiol groups seriously affected the following functionalization, we have investigated the reduction time with DTT using the retention factor (k) of toluene as standard. It is suggested from Fig. 3 that two hours of treatment would meet the following experiments because
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Fig. 6. Separation in HILIC mode on the MSA-modified hybrid monolith. Analytes: (1) dimethyl formamide, (2) benzene and (3) thiourea. Experimental conditions: effective length of 40 cm × 100 m i.d.; off column detection, 6 cm × 50 m i.d.; mobile phase as noted in Fig. 4a; flow rate, 150 L/min (before split); detection wavelength, 214 nm.
the retention factor of toluene did not further increase after 2 h reduction with DTT. It should be noted here that thiol-ene click reaction is generally conducted under radical conditions or nucleophilic catalysis. According to the reported method [36], the methacrylate is low reactive towards hydrothiolation under radical-mediated due to its electron-deficient feature, but such thiol-ene click reaction can be mediated under nucleophilic catalysis using primary/secondary amines or phosphines. What’s more, catalysis-mediated thiolene click reaction would be accomplished faster even at room temperature with insensitive to oxygen. So based on this point, DMPP was selected to catalyze thiol-ene click reaction between methacrylate and thiol group [37]. The grafting reaction was verified by FT-IR spectroscopic analysis (Fig. 4), which clearly showed the presence of an intense absorption band at 1734 cm−1 (C O), enhanced absorption at 2870 and 2950 cm−1 ( CH2 , CH3 ) (Fig. 4b), 1377 cm−1 (C N) (Fig. 4c) originating from the introduction of SMA or MSA. Due to the hydrophobicity of POSS-epoxy, the separation ability of POSS hybrid monolithic column was investigated in reversedphase mode using alkylbenzenes as probes. As shown in Fig. 5a,
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Fig. 8. Separation of phenols on SMA-modified monolithic column by cLC. Analytes: (1) hydroquinone, (2) resorcinol, (3) pyrocatechol, (4) phenol and (5) 4-cresol. Experimental conditions: effective length of 30 cm × 100 m i.d.; off column detection, 6 cm × 50 m i.d.; mobile phase, ACN/H2 O (55/45, v/v); flow rate, 150 L/min (before split); detection wavelength, 214 nm.
five alkylbenzenes were baseline-separated with good peak shapes under the mobile phase of ACN/H2 O (55/45, v/v). To evaluate the column efficiency of POSS monolithic column, the plate height–linear velocity curves were depicted in Fig. 5c. The lowest plate height 14 m was obtained, corresponding to ∼71,000 plates per meter. The influence of ACN content on the retention factors of alkylbenzenes is shown in Fig. 5e. The retention factors of alkylbenzenes decreased with an increase of ACN content from 45 to 75%, which suggested that the separation of these solutes on POSS hybrid monolithic column was based on typical reversedphase mechanism. However, when the ACN content was higher than 90%, the elution order of thiourea and benzene was reversed (Fig. S2). This result demonstrated a typical hydrophilic interaction retention mechanism, which may be attributed to the hydroxyl and amino groups on hybrid monolith surface. The same experiments were also performed on SMA-modified hybrid monolithic column (Fig. 5b,d,f). It was clear that the retention factors of alkylbenzenes were all increased significantly, confirming that SMA was successfully modified. The column efficiency was also enhanced after functionalization with SMA, especially in high linear velocity zone according to Fig. 5c and d.
Fig. 7. The chromatograms of 1-m-long columns with (a) 50 and (b) 75 m i.d. using alkylbenzenes as probes. The elution order is the same to Fig. 2a. Experimental conditions: off column detection, 6 cm × 50 m i.d.; mobile phase, ACN/H2 O (50/50, v/v); flow rate, (a) 120 L/min and (b) 200 L/min before split; detection wavelength, 214 nm; (a) unmodified and (b) SMA-modified monolithic columns, respectively.
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Fig. 9. Separations of EPA 610 on the (a) SMA- and (b) BMA-modified monolithic column by cLC with the same gradient elution. Solutes: (1) naphthalene, (2) acenaphthylene, (3) fluorene, (4) acenaphthene, (5) phenanthrene, (6) anthracene, (7) fluoranthene, (8) pyrene, (9) benzo(a)anthracene, (10) chrysene, (11) benzo(b)fluoranthene, (12) benzo(k)fluoranthene, (13) benzo(a)pyrene, (14) dibenzo(a,h)anthracene, (15) benzo(g,h,i)perylene and (16) indeno(1,2,3-cd)pyrene. Experimental conditions: effective length of 33 cm × 100 m i.d.; off column detection, 6 cm × 50 m i.d.; mobile phase, mobile phase A, water; mobile phase B, ACN; gradient, 60% B to 85% B in 25 min; flow rate, 150 L/min (before split); detection wavelength, 254 nm.
