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Coatings of molecularly imprinted polymers based on polyhedral oligomeric silsesquioxane for open tubular capillary electrochromatography
Talanta 152 (2016) 277–282
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Coatings of molecularly imprinted polymers based on polyhedral oligomeric silsesquioxane for open tubular capillary electrochromatography Qing-Li Zhao, Jin Zhou, Li-Shun Zhang, Yan-Ping Huang, Zhao-Sheng Liu n Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 November 2015 Received in revised form 3 February 2016 Accepted 7 February 2016 Available online 7 February 2016
Polyhedral oligomeric silsesquioxane (POSS) was successfully applied, for the first time, to prepare imprinted monolithic coating for capillary electrochromatography. The imprinted monolithic coating was synthesized with a mixture of PSS-(1-Propylmethacrylate)-heptaisobutyl substituted (MA 0702), S-amlodipine (template), methacrylic acid (functional monomer), and 2-methacrylamidopropyl methacrylate (crosslinker), in a porogenic mixture of toluene–isooctane. The influence of synthesis parameters on the imprinting effect and separation performance, including the amount of MA 0702, the ratio of template to monomer, and the ratio of monomer to crosslinker, was investigated. The greatest resolution for enantiomers separation on the imprinted monolithic column prepared with MA 0702 was up to 22.3, about 2 times higher than that prepared in absence of the POSS. Column efficiency on the POSS-based MIP coatings was beyond 30,000 plate m 1. The comparisons between MIP coating synthesized with the POSS and without the POSS were made in terms of selectivity, column efficiency, and resolution. POSSbased MIP capillaries with naproxen or zopiclone was also prepared and separation of enantiomers can be achieved. & 2016 Elsevier B.V. All rights reserved.
Keywords: Molecularly imprinted polymers Capillary electrochromatography Polyhedral oligomeric silsesquioxane Chiral separation Coating
1. Introduction Molecularly imprinted polymers (MIPs) are recognition materials with pre-determined selectivity based on template synthesis, which possess affinity complementary domains of the given molecule with good physicochemical stability. The template molecules are removed afterwards by extracting with solvents/hydrolysis from the polymer, leaving a three-dimensional porous polymer with recognition sites. The resultant MIPs have been found in applications of enzyme-like catalysis [1], bio-mimetic sensors [2], drug delivery system [3], solid-phase extraction [4], and chromatographic stationary phase [5]. One particular promising direction is the use of MIPs as stationary phases in capillary electrochromatography (CEC) since CEC is still with holding its popularity due to the numerous superiorities, such as low consumed reagents, high separation efficiencies, hybrid separation principle, etc. Many formats of MIP have been used in CEC analysis, including monolithic column [6–11], open tubular capillary [12–15] and particle-based technique [16,17]. n
Corresponding author. E-mail address: [email protected] (Z.-S. Liu).
http://dx.doi.org/10.1016/j.talanta.2016.02.019 0039-9140/& 2016 Elsevier B.V. All rights reserved.
In spite of successful separation achieved, there is still much room of improvement for MIP-based CEC. For example, polymeric MIPs have the advantage of simple polymerization procedure and easy tuning of porosity and surface chemistry, but may suffer from shrinking or swelling when exposed to different solvents [3]. In contrast, inorganic materials, e.g., silica-based MIPs, can offer excellent mechanical strength and good solvent resistance. However, low tolerance to pH value of the silica-based MIP often limit its use [1,2]. Thus, organic–inorganic hybrid MIPs are supposed to combine the merits of the organic polymer and inorganic-based materials. Earlier works on the hybrid MIPs for CEC focused on the MIP film anchored covalently on the surface of silica packings. Recently, Ou et al. developed another silica-based MIP coating in capillary column for CEC, in which the MIP coating was copolymerized and anchored onto the surface of a monolithic silica containing vinyl groups [8]. An alternative approach to the preparation of the hybrid MIP for CEC is using a nonhydrolytic sol–gel protocol based on room temperature ionic liquid [9]. In additional, graphene oxide hybrid MIPs for CEC has also been reported [14]. Polyhedral oligomeric silsesquioxanes (POSS) with the formula (RSiO1.5)n are nano-building blocks [18], which embody inorganic–organic cagelike architecture containing an inner
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inorganic framework made up of silicon and oxygen. The POSSpolymers have already been used for the creation of advanced porous materials due to the reinforcement in rigidity at the molecular level [19–21]. Their unique properties of facile chemical modification, good pH tolerance, high temperature and oxidation resistance, make POSS excellent building block for constructing multi-functional materials. In addition, POSS materials are thought as the truly molecularly dispersed nanocomposites, which can be easily incorporated into common polymers via copolymerization, grafting, or blending. Recently, it was found that the suppress of the non-selective binding sites was observed in the MIPs with vinyl-POSS from the study of binding sites [22]. In spite of this, so far there are no reports on the POSS-based MIPs for CEC. This paper described a novel method for the preparation of MIP coating for CEC based on POSS. The objective of the work was to take advantage of rigidity reinforcement at the molecular level from POSS to improve the performance of MIP. Such MIPs are expected to have higher imprinting efficiency than conventional POSS-free MIPs since the specific feature of the POSS network and architecture of MIP coating have been incorporated. The influence of polymerization parameters on the electroosmotic flow (EOF) and ability of enantiomer separation of the new MIP-coated capillary was investigated.
