Molecularly Imprinted Polymers for Selective Extraction of Crystal Violet from Natural Seawater coupled with High-Performance Liquid Chromatographic Determination

LIAN Ziru, and WANG Jiangtao

1) Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, P. R. China

2) Marine College, Shandong University at Weihai, Weihai 264209, P. R. China

Molecularly Imprinted Polymers for Selective Extraction of Crystal Violet from Natural Seawater coupled with High-Performance Liquid Chromatographic Determination

LIAN Ziru1),2), and WANG Jiangtao1),*

1) Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, P. R. China

2) Marine College, Shandong University at Weihai, Weihai 264209, P. R. China

Molecularly imprinted polymers (MIPs) were prepared by the bulk polymerization using crystal violet as the template molecule, and the methacrylic acid and ethylene glycol dimetheacrylate as functional monomer and cross-linker, respectively. Systematic investigations of synthetic conditions were conducted. The surface morphology and recognition mechanism of the obtained polymers were studied using scanning electron microscope and spectrophotometric analysis. MIPs showed high affinity to template molecule and were successfully applied as special solid-phase extraction sorbent for selective extraction of crystal violet from natural seawater. An off-line molecularly imprinted solid-phase extraction (MISPE) method followed by high-performance liquid chromatography with diodearray detection for the analysis of crystal violet was also established. MISPE columns have good recoveries for crystal violet standard solutions and good linearity was obtained over the concentration range of 0−200 μg L−1(R2> 0.99). Finally, two natural seawater samples were investigated. The recoveries of spiked seawater on the MISPE columns were from 44.47% to 62.34%, the relative standard deviation (n=3) being in the range of 2.89%−5.96%.

crystal violet; molecularly imprinted polymer; adsorption; solid-phase extraction; natural seawater

1 Introduction

Crystal violet (CV, Basic Violet 3 or Gentian violet) and malachite green (MG, Basic Green 4) belong to the group of triphenylmethane dyes (Fig.1). Both of them have found extensive use all over the world in the fish farming industry as a fungicide, ectoparasiticide and disinfectant (Rushing and Bowman, 1980; Guo et al., 2011; Oplatowska et al., 2011) .These compounds are normally present in chromatic forms, but they can be easily reduced to leuco (i.e., colorless) forms. Relevant studies have shown that the dyes and their leuco forms are potential carcinogens, teratogens and mutagens (Culp and Beland, 1996; Shivaji et al., 2004; Singh et al., 2011). In the U.S., the Food and Drug Administration (FDA) stipulates a minimum sensitivity of 1 ng g−1for regulatory testing (U.S. Food Drug Administration, 2003). In the European Union (EU), monitoring methodologies must meet the minimum required performance limit at 2 ng g−1for the sum of parent drugs and their leuco forms (European Commission, 2004).

In response to concerns regarding the health risks associated with the use of crystal violet, sensitive and selective analytical methods are needed for their determination in real samples. In recent years, high-peformance liquid chromato-graphy (HPLC) methods connected to mass spectrometric detection have been used for the determination of the dye (Rushing and Hansen, 1997; Villar-Pulido et al., 2011). But these methods cannot reach the trace level in the samples and time-consuming sample preparation is necessary. Solid-phase extraction (SPE) is an extraction method that uses a solid phase and a liquid phase to isolate one, or one type, of analyte from a solution. SPE is usually used to clean up a sample before using a chromatographic or other analytical method to quantitate the amount of analyte (s) in the sample (Stubbings et al., 2005; Stubbings and Bigwood, 2009). One of the main disadvantages of the classical SPE sorbents (C8, C18, etc.) is low selectivity. New sorbents such as molecularly imprinted polymers (MIPs) and immunosorbents have been increasingly developed to meet the need of selectivity. Coupling MIPs with SPE is possible to combine the advantages of both molecular recognition and traditional separation methods. Molecularly imprinted solid-phase extraction (MISPE) may achieve higher enrichment and clean-up efficiencies than traditional SPE cartridges, dueto the coupling of the high specificity, selectivity and sensitivity of the molecular recognition mechanism with the high resolving power of the separation methods (Puoci et al., 2008; Mei et al., 2011). To date, MISPE has become the most promising SPE method for trace analysis and has been extensively reviewed (Michailof et al., 2008; Wang et al., 2012).

Fig.1 Molecular structures of crystal violet (a) and malachite green (b).