For instance, when the linear velocity was at 1.0 mm/s, the column efficiencies were increased by 58% (benzene), 73% (toluene), 86% (ethylbenzene), 102% (propylbenzene) and 96% (butylbenzene), respectively. Additionally, the permeability decreased 1.6% after SMA modification. These results indicated the above modification would not destroy the 3D skeleton of POSS-based hybrid monolith. MSA as a zwitterionic monomer has been used for preparation or post-modification of hybrid monolithic column and successfully utilized in hydrophilic interaction liquid chromatography (HILIC). So we also attempted to modify MSA on surface of POSSbased hybrid monolithic column for HILIC separation. As shown in Fig. 6, the elution order of thiourea and benzene was reversed on MSA-modified monolithic column with mobile phase of 70% ACN, though thiourea was firstly eluted on unmodified POSS-based monolithic column with ACN content from 45% to 75% (as shown in Fig. 5e). According to peaks under mobile phase of 80% ACN in Fig. 6, the column efficiencies were calculated about 58,200 (DMF), 57,200 (benzene) and 76,700 (thiourea). However, the retention time of thiourea was obviously deferred with increasing ACN content. It’s worth noting that the analyte of DMF was always firstly eluted before benzene even ACN content higher than 90%. This phenomenon may result from the intrinsic hydrophobicity of POSS-based hybrid monolith, and that the hydrophilic interaction was still weak even though the POSS-based hybrid monolith was modified with MSA. In the LC separation and analysis of complex mixtures, there are often two different ways to improve the resolution power of columns by achieving extremely high column efficiency and using gradient elution instead of isocratic conditions. However, the general practice for the former method is to increase column length and/or decrease the particle sizes, which supervenes with high inlet pressure and long dead time [38–40]. Hybrid monolithic column would possess the potential for high efficiency separations by increasing its length, because hybrid monolithic column with through pores makes mobile phase at reasonable pressure drop [9]. Besides, using smaller i.d. columns could improve sensitivity in LC–MS analysis of enzymatic digests [41]. So achieving a long length and narrow-bore hybrid monolithic column would meet the separation of complex components to some extent. In this work, we demonstrated a 1-m-long column of 50 m i.d. with the highest column efficiency of ∼85,000 plates and a dead time of about 32 min at 24 MPa (Fig. 7a). By adjusting the proportion of prepolymerization mixture to improve the permeability, a 1-m-long column of
75 m i.d. was also prepared, which exhibited a dead time of 9 min at 24 MPa (Fig. 7b). Peak capacity (Pc ) is the maximum number of components resolvable under given √ condition as defined by the following equation [42]: Pc = 1 + N/4 ln tn /t0 for isocratic chromatography, where N is the average number of theoretical plates of all analytes, tn is the retention time of the nth component, and t0 is the retention time of void time marker. As noted above, the same separation conditions (column length, mobile phase and flow rate, etc.) were controlled for unmodified and SMA-modified monolithic columns in Fig. 5a and b. The peak capacities were calculated about 26 for unmodified and 73 for SMA-modified hybrid monolithic columns, respectively. The simultaneous increases of N and tn /t0 after SMA functionalization could account for an increase of peak capacity. Additionally, the ratio tn /t0 keeps a constant at a particular mobile phase ratio for SMA-modified monolithic column. So the change in Pc is due solely to the change in N, which could be regarded as length dependent. A 1-m-long SMA-modified POSS-based hybrid monolithic column of 75 m i.d. was evaluated using alkylbenzenes as probes under mobile phase of ACN/H2 O (50/50, v/v) (Fig. 7b). The peak capacity was calculated about 147. 3.3. Application of hybrid monolithic columns For further separation in reversed phase, the SMA-modified monolithic column was used to separate the mixture of phenols, exhibiting a column efficiency of 64,000–97,000 plates/m (Fig. 8). EPA 610, which consists of 16 priority pollutant PAHs, presents potential health hazards because of their toxic mutagenic and carcinogenic properties. Fig. 9a shows that the separation of EPA 610 on SMA-modified monolithic column by cLC with gradient elution. Except that benzo(a)anthracene (analyte 9) and chrysene (analyte 10) could not be separated, the others were well separated. In addition, the POSS-based hybrid monolith column was also modified with BMA to separate EPA 610 under the same gradient elution. As indicated in Fig. 9b, the result was similar to that on SMA-modified monolithic column. 4. Conclusions A facile approach to prepare a POSS-based hybrid monolith was successfully developed using POSS-epoxy and Cys·2HCl via ring-opening polymerization, in which the disulfide bonds were
