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Liquid crystal-based molecularly imprinted nanoparticles with low crosslinking for capillary electrochromatography
Journal of Chromatography A, 1309 (2013) 84–89
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Liquid crystal-based molecularly imprinted nanoparticles with low crosslinking for capillary electrochromatography Xiao Liu, Hai-Yan Zong, Yan-Ping Huang, Zhao-Sheng Liu ∗ Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
a r t i c l e
i n f o
Article history: Received 13 May 2013 Received in revised form 5 August 2013 Accepted 6 August 2013 Available online 9 August 2013 Keywords: Molecularly imprinted polymer Low crosslinking Liquid crystal Nanoparticles Zopiclone Enantiomers separation
a b s t r a c t In this work, molecularly imprinted polymer (MIP) nanoparticles were prepared at a low level of crosslinking in the presence of liquid crystalline monomer as physical crosslinker to replace the most of chemical crosslinker. The imprinted nanoparticles against d-zopiclone were synthesized by precipitation polymerization using a mixture of methacrylic acid, ethylene glycol dimethacrylate, and liquid crystalline monomers. The resulting d-zopiclone imprinted nanoparticles were evaluated in capillary electrochromatography by partial filling technique. The resolution of enantiomer separation achieved on the d-zopiclone-imprinted nanoparticles was up to 3.29 and the column efficiency of zopiclone enantiomer (up to 66,900 plates/m) with good peak symmetry was obtained. The imprinted nanoparticles prepared in the presence of liquid crystalline monomer can retain affinity and specificity for template even when prepared with a level of cross-linker as low as 5%. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Molecular imprinting is a means by which specific recognition sites for a target molecule may be incorporated into a polymeric phase. The resultant polymers, i.e., molecularly imprinted polymers (MIPs) are synthesized by an assembly of functional monomers around a chosen template [1,2]. The complex is then immobilized into a polymer matrix by copolymerization with cross-linking monomers. After extracting the template from the polymer matrix, complementary cavities with respect to shape and functional groups remain. Compared with bio-antibodies, MIPs possess many advantages especially of the low cost, good physical and chemical stability. Therefore, MIPs have been applied widely in many fields such as solid-phase extraction [3,4], antibody mimics [5], catalysis [6], drug delivery systems [7] and chromatographic stationary phases [8]. To preserve the memory of template in MIPs, a large amount of cross-linking agent has to be used (around 80–90%) to restrict the relaxation phenomena of the polymer backbones. As a result, the imprinted network is very stiff; this lowers the probability of interactions between the template and the network and hinders the mechanism of extraction of the template from the imprinted cavities. Therefore, only a part of the imprinted sites (around 10–20%)
∗ Corresponding author. Fax: +86 022 23536746. E-mail address: [email protected] (Z.-S. Liu). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.08.024
remained available, and the capacity of the network was drastically reduced [5,7]. Recently, the uses of liquid crystalline polymer networks for the molecular imprinting technique have shown encouraging perspectives [9]. In liquid crystalline-based imprinted materials, imprinting sites were preserved by virtue of the orientation imposed on the mesogenic side-groups and the coupling between the mesogenic side-groups and the polymer backbone. Mesomorphic order provides significant enhancement to the bonding between the template and the liquid crystalline network and reinforces the shape memory of the imprinted cavities. As a result, low levels of cross-linking (5–20 mol% of the monomer units) were sufficient to imprint the memory of a template. Because the liquid crystalline character avoids high cross-linking densities, the resultant MIPs show similar selectivity but much higher capacity compared with classical MIPs. Such liquid-crystalline MIPs at low crosslinking degree have found in successful application of catalysts [10] and sensors [11]. However, liquid-crystalline MIPs as stationary phase are still challenge due to the nature of elastomer of liquid crystal MIP, which is hard to resist the high pressure from HPLC. Capillary electrochromatography (CEC) is a micro-separation method which combines the advantage of the high separation efficiency of capillary electrophoresis (CE) and high selectivity offered by HPLC. In CEC, the driving force of mobile phase is electroosmotic flow (EOF) which results in an almost flat profile and higher column efficiency can be obtained. With EOF as flow driver instead
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of hydraulic flow, high pressure applied is avoided. Thus, the combination of liquid-crystalline MIP and CEC is expected to address the issues of liquid-crystalline MIP as stationary phase. Recently, a liquid crystal MIP coating with low level of EDMA was constructed in a capillary [12] and chiral separation is achieved in CEC mode, demonstrating that the CEC-based liquid crystal MIP is appealing as chiral stationary phase. Up to now, there are mainly three formats of MIP that can be applied to CEC, namely the monolith [13–19], the coating [20–24] and particle-based technique [25–33]. To extend the application of liquid crystal MIP, liquid crystalline MIP nanoparticles were tried for enantiomers separation in CEC mode. For this purpose, d-zopiclone imprinted nanoparticles were prepared under the low crosslinking density with liquid crystalline monomers as physical crosslinker to replace the most of chemical crosslinker. The liquid crystal MIP nanoparticles were prepared in precipitation polymerization mode and evaluated in CEC by partial filling technique (Fig. 1). Using the partial filling technique, the MIP nanoparticles are injected as a plug prior to the sample. The electrophoretic mobility of the enantiomers is in the same direction as the EOF, while the MIP nanoparticles have an electrophoretic mobility that is opposite to the EOF. Thus, if the length of the MIP nanoparticles plug is carefully chosen, the enantiomers will reach the detection window prior to the MIP nanoparticles plug to obtain chiral separation. Furthermore, the effects of polymerization variables and CEC parameters on separation of enantiomers were also investigated.
2. Materials and methods 2.1. Chemicals d- and rac-zopiclone (ZOP) were obtained from Kaiyuan Minsheng Sci. & Tech. Corp. (Suzhou, China). 4-Methyl phenyl dicyclohexyl ethylene (MPDE), 4-cyano phenyl cyclohexyl ethylene (CPCE), 4-cyano phenyl cyclohexyl propylene (MPDP), and 3, 4-difluorophenyl dicyclohexyl propylene (DFDP) were purchased from Hebei Meixin (Hebei, China). Methacrylic acid (MAA) was purchased from Beijing Pubo Biotech. (Beijing, China). Ethylene glycol dimethacrylate (EDMA) was from Sigma (St. Louis, MO, USA). 2,2-Azobis (2-isobutyronitrile) (AIBN) was purchased from Special Chemical Reagent Factory of Nankai University (Tianjin, China). Acetonitrile (ACN, HPLC grade) was from Fisher (NJ, USA). 3-(Trimethoxysilyl)propyl methacrylate (␥-MPS) was from Acros (Geel, Belgium). Other analytical reagents were obtained from Tianjin Chemical Reagent Co. Ltd. (Tianjin, China). Fused-silica capillaries with 100 m ID and 375 m OD were purchased from Xinnuo Optic Fiber Plant (Hebei, China).