was created at a distance of 10.5 cm from the outlet end of the MIP-coated capillary by burning out 2–3 mm segment of the polyimide outer coating. A procedures for the preparation of blank capillary column without the imprinted molecule was identical to that for the imprinted capillary. 2.3. Capillary electrochromatography Experiments of chiral separation were conducted using a K1050 system (Kaiao, Beijing, China) equipped with a CXTH-3000 software. The electrolyte used was composed of ACN/0.05 mol L 1 acetate (pH 4.2) (80/20, v/v). All the buffers were made using double distilled water and filtered with 0.2 mm membrane. Between runs the column was rinsed with mobile phase for 10 min. The separation voltage was 15 kV. UV detection was performed at 238 nm. In this paper, because some of analytes are eluted prior to EOF, separation factor is evaluated using α, which is calculated by
α = tR2/tR1
(1)
where tR1 and tR2 are the migration time of the first and second peaks. Normalized separation index, ΔtR/tR1, was used to assess the degree of enantiomer separation, where ΔtR is the difference in the elution times of the enantiomers at peak maximum.
2. Experimental 2.1. Reagents and chemicals 3-(Trimethoxysilyl) propyl methacrylate (γ-MPS, AR grade) was obtained from Acros (Geel, Belgium). Methacrylic acid (MAA, AR grade) was purchased from Beijing Donghuan Chemical Reagent (Beijing, China). Azobisisobutyronitrile (AIBN, AR grade) was from by Special Chemical Reagent Factory of Nankai University (Tianjin, China). Acetonitrile (ACN, HPLC grade) was obtained from Fisher (NJ, USA). Acetophenone (AR grade) were from Tianjin Bodi Chemical (Tianjin, China). 2-Methacrylamidopropyl methacrylate (MAM, AR grade) was synthesized using a previously published method [12]. Methacryllsobutyl POSS (MA 0702, AR grade) was purchased from Sigma–Aldrich (St. Louis, MO, USA). S-amlodipine (S-AML, AR grade) and racemic amlodipine (rac-AML, AR grade) were supplied by Hengshuo Sci. & Tech. Corp. (Hubei, China). SNaproxen (S-NAP, AR grade) and racemic naproxen (rac-NAP, AR grade) were purchased from Zhejiang Xianju Pharmaceutical Co. Ltd. (Zhejiang, China). d-Zopiclone (d-ZOP, AR grade) and racemic zopiclone (rac-ZOP, AR grade) were obtained from Kaiyuan Minsheng Sci. & Tech. Corp. (Suzhou, China). Bare fused-silica capillaries with 100 mm ID and 375 mm OD were supplied from Xinnuo Optic Fiber Plant (Hebei, China). 2.2. Preparation of POSS MIP capillary A bare fused-silica capillary was flushed with 1 mol L 1 NaOH solution followed by water for 30 min. Then the capillary was filled with 4 mL of γ-MPS solution (about 6 mmol L 1) in 1 mL acetic acid for 1.5 h. The capillary was then flushed with water and acetone, and dried with a flow of nitrogen. The imprinted molecules (0.0355 mmol), methacryllsobutyl POSS (0.166 mmol), MAA (0.142 mmol), MAM (0.142 mmol) and AIBN (14 mg) were dissolved in 1.232 mL toluene–isooctane (9/1, v/v). The pre-polymerization mixture was sonicated for 10 min and introduced into the capillary using a syringe. The capillary was sealed at both ends with a rubber septum and incubated in a water bath (50 °C) for polymerization. After polymerization, the capillary was immediately flushed using a hand-held syringe with acetonitrile and methanol/acetic acid (9:1, v/v), respectively. A detection window
3. Results and discussion 3.1. Preparation of POSS-based MIP coatings The preparation of the MIP film was based on the work by Wei et al. with some important adaptations [12]. To obtain a sufficient EOF in CEC, anionizable moiety MAA was chosen as functional monomer in place of γ-MPS. Porogen is one of the most important parameter of polymerization to form a successful polymer coating, since the affinity and morphology of the final MIP film is highly related to the nature of the porogen [12]. In the present study, it was found that binary porogens can not only dissolve enough POSS but also MAM. For example, toluene/isooctane mixture was better than ACN/isooctane on the preparation of POSS-based MIP. However, high proportion of isooctane result in lower column efficiency due to unfavorable morphology of the resultant MIP coating. In the present work, the optimum porogens was 10% (v/v) of isooctane in solvent mixture. Three different cross-linking monomers, i.e., trimethylolpropane trimethacrylate (TRIM), MAM and ethylene glycoldimethacrylate (EDMA), were investigated for their ability to determine imprinting effect on the resulting MIP (Table 1). The use of both EDMA and TRIM led to the unsuccessful MIP-coated capillaries. For example, TRIMbased MIP (C3) indicated smaller normalized separation index than POSS-free MIP. When preparing MIP with MAM as the cross-linker, however, excellent MIP capillary coatings were obtained with strong recognition ability and high column performance of the template. As a result, MAM was chosen for the crosslinker to prepare POSS-based MIP capillaries in the following study. The polymerization time provides a control over the pore property of the resulting MIP coating depending on the conversion. Generally, the specific surface area of MIP monolith decreases with the polymerization time. Although the conversion of monomers to polymer was close to quantitative after about 1.5 h, some additional structural changes still occur within the MIP coating if the system was kept longer. The normalized separation index reached a maximum after about 2 h of polymerization. Further increasing polymerization time, however, led to a blocked capillary.