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simply and directly introduced. The thiol groups were produced on the porous surface of hybrid monolith after reduction of disulfide bonds, and subsequently reacted with various methacrylate monomers via thiol-ene click chemistry. The results demonstrated that the thiol-ene reaction was a facile and effective approach to introduce various methacrylate monomers to POSS-based hybrid monolith with thiol groups. In fact, thiol group can also react with alkene, epoxy, alkynyl, isocyanate etc. via click chemistry, which broadens the scope of modification of thiol-containing hybrid macroporous materials. Furthermore, the obtained hybrid monolithic column has advantages in separation of complex biosamples due to its good permeability and high peak capacity. So we would like to focus attention on application of longer hybrid column in analysis of complex biosamples in our future works. Acknowledgments 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) and the Hundred Talents Program of the Dalian Institute of Chemical Physics of Chinese Academy of Sciences to J. Ou. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.03.058. References [1] F. Svec, J.M. Frechet, Anal. Chem. 64 (1992) 820. [2] H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal. Chem. 68 (1996) 3498. [3] J.D. Hayes, A. Malik, Anal. Chem. 72 (2000) 4090.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Preparation of polyhedral oligomeric silsesquioxane-based hybrid monolith by ring-opening polymerization and post-functionalization via thiol-ene click reaction夽 Zhongshan Liu a,b , Junjie Ou a,∗ , Hui Lin a,b , Hongwei Wang a , Jing Dong a , Hanfa Zou a,∗ a b
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Graduate School of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 6 January 2014 Received in revised form 17 March 2014 Accepted 20 March 2014 Available online 28 March 2014 Keywords: Hybrid monolith Thiol-ene reaction Disulfide bond Ring-opening polymerization Polyhedral oligomeric silsesquioxane
a b s t r a c t A polyhedral oligomeric silsesquioxane (POSS) hybrid monolith was simply prepared by using octaglycidyldimethylsilyl POSS (POSS-epoxy) and cystamine dihydrochloride as monomers via ring-opening polymerization. The effects of composition of prepolymerization solution and polycondensation temperature on the morphology and permeability of monolithic column were investigated in detail. The obtained POSS hybrid monolithic column showed 3D skeleton morphology and exhibited high column efficiency of ∼71,000 plates per meter in reversed-phase mechanism. Owing to this POSS hybrid monolith essentially possessing a great number of disulfide bonds, the monolith surface would expose thiol groups after reduction with dithiothreitol (DTT), which supplied active sites to functionalize with various alkene monomers via thiol-ene click reaction. The results indicated that the reduction with DTT could not destroy the 3D skeleton of hybrid monolith. Both stearyl methylacrylate (SMA) and benzyl methacrylate (BMA) were selected to functionalize the hybrid monolithic columns for reversed-phase liquid chromatography (RPLC), while [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (MSA) was used to modify the hybrid monolithic column in hydrophilic interaction chromatography (HILIC). These modified hybrid monolithic columns could be successfully applied for separation of small molecules with high efficiency. It is demonstrated that thiol-ene click reaction supplies a facile way to introduce various functional groups to the hybrid monolith possessing thiol groups. Furthermore, due to good permeability of the resulting hybrid monoliths, we also prepared long hybrid monolithic columns in narrow-bore capillaries. The highest column efficiency reached to ∼70,000 plates using a 1-m-long column of 75 m i.d. with a peak capacity of 147 for isocratic chromatography, indicating potential application in separation and analysis of complex biosamples. © 2014 Published by Elsevier B.V.
1. Introduction Up till now, monolithic column, as called continuous bed, has achieved much progress in preparation, characterization and application in separation of small molecules and biomacromolecules [1–14]. Generally, the monolithic columns are classified into organic polymer-based [15–17], inorganic silica-based [18] and
夽 Presented at the 40th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2013 Hobart), Hobart, Tasmania, Australia, 18–21 November 2013. ∗ Corresponding author at: Chinese Academy of Sciences, Dalian Institute of Chemical Physics, Key Laboratory of Separation Science for Analytical Chemistry, Dalian 116023, China. Tel.: +86 411 84379576/+86 411 84379610; fax: +86 411 84379620. E-mail addresses: [email protected] (J. Ou), [email protected] (H. Zou). http://dx.doi.org/10.1016/j.chroma.2014.03.058 0021-9673/© 2014 Published by Elsevier B.V.