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polymer nanoparticles were dried and stored at room temperature until use. 2.3. CEC All CEC experiments were carried out on a K1050 system (Kaiao, Beijing, China) equipped with a UV detector. A Lenovo personal computer was used for data processing. In our work, in order to allow the analyte to reach the detection window prior to the MIP plug, a capillary was derivatized with ␥-MPS to reduce EOF [28]. The EOF of such a capillary was reduced two to threefold compared to an uncoated fused-silica capillary (from 7.67 × 10−8 m2 V−1 s−1 to 3.07 × 10−8 m2 V−1 s−1 ). The derivatization was performed by successively flushing the capillary with 1 mol/L NaOH, water, 0.1 mol/L HCl and water for 2 h, respectively, followed by drying with a stream of nitrogen gas. Then a solution of toluene/␥-MPS (85:15, v/v) was filled into the capillary and kept overnight. Finally the capillary was flushed with toluene and dried. A ␥-MPS-derivatized capillary (74.5 cm total length, 64.5 cm effective length or 50 cm total length, 40 cm effective length) was used in CEC separation. In this work, longer capillary was used to avoid the co-elution of sample and MIP particles on shorter column at some buffer system. The electrolyte used was composed of ACN/20 mmol/L acetate-sodium acetate buffer solution (pH 3.6) (85:15 or 80:20, v/v). All the electrolyte was made using double distilled water and filtered with 0.2 m microporous film. The MIP microparticles were suspended in the electrolyte to a maximum amount of 5 mg/mL to get stable suspensions since increasing the MIP content further resulted in sedimentation due to the aggregation of the particles. The samples of 40 mol/L concentrations were prepared from 5 mmol/L ACN solutions diluted with electrolyte. All solutions were degassed by sonication. Before the CEC analysis every day, the capillary was rinsed with water and electrolyte for 10 min respectively. Between consecutive runs, the capillary was rinsed with electrolyte for 1 min. The MINP suspensions and the samples were introduced hydrodynamically at 15 mbar for 5.0 s and 3.0 s, respectively. In this paper, because some of analytes are eluted prior EOF, separation factor is evaluated using ˛, which is calculated by [18],˛ = t2 /t1 where t1 and t2 are the retention time of the first and second peak. The degree of enantiomer separation was represented by a normalized separation index tR /tR1 , where tR is the difference in the elution time of the enantiomers at peak maximum and tR1 is the retention time of the first eluted enantiomer. The resolution (Rs ) was calculated according to Rs = (t2 − t1 )/0.5(W2 + W1 ), W is the width at the baseline between tangents drawn to inflection points for the peak. 2.4. Characterization of the liquid crystal MIP nanoparticles
2.2. Preparation of liquid crystal MIP nanoparticles The template molecule d-zopiclone, the radical initiator AIBN (4.3 mg/mL), liquid crystalline monomer, functional monomers (MAA) and cross-linking monomers (EDMA) were dissolved in solvents in proportions stated in Table 1. The pre-polymerization mixture was sonicated for 15 min followed by degassing by a stream of nitrogen for 5 min. The flask was sealed and put into a water bath and heated at 55 ◦ C for 1 h. After polymerization, the obtained suspension was recovered by successive centrifugation (13,000 rpm for 15 min), after which the solid phase was resuspended in methanol/acetic acid (7:3, v/v), sonicated for 10 min in an ultrasonic bath, agitated for 30 min, and was repeated twice to remove the template. At last, only ACN was used for suspension of the particles to wash acetic acid. The corresponding blank polymers were prepared in the absence of the template. The resultant
Transmission electron microscopy (TEM) was performed on a JEM100CXII UHR microscope from JEOL, with a 100 kV acceleration tension. The porosity of the MIP particles was measured at 77 K by nitrogen adsorption–desorption isotherms using a V-Sorb 2800TP Surface Area and Pore Distribution Analyzer instrument (Gold APP Instruments Corporation China, Beijing, China). 3. Results and discussion 3.1. Synthesis of liquid crystal-based imprinted nanoparticles with d-zopiclone imprints Conventional approaches for the preparation of spherical MIP nanoparticles are based on emulsion polymerization techniques. This process is a heterophase polymerization in which the use of surfactants as well as co-stabilizers, two non-miscible liquid
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Fig. 1. The schematic images of the preparation of nano-MIP and CEC with the MIP.
phases are emulsified to become homogeneous and stable. The nanodroplets produced in this way from the monomer, the template molecule and the osmotic reagent form nanoreactors in which the polymerization takes place. Using precipitation polymerization protocol, in contrast, small spherical polymer particles were successfully synthesized without any need for stabilizing surfactants, which is beneficial in terms of particle cleanup. The first step in the present experiments was to choose an appropriate solvent or solvent mixture that would allow for narrow disperse particles. Toluene and ACN are the common porogens in the preparation of MIP nanoparticles and was used to prepare d-ZOP-imprinted nanoparticles in scouting experiments about polymerization parameters. In our study, it was found that the liquid-crystal monomer could be dissolved well in toluene. In contrast, d-ZOP and AIBN could not be dissolved completely in toluene. Thus, a mixture of toluene/ACN was used as the porogen to prepare MIP nanoparticles. Lower ratio of toluene/ACN (e.g., 5:5) led to a limited amount of MPDE dissolved. In contrast, when higher ratio of toluene/ACN (e.g., 9:1) was used, d-ZOP could not be dissolved completely. It was found that the optimized ratio of toluene/ACN was 7:3. The visualization and nanometer size of the resultant MIP nanoparticles (prepared with liquid crystalline monomer MPDE) were demonstrated by TEM. As shown in Fig. 2, the resulting particles indicate a size distribution of 150–180 nm. Compared to previous MIP [33] prepared without liquid crystalline monomer, there was not agglomeration between the MIP nanoparticles. In contrast, the liquid crystal MIP nanoparticles were more uniform and narrow-disperse. Such ability to synthesize monodisperse MIP beads may be attributed to the decrease in the cross-linking density (20%) from the liquid crystal MIP, as observed by Yoshimatsu et al. [34].
Fig. 2. Transmission electron microscope of MIP 1 using d-ZOP as template molecule. (A) High magnification; (B) low magnification.
Table 1 Recipes of preparation for liquid crystal MIP nanoparticles. MIP
d-ZOP (mmol)
MIP 1 MIP 2 MIP 3 MIP 4 MIP 5 MIP 6 MIP 7 MIP 8 MIP 9 MIP10 MIP11 NIP1
0.08 0.08 0.08 0.08 0.08 0.16 0.107 0.064 0.08 0.08 0.08
Liquid crystalline monomer (mmol) MPDE
CPCE
MPDP
EDMA (mmol)
0. 64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64
0.64 0.16 0.32 0.48 0.80 0.64 0.64 0.64 0.64 0.64 0.64 0.64
DFDP
1.92 2.40 2.24 2.08 1.76 1.92 1.92 1.92 1.92 1.92 1.92 1.92
MAA (mmol)
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our MIP-coated capillary with d-ZOP imprinting with comparable enantiomers resolution [28]. 3.3. Influence of polymerization parameters on chiral separation
Fig. 3. CEC analysis of rac-ZOP (A) and d-ZOP (B) on MIP nanoparticles and rac-ZOP on NIP nanoparticles (C) demonstrating the imprinting effect and identifying the peaks. Conditions: separation voltage, 15 kV; UV–vis detector, 254 nm; ACN/20 mmol/L acetate (pH 3.6) (80/20, v/v); capillary length, 50 cm.
The d-ZOP-imprinted nanoparticles with a pore volume of 0.31 cm3 /g showed “type IV” isotherms which are usually related to meso-macroporous materials. The hysteresis loops resemble H3 types, suggesting that the nanoparticles are porous materials with specific structure of slit-shapes pores [35]. BET area of the polymer materials (SBET = 33.065 m2 /g) is calculated from the adsorption data using 0.162 nm2 as the molecular cross-sectional area for adsorbed nitrogen molecules. This isotherm of adsorption versus relative pressure is transformed to average pore diameter of 46.3 nm calculated by Barrett–Joyner–Halenda (BJH) method [35]. 3.2. Confirming the imprinting effect of MIP nanoparticles by CEC The imprinting effect of the low crosslinking MIP nanoparticles made with liquid crystalline monomers was evaluated by enantiomer separation in CEC mode. Fig. 3A and B illustrated the separation of rac-ZOP on the d-ZOP-imprinted nanoparticles with liquid crystalline monomer MPDE, depicting the characteristic elution order and the selective retention of the template. The resolution of two enantiomers was up to 3.29 and high column performance of the template (up to 26,200 plates/m) was obtained. Compared with MIPs nanoparticles with high level crosslinking [33], the liquid crystal MIPs with low crosslinking degree have comparable selectivity. The non-imprinted polymer (NIP) nanoparticle prepared with liquid crystalline monomers showed no enantiomeric separation (Fig. 3C). The fact that the reference polymer prepared in the absence of the template was unable to resolve the enantiomers of rac-ZOP (Table 2) and the d-enantiomer always was the last eluted when the enantiomers were separated confirms that the separation is based on molecular recognition achieved through molecular imprinting. In addition, even if the capillary is deactivated, this work indicates shorter analysis time than Table 2 Effect of the different levels of crosslinking on d-ZOP imprinted nanoparticles. Level of crosslinking 5% 10% 15% 20% 25%
Nl (m−1 )
Nd (m−1 )
48,600 24,400 46,500 66,900 25,000
28,700 14,900 12,600 26,200 16,700
˛
tR /tR1
Rs
1.12 1.10 1.09 1.07 1. 10
0.12 0.10 0.09 0.07 0.10
5.21 3.05 2.99 3.29 3.32
CEC conditions: separation voltage, 15 kV; UV–vis detector, 254 nm; ACN/20 mM acetate (pH 3.6) (85/15, v/v), capillary length, 75 cm.