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Table 1 Recipes of POSS-based MIP capillary coatings. Capillaries
S-AML (mmol)
MAA (mmol)
MA0702 (mmol)
MAM (mmol)
toluene (mL)
isooctane (mL)
time (h)
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17
0.0355 0.0355 0.0355 0.0355 0.0355 0.0355 0.0355 0.0355 0.071 0.047 0.028 0.024 0.0355c 0.0355d 0.0355 0.0355 0.0355
0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142
0.166 – 0.166 0.166 0.071 0.142 0.284 0.568 0.166 0.166 0.166 0.166 0.166 0.166 0.162 0.162 0.162
0.142 0.142 0.142a 0.142b 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.284 0.142 0.142 0.142 0.142
1108.9 1108.9 1108.9 1109 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1232.1 1170.5 1047.3
123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 61.6 184.8
1.75 2 2 – 1.75 1.75 2 3 2 1.75 2 2 2 2.25 1.5 1.75 1.75
a
TRIM. EDMA. c S-NAP. d d-ZOP. b
Fig. 1. Electrochromatograms of rac-AML (A), S-AML (B) on S-AML imprinted capillary (C1) and rac-AML on non-imprinted capillary (C) demonstrating the imprinting effect. Conditions: capillary, 100-mm inner diameter, 47.5 cm total length, and 37.0 cm effective length; separation voltage, 15 kV; UV detector, 238 nm; acetonitrile/50 mM acetate (pH 4.2) (80/20, v/v). Elution order of two enantiomers was identificated by injecting S-AML on the imprinted capillary.
Imprinting effect of the POSS-based MIP coatings was assessed by CEC. The resolution of two enantiomers of AML was 14.8 (Fig. 1). In contrast, a nonimprinted blank capillary prepared with POSS showed no enantiomeric separation. In contrast to previous report about MIP coating with AML imprints [14,15], clear improvement in resolution and column efficiency was recorded. This can be observed from greater selectivity factor (2.05) and higher column performance of the template (up to 30,000 plates/m). Fig. 2 shows microstructure of the POSS-based films on capillary segments. Through scanning electron microscopic investigations, a layer of MIP with a thickness around 0.1–0.2 mm covering the inner surface of the capillary was observed. The Fourier-transform infrared (FTIR) spectra of the POSSbased (C1) and POSS-free MIP (C2) are shown in Fig. S1. The very strong absorption band of C1 at 1110 cm 1 for the POSS-based MIP (asymmetric nSi–O–Si) was observed, suggesting that POSS groups
Fig. 2. Image of scanning electron micrograph for POSS-based MIP-coated capillary (C1) using S-AML as template molecule. Table 2 Pore properties of POSS-based MIP and POSS-free MIPs. Polymers
SBET (m2 g 1)
St (m2 g 1)
Vp (10 3 cm3 g 1)
Dmean (nm)
POSS-based MIPs (C1) POSS-free MIPs (C2)
5.32 7.96
6.24 9.19
26.99 31.46
17.3 13.69
have been integrated into MIP matrix. The peaks at 1170 cm 1 for the POSS-free MIP were assigned to the asymmetric ester stretching bands of C–O for MAM. Table 2 summarizes the pore parameters of the POSS-based (C1) and POSS-free MIP (C2) based on the nitrogen adsorption/ desorption. The POSS-based MIP showed smaller surface areas and pore volumes but greater pore sizes than the POSS-free MIP. The pore size distribution curves obtained from N2 adsorption (Fig. S2) for the POSS-based and POSS-free MIP gave rise to the maxima, indicating a narrow distribution of pore sizes of the MIPs. In addition, Fig. 3 shows incomplete type IV of N2 adsorption–
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Fig. 3. Nitrogen adsorption–desorption isotherms of POSS-based (C1) and POSSfree MIP (C2).
Fig. 4. Comparison of the chiral separation of rac-AML on the POSS (C1) (A) and free-POSS (C2) (B) MIP coating columns. Conditions: separation voltage, 15 kV; UV detector, 238 nm; acetonitrile/50 mM acetate (pH 4.2) (80/20, v/v). Elution order of two enantiomers was identificated by injecting S-AML on the imprinted capillary.