At present, most methods for crystal violet determination are meant for the analysis of food, drinking water or river water samples (Safarik and Safarikova, 2002; Dowling et al., 2007; Shen et al., 2011) , and few methods have been devoted to natural seawater samples. In this work, bulk polymerization was employed to prepare crystal violet molecularly imprinted polymers using methacrylic acid as functional monomer. The influences of porogen and cross-linker on the adsorption specificity for the template molecules were investigated. The characteristics of the obtained polymers were analyzed through scanning electron microscope. The imprinted polymers showed high affinity and were successfully applied as special SPE sorbent for selective extraction of crystal violet from natural seawater samples. To our knowledge, this work represents the first attempt of using MIPs as selective SPE sorbents for the determination of crystal violet in natural seawater samples.

2 Materials and Methods

2.1 Materials and Reagents

Crystal violet (CV) and malachite green (MG) were purchased from Sinopharm Chemical Reagent Company (Chengdu, China). Methacrylic acid (MAA) and 2, 2-azoisobutyronitrile (AIBN) were obtained from Kermel Chemical Company (Tianjin, China). Ethylene glycol dimethacrylate (EGDMA) were from Alfa. MAA and EGDMA were purified prior to use to remove the polymerization inhibitor, and AIBN was recrystallized prior to use. Acetonitrile and methanol are all of HPLC grade and from Merck. Unless specified, all reagents were of analytical reagent grade. All water used was obtained from a Millipore Milli-Q purification system (Millipore, Bedford, MA, USA). A 1.0 g L−1CV stock solution (Aladdin Company, Shanghai) was prepared in methanol-water (40:60) and placed in the dark at 4℃.

2.2 Preparation of MIPs and NIPs

MIPs were prepared using bulk polymerization method by dissolving the template molecule CV, functional monomer MAA and cross-linker EGDMA in 5 mL porogen (methanol or chloroform) in a 50 mL borosilicate glass bottle equipped with a rubber cap. This mixture was rotated at 150 r min−1for 6 h at 50℃ for the formation of a complex of imprint molecule and monomers. After adding 20 mg of AIBN, the solution was saturated with dry nitrogen for 15 min and the bottle was placed in a thermostated water bath at 60℃ for 24 h. After polymerization, the polymer was ground with a mortar and pestle, and sieved to give particles with size dimensions of 106μm or less. The product was extracted with methanol containing 5% acetic acid using a Soxhlet apparatus until no template was detected. Then the product was washed with methanol three times and dried under vacuum at room temperature.

The non-imprinted polymers (NIPs) particles were prepared and washed using the same recipe but without the addition of the template. The compositions of MIPs and NIPs are shown in Table 1.

2.3 Morphology Observation

The surface morphology of the particles was observed by a S-4800 cold field emission scanning electron micro scope (Hitachi, Japan). All samples were prepared by wetting the slide glass with a small drop of diluted particle dispersion. Before scanning electron microscopy experiments, the dried specimen was coated under vacuum with a thin layer of gold.

Table 1 Composition of the polymerization mixture for MIPs and NIPs†

2.4 Spectrophotometric Analysis

A series of solutions prepared in methanol was used for spectrophotometric analysis. In the solutions, the CV concentration was fixed at 1×10−5mol L−1, while the concentrations of functional monomer MAA were varied. Five different molar ratios of template to monomer were set at 1:4, 1:8, 1:12, 1:16 and 1:20. The changes in absorbanceand difference absorption spectra of their solutions were determined with Spectrophotometer UV-2550 (SHIMADZU Com., Japan) using pure methanol as reference.

2.5 Steady-State Binding Studies

An amount of 20 mg of the bulk polymer particles was weighed and put into a 4 mL vial. Then 2 mL of CV methanol-water (40:60) standard solution with known concentration was mixed with the polymer. The mixture was slightly shaken on a horizontal shaker for 24 h at 25℃ and then centrifuged for 5 min at 4000 rmin−1. Final CV concentrations were determined by HPLC using diodearray detector at 588 nm. The amount of CV bound to the polymer was calculated by subtracting the amount of free CV from the initial amount added to the mixture.

2.6 HPLC Conditions

HPLC analysis was performed on a Hitachi L-2000 HPLC system containing an L-2130 binary pump, an L-2200 autosampler, an L-2300 column compartment, and an L-2455 diodearray detector monitoring the effluent at 588 nm. The analytical column was a 250 mm×4.6 mm, 5 μm LaChrom C18 column (Hitachi, Japan). The column thermostat was set at 25℃. The mobile phase was HAc/NH4Ac (0.1mmol L−1, pH 4.5)- acetonitrile (3:7), and its flow rate was set at 1.0 mL min−1.