3.3.1. Role of liquid crystalline monomers To investigate the function of liquid crystalline monomers on the selectivity, the synthesis of MIP nanoparticles with varied amounts of cross-linker in the presence of liquid crystalline monomers was conducted. All the MIP nanoparticles were prepared at a fixed ratio of MAA (functional monomer, 20%, molar ratios). Variation of cross-linking degree was performed by altering the relative ratio of liquid crystalline monomers to cross-linker, EDMA. In general, the increase in the content of cross-linking monomers could cause an increase in selectivity of resulting MIP and there will be no imprinting effect when the concentration of cross-linker approaches 12% [1]. In the present study, even if the level of crosslinking was as low as 5%, enantiomer selectivity can be realized on the resulting MIP nanoparticles. Interestingly, the d-ZOP-MIP nanoparticles could still provide baseline separation of rac-ZOP even with an increased resolution than the liquid crystal MIP with higher level of crosslinker (Table 2). The reason for this behavior could be an effect originating in molecule chiralty caused by the liquid crystal monomers [10,11], which comes from a helical twist of the liquid crystalline structure due to the presence of a small percentage of chiral dopant in a nematic phase leading to a cholesteric phase. However, further decrease in the content of EDMA led to very low enantiomer selectivity. It is well known that the presence of a small percentage of chiral dopant in a nematic phase induces a helical twist of the liquid crystalline structure leading to a cholesteric phase. Therefore, the introduction of a chiral template during the synthesis of the liquid crystal-based MIP leads to two different levels of chirality. The first one is at the cavity level, exactly like in regular MIPs, and the second one is due to the presence of liquid crystal moieties. To investigate what mechanism of an enantiomer separation process comes from the chiral cavity or from the chiral helix owing to the cholesteric phase, we also prepared liquid crystal polymer without functional monomer, MAA, in the presence of the template. However, chiral separation for rac-ZOP was not achieved. It seemed that a non-imprinted material adsorbs equally both enantiomers, the doped material, presenting only chirality at the mesoscopic level coming from the cholesteric helix, showed only a limited effect in the recognition process. Thus, the helical structure, induced by the mesogenic groups appeared to increase the accessibility of binding sites and the mass transfer of liquid crystal MIP compared to non-liquid crystal one. 3.3.2. Template to monomer ratio The influence of template to monomer molar ratio (t/m-ratio) on the imprinting effect of the liquid crystal MIP nanoparticles was investigated by varying t/m-ratio with four levels. The four MIP nanoparticles all indicated the ability to separate rac-ZOP. The separation factors varied little and ranged from 1.18 to 1.23, which can be assumed that this preparation protocol yielded MIP particles with similar affinity. As shown in Fig. 4, as the t/m-ratio decreased to 1:8, higher resolution and column efficiency of d-ZOP was obtained and the peak shape of d-ZOP was improved. This result may be due to the pronounced influence of the presence of the template on the size of the obtained particles rather than selective interaction. While t/m-ratio decreased to 1:10, the greatest resolution (6.23) was observed due to significant increase in column efficiency of l-ZOP. Previously, enantiomer separation even on MIPs with an extremely low template-to-monomer (1:80) has been demonstrated [30]. However, baseline separation was not achieved due to
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X. Liu et al. / J. Chromatogr. A 1309 (2013) 84–89 Table 4 Reproducibility of the enantiomer separation of rac-ZOP using liquid crystal MIP nanoparticles.
Retention time (RSD%) Column efficiency (RSD%) Resolution (RSD%) ˛ (RSD%)
l-ZOP
d-ZOP
0.77 7.71 3.45 1.47
2.59 8.99
Separation voltage: 5 kV; UV–vis detector: 230 nm; ACN/30 mmol/L acetate (pH 3.6) (80/20, v/v); capillary length, 50 cm.
Fig. 4. The enantiomer separations with MIP nanoparticles of different t/m ratio. Conditions: separation voltage, 15 kV; UV–vis detector, 254 nm; ACN/20 mmol/L acetate (pH 3.6) (80/20, v/v); capillary length, 50 cm.
lower chain of liquid crystal groups are toughen enough to keep the memory to the template. Furthermore, it seemed that the bulkiness of the pending groups and their chemical interactions play no role to toughen the network. Variations of functional groups in liquid crystalline also showed little effect on the selectivity of the resulting MIP nanoparticles. 3.4. Effect of CEC parameters on separation of enantiomers
Fig. 5. The enantiomer separations with MIP nanoparticles of different liquid crystalline monomers. Conditions: separation voltage, 15 kV; UV–vis detector, 254 nm; ACN/20 mmol/L acetate (pH 3.6) (80/20, v/v); capillary length, 50 cm.
