Chem. Res. Chin. Univ., 2014, 30(3), 374—378 doi: 10.1007/s40242-014-3420-8

——————————— *Corresponding authors. E-mail: zhangcan@mail.ujs.edu.cn; xuliang@tmu.edu.cn Received Octorber 8, 2013; accepted Jannary 21, 2014. Supported by the National Natural Science Foundation of China(Nos.31000783, 31201456), the Jiangsu Planned Projects for

Postdoctoral Research Funds, China(No.1202010B) and the Talent Foundation of Jiangsu University, China(No.09JDG052). © Jilin University, The Editorial Department of Chemical Research in Chinese Universities and Springer-Verlag GmbH

Synthesis, Characterization and Application of Organic-inorganic Hybrid and Carbaryl-imprinted Capillary Monolithic Column

ZHANG Can1*, CAI Jianrong1, DUAN Yuqing1, XU Liang2*, FANG Guozhen3 and WANG Shuo3 1. School of Food & Biological Engineering, Jiangsu University, Zhenjiang 212013, P. R. China;

2. Tianjin Key Laboratory on Technologies Enabling Development Clinical Therapeutics and Diagnostics(Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, P. R. China;

3. Faculty of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P. R. China

Abstract Organic-inorganic hybrid and carbaryl-imprinted capillary monolith was synthesized via methacrylic acid(MAA) as functional monomer, γ-methacryloxypropyltrimethoxysilane(γ-MAPS) as crosslinker and carbaryl as template molecule in an acetonitrile/dichloromethane mixture(1:4, volume ratio). With the capillary column obtained from this monolith, three carbamates(carbaryl, fenobucarb and metolcarb) were separated effectively by electro- chromatography with the kMIP/kNIP values of 7.57, 1.27 and 1.64, respectively. In 20 mmol/L phosphate buffer solu-tion(pH=3.5) with 30%(volume fraction) of acetonitrile, carbaryl was separated directly from the three-carbamate mixture(carbaryl, fenobucarb and metolcarb) with an effective 15 cm-length imprinted column. Keywords Organic-inorganic hybrid; Carbaryl-imprinted capillary; Monolith; Separate

1 Introduction

Capillary electrochromatography(CEC) is an exciting mi-niaturized separation technique combining the high separation efficiency of CE and the various retention mechanisms and selectivity of high performance liquid chromatography(HPLC). The further coupling of molecularly imprinted polymer(MIP) and CEC(or HPLC) has already shown a bright prospect in the separation of target compound from its analogs with good se-lectivity and high efficiency[1―8]. Among various MIP struc-tures studied, only monolithic columns have good permeability and an easy tuning pore structure. Therefore, the combination of MIP monolithic column with CEC could be very promising to recognize and separate small molecules and biomacromole-cules efficiently. Both organic polymer- and silica-based mono-liths have been widely used in CEC. Organic polymer-based monolith has the various monomer availability and excellent stability in different environments with various pH. However, it may shrink or swell in different organic solvents. The shrinking or swelling of the monolith can cause considerable deformation of the MIP receptors and thus decrease its recognition ability towards the template. Although the silica-based MIP monolith can offer excellent mechanical strength and good solvent resis-tance, the cracking and shrinking of the bed during its drying process become a drawback of it. Organic-inorganic hybrid technology can avoid the shortcomings of both organic and inorganic matrix monoliths. Recently, organic-inorganic hybrid MIP-monolith coupling with CEC has been applied in the chi-

ral separation and screening of target compound from its struc-tural analogs. For example, Yan et al.[9,10] reported the molecu-larly imprinted silica monolithic column obtained from the non-hydrolysis of organic-inorganic hybrid and the use of it for the chiral CEC separation of (S)-naproxen and (S)-zolmitriptan with excellent chiral recognition ability and high resolution. Wang et al.[11] prepared a molecularly imprinted capillary mo-nolithic column via organic-inorganic hybrid approach with four ractopamine isomers as templates, and found that the as-prepared MIP capillary monolithic column had high selec-tivity to the four ractopamine isomers in CEC. Wang et al.[12] also prepared a molecularly imprinted silica monolithic column via a two-step approach and applied it in the selectively recog-nization of sulfamethazine from the sulfonamide mixtures with improved selectivity and good stability in CEC.