2.7 Preparation of MISPE Column

MISPE column was prepared by packing the dry MIPs (50 mg) in a 1.0 mL glass syringe (2 cm×0.9 cm i.d.). The syringe tube was thoroughly cleaned and dried, and attached with two sieve plates at the bottom and top ends respectively. The MISPE column was previously conditioned with 2 mL of Milli-Q water and methanol successively. Prior to loading the sample, the MISPE column was washed with methanol containing 5% acetic acid to remove residual template molecules from the synthetic procedure until the template could not be detected in the filtrate.

2.8 MISPE for Standard Solutions

CV methanol-water (40:60) standard solutions at different concentrations were passed through the columns at a flow rate of 0.25 mL min−1, and the columns were washed with 2 mL methanol-water (70:30) at the same flow rate. The analyte retained on the sorbent was eluted with 2 mL methanol containing 5% acetic acid for further HPLC analysis.

2.9 MISPE for Natural Seawater Samples

Natural seawater samples were collected from two stations: No.1, located at Qingdao coastal sea (120.29°E, 36.01°N); No.2, located at the northwest Pacific (117.40°E，20.06°N). 20 mL of seawater samples were filtered through 0.22 μm filter before use, and were spiked with CV at concentrations of 5, 10, 15 and 20 μg L−1for loading. The MISPE columns were washed with 2 mL methanol-water (70:30) at a flow rate of 0.25 mL min−1. The analyte retained on the sorbent was eluted with 2 mL methanol containing 5% acetic acid for further HPLC analysis.

3 Results and Discussion

3.1 Preparation of MIPs and NIPs

Fig.2 Schematic representation of crystal violet imprinted polymer.

For a successful imprinting, an adequate molar ratio of template to functional monomer has to be used (Baggiani et al., 2004). In most of cases, the molar ratio of template to functional monomer could be approximately set from 1:3 to 1:5 (the molar ratio was at 1:4, in this experiment). MAA was selected as the functional monomer because it was favorable for hydrogen bond or ionic bond interacttion in the porogen prior to polymerization. A stable donor-receptor complex between template and functional monomer is formed in the imprinting process (see Fig.2).The existence of such a complex leads to the formation of well-defined specific binding sites in the MIPs. So a thermal polymerization was followed, in which AIBN was used as free radical initiator and its half-life at 60℃was around 10 h and proper for polymerization. The cross-linker EDGMA formed a ‘frozen’ spatial structure. In this study, the carboxylic group in MAA can form ionic bond with the amine in the template molecule (Attardi et al., 2000), and the specific and positioned interactions would contribute to the selective affinity of the imprinted polymers.

Fig.3 shows the equilibrium adsorption amounts of MIPs and NIPs for CV under different polymerization conditions. It is obvious that the equilibrium adsorption amount of CV on MIPs was higher when they were in methanol as the porogen instead of being in chloroform, and the highest amount of CV on the M4 was observed.

Fig.3 Binding of MIPs and NIPs for CV under different polymerization conditions (Initial concentration 100 mg L−1).

Table 2 Imprinting factor (α) of polymers in different synthetic conditions

To assess the template recognition performance of the MIPs, imprint factors (α) are calculated, which is defined as α= BMIP/BNIP, where B is the saturation binding amount of template molecule (Lin et al., 2003; Song et al., 2009). The α value is a measurement of the binding specificity and a high α value indicates a higher performing material. The obtained results (see Table 2) show that the α values were found to be obviously higher than those of other polymers when the molar ratio of template to cross-linker was at 1: 40.

3.2 Morphological Features

Different morphologies of the particles prepared in chloroform or methanol as porogen were characterized by using SEM (Fig.4). Compared with the MIPs, the surface of the NIPs was irregular and globular without dense three-dimensional cavities. Although MIPs have rough surfaces, M4 in methanol exhibited more dense and homogenous pores than M3 in chloroform. The uniform and more open structure was obviously favorable for the embedding of the template molecules. Therefore, the optimized polymerization condition was that the molar ratio of template to cross-linker was at 1:40 in methanol as porogen. In particular, M4 and N4 were selected in the following discussion.

Fig.4 SEM photograph of MIPs and NIPs.

3.3 Recognition Mechanism

The principle of molecular imprinting technology is the fixation of the host-guest structure formed by the interaction between template and monomer through hydrogen bond, ionic bond or other interaction forces. Spectrophotometric analysis was employed to elucidate the recogni-tion mechanism on a molecular level. The research into the recognition mechanism of the polymers could be important to understand the imprinting and rebinding phenomena.

The plot of ΔA/b0versus ΔA between CV and MAA was linear with the correlation coefficient of 0.96 (ΔA/b0= −1.20×104ΔA+980.93). This indicates that 1:1 complexes were formed in the mixture solution prior to polymerization, as shown in Fig.2. From the slope, the association constant K was calculated to be 1.20×104mol L−1. The high value of K might contribute to the strong ionic bond between the positive amine in template molecule and the negatively charged carboxylic group in MAA monomer.