the phenomenon of peak tailing. The results here obtained clearly show higher resolution and column efficiency, probably owing to the homogeneous and monodispersed nature derived from the liquid crystal MIP nanoparticles. A t/m-ratio of 1:8 was chosen for the following studies in terms of shorter separation time with adequate resolution. 3.3.3. Liquid crystalline monomer To study the role of the pending mesogenic groups of liquid crystalline on the imprinting of the low crosslinking MIP, four different liquid crystalline monomers (MPDE, CPCE, MPDP, and DFDP) were used as physical crosslinker to prepare MIPs, respectively. As shown in Fig. 5, they all had the ability to separate rac-ZOP. From a comparison of Table 3, the length variation of liquid crystal groups indicates little effect on the selectivity of liquid crystal MIP, which suggests that the liquid crystalline interactions with Table 3 Effect of the different liquid crystalline monomer on d-ZOP imprinted nanoparticles. Liquid crystalline monomers
Nl (m−1 )
Nd (m−1 )
˛
tR /tR1
Rs
MPDE CPCE MPDP DFDP
14,500 10,000 21,100 7200
15,800 6400 10,600 6100
1.20 1.22 1.21 1.23
0.20 0.22 0.21 0.23
5.60 4.32 4.33 4.28
To study weather the ability of liquid crystalline to stable the imprinted cavities may be varied in different solvents, a serious of experiments for the separation of ZOP enantiomers with different mobile phase was performed. A high organic content in the electrolyte has been proved to be optimal for MIP-based CEC separations [28]. In this work, ACN was chosen as the organic modifier and the level of ACN content on enantiomers separation was investigated. Increasing ACN content led to the increased column efficiency of d-ZOP and shorter elution time, while the resolution (Rs ) for zopiclone enantiomers and separation factor (␣) were decreased. This trend is in line with the results of non-liquid crystal MIPs [30]. The result suggested that the mechanism is not shifted when the groups of liquid crystalline are incorporated into MIP matrix, compared to MIPs without liquid crystalline. Baseline separation for rac-ZOP could not be achieved when ACN content was increased to 90%. In 70% ACN, the elution time of liquid crystal MIP nanoparticles was too long (approximately 30 min). 85% ACN in the electrolyte was applied in following experiments as a compromise between the resolution for zopiclone enantiomers and the elution time of liquid crystal MIP nanoparticles. The pH effect in mobile phase on enantiomers separation with liquid crystal MIP nanoparticles was examined from 3.0 to 4.8. When the value of pH increased from 3.0 to 3.6, the column efficiency of d-ZOP increased, but the resolution (Rs ), separation factor (˛) and normalized separation index (tR /tR1 ) decreased. There was no enantiomer separation as the pH value was increased to 4.8. This result is different from previously reported non-liquid crystal MIP [33], suggesting a faster increase in nonselective sites with increasing pH value than selective sites on the liquid crystal-based MIP [36]. The highest column efficiency of d-ZOP was obtained when pH value was 3.6. 3.5. Reproducibility The reproducibility of various CEC parameters is a critical consideration in the field of the preparation and the application of MIP nanoparticles. In our study, a number of parameters such as retention time and Rs were measured to test the reproducibility of the liquid crystal MIP with d-ZOP imprinting. The results of retention time, column efficiency and resolution of d- and l-ZOP and Rs of two enantiomers are shown in Table 4. The injection reproducibility is averaged from the results of continuously repeating five injections. RSD for the resolution of two enantiomers is lower than 3%. Compared to MIP nanoparticles prepared with conventional crosslinking agent [33], it seemed that the MIP nanoparticles using liquid crystal monomer had better reproducibility. In addition, the
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dissolving of derivatized capillary was not observed after 100 runs in our investigation. This result demonstrated the advantage of monodisperse nanoparticles again. 3.6. Loading capacity of liquid crystal MIP Thanks to the low content of cross-linker, the liquid crystalline networks exhibit a high molecular trapping capacity. By increasing the sample load of the template, the separation factor typically decreased rapidly, leveling off at a sample load of 20 mol/L. It should be noted that the saturation capacity (number of available binding sites) of the polymers in this study is high in relation to the amount of template added to the monomer mixture (theoretical maximum number of sites), which can be estimated from the loading factor giving column overloading. The amount of template incorporated during the synthesis of MIP corresponds to 8 mol/g of polymer. The amounts are notably higher than those of the usual imprinted materials (around 2.5 mol/g of polymer) [33] in which high cross-linker rates are needed.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
[12] [13] [14] [15] [16] [17] [18]
4. Conclusion [19]
The low cross-linked MIP nanoparticles based on liquid crystalline monomers were successfully prepared for the first time. Separation of racemic zopiclone was achieved in CEC mode with good peak symmetry. Different liquid crystalline monomers were able to form MIP nanoparticles with low crosslinking degree. Even if the level of crosslinking of chemical crosslinkers was as low as 5%, enantiomer selectivity was achieved. Compared to previous prepared MIP nanoparticles [30] at high level of crosslinker, the liquid crystal MIP nanoparticles were more uniform and showed higher reproducibility of CEC. We believe that the results presented here should be valuable for future research toward development of new MIP-based separation systems at low crosslinking. Acknowledgments This work was supported by the Hundreds Talents Program of the Chinese Academy of Sciences and supported by the National Natural Science Foundation of China (Grant No. 21075090).
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[20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]
L. Levi, V. Raim, S. Srebnik, J. Mol. Recognit. 24 (2011) 883. S. Tokonami, H. Shiigi, T. Nagaoka, Anal. Chim. Acta 641 (2009) 7. F. Barahona, E. Turiel, A. Martín-Esteban, J. Chromatogr. A 1218 (2011) 7065. E. Turiel, A. Martín-Esteban, J. Sep. Sci. 32 (2009) 3278. K.D. Shimizu, C.J. Stephenson, Curr. Opin. Chem. Biol. 14 (2010) 743. M. Resmini, Anal. Bioanal. Chem. 402 (2012) 3021. D. Cunliffe, A. Kirby, C. Alexander, Adv. Drug Deliv. Rev. 57 (2005) 1836. J. Haginaka, J. Sep. Sci. 32 (2009) 1548. J.-D. Marty, M. Tizra, M. Mauzac, I. Rico-Lattes, A. Lattes, Macromolecules 32 (1999) 8674. M. Weyland, S. Ferrere, A. Lattes, A.-F. Mingotaud, M. Mauzac, Liq. Cryst. 35 (2008) 219. C. Binet, D. Bourrier, M. Dilhan, D. Esteve, S. Ferrere, J.C. Garrigue, H. Granier, A. Lattes, A.M. Gue, M. Mauzac, A.F. Mingotaud, Talanta 69 (2006) 757. Z.-H. Wei, L.-N. Mu, Y.-P. Huang, Z.-S. Liu, J. Chromatogr. A 1273 (2012) 115. Z.-H. Wei, L.-N. Mu, Q.-Q. Pang, Y.-P. Huang, Z.-S. Liu, Electrophoresis 33 (2012) 3021. J. He, G. Fang, Q. Deng, S. Wang, Anal. Chim. Acta 692 (2011) 57. B.-Y. Huang, Y.-C. Chen, G.-R. Wang, C.-Y. Liu, J. Chromatogr. A 1218 (2011) 849. S. Liao, X. Wang, X. Lin, Z. Xie, Electrophoresis 31 (2010) 2822. L. Schweitz, L.I. Andersson, S. Nilsson, Anal. Chem. 69 (1997) 1179. C. Zheng, Z.-S. Liu, R.-Y. Gao, L.-H. Zhang, Y.-K. Zhang, Anal. Bioanal. Chem. 388 (2007) 5. J. Ou, X. Li, S. Feng, J. Dong, X. Dong, L. Kong, M. Ye, H. Zou, Anal. Chem. 79 (2007) 639. Z.-H. Wei, X. Wu, B. Zhang, R. Li, Y.-P. Huang, Z.-S. Liu, J. Chromatogr. A 1218 (2011) 6498. Y.C. Huang, C.C. Lin, C.Y. Liu, Electrophoresis 25 (2004) 554. S.A. Zaidi, W.J. Cheong, J. Chromatogr. A 1216 (2009) 2947. O. Brüggermann, R. Freitag, M.J. Whitcombe, E.N. Vulfson, J. Chromatogr. A 781 (1997) 43. J.Z. Tan, V.T. Remcho, Electrophoresis 19 (1998) 2055. L. Schweitz, Anal. Chem. 74 (2002) 1192. L. Schweitz, P. Spégel, S. Nilsson, Analyst 125 (2000) 1899. X.X. Shi, L. Xu, H.Q. Duan, Y.P. Huang, Z.S. Liu, Electrophoresis 32 (2011) 1348. X. Wu, Z.-H. Wei, Y.-P. Huang, Z.-S. Liu, Chromatographia 72 (2010) 101. F. Priego-Capote, L. Ye, S. Shakil, S.A. Shamsi, S. Nilsson, Anal. Chem. 80 (2008) 2881. P. Spégel, L. Schweitz, S. Nilsson, Anal. Chem. 75 (2003) 6608. P. Spégel, L. Schweitz, S. Nilsson, Electrophoresis 22 (2001) 3833. J.M. Lin, K. Uchiyama, T. Hobo, Chromatographia 47 (1998) 625. X. Liu, Z.-H. Wei, Y.-P. Huang, J.-R. Yang, Z.-S. Liu, J. Chromatogr. A 1264 (2012) 137. K. Yoshimatsu, K. Reimhult, A. Krozer, K. Mosbach, K. Sode, L. Ye, Anal. Chim. Acta 584 (2007) 112. K.S.W. Sing, Pure Appl. Chem. 54 (1982) 2201. B. Sellergren, K.J. Shea, J. Chromatogr. A 654 (1993) 17.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Liquid crystal-based molecularly imprinted nanoparticles with low crosslinking for capillary electrochromatography Xiao Liu, Hai-Yan Zong, Yan-Ping Huang, Zhao-Sheng Liu ∗ Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