hybrid organic–inorganic [6] monolithic columns. Polymer-based monolithic columns are prepared by in situ polymerization using organic monomers and crosslinkers in the presence of porogenic solvents to form organic polymers, such as polymethacrylates, polyacrylamides and polystyrenes [16]. Silica-based monolithic columns are commonly fabricated via sol–gel technique following a chemical modification on surface of matrix with silylation reagents. Compared with the former two, the hybrid monolith may somewhat combine the advantages of organic polymer-based and silica-based monoliths, such as mechanical stability, low shrinkage and controlling porous structure easily. Therefore, the hybrid monolith, mainly organic-silica monolith, has attracted more and more attentions. Since Hayes and Malik [3] incorporated organic functional moieties into inorganic silica monolithic matrices via sol–gel chemistry, demonstrating good separation efficiency in capillary electrochromatography (CEC), various types
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of organic-silica based monoliths have been reported by changing the siloxanes with different organic moieties, such as allyl, propyl, aminopropyl, vinyl, etc. [6,19–21]. However, the limited types of organic-trialkoxysilanes restricted the development of hybrid monolithic columns. To overcome this limitation, we have developed the “one-pot” approach for incorporation of organic moieties into the silica monolithic matrix in our previous works [7,22]. To some extent, it turns out that the “one-pot” approach is a facile method to prepare various functionalized hybrid monolithic columns. In our recent works, we have introduced polyhedral oligomeric silsesquioxanes (POSS) monomer, which can be regarded as the smallest possible particles of silica with sizes of from 1 to 3 nm in diameter, into monolithic column matrix via free radical polymerization [10,23–25]. This method has some merits comparing with the sol–gel method, such as without hydrolysis, condensation reactions of siloxane and good pH stability over a wide pH range. Furthermore, different from free radical polymerization, we selected octaglycidyldimethylsilyl POSS (POSS-epoxy) and diamines as monomers to prepare a series of hybrid monoliths with well-controlled 3D skeletons via ring-opening polymerization, gaining excellent chromatographic performance [11,26,27]. Herein, we prepared hybrid monolithic column via ring-opening polymerization using POSS-epoxy and cystamine dihydrochloride (Cys·2HCl) as monomers. Due to the introduction of cystamine, the skeletal surface of monolithic column possesses a lot of disulfide bonds. After simple reduction with dithiothreitol (DTT), thiol groups were generated as the active sites and subsequently reacted with methylacrylate monomers via thiol-ene click chemistry, which is famous for its merits of high selectivity and high conversion under a variety of mild conditions [28–32]. The resulting hybrid monoliths were successfully applied for capillary liquid chromatography (cLC). 2. Materials and methods 2.1. Chemicals and reagents (3-Aminopropyl)triethoxysilane (APTES), POSS-epoxy, Cys·2HCl, [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)ammonium hydroxide (MSA) and stearyl methylacrylate (SMA) were purchased from Aldrich (Milwaukee, WI, USA). DTT, cetyltrimethyl ammonium bromide (CTAB), benzyl methacrylate (BMA) and EPA610 were purchased from Sigma Chemical Co. (St Louis, Mo, USA). Dimethylphenylphosphine (DMPP) was obtained from J&K Scientific Ltd. (Beijing, China). The fused-silica capillaries with dimension of 50, 75 and 100 m i.d. were obtained from the Refine Chromatography Ltd. (Yongnian, Hebei, China). Benzene and butylbenzene were purchased from Beijing Chemical Works (Beijing, China). Ammonium bicarbonate, sodium hydroxide (NaOH), ethylbenzene, dimethyl formamide (DMF), hydroquinone and other standard compounds were obtained from Tianjin Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). HPLC-grade acetonitrile (ACN) was used for mobile phase and obtained from Yuwang Group (Shandong, China). The water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore Inc., Milford, MA, USA). Other chemical reagents were all of analytical grade. 2.2. Preparation of POSS-based hybrid monoliths Prior to use, the inner wall of fused-silica capillary was pretreated and modified with a layer of amino groups for anchoring monolith matrix according to the method described by Lin et al. [11]. Briefly, the capillary was rinsed by 1.0 mol/L NaOH, water,
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1.0 mol/L HCl and water, successively, which was later dried by nitrogen stream at room temperature. Then, the capillary was filled with APTES solution in methanol (50%, v/v), sealed with rubbers at both ends and submerged in water bath at 50 ◦ C for 12 h. Finally, the capillary was rinsed with methanol to flush out the residual reagent and dried under nitrogen flow. For preparation of POSS-based hybrid monolithic capillary column, the prepolymerization mixture with different composition as listed in Table 1 was introduced into the above-mentioned pretreated capillaries with a syringe. After sealing both ends with rubbers, the capillary was immersed in a water bath at different temperature for 12 h. The obtained POSS-based hybrid monolith column was then flushed with methanol/H2 O (60/40, v/v) to remove residuals. For preparing of bulk hybrid monoliths, to a centrifuge tube the prepolymerization mixture was added and reacted at 50 ◦ C for 12 h. Then the bulk hybrid monolith was cut into smaller pieces and extracted with methanol/H2 O (60/40, v/v) in a Soxhlet apparatus and dried in a vacuum. For the following reduction of disulfide bonds and modification, the monolith was immersed in 0.2 mol/L DTT in 0.1 mol/L aqueous ammonium