3.4 Evaluation of the Recognition Efficiency of MIPs

A primary and indispensable requirement of MIPs is the selectivity of the binding cavities toward the template molecule, and the selectivity properties of MIPs were evaluated by performing the cross-reactivity tests. Mala chite green was structurally similar to crystal violet. Fig.5 shows experimental binding isotherms of M4 and N4. In the 0.5−150 mg L−1concentration range, the M4 exhibited a higher capacity for crystal violet than N4, while the binding for malachite green was obviously lower than template molecule. The weak adsorption of template on NIPs is due to non-specific interaction with the polymer matrix. There are no selective recognition sites on the NIPs and the adsorption for substrates is non-selective.

The molecular recognition of MIPs mainly depends on two factors, molecular dimension of template and matching degree of the bonding sites (Wang and Song, 2010). The spatial diameter of malachite green is a little smaller than that of CV, for there is only one –N(CH3)2in malachite green compared to the CV. The molecular dimension resulted in weaker binding interaction and lower selectivity on M4 for malachite green.

Fig.5 Binding isotherms of M4 for CV (￭, the analyte was crystal violet) or MG (▲, the analyte was malachite green), N4 for CV (♦, the analyte was crystal violet) (n=3).

3.5 MISPE for Standard Solutions

The recoveries of standard solutions containing CV (0−200 μg L−1) were calculated. The results in Fig.6 show that MISPE columns have good recoveries (>88.56%) for template molecule at different concentrations. A standard calibration curve for CV analysis (see Fig.7) was constructed, which was linear (R2> 0.99) over the concentration range of 0−200 μg L−1. Thus, this method could be used to detect some polluted real samples.To minimize the non-specific component of the interaction between CV and polymeric matrices, methanol with different percentages of water was tested as the washing solution, data being not shown here. The results indicated that methanol-water (70:30) and methanol-acetic acid (95:5) were the optimal washing and eluting solution, with the highest eluting effect achieved.

Fig.6 The recoveries of crystal violet in standard solutions. (n=3).

Fig.7 Calibration curve for quantitative analysis of crystal violet.

3.6 MISPE in Natural Seawater Samples

Fig.8a is the chromatogram of No.1 sample after addition of 10 μg L−1of CV, in which the complexity of the seawater matrix background was evident and there was no signal in HPLC chromatograms. The extraction on MISPE column can successfully clean up the natural seawater matrix, thus allowing the extraction of CV with high selectivity (Fig.8b). Contrary to this, NIPs column showed no such selectivity and there was little crystal violet in the elutions (Fig.8c). Hence MIPs offer itself as a simple and straightforward technique for the direct analysis of crystal violet in natural seawater without lengthy sample cleanup.

Fig.8 HPLC chromatograms of No.1 seawater extract spiked with 10 μg L−1crystal violet before (a) and after extracted by MISPE column (b) and NISPE (c) with enrichment factor of 10.

Table 3 Recovery and R.S.D. of crystal violet on MISPE columns from 20 mL spiked seawater samples with an enrichment factor of 10 (n=3)

The result from Table 3 shows that the recoveries of No.1 and No.2 spiked samples on MISPE columns were respectively 44.47%−55.36% and 59.58%−62.34%, and the values of the relative standard deviation (n=3) were in the range of 2.89%−5.12% and 3.20%−5.96%. Possibly, the factors of salinity, pH and dissovled organic matter in natural seawater have influences on the recovery. No.1 samples collected from the coastal sea in Qingdao with matrices more complicated than those of No.2 sample from northwest Pacific, were seriously affected by anthropogenic activities. This may be the crucial factor for different recoveries in different areas.

4 Conclusions

The crystal violet imprinted polymers were prepared by bulk polymerization using MAA and EGDMA as the functional monomer and cross-linker respectively. The obtained MIPs showed good selectivity and affinity for crystal violet. Accordingly, a method was successfully developed for analysis of crystal violet in natural seawater by using the obtained MIPs as the SPE sorbent coupled with HPLC. An off-line MISPE method followed by high-performance liquid chromatography with diodearray detection for the measurement of crystal violet was also established. The recoveries and satisfied precision prove that the proposed method is valid for the analysis of crystal violet in natural seawater sample.

Acknowledgements

This project was supported by the Natural Science Foundation of China (41076065) and the Major State Basic Research Development Program of China (973 Program) (2010CB428701).

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(Edited by Ji Dechun)

(Received July 1, 2012; revised March 3, 2013; accepted March 17, 2013)

© Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2014

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E-mail: jtwang@ouc.edu.cn