a r t i c l e
i n f o
Article history: Received 13 May 2013 Received in revised form 5 August 2013 Accepted 6 August 2013 Available online 9 August 2013 Keywords: Molecularly imprinted polymer Low crosslinking Liquid crystal Nanoparticles Zopiclone Enantiomers separation
a b s t r a c t In this work, molecularly imprinted polymer (MIP) nanoparticles were prepared at a low level of crosslinking in the presence of liquid crystalline monomer as physical crosslinker to replace the most of chemical crosslinker. The imprinted nanoparticles against d-zopiclone were synthesized by precipitation polymerization using a mixture of methacrylic acid, ethylene glycol dimethacrylate, and liquid crystalline monomers. The resulting d-zopiclone imprinted nanoparticles were evaluated in capillary electrochromatography by partial filling technique. The resolution of enantiomer separation achieved on the d-zopiclone-imprinted nanoparticles was up to 3.29 and the column efficiency of zopiclone enantiomer (up to 66,900 plates/m) with good peak symmetry was obtained. The imprinted nanoparticles prepared in the presence of liquid crystalline monomer can retain affinity and specificity for template even when prepared with a level of cross-linker as low as 5%. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Molecular imprinting is a means by which specific recognition sites for a target molecule may be incorporated into a polymeric phase. The resultant polymers, i.e., molecularly imprinted polymers (MIPs) are synthesized by an assembly of functional monomers around a chosen template [1,2]. The complex is then immobilized into a polymer matrix by copolymerization with cross-linking monomers. After extracting the template from the polymer matrix, complementary cavities with respect to shape and functional groups remain. Compared with bio-antibodies, MIPs possess many advantages especially of the low cost, good physical and chemical stability. Therefore, MIPs have been applied widely in many fields such as solid-phase extraction [3,4], antibody mimics [5], catalysis [6], drug delivery systems [7] and chromatographic stationary phases [8]. To preserve the memory of template in MIPs, a large amount of cross-linking agent has to be used (around 80–90%) to restrict the relaxation phenomena of the polymer backbones. As a result, the imprinted network is very stiff; this lowers the probability of interactions between the template and the network and hinders the mechanism of extraction of the template from the imprinted cavities. Therefore, only a part of the imprinted sites (around 10–20%)
∗ Corresponding author. Fax: +86 022 23536746. E-mail address: [email protected] (Z.-S. Liu). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.08.024
remained available, and the capacity of the network was drastically reduced [5,7]. Recently, the uses of liquid crystalline polymer networks for the molecular imprinting technique have shown encouraging perspectives [9]. In liquid crystalline-based imprinted materials, imprinting sites were preserved by virtue of the orientation imposed on the mesogenic side-groups and the coupling between the mesogenic side-groups and the polymer backbone. Mesomorphic order provides significant enhancement to the bonding between the template and the liquid crystalline network and reinforces the shape memory of the imprinted cavities. As a result, low levels of cross-linking (5–20 mol% of the monomer units) were sufficient to imprint the memory of a template. Because the liquid crystalline character avoids high cross-linking densities, the resultant MIPs show similar selectivity but much higher capacity compared with classical MIPs. Such liquid-crystalline MIPs at low crosslinking degree have found in successful application of catalysts [10] and sensors [11]. However, liquid-crystalline MIPs as stationary phase are still challenge due to the nature of elastomer of liquid crystal MIP, which is hard to resist the high pressure from HPLC. Capillary electrochromatography (CEC) is a micro-separation method which combines the advantage of the high separation efficiency of capillary electrophoresis (CE) and high selectivity offered by HPLC. In CEC, the driving force of mobile phase is electroosmotic flow (EOF) which results in an almost flat profile and higher column efficiency can be obtained. With EOF as flow driver instead
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of hydraulic flow, high pressure applied is avoided. Thus, the combination of liquid-crystalline MIP and CEC is expected to address the issues of liquid-crystalline MIP as stationary phase. Recently, a liquid crystal MIP coating with low level of EDMA was constructed in a capillary [12] and chiral separation is achieved in CEC mode, demonstrating that the CEC-based liquid crystal MIP is appealing as chiral stationary phase. Up to now, there are mainly three formats of MIP that can be applied to CEC, namely the monolith [13–19], the coating [20–24] and particle-based technique [25–33]. To extend the application of liquid crystal MIP, liquid crystalline MIP nanoparticles were tried for enantiomers separation in CEC mode. For this purpose, d-zopiclone imprinted nanoparticles were prepared under the low crosslinking density with liquid crystalline monomers as physical crosslinker to replace the most of chemical crosslinker. The liquid crystal MIP nanoparticles were prepared in precipitation polymerization mode and evaluated in CEC by partial filling technique (Fig. 1). Using the partial filling technique, the MIP nanoparticles are injected as a plug prior to the sample. The electrophoretic mobility of the enantiomers is in the same direction as the EOF, while the MIP nanoparticles have an electrophoretic mobility that is opposite to the EOF. Thus, if the length of the MIP nanoparticles plug is carefully chosen, the enantiomers will reach the detection window prior to the MIP nanoparticles plug to obtain chiral separation. Furthermore, the effects of polymerization variables and CEC parameters on separation of enantiomers were also investigated.
2. Materials and methods 2.1. Chemicals d- and rac-zopiclone (ZOP) were obtained from Kaiyuan Minsheng Sci. & Tech. Corp. (Suzhou, China). 4-Methyl phenyl dicyclohexyl ethylene (MPDE), 4-cyano phenyl cyclohexyl ethylene (CPCE), 4-cyano phenyl cyclohexyl propylene (MPDP), and 3, 4-difluorophenyl dicyclohexyl propylene (DFDP) were purchased from Hebei Meixin (Hebei, China). Methacrylic acid (MAA) was purchased from Beijing Pubo Biotech. (Beijing, China). Ethylene glycol dimethacrylate (EDMA) was from Sigma (St. Louis, MO, USA). 2,2-Azobis (2-isobutyronitrile) (AIBN) was purchased from Special Chemical Reagent Factory of Nankai University (Tianjin, China). Acetonitrile (ACN, HPLC grade) was from Fisher (NJ, USA). 3-(Trimethoxysilyl)propyl methacrylate (␥-MPS) was from Acros (Geel, Belgium). Other analytical reagents were obtained from Tianjin Chemical Reagent Co. Ltd. (Tianjin, China). Fused-silica capillaries with 100 m ID and 375 m OD were purchased from Xinnuo Optic Fiber Plant (Hebei, China).