desorption isotherms with type H4 hysteresis loops [23], suggesting narrow slit-like pores. To demonstrate the superiority of the POSS-based MIP coatings than conventional one, the POSS-free MIP coating with 80% (molar ratio) MAM was synthesized. It was observed that the POSS-free MIP showed lower selectivity (α ¼1.68) than the POSS-based MIP (α ¼ 2.05) (Fig. 4). Higher resolution of the enantiomers (Rs ¼14.8) were also recorded for the POSS-based MIP, which were about 2 times than that of the POSS-free MIP. Thus, not only affinity but also morphology on the POSS-based MIP were better than the POSS-free MIP. To look into the scope of the POSS-based strategy and demonstrate its general applicability, the MIP-coated capillaries using S-NAP and d-ZOP as templates was further synthesized, respectively. The resolutions of rac-NAP and rac-ZOP were 2.68 and 2.49, respectively, in their respective POSS-based MIP columns (Figs. 5 and 6). It was also found that in the separation of rac-NAP, resolution on the POSS-based MIP coating column was higher than the POSS-free MIP coating column [12]. As a conclusion, improved
Fig. 5. Separation of rac-naproxen on S-NAP-imprinted POSS coating capillary (C13). Conditions: separation voltage, 15 kV; UV detector, 254 nm; acetonitrile/ 10 mM acetate (pH 3.6) (80/20, v/v). Elution order of two enantiomers was identificated by injecting S-NAP on the imprinted capillary.
Fig. 6. Separation of rac-ZOP on d-ZOP-imprinted POSS coating capillary (C14). Conditions: separation voltage, 15 kV; UV detector, 254 nm; acetonitrile/10 mM acetate (pH 3.6) (80/20, v/v). Elution order of two enantiomers was identificated by injecting d-ZOP on the imprinted capillary.
performance of the resulting MIP with incorporation of POSS unit is not limited to the imprinting molecule like AML. 3.2. Effect of polymerization variables The effect of MA0702 amount on the affinity of the resulting SAML MIPs was further investigated to understand the function of POSS. The hydrophobicity inside the POSS-based MIPs significantly depend on the the monomer concentrations in the reaction solutions [21]. The shift in percent of POSS monomers was adjusted by changing the relative ratio of synthesis of MA0702 to MAM at a fixed ratio of MAA (functional monomer, 20%, molar ratio). As shown in Table S1, an increased trend of selectivity with increase in the amount of POSS monomer on the POSS-based MIPs was observed. Previous NMR study on the pseudo-pre-polymerization mixture consisting of MA0702 and template molecules showed that the interaction between POSS monomer and the template was not involved in the recognition of the resulting MIP [22]. Thus, the POSS core introduced into MIP matrix might work as a host for
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Table 3 Relative standard deviation (RSD) of reproducibility on POSS-based capillaries with S-AML imprints (C1).
Retention time (RSD%) Column efficiency (RSD%) Resolution (RSD%) α (RSD%)
Intra-capillary
Inter-capillary
The first peak
The second peak
The first peak
The second peak
0.58 2.76
0.18 3.32
0.46 2.55
1.14 3.01
2.89 0.56
1.66 0.51
encapsulating various kinds of hydrophobic molecules [19]. However, a decreased trend of the selectivity was found when the ratio of POSS monomer was beyond 50%. Because there is a minimum amount of crosslinker necessary to form a polymer network rigid enough to maintain the fidelity of the binding site, the decrease in relative ratio of the crosslinker in synthesis, MAM, will cause the decrease in imprinting effect and selectivity. Varying the molar ratio of MAA to the template molecule was performed by adjusting the amount of imprinted molecule added to an otherwise constant prepolymerization mixture. The enantiomer selectivity, in terms of a normalized separation index (ΔtR/tR1), on the POSS-based MIP ranged from 0.56 to 1.05. As expected, in our work, an increase in molar ratio of MAA to S-AML raised the selectivity of the MIP coatings due to the increase in the concentration of prepolymerization complex thus more recognition sites. Higher S-AML/MAA ratio (1:3 or 1:2), i.e., higher levels of template, in this case might give rise to an incomplete preorganization of functional groups, leading to decreased selectivity of the resultant MIPs. The best resolution for AML enantiomers concured with the maxima in selectivity. The best MIP coating obtained was C1 in terms of selectivity and resolution, which was prepared with MAA/S-AML ratio of 4:1 (Table S2). pH effect (3.6 to 6.0), ACN content and buffer concentration in mobile phase on the separation of enantiomers can be futher optimized at the POSSbased coating containing S-AML imprints (Table S3–,5). 3.3. Stability and reproducuibility of the POSS-based columns The stability and reproducuibility of the POSS-based columns was tested by measuring the separation of of AML enantiomers. The chromatographic behavior of the AML enantiomers in the sample was compared at the beginning of a new column and after 200 injections, no untoward changes were observed in the chromatograms. The relative standard deviation (RSD) of intra-day and batch-to-batch reproducibility for the resolution of two enantiomers on the POSSbased MIP capillary was lower than 3% (Table 3).
4. Conclusion We described here a new method to prepare POSS-based MIP film inside capillaries. Separation of enantiomers of naproxen, amlodipine, and zopiclone on the POSS-based MIP capillaries can be achieved in CEC mode. Column efficiency on the POSS-based MIP coatings was beyond 30,000 plate m 1 and comparable to that recorded for the POSS-free coatings [12,13]. The optimized preparation conditions led to a resolution of enantiomers separation up to 22.3. The study displayed that POSS-based MIP is much more selective and efficient chiral stationary phase compared to conventional MIP.
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Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos. U1303202 and 21375096) and by the Hundreds Talents Program of the Chinese Academy of Science (Y12H971401).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2016.02.019.