bicarbonate for 2 h, and then rinsed with methanol/H2 O (60/40, v/v). The monomer solutions (SMA, BMA or MSA, the precise proportion as seen below) were added to the above monolith for modification. The bulk hybrid monoliths were rinsed with methanol and dried for measuring in FT-IR. 2.3. Modification of hybrid columns via reduction of disulfide bonds and thiol-ene click reaction (Fig. 1) A 0.2 mol/L DTT in 0.1 mol/L aqueous ammonium bicarbonate was flushed through POSS-based hybrid monolith column by nitrogen pressure with 4 MPa for 2 h, and then rinsed with methanol/H2 O (60/40, v/v). Then the monomer solution of SMA/DMPP/ethanol (10/1/100, v/v/v) was flushed through hybrid monolith under nitrogen pressure for 2 h, and rinsed with methanol for chromatographic experiments. Similarly, the hybrid columns were also functionalized with BMA and MSA according to aforementioned procedures. The precise proportions of monomer solution were BMA/DMPP/ethanol = 10/1/100 (v/v/v) for BMA modification and MSA/DMPP/ethanol/H2 O = 20/2/400/100 (w/v/v/v) for MSA modification, respectively. 2.4. Instruments and methods The microscopic morphology of hybrid monoliths was obtained by scanning electron microscopy (SEM) (JEOL JSM-5600, Tokyo, Japan). Fourier-transformed infrared spectroscopy (FT-IR) characterization was carried out on Thermo Nicolet 380 spectrometer using KBr pellets (Nicolet, Wisconsin, USA). The cLC experiments were performed on LC system equipped with an Agilent 1100 micropump, a K-2501 UV detector (Knauer, Berlin, Germany) and a 7725i injector with a 20 L sample loop. A T-union connector was used as a splitter, with one end connected to a blank capillary (200 cm × 50 m i.d.) and the other connected to the monolithic column. The detection window was made by removing the polyimide coating of fused-silica capillary tubing. All chromatographic data were collected and evaluated using the software program HW-2000 from Qianpu Software (Shanghai, China). For illustrating the effects of all parameters more intuitively, permeability was calculated according to Darcy’s law [33] by the following, B0 = FL/(r2 P), where F (m3 /s) is the flow rate of mobile phase, is the viscosity of water (1.0 × 10−3 Pa s), L and r (m) are effective length and inner radius of the column, P (Pa) is the pressure drop of column. The data of P and F was measured
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Table 1 Detail composition of prepolymerization mixture for preparing POSS-based hybrid monolithic columns. Column
A B C D E #
POSS-epoxy (mg)
50 50 50 50 50
CTAB (mg)
14 16 18 16 16
Cys·2HCl/NaOH/H2 O (L)#
130 130 130 125 135
Ethanol (L)
125 125 125 125 125
Permeability/10−14 m2 45 ◦ C
50 ◦ C
55 ◦ C
13.7 12.2 0.001 0.15 142
3.1 3.0 – 0.07 55
2.4 2.0 – – 12
Cys·2HCl/NaOH/H2 O: 1080/383/7200, mg/mg/L. (Cys·2HCl, 12.47%; NaOH, 4.42%, wt%)
on an Eksigent one dimensional Plus Nano-HPLC system (Eksigent, Dublin). The mobile phase was water, and flow rate was set at 200–1500 nL/min.
3. Results and discussion 3.1. Preparation of POSS-based hybrid monolithic column The preparation of POSS hybrid monolith is outlined in Fig. 1. As expected, the morphology and permeability of hybrid monoliths were significantly affected by composition of prepolymerization solution and polycondensation temperature, which were investigated in detail. The mixture of propanol/1,4-butanediol and PEG10,000 was initially selected as porogenic solvents following the approach in our previous works [11,26,27]. However, it was found that the commercial reagent of Cys·2HCl could not react with POSS-epoxy because of low reaction activity and insolubility of Cys·2HCl in this porogenic system. We have attempted using anhydrous triethylamine and sodium hydroxide to generate free cystamine, and finally chose sodium hydroxide solution to neutralize hydrogen chloride in Cys·2HCl and form Cys·2HCl/NaOH/H2 O solution (Cys·2HCl/NaOH/H2 O, 1080/383/7200, mg/mg/L), and the ring-opening polymerization between the free cystamine (Cys) and POSS-epoxy could be carried out in the ethanolCTAB-Cys·2HCl/NaOH/H2 O solution. However, the content of
Cys·2HCl/NaOH/H2 O solution has a great influence on the morphology and permeability. A little change of Cys·2HCl/NaOH/H2 O content could result in a transformation of monoliths from dense to loose (Fig. 2d, b, e), which was also confirmed by column permeability changing from 1.5 × 10−15 to 1.42 × 10−12 m2 with increase content of Cys·2HCl/NaOH/H2 O solution at 45 ◦ C. Therefore, the Cys·2HCl/NaOH/H2 O solution was selected at 130 L for the following experiments. Owing to the hydrophobic property of POSS-epoxy, CTAB was needed to form a homogenous mixture. As discussed above, water was a poor solvent that led to earlier phase separation, supervening with large pores in monolithic material (Fig. 2e). CTAB may form micelles and enhance the uniformity and stability of prepolymerization mixture due to the interaction between water molecules and hydrophilic heads of CTAB. As a result, the phase separation may be postponed, and the average pore diameter of monolith is decreased with an increase of CTAB content. As shown in Table 1 (Columns A–C), increasing CTAB content from 14 to 18 mg, the permeability of monolithic columns was remarkably decreased from 1.37 × 10−13 to 1.0 × 10−17 m2 , which hardly allowed the mobile phase to pass through (Table 1, Column C). The SEM micrographs also verified that the pore size became smaller, especially when CTAB content was up to 18 mg in Fig. 2c. What’s worse, high CTAB content would lead to monolith matrix detaching from the inner wall of fused-silica capillary. This phenomenon was also observed as raising the content of ethanol in
Fig. 1. Preparation of POSS-based hybrid monolith via ring-opening polymerization and post-functionalization based on thiol-ene click reaction.