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polymer nanoparticles were dried and stored at room temperature until use. 2.3. CEC All CEC experiments were carried out on a K1050 system (Kaiao, Beijing, China) equipped with a UV detector. A Lenovo personal computer was used for data processing. In our work, in order to allow the analyte to reach the detection window prior to the MIP plug, a capillary was derivatized with ␥-MPS to reduce EOF [28]. The EOF of such a capillary was reduced two to threefold compared to an uncoated fused-silica capillary (from 7.67 × 10−8 m2 V−1 s−1 to 3.07 × 10−8 m2 V−1 s−1 ). The derivatization was performed by successively flushing the capillary with 1 mol/L NaOH, water, 0.1 mol/L HCl and water for 2 h, respectively, followed by drying with a stream of nitrogen gas. Then a solution of toluene/␥-MPS (85:15, v/v) was filled into the capillary and kept overnight. Finally the capillary was flushed with toluene and dried. A ␥-MPS-derivatized capillary (74.5 cm total length, 64.5 cm effective length or 50 cm total length, 40 cm effective length) was used in CEC separation. In this work, longer capillary was used to avoid the co-elution of sample and MIP particles on shorter column at some buffer system. The electrolyte used was composed of ACN/20 mmol/L acetate-sodium acetate buffer solution (pH 3.6) (85:15 or 80:20, v/v). All the electrolyte was made using double distilled water and filtered with 0.2 m microporous film. The MIP microparticles were suspended in the electrolyte to a maximum amount of 5 mg/mL to get stable suspensions since increasing the MIP content further resulted in sedimentation due to the aggregation of the particles. The samples of 40 mol/L concentrations were prepared from 5 mmol/L ACN solutions diluted with electrolyte. All solutions were degassed by sonication. Before the CEC analysis every day, the capillary was rinsed with water and electrolyte for 10 min respectively. Between consecutive runs, the capillary was rinsed with electrolyte for 1 min. The MINP suspensions and the samples were introduced hydrodynamically at 15 mbar for 5.0 s and 3.0 s, respectively. In this paper, because some of analytes are eluted prior EOF, separation factor is evaluated using ˛, which is calculated by [18],˛ = t2 /t1 where t1 and t2 are the retention time of the first and second peak. The degree of enantiomer separation was represented by a normalized separation index tR /tR1 , where tR is the difference in the elution time of the enantiomers at peak maximum and tR1 is the retention time of the first eluted enantiomer. The resolution (Rs ) was calculated according to Rs = (t2 − t1 )/0.5(W2 + W1 ), W is the width at the baseline between tangents drawn to inflection points for the peak. 2.4. Characterization of the liquid crystal MIP nanoparticles
2.2. Preparation of liquid crystal MIP nanoparticles The template molecule d-zopiclone, the radical initiator AIBN (4.3 mg/mL), liquid crystalline monomer, functional monomers (MAA) and cross-linking monomers (EDMA) were dissolved in solvents in proportions stated in Table 1. The pre-polymerization mixture was sonicated for 15 min followed by degassing by a stream of nitrogen for 5 min. The flask was sealed and put into a water bath and heated at 55 ◦ C for 1 h. After polymerization, the obtained suspension was recovered by successive centrifugation (13,000 rpm for 15 min), after which the solid phase was resuspended in methanol/acetic acid (7:3, v/v), sonicated for 10 min in an ultrasonic bath, agitated for 30 min, and was repeated twice to remove the template. At last, only ACN was used for suspension of the particles to wash acetic acid. The corresponding blank polymers were prepared in the absence of the template. The resultant
Transmission electron microscopy (TEM) was performed on a JEM100CXII UHR microscope from JEOL, with a 100 kV acceleration tension. The porosity of the MIP particles was measured at 77 K by nitrogen adsorption–desorption isotherms using a V-Sorb 2800TP Surface Area and Pore Distribution Analyzer instrument (Gold APP Instruments Corporation China, Beijing, China). 3. Results and discussion 3.1. Synthesis of liquid crystal-based imprinted nanoparticles with d-zopiclone imprints Conventional approaches for the preparation of spherical MIP nanoparticles are based on emulsion polymerization techniques. This process is a heterophase polymerization in which the use of surfactants as well as co-stabilizers, two non-miscible liquid
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Fig. 1. The schematic images of the preparation of nano-MIP and CEC with the MIP.
phases are emulsified to become homogeneous and stable. The nanodroplets produced in this way from the monomer, the template molecule and the osmotic reagent form nanoreactors in which the polymerization takes place. Using precipitation polymerization protocol, in contrast, small spherical polymer particles were successfully synthesized without any need for stabilizing surfactants, which is beneficial in terms of particle cleanup. The first step in the present experiments was to choose an appropriate solvent or solvent mixture that would allow for narrow disperse particles. Toluene and ACN are the common porogens in the preparation of MIP nanoparticles and was used to prepare d-ZOP-imprinted nanoparticles in scouting experiments about polymerization parameters. In our study, it was found that the liquid-crystal monomer could be dissolved well in toluene. In contrast, d-ZOP and AIBN could not be dissolved completely in toluene. Thus, a mixture of toluene/ACN was used as the porogen to prepare MIP nanoparticles. Lower ratio of toluene/ACN (e.g., 5:5) led to a limited amount of MPDE dissolved. In contrast, when higher ratio of toluene/ACN (e.g., 9:1) was used, d-ZOP could not be dissolved completely. It was found that the optimized ratio of toluene/ACN was 7:3. The visualization and nanometer size of the resultant MIP nanoparticles (prepared with liquid crystalline monomer MPDE) were demonstrated by TEM. As shown in Fig. 2, the resulting particles indicate a size distribution of 150–180 nm. Compared to previous MIP [33] prepared without liquid crystalline monomer, there was not agglomeration between the MIP nanoparticles. In contrast, the liquid crystal MIP nanoparticles were more uniform and narrow-disperse. Such ability to synthesize monodisperse MIP beads may be attributed to the decrease in the cross-linking density (20%) from the liquid crystal MIP, as observed by Yoshimatsu et al. [34].
Fig. 2. Transmission electron microscope of MIP 1 using d-ZOP as template molecule. (A) High magnification; (B) low magnification.
Table 1 Recipes of preparation for liquid crystal MIP nanoparticles. MIP
d-ZOP (mmol)
MIP 1 MIP 2 MIP 3 MIP 4 MIP 5 MIP 6 MIP 7 MIP 8 MIP 9 MIP10 MIP11 NIP1
0.08 0.08 0.08 0.08 0.08 0.16 0.107 0.064 0.08 0.08 0.08
Liquid crystalline monomer (mmol) MPDE
CPCE
MPDP
EDMA (mmol)
0. 64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0.64
0.64 0.16 0.32 0.48 0.80 0.64 0.64 0.64 0.64 0.64 0.64 0.64
DFDP
1.92 2.40 2.24 2.08 1.76 1.92 1.92 1.92 1.92 1.92 1.92 1.92
MAA (mmol)
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our MIP-coated capillary with d-ZOP imprinting with comparable enantiomers resolution [28]. 3.3. Influence of polymerization parameters on chiral separation
Fig. 3. CEC analysis of rac-ZOP (A) and d-ZOP (B) on MIP nanoparticles and rac-ZOP on NIP nanoparticles (C) demonstrating the imprinting effect and identifying the peaks. Conditions: separation voltage, 15 kV; UV–vis detector, 254 nm; ACN/20 mmol/L acetate (pH 3.6) (80/20, v/v); capillary length, 50 cm.
The d-ZOP-imprinted nanoparticles with a pore volume of 0.31 cm3 /g showed “type IV” isotherms which are usually related to meso-macroporous materials. The hysteresis loops resemble H3 types, suggesting that the nanoparticles are porous materials with specific structure of slit-shapes pores [35]. BET area of the polymer materials (SBET = 33.065 m2 /g) is calculated from the adsorption data using 0.162 nm2 as the molecular cross-sectional area for adsorbed nitrogen molecules. This isotherm of adsorption versus relative pressure is transformed to average pore diameter of 46.3 nm calculated by Barrett–Joyner–Halenda (BJH) method [35]. 3.2. Confirming the imprinting effect of MIP nanoparticles by CEC The imprinting effect of the low crosslinking MIP nanoparticles made with liquid crystalline monomers was evaluated by enantiomer separation in CEC mode. Fig. 3A and B illustrated the separation of rac-ZOP on the d-ZOP-imprinted nanoparticles with liquid crystalline monomer MPDE, depicting the characteristic elution order and the selective retention of the template. The resolution of two enantiomers was up to 3.29 and high column performance of the template (up to 26,200 plates/m) was obtained. Compared with MIPs nanoparticles with high level crosslinking [33], the liquid crystal MIPs with low crosslinking degree have comparable selectivity. The non-imprinted polymer (NIP) nanoparticle prepared with liquid crystalline monomers showed no enantiomeric separation (Fig. 3C). The fact that the reference polymer prepared in the absence of the template was unable to resolve the enantiomers of rac-ZOP (Table 2) and the d-enantiomer always was the last eluted when the enantiomers were separated confirms that the separation is based on molecular recognition achieved through molecular imprinting. In addition, even if the capillary is deactivated, this work indicates shorter analysis time than Table 2 Effect of the different levels of crosslinking on d-ZOP imprinted nanoparticles. Level of crosslinking 5% 10% 15% 20% 25%
Nl (m−1 )
Nd (m−1 )
48,600 24,400 46,500 66,900 25,000
28,700 14,900 12,600 26,200 16,700
˛
tR /tR1
Rs
1.12 1.10 1.09 1.07 1. 10
0.12 0.10 0.09 0.07 0.10
5.21 3.05 2.99 3.29 3.32
CEC conditions: separation voltage, 15 kV; UV–vis detector, 254 nm; ACN/20 mM acetate (pH 3.6) (85/15, v/v), capillary length, 75 cm.