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Coatings of molecularly imprinted polymers based on polyhedral oligomeric silsesquioxane for open tubular capillary electrochromatography Qing-Li Zhao, Jin Zhou, Li-Shun Zhang, Yan-Ping Huang, Zhao-Sheng Liu n Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 November 2015 Received in revised form 3 February 2016 Accepted 7 February 2016 Available online 7 February 2016
Polyhedral oligomeric silsesquioxane (POSS) was successfully applied, for the first time, to prepare imprinted monolithic coating for capillary electrochromatography. The imprinted monolithic coating was synthesized with a mixture of PSS-(1-Propylmethacrylate)-heptaisobutyl substituted (MA 0702), S-amlodipine (template), methacrylic acid (functional monomer), and 2-methacrylamidopropyl methacrylate (crosslinker), in a porogenic mixture of toluene–isooctane. The influence of synthesis parameters on the imprinting effect and separation performance, including the amount of MA 0702, the ratio of template to monomer, and the ratio of monomer to crosslinker, was investigated. The greatest resolution for enantiomers separation on the imprinted monolithic column prepared with MA 0702 was up to 22.3, about 2 times higher than that prepared in absence of the POSS. Column efficiency on the POSS-based MIP coatings was beyond 30,000 plate m 1. The comparisons between MIP coating synthesized with the POSS and without the POSS were made in terms of selectivity, column efficiency, and resolution. POSSbased MIP capillaries with naproxen or zopiclone was also prepared and separation of enantiomers can be achieved. & 2016 Elsevier B.V. All rights reserved.
Keywords: Molecularly imprinted polymers Capillary electrochromatography Polyhedral oligomeric silsesquioxane Chiral separation Coating
1. Introduction Molecularly imprinted polymers (MIPs) are recognition materials with pre-determined selectivity based on template synthesis, which possess affinity complementary domains of the given molecule with good physicochemical stability. The template molecules are removed afterwards by extracting with solvents/hydrolysis from the polymer, leaving a three-dimensional porous polymer with recognition sites. The resultant MIPs have been found in applications of enzyme-like catalysis [1], bio-mimetic sensors [2], drug delivery system [3], solid-phase extraction [4], and chromatographic stationary phase [5]. One particular promising direction is the use of MIPs as stationary phases in capillary electrochromatography (CEC) since CEC is still with holding its popularity due to the numerous superiorities, such as low consumed reagents, high separation efficiencies, hybrid separation principle, etc. Many formats of MIP have been used in CEC analysis, including monolithic column [6–11], open tubular capillary [12–15] and particle-based technique [16,17]. n
Corresponding author. E-mail address: [email protected] (Z.-S. Liu).
http://dx.doi.org/10.1016/j.talanta.2016.02.019 0039-9140/& 2016 Elsevier B.V. All rights reserved.
In spite of successful separation achieved, there is still much room of improvement for MIP-based CEC. For example, polymeric MIPs have the advantage of simple polymerization procedure and easy tuning of porosity and surface chemistry, but may suffer from shrinking or swelling when exposed to different solvents [3]. In contrast, inorganic materials, e.g., silica-based MIPs, can offer excellent mechanical strength and good solvent resistance. However, low tolerance to pH value of the silica-based MIP often limit its use [1,2]. Thus, organic–inorganic hybrid MIPs are supposed to combine the merits of the organic polymer and inorganic-based materials. Earlier works on the hybrid MIPs for CEC focused on the MIP film anchored covalently on the surface of silica packings. Recently, Ou et al. developed another silica-based MIP coating in capillary column for CEC, in which the MIP coating was copolymerized and anchored onto the surface of a monolithic silica containing vinyl groups [8]. An alternative approach to the preparation of the hybrid MIP for CEC is using a nonhydrolytic sol–gel protocol based on room temperature ionic liquid [9]. In additional, graphene oxide hybrid MIPs for CEC has also been reported [14]. Polyhedral oligomeric silsesquioxanes (POSS) with the formula (RSiO1.5)n are nano-building blocks [18], which embody inorganic–organic cagelike architecture containing an inner
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inorganic framework made up of silicon and oxygen. The POSSpolymers have already been used for the creation of advanced porous materials due to the reinforcement in rigidity at the molecular level [19–21]. Their unique properties of facile chemical modification, good pH tolerance, high temperature and oxidation resistance, make POSS excellent building block for constructing multi-functional materials. In addition, POSS materials are thought as the truly molecularly dispersed nanocomposites, which can be easily incorporated into common polymers via copolymerization, grafting, or blending. Recently, it was found that the suppress of the non-selective binding sites was observed in the MIPs with vinyl-POSS from the study of binding sites [22]. In spite of this, so far there are no reports on the POSS-based MIPs for CEC. This paper described a novel method for the preparation of MIP coating for CEC based on POSS. The objective of the work was to take advantage of rigidity reinforcement at the molecular level from POSS to improve the performance of MIP. Such MIPs are expected to have higher imprinting efficiency than conventional POSS-free MIPs since the specific feature of the POSS network and architecture of MIP coating have been incorporated. The influence of polymerization parameters on the electroosmotic flow (EOF) and ability of enantiomer separation of the new MIP-coated capillary was investigated.