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Fig. 2. SEM micrographs of POSS-based hybrid monolithic columns with (a) Column A, (b) Column B, (c) Column C, (d) Column D and (e) Column E prepared at 45 ◦ C, (f) Column E prepared at 50 ◦ C, (g) Column E prepared at 55 ◦ C. The composition of prepolymerization mixture as shown in Table 1.
prepolymerization mixture (Fig. S1). Therefore, the CTAB content of 16 mg was controlled. Another important effect on formation of monolithic column was polycondensation temperature. In fact, prepolymerization mixture would transform to opaque monolithic materials in 2 h at temperatures above 45 ◦ C, which implied ring-opening reaction basically finished. As indicated in Table 1, the permeability was decreased with an increase of polycondensation temperature for all prepolymerization mixtures with different ratios. For example, as for column A, the permeability was decreased from 1.37 × 10−13 to 2.4 × 10−14 m2 as polycondensation temperature increasing from 45 to 50 ◦ C. Additionally, they all exhibited more uniform and smaller pore size at high temperature (Fig. 2e–g). This trend may result from high temperature accelerating the ringopening polymerization, which lead to little diffusion of monomers
and phase separation in situ rapidly. Concretely, low polycondensation temperature made the column good permeability but low column efficiency, while too high temperature resulted in the monolith hard to flush through and lowered the reproducibility. As for Column B (Table 1) being prepared at 55 ◦ C, the values of reproducibility were 9.4% (column-to-column) and 12.1% (batch-to-batch), respectively. So we finally controlled the polycondensation temperature at 50 ◦ C, which also significantly improved column reproducibility. The reproducibility of hybrid monolith was evaluated through the relative standard deviation (RSD) for the retention factor (k) of toluene as model analyte (thiourea as the void time marker). The run-to-run (n = 4), column-to-column (n = 4) and batch-to-batch (n = 4) were 0.17%, 0.33% and 0.50%, respectively. All results above indicated the good reproducibility of POSS-based hybrid monolith.
Fig. 3. The retention factor (k) of toluene on unmodified and SMA-modified hybrid monolithic columns with different DTT reduction time.
Fig. 4. FT-IR spectra of (a) unmodified monolith, (b) SMA-modified monolith and (c) MSA-modified monolith.
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Fig. 5. (a) and (b): separation of alkylbenzenes on the hybrid monolith by cLC. Analytes: (1) thiourea, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene and (6) butylbenzene. (c) and (d): Dependence of the plate height of analytes on the linear velocity of mobile phase by the hybrid monolith capillary column. (e) and (f): The effect of ACN content in mobile phase on retention factor of alkylbenzenes. Experimental conditions: effective length of 40.7 cm × 100 m i.d.; off column detection, 6 cm × 50 m i.d.; mobile phase, ACN/H2 O (55/45, v/v, for a, b, c, d); flow rate, 160 L/min (before split, for a, b, e, f); detection wavelength, 214 nm. (Note: a, c, e for unmodified and b, d, f for SMA-modified monolithic columns, respectively.)
3.2. Modification and characterization of hybrid monoliths In recent reports, Svec et al. has prepared polymeric monolith using glycidyl methacrylate and ethylene dimethacrylate, to which thiol groups were introduced via post-modification and disulfide bond reduction of cystamine dihydrochloride [30,34]. In present work, POSS-based hybrid monolithic column, which intrinsically
contained disulfide bonds, was facilely prepared by “one-step” method. Thiol groups were produced on surface of hybrid monolith after reduction with DTT [35]. Considering that the amount of thiol groups seriously affected the following functionalization, we have investigated the reduction time with DTT using the retention factor (k) of toluene as standard. It is suggested from Fig. 3 that two hours of treatment would meet the following experiments because
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Fig. 6. Separation in HILIC mode on the MSA-modified hybrid monolith. Analytes: (1) dimethyl formamide, (2) benzene and (3) thiourea. Experimental conditions: effective length of 40 cm × 100 m i.d.; off column detection, 6 cm × 50 m i.d.; mobile phase as noted in Fig. 4a; flow rate, 150 L/min (before split); detection wavelength, 214 nm.