Furthermore, several quantitative and qualitative methods, including the chromatographic assay[13], enzyme-linked immu-no-sorbent assay(ELISA)[14], biosensor[15], and capillary elec-trophoresis-based immunoassay(CEIA)[16], have been used to detect and screen carbaryl(1-naphthol-N-methylcarbamate), the first successful carbamate insecticide owing to its broad-spectrum efficacy. Because anti-carbaryl antibody was used as the sensing material in some assay such as ELISA and CEIA, carbaryl can be recognized rapidly and sensitively. However, with the assays mentioned above, the mixture of three carbamate insecticides(carbaryl, fenobucarb and metol-carb) cannot be separated simultaneously without the aid of anti-carbaryl antibody. With the development of molecular

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imprinting technology, researchers focus their attention on the application of molecular imprinted technology to prepare bio-mimetic antibody that can imitate the molecular recognition ability of biological antibodies. In this study, carbaryl was used as template to prepare molecularly imprinted capillary mono-lithic column via organic-inorganic hybrid approach. CEC with carbaryl-MIP monolith not only had good recognition capabili-ty for carbaryl, but also could separate the three-carbamate insecticide mixture(carbaryl, fenobucarb and metolcarb) com-pletely. Therefore, combined with CEC, carbaryl-MIP monolith can be used to screen carbaryl from structure similar com-pounds efficiently and the clean-up steps that are usually man-datory to analyze complicate samples can be avoided.

2 Materials and Methods

2.1 Materials

Carbaryl, fenobucarb and metolcarb were purchased from Sigma(St Louis, USA); methacrylic acid(MAA), 2,2′-azobisi- sobutyronitrile(AIBN) and γ-methacryloxypropyltrimethoxy- silane(γ-MAPS) were purchased from Shanghai Chemical Plant (Shanghai, China); MAA was distilled under vacuum and AIBN was re-crystallized in ethanol before use; fused-silica capillary(100 µm×375 µm) was purchased from Yongnian Op-tic Fiber Plant(Hebei, China); acetonitrile of HPLC grade and other reagents were all of analytical grade; doubly deionized water(DDW, 18.2 MΩ·cm–1) was obtained from WaterPro sys-tem(Labconco Corporation, Kansas, USA).

2.2 Instruments and Methods

CEC experiments were performed on a P/ACE MDQ CE system(Beckman-Coulter, Fullerton, USA) with a UV detector (220 nm). The monolith column was rinsed under a pressure of 413.7 kPa and was further electro-kinetically conditioned at 15 kV in the instrument with the separation buffer solution until a stable current was obtained. All the phosphate buffers were prepared with doubly deionized water and filtered through a 0.22-µm filter before use. The UV adsorption spectra of car-baryl were measured on a Varian Cary 100 UV spectrometer (Varian, Maryland, USA). Scanning electron microscopy(SEM) images of the synthesized monoliths were taken at 15.0 kV on a JSM-7001F scanning electron microscope(Joel, Tokyo, Japan). FTIR spectra(4000―400 cm–1) in KBr were recorded on a Magna-560 spectrometer(Nicolet, Madison, USA).

2.3 Preparation of Carbaryl Molecularly Im-printed Capillary Monolithic Column

Before the preparation of monolithic, fused-silica capillary was pretreated as the following. The capillary was rinsed with 1.0 mol/L NaOH for 12 h, then with water for 30 min and 0.1 mol/L HCl for 30 min, after that it was washed with water again until the pH value of the outflow reached 7.0. Finally, the capillary was blew with nitrogen and dried at 110 °C in a gas chromatographic oven overnight.