was created at a distance of 10.5 cm from the outlet end of the MIP-coated capillary by burning out 2–3 mm segment of the polyimide outer coating. A procedures for the preparation of blank capillary column without the imprinted molecule was identical to that for the imprinted capillary. 2.3. Capillary electrochromatography Experiments of chiral separation were conducted using a K1050 system (Kaiao, Beijing, China) equipped with a CXTH-3000 software. The electrolyte used was composed of ACN/0.05 mol L 1 acetate (pH 4.2) (80/20, v/v). All the buffers were made using double distilled water and filtered with 0.2 mm membrane. Between runs the column was rinsed with mobile phase for 10 min. The separation voltage was 15 kV. UV detection was performed at 238 nm. In this paper, because some of analytes are eluted prior to EOF, separation factor is evaluated using α, which is calculated by
α = tR2/tR1
(1)
where tR1 and tR2 are the migration time of the first and second peaks. Normalized separation index, ΔtR/tR1, was used to assess the degree of enantiomer separation, where ΔtR is the difference in the elution times of the enantiomers at peak maximum.
2. Experimental 2.1. Reagents and chemicals 3-(Trimethoxysilyl) propyl methacrylate (γ-MPS, AR grade) was obtained from Acros (Geel, Belgium). Methacrylic acid (MAA, AR grade) was purchased from Beijing Donghuan Chemical Reagent (Beijing, China). Azobisisobutyronitrile (AIBN, AR grade) was from by Special Chemical Reagent Factory of Nankai University (Tianjin, China). Acetonitrile (ACN, HPLC grade) was obtained from Fisher (NJ, USA). Acetophenone (AR grade) were from Tianjin Bodi Chemical (Tianjin, China). 2-Methacrylamidopropyl methacrylate (MAM, AR grade) was synthesized using a previously published method [12]. Methacryllsobutyl POSS (MA 0702, AR grade) was purchased from Sigma–Aldrich (St. Louis, MO, USA). S-amlodipine (S-AML, AR grade) and racemic amlodipine (rac-AML, AR grade) were supplied by Hengshuo Sci. & Tech. Corp. (Hubei, China). SNaproxen (S-NAP, AR grade) and racemic naproxen (rac-NAP, AR grade) were purchased from Zhejiang Xianju Pharmaceutical Co. Ltd. (Zhejiang, China). d-Zopiclone (d-ZOP, AR grade) and racemic zopiclone (rac-ZOP, AR grade) were obtained from Kaiyuan Minsheng Sci. & Tech. Corp. (Suzhou, China). Bare fused-silica capillaries with 100 mm ID and 375 mm OD were supplied from Xinnuo Optic Fiber Plant (Hebei, China). 2.2. Preparation of POSS MIP capillary A bare fused-silica capillary was flushed with 1 mol L 1 NaOH solution followed by water for 30 min. Then the capillary was filled with 4 mL of γ-MPS solution (about 6 mmol L 1) in 1 mL acetic acid for 1.5 h. The capillary was then flushed with water and acetone, and dried with a flow of nitrogen. The imprinted molecules (0.0355 mmol), methacryllsobutyl POSS (0.166 mmol), MAA (0.142 mmol), MAM (0.142 mmol) and AIBN (14 mg) were dissolved in 1.232 mL toluene–isooctane (9/1, v/v). The pre-polymerization mixture was sonicated for 10 min and introduced into the capillary using a syringe. The capillary was sealed at both ends with a rubber septum and incubated in a water bath (50 °C) for polymerization. After polymerization, the capillary was immediately flushed using a hand-held syringe with acetonitrile and methanol/acetic acid (9:1, v/v), respectively. A detection window
3. Results and discussion 3.1. Preparation of POSS-based MIP coatings The preparation of the MIP film was based on the work by Wei et al. with some important adaptations [12]. To obtain a sufficient EOF in CEC, anionizable moiety MAA was chosen as functional monomer in place of γ-MPS. Porogen is one of the most important parameter of polymerization to form a successful polymer coating, since the affinity and morphology of the final MIP film is highly related to the nature of the porogen [12]. In the present study, it was found that binary porogens can not only dissolve enough POSS but also MAM. For example, toluene/isooctane mixture was better than ACN/isooctane on the preparation of POSS-based MIP. However, high proportion of isooctane result in lower column efficiency due to unfavorable morphology of the resultant MIP coating. In the present work, the optimum porogens was 10% (v/v) of isooctane in solvent mixture. Three different cross-linking monomers, i.e., trimethylolpropane trimethacrylate (TRIM), MAM and ethylene glycoldimethacrylate (EDMA), were investigated for their ability to determine imprinting effect on the resulting MIP (Table 1). The use of both EDMA and TRIM led to the unsuccessful MIP-coated capillaries. For example, TRIMbased MIP (C3) indicated smaller normalized separation index than POSS-free MIP. When preparing MIP with MAM as the cross-linker, however, excellent MIP capillary coatings were obtained with strong recognition ability and high column performance of the template. As a result, MAM was chosen for the crosslinker to prepare POSS-based MIP capillaries in the following study. The polymerization time provides a control over the pore property of the resulting MIP coating depending on the conversion. Generally, the specific surface area of MIP monolith decreases with the polymerization time. Although the conversion of monomers to polymer was close to quantitative after about 1.5 h, some additional structural changes still occur within the MIP coating if the system was kept longer. The normalized separation index reached a maximum after about 2 h of polymerization. Further increasing polymerization time, however, led to a blocked capillary.