the retention factor of toluene did not further increase after 2 h reduction with DTT. It should be noted here that thiol-ene click reaction is generally conducted under radical conditions or nucleophilic catalysis. According to the reported method [36], the methacrylate is low reactive towards hydrothiolation under radical-mediated due to its electron-deficient feature, but such thiol-ene click reaction can be mediated under nucleophilic catalysis using primary/secondary amines or phosphines. What’s more, catalysis-mediated thiolene click reaction would be accomplished faster even at room temperature with insensitive to oxygen. So based on this point, DMPP was selected to catalyze thiol-ene click reaction between methacrylate and thiol group [37]. The grafting reaction was verified by FT-IR spectroscopic analysis (Fig. 4), which clearly showed the presence of an intense absorption band at 1734 cm−1 (C O), enhanced absorption at 2870 and 2950 cm−1 ( CH2 , CH3 ) (Fig. 4b), 1377 cm−1 (C N) (Fig. 4c) originating from the introduction of SMA or MSA. Due to the hydrophobicity of POSS-epoxy, the separation ability of POSS hybrid monolithic column was investigated in reversedphase mode using alkylbenzenes as probes. As shown in Fig. 5a,
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Fig. 8. Separation of phenols on SMA-modified monolithic column by cLC. Analytes: (1) hydroquinone, (2) resorcinol, (3) pyrocatechol, (4) phenol and (5) 4-cresol. Experimental conditions: effective length of 30 cm × 100 m i.d.; off column detection, 6 cm × 50 m i.d.; mobile phase, ACN/H2 O (55/45, v/v); flow rate, 150 L/min (before split); detection wavelength, 214 nm.
five alkylbenzenes were baseline-separated with good peak shapes under the mobile phase of ACN/H2 O (55/45, v/v). To evaluate the column efficiency of POSS monolithic column, the plate height–linear velocity curves were depicted in Fig. 5c. The lowest plate height 14 m was obtained, corresponding to ∼71,000 plates per meter. The influence of ACN content on the retention factors of alkylbenzenes is shown in Fig. 5e. The retention factors of alkylbenzenes decreased with an increase of ACN content from 45 to 75%, which suggested that the separation of these solutes on POSS hybrid monolithic column was based on typical reversedphase mechanism. However, when the ACN content was higher than 90%, the elution order of thiourea and benzene was reversed (Fig. S2). This result demonstrated a typical hydrophilic interaction retention mechanism, which may be attributed to the hydroxyl and amino groups on hybrid monolith surface. The same experiments were also performed on SMA-modified hybrid monolithic column (Fig. 5b,d,f). It was clear that the retention factors of alkylbenzenes were all increased significantly, confirming that SMA was successfully modified. The column efficiency was also enhanced after functionalization with SMA, especially in high linear velocity zone according to Fig. 5c and d.
Fig. 7. The chromatograms of 1-m-long columns with (a) 50 and (b) 75 m i.d. using alkylbenzenes as probes. The elution order is the same to Fig. 2a. Experimental conditions: off column detection, 6 cm × 50 m i.d.; mobile phase, ACN/H2 O (50/50, v/v); flow rate, (a) 120 L/min and (b) 200 L/min before split; detection wavelength, 214 nm; (a) unmodified and (b) SMA-modified monolithic columns, respectively.
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Fig. 9. Separations of EPA 610 on the (a) SMA- and (b) BMA-modified monolithic column by cLC with the same gradient elution. Solutes: (1) naphthalene, (2) acenaphthylene, (3) fluorene, (4) acenaphthene, (5) phenanthrene, (6) anthracene, (7) fluoranthene, (8) pyrene, (9) benzo(a)anthracene, (10) chrysene, (11) benzo(b)fluoranthene, (12) benzo(k)fluoranthene, (13) benzo(a)pyrene, (14) dibenzo(a,h)anthracene, (15) benzo(g,h,i)perylene and (16) indeno(1,2,3-cd)pyrene. Experimental conditions: effective length of 33 cm × 100 m i.d.; off column detection, 6 cm × 50 m i.d.; mobile phase, mobile phase A, water; mobile phase B, ACN; gradient, 60% B to 85% B in 25 min; flow rate, 150 L/min (before split); detection wavelength, 254 nm.