The pre-polymerization mixture, including 100.5 mg of carbaryl and 0.258 mL of MAA, was dissolved in 5 mL of

acetonitrile/dichloromethane mixture(1:4, volume ratio). One hour later, 0.95 mL of γ-MAPS and 30 mg of AIBN were added to the above mixture and dissolved homogeneously with stirring. The mixture was purged with nitrogen for 5 min and sonicated for 5 min to remove oxygen. The obtained transpa-rent solution was carefully introduced into the activated capillary to fill about 20 cm with a 0.5 mL disposable syringe. The capillary was submerged into a water bath at 50 °C for 12 h for polymerization after both ends of it were plugged with silicon rubber. Following the polymerization, the prepared capillary monolith was eluted with acetonitrile by an HPLC pump to remove the template molecule and un-reacted mono-mers, dried with nitrogen, and dried at 80 °C in a vacuum oven for 10 h. After the monnolith segment, a detection window was created by burning off a 2-mm segment of the polyimide coa- ting. The distance from the detection window to the outlet end of capillary was 10 cm. Once fabricated, the capillary was in-stalled in the cartridge from the inlet to the detection window with a monolith of 15 cm. The total length of capillary was 30.2 cm. A non-imprinted polymer(NIP) capillary monolith without template was also prepared by the same procedure.

The imprinted factor(IF=kMIP/kNIP) was defined as the ratio of the retention factor of template in MIP monolith to that in NIP monolith, which was adopted to evaluate the imprinting ability in this study.

3 Results and Discussion

3.1 Molecular Interaction Between Template and Functional Monomer

The molar ratio of the template to the functional monomer has a significant influence on the preparation of MIPs. In order to prepare highly selective affinity sites, the formation of stable complexes is essential in the early period of polymerization. Therefore, it is important to study the intermolecular interaction between the template and the functional monomer. The impact of different molar ratios of carbaryl to MAA on the UV absorp-tion spectra of carbaryl is shown in Fig.1. It is found that the carbaryl absorption has obvious blue-shift with the increase of MAA concentration. When the molar ratio of carbaryl to MAA is 1:6, the maximum blue-shift from 220 nm to 203 nm is reached. The peak does not further change when the molar ratio of carbaryl to MAA is more than 1:6. This could be due to the formation of hydrogen bonds between carbaryl and MAA.

Fig.1 UV spectra of carbaryl with different molar ratios of carbaryl to MAA in acetonitrile

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Therefore, the molar ratio of the template to the monomer is kept at 1:6 in the polymerization of this study.

3.2 Preparation of Carbaryl Molecularly Im-printed Capillary Monolithic Column

Porogenic solvent plays a dual role in the preparation of MIP. First, the porogen can produce large pores to assure good flow-through properties of the resultant MIP. Second, the po-rogenic solvent governs the strength of non-covalent interac-tions in addition to its influence on the polymer morphology. The best imprinting porogens, accentuating the binding strengths, are solvents with very low dielectric constant, such as toluene and dichloromethane. Acetonitrile is also selected as the solvent/porogen because of its good solubility for a large number of polar template molecules and its wide application as a component of electrolyte buffers. It is observed that low (below 40 °C) or high polymerized temperature(above 60 °C) might induce the incomplete-polymerization or over-polyme- rization. Therefore, the impacts of different volume ratios of acetonitrile to dichloromethane(0:5, 1:4, 2:3, 3:2) and different temperatures(40, 50, 60 °C) on the permeability were studied by Wang et al.[11]. It was also found that if the columns were prepared via polymerization at higher temperature or with longer time, its flow-through property became lower. The pore structure of molecularly imprinted polymers with permeability was measured and listed in Table 1. Based on the permeability and affinity, the porogenic solvent of acetonitrile/ dichloromethane(1:4, volume ratio) and the temperature of 50 °C(M2) are chosen to prepare the capillary monolith.