3.3.1. Role of liquid crystalline monomers To investigate the function of liquid crystalline monomers on the selectivity, the synthesis of MIP nanoparticles with varied amounts of cross-linker in the presence of liquid crystalline monomers was conducted. All the MIP nanoparticles were prepared at a fixed ratio of MAA (functional monomer, 20%, molar ratios). Variation of cross-linking degree was performed by altering the relative ratio of liquid crystalline monomers to cross-linker, EDMA. In general, the increase in the content of cross-linking monomers could cause an increase in selectivity of resulting MIP and there will be no imprinting effect when the concentration of cross-linker approaches 12% [1]. In the present study, even if the level of crosslinking was as low as 5%, enantiomer selectivity can be realized on the resulting MIP nanoparticles. Interestingly, the d-ZOP-MIP nanoparticles could still provide baseline separation of rac-ZOP even with an increased resolution than the liquid crystal MIP with higher level of crosslinker (Table 2). The reason for this behavior could be an effect originating in molecule chiralty caused by the liquid crystal monomers [10,11], which comes from a helical twist of the liquid crystalline structure due to the presence of a small percentage of chiral dopant in a nematic phase leading to a cholesteric phase. However, further decrease in the content of EDMA led to very low enantiomer selectivity. It is well known that the presence of a small percentage of chiral dopant in a nematic phase induces a helical twist of the liquid crystalline structure leading to a cholesteric phase. Therefore, the introduction of a chiral template during the synthesis of the liquid crystal-based MIP leads to two different levels of chirality. The first one is at the cavity level, exactly like in regular MIPs, and the second one is due to the presence of liquid crystal moieties. To investigate what mechanism of an enantiomer separation process comes from the chiral cavity or from the chiral helix owing to the cholesteric phase, we also prepared liquid crystal polymer without functional monomer, MAA, in the presence of the template. However, chiral separation for rac-ZOP was not achieved. It seemed that a non-imprinted material adsorbs equally both enantiomers, the doped material, presenting only chirality at the mesoscopic level coming from the cholesteric helix, showed only a limited effect in the recognition process. Thus, the helical structure, induced by the mesogenic groups appeared to increase the accessibility of binding sites and the mass transfer of liquid crystal MIP compared to non-liquid crystal one. 3.3.2. Template to monomer ratio The influence of template to monomer molar ratio (t/m-ratio) on the imprinting effect of the liquid crystal MIP nanoparticles was investigated by varying t/m-ratio with four levels. The four MIP nanoparticles all indicated the ability to separate rac-ZOP. The separation factors varied little and ranged from 1.18 to 1.23, which can be assumed that this preparation protocol yielded MIP particles with similar affinity. As shown in Fig. 4, as the t/m-ratio decreased to 1:8, higher resolution and column efficiency of d-ZOP was obtained and the peak shape of d-ZOP was improved. This result may be due to the pronounced influence of the presence of the template on the size of the obtained particles rather than selective interaction. While t/m-ratio decreased to 1:10, the greatest resolution (6.23) was observed due to significant increase in column efficiency of l-ZOP. Previously, enantiomer separation even on MIPs with an extremely low template-to-monomer (1:80) has been demonstrated [30]. However, baseline separation was not achieved due to
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X. Liu et al. / J. Chromatogr. A 1309 (2013) 84–89 Table 4 Reproducibility of the enantiomer separation of rac-ZOP using liquid crystal MIP nanoparticles.
Retention time (RSD%) Column efficiency (RSD%) Resolution (RSD%) ˛ (RSD%)
l-ZOP
d-ZOP
0.77 7.71 3.45 1.47
2.59 8.99
Separation voltage: 5 kV; UV–vis detector: 230 nm; ACN/30 mmol/L acetate (pH 3.6) (80/20, v/v); capillary length, 50 cm.
Fig. 4. The enantiomer separations with MIP nanoparticles of different t/m ratio. Conditions: separation voltage, 15 kV; UV–vis detector, 254 nm; ACN/20 mmol/L acetate (pH 3.6) (80/20, v/v); capillary length, 50 cm.
lower chain of liquid crystal groups are toughen enough to keep the memory to the template. Furthermore, it seemed that the bulkiness of the pending groups and their chemical interactions play no role to toughen the network. Variations of functional groups in liquid crystalline also showed little effect on the selectivity of the resulting MIP nanoparticles. 3.4. Effect of CEC parameters on separation of enantiomers
Fig. 5. The enantiomer separations with MIP nanoparticles of different liquid crystalline monomers. Conditions: separation voltage, 15 kV; UV–vis detector, 254 nm; ACN/20 mmol/L acetate (pH 3.6) (80/20, v/v); capillary length, 50 cm.