For instance, when the linear velocity was at 1.0 mm/s, the column efficiencies were increased by 58% (benzene), 73% (toluene), 86% (ethylbenzene), 102% (propylbenzene) and 96% (butylbenzene), respectively. Additionally, the permeability decreased 1.6% after SMA modification. These results indicated the above modification would not destroy the 3D skeleton of POSS-based hybrid monolith. MSA as a zwitterionic monomer has been used for preparation or post-modification of hybrid monolithic column and successfully utilized in hydrophilic interaction liquid chromatography (HILIC). So we also attempted to modify MSA on surface of POSSbased hybrid monolithic column for HILIC separation. As shown in Fig. 6, the elution order of thiourea and benzene was reversed on MSA-modified monolithic column with mobile phase of 70% ACN, though thiourea was firstly eluted on unmodified POSS-based monolithic column with ACN content from 45% to 75% (as shown in Fig. 5e). According to peaks under mobile phase of 80% ACN in Fig. 6, the column efficiencies were calculated about 58,200 (DMF), 57,200 (benzene) and 76,700 (thiourea). However, the retention time of thiourea was obviously deferred with increasing ACN content. It’s worth noting that the analyte of DMF was always firstly eluted before benzene even ACN content higher than 90%. This phenomenon may result from the intrinsic hydrophobicity of POSS-based hybrid monolith, and that the hydrophilic interaction was still weak even though the POSS-based hybrid monolith was modified with MSA. In the LC separation and analysis of complex mixtures, there are often two different ways to improve the resolution power of columns by achieving extremely high column efficiency and using gradient elution instead of isocratic conditions. However, the general practice for the former method is to increase column length and/or decrease the particle sizes, which supervenes with high inlet pressure and long dead time [38–40]. Hybrid monolithic column would possess the potential for high efficiency separations by increasing its length, because hybrid monolithic column with through pores makes mobile phase at reasonable pressure drop [9]. Besides, using smaller i.d. columns could improve sensitivity in LC–MS analysis of enzymatic digests [41]. So achieving a long length and narrow-bore hybrid monolithic column would meet the separation of complex components to some extent. In this work, we demonstrated a 1-m-long column of 50 m i.d. with the highest column efficiency of ∼85,000 plates and a dead time of about 32 min at 24 MPa (Fig. 7a). By adjusting the proportion of prepolymerization mixture to improve the permeability, a 1-m-long column of
75 m i.d. was also prepared, which exhibited a dead time of 9 min at 24 MPa (Fig. 7b). Peak capacity (Pc ) is the maximum number of components resolvable under given √ condition as defined by the following equation [42]: Pc = 1 + N/4 ln tn /t0 for isocratic chromatography, where N is the average number of theoretical plates of all analytes, tn is the retention time of the nth component, and t0 is the retention time of void time marker. As noted above, the same separation conditions (column length, mobile phase and flow rate, etc.) were controlled for unmodified and SMA-modified monolithic columns in Fig. 5a and b. The peak capacities were calculated about 26 for unmodified and 73 for SMA-modified hybrid monolithic columns, respectively. The simultaneous increases of N and tn /t0 after SMA functionalization could account for an increase of peak capacity. Additionally, the ratio tn /t0 keeps a constant at a particular mobile phase ratio for SMA-modified monolithic column. So the change in Pc is due solely to the change in N, which could be regarded as length dependent. A 1-m-long SMA-modified POSS-based hybrid monolithic column of 75 m i.d. was evaluated using alkylbenzenes as probes under mobile phase of ACN/H2 O (50/50, v/v) (Fig. 7b). The peak capacity was calculated about 147. 3.3. Application of hybrid monolithic columns For further separation in reversed phase, the SMA-modified monolithic column was used to separate the mixture of phenols, exhibiting a column efficiency of 64,000–97,000 plates/m (Fig. 8). EPA 610, which consists of 16 priority pollutant PAHs, presents potential health hazards because of their toxic mutagenic and carcinogenic properties. Fig. 9a shows that the separation of EPA 610 on SMA-modified monolithic column by cLC with gradient elution. Except that benzo(a)anthracene (analyte 9) and chrysene (analyte 10) could not be separated, the others were well separated. In addition, the POSS-based hybrid monolith column was also modified with BMA to separate EPA 610 under the same gradient elution. As indicated in Fig. 9b, the result was similar to that on SMA-modified monolithic column. 4. Conclusions A facile approach to prepare a POSS-based hybrid monolith was successfully developed using POSS-epoxy and Cys·2HCl via ring-opening polymerization, in which the disulfide bonds were
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simply and directly introduced. The thiol groups were produced on the porous surface of hybrid monolith after reduction of disulfide bonds, and subsequently reacted with various methacrylate monomers via thiol-ene click chemistry. The results demonstrated that the thiol-ene reaction was a facile and effective approach to introduce various methacrylate monomers to POSS-based hybrid monolith with thiol groups. In fact, thiol group can also react with alkene, epoxy, alkynyl, isocyanate etc. via click chemistry, which broadens the scope of modification of thiol-containing hybrid macroporous materials. Furthermore, the obtained hybrid monolithic column has advantages in separation of complex biosamples due to its good permeability and high peak capacity. So we would like to focus attention on application of longer hybrid column in analysis of complex biosamples in our future works. Acknowledgments 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) and the Hundred Talents Program of the Dalian Institute of Chemical Physics of Chinese Academy of Sciences to J. Ou. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.03.058. References [1] F. Svec, J.M. Frechet, Anal. Chem. 64 (1992) 820. [2] H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal. Chem. 68 (1996) 3498. [3] J.D. Hayes, A. Malik, Anal. Chem. 72 (2000) 4090.
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