Table 1 Pore structure of the molecularly imprinted polymers

Method BET surface area/ (cm2·g–1)

Pore volume/ (m3·g–1) Pore size/nm

M1 7.04 0.0272 6.00 M2 5.72 0.0152 4.98 M3 4.44 0.0116 4.27 M4 3.06 0.0080 5.46 M5 3.92 0.0021 4.85

3.3 Characterization of Carbaryl Molecularly Imprinted Capillary Monolithic Column

3.3.1 FTIR Spectra of Carbaryl Molecularly Im-printed Capillary Monolithic Column

To further determine the characteristics of the silica-based network, FTIR spectra of carbaryl MIP monolith before and after the extraction of template were compared, and the non-imprinted monolith was used as the control to verify the complete removal of template molecules. As shown in Fig.2, the peaks of carbaryl at 3300(N―H stretch), 1418(C―N stretch), 1541(C=C skeleton vibration in naphthalene) and 3061 cm–1(C―H stretch in naphthalene) are only presented on the molecularly imprinted capillary monolithic column before washed, which indicates that the template is completely re-moved from the MIP. The disappearance of the peak at 1635 cm–1(C=C stretch in MAA) on MIP indicates that the double bonds of MAA participate the polymerized reaction. The peaks

of MAA at 1299(C―O stretch) and 947 cm–1(O―H bending) have red-shifts on the MIP, and change to 1270 and 913 cm–1, respectively, possibly due to the interaction of MAA and car-baryl or the formation of polymers. The peaks near 1088 and 820 cm–1 can be assigned to the Si―O―Si asymmetric stretch and symmetric stretch, respectively. The peak at 3447 cm–1 is assigned to the ―OH vibration in the bulk gels.

Fig.2 FTIR spectra of methacrylic acid(a), carbaryl (b), carbaryl-imprinted polymers before(c) and after extraction(d) and non-imprinted polymers(e)

3.3.2 Effects of Functional Monomer and Cross- linker

A number of MIP monolith-based capillary columns were prepared with different molar ratios of monomer to cross-linker. It is found that γ-MAPS with small percentage cannot react with MAA completely and the stable matrix cannot be formed because only γ-MAPS with high percentage can induce its self-hydrolysis to form the dense polymer. As shown in Fig.3, with the increase of the amount of γ-MAPS, the pore size of the

Fig.3 SEM images(A―C) and partial enlarged SEM images(A′―C′) of molecularly imprinted ca-pillary monolithic columns prepared with dif-ferent amounts of γ-MAPS Amount of γ-MAPS/mL: (A, A′) 1.0; (B, B′) 0.75; (C, C′) 0.5.

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MIP monolith-based capillary column becomes smaller and the flow-through property of it diminishes slowly, which can further affect the selectivity of the capillary monolithic column.

Additionally, molar ratios of carbaryl:MAA:γ-MAPS of 1:6:6 and 1:6:8 are compared as shown in Fig.4 and the ability of carbaryl-imprinted capillary monolith with two different molar ratio to recognize carbaryl in CEC is recorded. It is found that the ratio of 1:6:8 is better for the detection of car-baryl with kMIP/kNIP of 7.57 for the optimum capillary.

Fig.4 CEC chromatograms of carbaryl-imprinted capillary monolith at different molar ratios of monomer to cross-linker

Peaks: 1. thiourea; 2. carbaryl. Molar ratio of carbaryl:MAA:γ-MAPS: a. 1:6:6; b. 1:6:8. CEC separations were performed at 15 kV with 20 mmol/L phosphate buffer(pH=3.5) which contained 30%(volume fraction) of acetonitrile.

3.3.3 Impact of pH Value of Separation Buffer on Carbaryl Identification and Separation Behavior of Prepared Molecularly Imprinted Capillary Monolithic Column

The pH value of the separation buffer is important because it can alter the charges of both the analyte and the polymer, thus can affect the molecular recognition ability of the MIP and the electrophoretic mobility of the analyte. The electroosmotic flow(EOF) is generated by the ionization of the surface groups of the stationary phase. Analytes are carried through the column by the mobile phase driven with the EOF as well as the self-electrophoretic mobility of analyte. The impact of pH value on the separation of carbaryl via the prepared monolith column at different pH values of 3.0, 3.5, 4.0, 4.5 and 5.0 was investigated. It is found that the migration time of carbaryl decreases with the increase of pH value, which may be ascribed to the higher EOF generated by not only the carboxyl groups of MAA, but the silanol groups on the surface of MIP-monolith. The presence of silanol groups is due to that the surface of the silica monolith could not be completely covered by the MIP film. 3.3.4 Effect of Acetonitrile Concentration in Separa-tion Buffer