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Table 1 Recipes of POSS-based MIP capillary coatings. Capillaries
S-AML (mmol)
MAA (mmol)
MA0702 (mmol)
MAM (mmol)
toluene (mL)
isooctane (mL)
time (h)
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17
0.0355 0.0355 0.0355 0.0355 0.0355 0.0355 0.0355 0.0355 0.071 0.047 0.028 0.024 0.0355c 0.0355d 0.0355 0.0355 0.0355
0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142
0.166 – 0.166 0.166 0.071 0.142 0.284 0.568 0.166 0.166 0.166 0.166 0.166 0.166 0.162 0.162 0.162
0.142 0.142 0.142a 0.142b 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.142 0.284 0.142 0.142 0.142 0.142
1108.9 1108.9 1108.9 1109 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1108.9 1232.1 1170.5 1047.3
123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 123.2 61.6 184.8
1.75 2 2 – 1.75 1.75 2 3 2 1.75 2 2 2 2.25 1.5 1.75 1.75
a
TRIM. EDMA. c S-NAP. d d-ZOP. b
Fig. 1. Electrochromatograms of rac-AML (A), S-AML (B) on S-AML imprinted capillary (C1) and rac-AML on non-imprinted capillary (C) demonstrating the imprinting effect. Conditions: capillary, 100-mm inner diameter, 47.5 cm total length, and 37.0 cm effective length; separation voltage, 15 kV; UV detector, 238 nm; acetonitrile/50 mM acetate (pH 4.2) (80/20, v/v). Elution order of two enantiomers was identificated by injecting S-AML on the imprinted capillary.
Imprinting effect of the POSS-based MIP coatings was assessed by CEC. The resolution of two enantiomers of AML was 14.8 (Fig. 1). In contrast, a nonimprinted blank capillary prepared with POSS showed no enantiomeric separation. In contrast to previous report about MIP coating with AML imprints [14,15], clear improvement in resolution and column efficiency was recorded. This can be observed from greater selectivity factor (2.05) and higher column performance of the template (up to 30,000 plates/m). Fig. 2 shows microstructure of the POSS-based films on capillary segments. Through scanning electron microscopic investigations, a layer of MIP with a thickness around 0.1–0.2 mm covering the inner surface of the capillary was observed. The Fourier-transform infrared (FTIR) spectra of the POSSbased (C1) and POSS-free MIP (C2) are shown in Fig. S1. The very strong absorption band of C1 at 1110 cm 1 for the POSS-based MIP (asymmetric nSi–O–Si) was observed, suggesting that POSS groups
Fig. 2. Image of scanning electron micrograph for POSS-based MIP-coated capillary (C1) using S-AML as template molecule. Table 2 Pore properties of POSS-based MIP and POSS-free MIPs. Polymers
SBET (m2 g 1)
St (m2 g 1)
Vp (10 3 cm3 g 1)
Dmean (nm)
POSS-based MIPs (C1) POSS-free MIPs (C2)
5.32 7.96
6.24 9.19
26.99 31.46
17.3 13.69
have been integrated into MIP matrix. The peaks at 1170 cm 1 for the POSS-free MIP were assigned to the asymmetric ester stretching bands of C–O for MAM. Table 2 summarizes the pore parameters of the POSS-based (C1) and POSS-free MIP (C2) based on the nitrogen adsorption/ desorption. The POSS-based MIP showed smaller surface areas and pore volumes but greater pore sizes than the POSS-free MIP. The pore size distribution curves obtained from N2 adsorption (Fig. S2) for the POSS-based and POSS-free MIP gave rise to the maxima, indicating a narrow distribution of pore sizes of the MIPs. In addition, Fig. 3 shows incomplete type IV of N2 adsorption–
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Fig. 3. Nitrogen adsorption–desorption isotherms of POSS-based (C1) and POSSfree MIP (C2).
Fig. 4. Comparison of the chiral separation of rac-AML on the POSS (C1) (A) and free-POSS (C2) (B) MIP coating columns. Conditions: separation voltage, 15 kV; UV detector, 238 nm; acetonitrile/50 mM acetate (pH 4.2) (80/20, v/v). Elution order of two enantiomers was identificated by injecting S-AML on the imprinted capillary.