the phenomenon of peak tailing. The results here obtained clearly show higher resolution and column efficiency, probably owing to the homogeneous and monodispersed nature derived from the liquid crystal MIP nanoparticles. A t/m-ratio of 1:8 was chosen for the following studies in terms of shorter separation time with adequate resolution. 3.3.3. Liquid crystalline monomer To study the role of the pending mesogenic groups of liquid crystalline on the imprinting of the low crosslinking MIP, four different liquid crystalline monomers (MPDE, CPCE, MPDP, and DFDP) were used as physical crosslinker to prepare MIPs, respectively. As shown in Fig. 5, they all had the ability to separate rac-ZOP. From a comparison of Table 3, the length variation of liquid crystal groups indicates little effect on the selectivity of liquid crystal MIP, which suggests that the liquid crystalline interactions with Table 3 Effect of the different liquid crystalline monomer on d-ZOP imprinted nanoparticles. Liquid crystalline monomers
Nl (m−1 )
Nd (m−1 )
˛
tR /tR1
Rs
MPDE CPCE MPDP DFDP
14,500 10,000 21,100 7200
15,800 6400 10,600 6100
1.20 1.22 1.21 1.23
0.20 0.22 0.21 0.23
5.60 4.32 4.33 4.28
To study weather the ability of liquid crystalline to stable the imprinted cavities may be varied in different solvents, a serious of experiments for the separation of ZOP enantiomers with different mobile phase was performed. A high organic content in the electrolyte has been proved to be optimal for MIP-based CEC separations [28]. In this work, ACN was chosen as the organic modifier and the level of ACN content on enantiomers separation was investigated. Increasing ACN content led to the increased column efficiency of d-ZOP and shorter elution time, while the resolution (Rs ) for zopiclone enantiomers and separation factor (␣) were decreased. This trend is in line with the results of non-liquid crystal MIPs [30]. The result suggested that the mechanism is not shifted when the groups of liquid crystalline are incorporated into MIP matrix, compared to MIPs without liquid crystalline. Baseline separation for rac-ZOP could not be achieved when ACN content was increased to 90%. In 70% ACN, the elution time of liquid crystal MIP nanoparticles was too long (approximately 30 min). 85% ACN in the electrolyte was applied in following experiments as a compromise between the resolution for zopiclone enantiomers and the elution time of liquid crystal MIP nanoparticles. The pH effect in mobile phase on enantiomers separation with liquid crystal MIP nanoparticles was examined from 3.0 to 4.8. When the value of pH increased from 3.0 to 3.6, the column efficiency of d-ZOP increased, but the resolution (Rs ), separation factor (˛) and normalized separation index (tR /tR1 ) decreased. There was no enantiomer separation as the pH value was increased to 4.8. This result is different from previously reported non-liquid crystal MIP [33], suggesting a faster increase in nonselective sites with increasing pH value than selective sites on the liquid crystal-based MIP [36]. The highest column efficiency of d-ZOP was obtained when pH value was 3.6. 3.5. Reproducibility The reproducibility of various CEC parameters is a critical consideration in the field of the preparation and the application of MIP nanoparticles. In our study, a number of parameters such as retention time and Rs were measured to test the reproducibility of the liquid crystal MIP with d-ZOP imprinting. The results of retention time, column efficiency and resolution of d- and l-ZOP and Rs of two enantiomers are shown in Table 4. The injection reproducibility is averaged from the results of continuously repeating five injections. RSD for the resolution of two enantiomers is lower than 3%. Compared to MIP nanoparticles prepared with conventional crosslinking agent [33], it seemed that the MIP nanoparticles using liquid crystal monomer had better reproducibility. In addition, the
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dissolving of derivatized capillary was not observed after 100 runs in our investigation. This result demonstrated the advantage of monodisperse nanoparticles again. 3.6. Loading capacity of liquid crystal MIP Thanks to the low content of cross-linker, the liquid crystalline networks exhibit a high molecular trapping capacity. By increasing the sample load of the template, the separation factor typically decreased rapidly, leveling off at a sample load of 20 mol/L. It should be noted that the saturation capacity (number of available binding sites) of the polymers in this study is high in relation to the amount of template added to the monomer mixture (theoretical maximum number of sites), which can be estimated from the loading factor giving column overloading. The amount of template incorporated during the synthesis of MIP corresponds to 8 mol/g of polymer. The amounts are notably higher than those of the usual imprinted materials (around 2.5 mol/g of polymer) [33] in which high cross-linker rates are needed.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
[12] [13] [14] [15] [16] [17] [18]
4. Conclusion [19]
The low cross-linked MIP nanoparticles based on liquid crystalline monomers were successfully prepared for the first time. Separation of racemic zopiclone was achieved in CEC mode with good peak symmetry. Different liquid crystalline monomers were able to form MIP nanoparticles with low crosslinking degree. Even if the level of crosslinking of chemical crosslinkers was as low as 5%, enantiomer selectivity was achieved. Compared to previous prepared MIP nanoparticles [30] at high level of crosslinker, the liquid crystal MIP nanoparticles were more uniform and showed higher reproducibility of CEC. We believe that the results presented here should be valuable for future research toward development of new MIP-based separation systems at low crosslinking. Acknowledgments This work was supported by the Hundreds Talents Program of the Chinese Academy of Sciences and supported by the National Natural Science Foundation of China (Grant No. 21075090).
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[20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]
L. Levi, V. Raim, S. Srebnik, J. Mol. Recognit. 24 (2011) 883. S. Tokonami, H. Shiigi, T. Nagaoka, Anal. Chim. Acta 641 (2009) 7. F. Barahona, E. Turiel, A. Martín-Esteban, J. Chromatogr. A 1218 (2011) 7065. E. Turiel, A. Martín-Esteban, J. Sep. Sci. 32 (2009) 3278. K.D. Shimizu, C.J. Stephenson, Curr. Opin. Chem. Biol. 14 (2010) 743. M. Resmini, Anal. Bioanal. Chem. 402 (2012) 3021. D. Cunliffe, A. Kirby, C. Alexander, Adv. Drug Deliv. Rev. 57 (2005) 1836. J. Haginaka, J. Sep. Sci. 32 (2009) 1548. J.-D. Marty, M. Tizra, M. Mauzac, I. Rico-Lattes, A. Lattes, Macromolecules 32 (1999) 8674. M. Weyland, S. Ferrere, A. Lattes, A.-F. Mingotaud, M. Mauzac, Liq. Cryst. 35 (2008) 219. C. Binet, D. Bourrier, M. Dilhan, D. Esteve, S. Ferrere, J.C. Garrigue, H. Granier, A. Lattes, A.M. Gue, M. Mauzac, A.F. Mingotaud, Talanta 69 (2006) 757. Z.-H. Wei, L.-N. Mu, Y.-P. Huang, Z.-S. Liu, J. Chromatogr. A 1273 (2012) 115. Z.-H. Wei, L.-N. Mu, Q.-Q. Pang, Y.-P. Huang, Z.-S. Liu, Electrophoresis 33 (2012) 3021. J. He, G. Fang, Q. Deng, S. Wang, Anal. Chim. Acta 692 (2011) 57. B.-Y. Huang, Y.-C. Chen, G.-R. Wang, C.-Y. Liu, J. Chromatogr. A 1218 (2011) 849. S. Liao, X. Wang, X. Lin, Z. Xie, Electrophoresis 31 (2010) 2822. L. Schweitz, L.I. Andersson, S. Nilsson, Anal. Chem. 69 (1997) 1179. C. Zheng, Z.-S. Liu, R.-Y. Gao, L.-H. Zhang, Y.-K. Zhang, Anal. Bioanal. Chem. 388 (2007) 5. J. Ou, X. Li, S. Feng, J. Dong, X. Dong, L. Kong, M. Ye, H. Zou, Anal. Chem. 79 (2007) 639. Z.-H. Wei, X. Wu, B. Zhang, R. Li, Y.-P. Huang, Z.-S. Liu, J. Chromatogr. A 1218 (2011) 6498. Y.C. Huang, C.C. Lin, C.Y. Liu, Electrophoresis 25 (2004) 554. S.A. Zaidi, W.J. Cheong, J. Chromatogr. A 1216 (2009) 2947. O. Brüggermann, R. Freitag, M.J. Whitcombe, E.N. Vulfson, J. Chromatogr. A 781 (1997) 43. J.Z. Tan, V.T. Remcho, Electrophoresis 19 (1998) 2055. L. Schweitz, Anal. Chem. 74 (2002) 1192. L. Schweitz, P. Spégel, S. Nilsson, Analyst 125 (2000) 1899. X.X. Shi, L. Xu, H.Q. Duan, Y.P. Huang, Z.S. Liu, Electrophoresis 32 (2011) 1348. X. Wu, Z.-H. Wei, Y.-P. Huang, Z.-S. Liu, Chromatographia 72 (2010) 101. F. Priego-Capote, L. Ye, S. Shakil, S.A. Shamsi, S. Nilsson, Anal. Chem. 80 (2008) 2881. P. Spégel, L. Schweitz, S. Nilsson, Anal. Chem. 75 (2003) 6608. P. Spégel, L. Schweitz, S. Nilsson, Electrophoresis 22 (2001) 3833. J.M. Lin, K. Uchiyama, T. Hobo, Chromatographia 47 (1998) 625. X. Liu, Z.-H. Wei, Y.-P. Huang, J.-R. Yang, Z.-S. Liu, J. Chromatogr. A 1264 (2012) 137. K. Yoshimatsu, K. Reimhult, A. Krozer, K. Mosbach, K. Sode, L. Ye, Anal. Chim. Acta 584 (2007) 112. K.S.W. Sing, Pure Appl. Chem. 54 (1982) 2201. B. Sellergren, K.J. Shea, J. Chromatogr. A 654 (1993) 17.