Acetonitrile, as an organic modifier in the separation buf-fer, is the most extensively used electrolyte due to its low vis-cosity. CEC results show that the recognition ability of MIPs is a function of acetonitrile concentration(10%―40%, volume fraction) in the mobile phase(20 mmol/L phosphate buffer con-taining acetonitrile with pH value of 3.5). It is also found that

increasing the acetonitrile concentration can reduce the reten-tion time of carbaryl. The possible reason is that the retention of carbaryl is mainly determined by the hydrophobic interac-tion except the hydrogen bonding interaction with the im-printed cavities in the polymer. Additionally, the configuration of carbaryl peak becomes more symmetrical and the width of peak becomes narrower with the increase of the concentration of acetonitrile in the separation buffer. However, too high con-centration of acetonitrile in the buffer(above 50%, volume frac-tion) can easily produce bubbles in CEC, which can disrupt the separation. All of these results suggest that, during the prepara-tion of MIP monolith, 30% of acetonitrile can be chosen to favor the recognition and separation of carbaryl.

3.4 Comparison of MIP Monolithic Column with NIP Monolithic Column

The carbaryl-imprinted capillary monolith was used to separate three carbamate insecticides(carbaryl, fenobucarb and metolcarb) in a mixture. As shown in Fig.5, on NIP monolith column(curve a), three carbamates cannot be well separated; however, they can be separated completely on MIP monolith column(curve b) with kMIP/kNIP of 7.57, 1.27 and 1.64 for car-baryl, fenobucarb and metolcarb, respectively. Additionally, carbaryl was separated after fenobucarb and metolcarb because of the specific recognition of molecular imprinted cavity for it. For MIP monolith column, the separation factors (α=kCarbaryl/kAnalogs) are 5.96(kCarbaryl/kFenobucarb) and 4.62 (kCarbaryl/kMetolcarb), respectively. During the effective separation of the mixture of the three insecticides(carbaryl, fenobucarb and metolcarb) and the screening of carbaryl with CEC, the MIP-capillary monolith column is preferred because of the molecularly designed cavities which show a special affinity for carbaryl over other structurally similar compounds.

Fig.5 Comparison of NIP(a) and MIP monolith(b) for the separation of fenobucarb(peak 1), me-tolcarb(peak 2) and carbaryl(peak 3) by CEC

The CEC separations were performed at 15 kV with 20 mmol/L phosphate buffer(pH=3.5) which contained 30%(volume fraction) of acetonitrile. The analyte solution was injected under pressure of 68.95 kPa for 5 s and de-tected at 220 nm with a UV detector.

3.5 Repeatability and Reproducibility

The repeatability of the MIP monolith column was as-sessed in terms of the migration time of carbaryl, fenobucarb and metolcarb and the separation factors under the same condi-tions as shown in Fig.5. The reproducible migration time for all

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analytes is not more than 2.1%(relative standard deviation, RSD, run-to-run, n=3), and no general trend in the migration time change is found. The RSD values of the separation factors are less than 5.1%. The column-to-column reproducibility was also examined(n=3). The RSD values of the migration time and separation factors are not higher than 4.5% and 10.3%, respec-tively.

4 Conclusions In this study, carbaryl-imprinted capillary monolithic

column was prepared via the organic-inorganic hybrid method. The preparation method is simple, and the prepared MIP capil-lary monolithic column has high selectivity for carbaryl. The mixture of three carbamates(carbaryl, fenobucarb and metol-carb) can be separated effectively by the combination of MIP monolith with CEC, which is good for the screening of carbaryl from a group of structural analogs. This method can also be used to separate the analyte directly from the matrix compo-nents, avoiding the clean-up steps that were usually mandatory to analyze the complicate samples. The combination of mono-lithic MIP column with CEC would be very useful for the screening and development of insecticides and medicines, in particular, during the initial screening of large numbers of compounds.

Acknowledgement

The authors thank Dr. SONG Wei(School of Food Science and Engineering, Harbin Institute of Technology, China) for the measurement of the BET surface area.

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