desorption isotherms with type H4 hysteresis loops [23], suggesting narrow slit-like pores. To demonstrate the superiority of the POSS-based MIP coatings than conventional one, the POSS-free MIP coating with 80% (molar ratio) MAM was synthesized. It was observed that the POSS-free MIP showed lower selectivity (α ¼1.68) than the POSS-based MIP (α ¼ 2.05) (Fig. 4). Higher resolution of the enantiomers (Rs ¼14.8) were also recorded for the POSS-based MIP, which were about 2 times than that of the POSS-free MIP. Thus, not only affinity but also morphology on the POSS-based MIP were better than the POSS-free MIP. To look into the scope of the POSS-based strategy and demonstrate its general applicability, the MIP-coated capillaries using S-NAP and d-ZOP as templates was further synthesized, respectively. The resolutions of rac-NAP and rac-ZOP were 2.68 and 2.49, respectively, in their respective POSS-based MIP columns (Figs. 5 and 6). It was also found that in the separation of rac-NAP, resolution on the POSS-based MIP coating column was higher than the POSS-free MIP coating column [12]. As a conclusion, improved
Fig. 5. Separation of rac-naproxen on S-NAP-imprinted POSS coating capillary (C13). Conditions: separation voltage, 15 kV; UV detector, 254 nm; acetonitrile/ 10 mM acetate (pH 3.6) (80/20, v/v). Elution order of two enantiomers was identificated by injecting S-NAP on the imprinted capillary.
Fig. 6. Separation of rac-ZOP on d-ZOP-imprinted POSS coating capillary (C14). Conditions: separation voltage, 15 kV; UV detector, 254 nm; acetonitrile/10 mM acetate (pH 3.6) (80/20, v/v). Elution order of two enantiomers was identificated by injecting d-ZOP on the imprinted capillary.
performance of the resulting MIP with incorporation of POSS unit is not limited to the imprinting molecule like AML. 3.2. Effect of polymerization variables The effect of MA0702 amount on the affinity of the resulting SAML MIPs was further investigated to understand the function of POSS. The hydrophobicity inside the POSS-based MIPs significantly depend on the the monomer concentrations in the reaction solutions [21]. The shift in percent of POSS monomers was adjusted by changing the relative ratio of synthesis of MA0702 to MAM at a fixed ratio of MAA (functional monomer, 20%, molar ratio). As shown in Table S1, an increased trend of selectivity with increase in the amount of POSS monomer on the POSS-based MIPs was observed. Previous NMR study on the pseudo-pre-polymerization mixture consisting of MA0702 and template molecules showed that the interaction between POSS monomer and the template was not involved in the recognition of the resulting MIP [22]. Thus, the POSS core introduced into MIP matrix might work as a host for
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Table 3 Relative standard deviation (RSD) of reproducibility on POSS-based capillaries with S-AML imprints (C1).
Retention time (RSD%) Column efficiency (RSD%) Resolution (RSD%) α (RSD%)
Intra-capillary
Inter-capillary
The first peak
The second peak
The first peak
The second peak
0.58 2.76
0.18 3.32
0.46 2.55
1.14 3.01
2.89 0.56
1.66 0.51
encapsulating various kinds of hydrophobic molecules [19]. However, a decreased trend of the selectivity was found when the ratio of POSS monomer was beyond 50%. Because there is a minimum amount of crosslinker necessary to form a polymer network rigid enough to maintain the fidelity of the binding site, the decrease in relative ratio of the crosslinker in synthesis, MAM, will cause the decrease in imprinting effect and selectivity. Varying the molar ratio of MAA to the template molecule was performed by adjusting the amount of imprinted molecule added to an otherwise constant prepolymerization mixture. The enantiomer selectivity, in terms of a normalized separation index (ΔtR/tR1), on the POSS-based MIP ranged from 0.56 to 1.05. As expected, in our work, an increase in molar ratio of MAA to S-AML raised the selectivity of the MIP coatings due to the increase in the concentration of prepolymerization complex thus more recognition sites. Higher S-AML/MAA ratio (1:3 or 1:2), i.e., higher levels of template, in this case might give rise to an incomplete preorganization of functional groups, leading to decreased selectivity of the resultant MIPs. The best resolution for AML enantiomers concured with the maxima in selectivity. The best MIP coating obtained was C1 in terms of selectivity and resolution, which was prepared with MAA/S-AML ratio of 4:1 (Table S2). pH effect (3.6 to 6.0), ACN content and buffer concentration in mobile phase on the separation of enantiomers can be futher optimized at the POSSbased coating containing S-AML imprints (Table S3–,5). 3.3. Stability and reproducuibility of the POSS-based columns The stability and reproducuibility of the POSS-based columns was tested by measuring the separation of of AML enantiomers. The chromatographic behavior of the AML enantiomers in the sample was compared at the beginning of a new column and after 200 injections, no untoward changes were observed in the chromatograms. The relative standard deviation (RSD) of intra-day and batch-to-batch reproducibility for the resolution of two enantiomers on the POSSbased MIP capillary was lower than 3% (Table 3).
4. Conclusion We described here a new method to prepare POSS-based MIP film inside capillaries. Separation of enantiomers of naproxen, amlodipine, and zopiclone on the POSS-based MIP capillaries can be achieved in CEC mode. Column efficiency on the POSS-based MIP coatings was beyond 30,000 plate m 1 and comparable to that recorded for the POSS-free coatings [12,13]. The optimized preparation conditions led to a resolution of enantiomers separation up to 22.3. The study displayed that POSS-based MIP is much more selective and efficient chiral stationary phase compared to conventional MIP.
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Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos. U1303202 and 21375096) and by the Hundreds Talents Program of the Chinese Academy of Science (Y12H971401).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2016.02.019.
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