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Analytica Chimica Acta 639 (2009) 42–50

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l homepage: www.e lsev ier .com/ locate /aca

tudy of molecularly imprinted solid-phase extraction of diphenylguanidine andts structural analogs

ing Fana,∗, Yafang Weia, Jianji Wanga, Chunlai Wub, Huili Shia

School of Chemistry and Environmental Science, Key Laboratory of Water Environment and Pollution Control for Yellow and Huai Rivers, Ministry of Education,enan Key Laboratory for Environmental Pollution Control, Henan Normal University, Xinxiang, Henan 453007, PR ChinaDepartment of Environmental and Chemical Engineering, Luoyang Institute of Science and Technology, Luoyang, Henan 471023, PR China

r t i c l e i n f o

rticle history:eceived 20 November 2008eceived in revised form 6 February 2009ccepted 25 February 2009vailable online 9 March 2009

eywords:iphenylguanidineolecular imprinting

a b s t r a c t

A new molecularly imprinted polymer was prepared as a solid-phase extractant by bulk polymerizationwith diphenylguanidine (DPG) as template molecules. Its specific recognition characteristics were studiedin aqueous buffer and in acetonitrile. It was shown that in acetonitrile, the maximum binding capacitiesof the imprinted and non-imprinted polymers were 474 and 389 �mol g−1, respectively. There exist twokinds of binding sites on the imprinted polymer, the static binding equilibrium could be reached in 9 h, andthe DPG could be completely separated from its structural analogs, including diphenylthiourea, diphenyl-carbazide and 1,4-diphenylsemicarbazide after washing with 0.5% acetic acid/acetonitrile. In aqueousbuffer medium, the influence of pH, phosphate buffer concentration, and ionic strength on the binding

olid-phase extractionelectivityeparation

capacities of DPG on the polymers were investigated. The results indicated that DPG could be separatedfrom moroxydine hydrochloride after washing with a solution of phosphate buffer (0.067 mol L−1, pH4.5)/methanol (90:10, v/v). It is suggested that hydrogen bonding and ionic bonding between the bindingsites and the DPG played an important role in molecular recognition in acetonitrile, whereas ionic bondingand hydrophobic interactions were predominant in aqueous buffer. The prepared molecularly imprintedpolymer has been used for the pre-concentration and selective separation of DPG from environmental

water samples.

. Introduction

Diphenylguanidine (DPG) is used as a primary accelera-or in vulcanization of rubber, as a secondary accelerator forulfur-containing compounds such as thiazoles, sulfonamides andhiurams and as a minor use for standardizing acids. Due to theelatively high solubility at environmental pHs, low octanol waterartition coefficient (<3) and low volatility, DPG is not expected toe adsorbed on sediment and will mainly be present in the aqueous.uman exposure to DPG may occur as a result of dermal contact,

nhalation of particulates, or unintended oral ingestion. AlthoughPG has been reported to cause contact dermatitis, it is poorlybsorbed through skin and can easily be absorbed through the gas-rointestinal tract. The toxicity of DPG has attracted attention from980s [1,2]. It has been shown that DPG is toxic to fish and algae,

nd harmful to daphnia in several acute studies. In man, earliernd unconfirmed studies described the following symptoms afterorkplace exposures to DPG: eye and mucous membrane irritation,

astric and bilious complaints and disturbed liver metabolism. Lab-

∗ Corresponding author. Tel.: +86 373 3325971; fax: +86 373 3326445.E-mail address: fanjing@henannu.edu.cn (J. Fan).

003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2009.02.045

© 2009 Elsevier B.V. All rights reserved.

oratory tests indicated that this material may be a teratogen (Amestest). Therefore, diphenylguanidine is a candidate for further workdue to its high toxicity profile for human health and environment[3,4].

So far as we known, methods for the determination of DPGinclude high performance liquid chromatography (HPLC), thin-layer chromatography, and liquid chromatography coupled withmass spectrometry (LC–MS). However, these methods are used foreither purity analysis [5] or qualitative analysis in real samples [6].The quantitative analysis of DPG in environmental samples has notbeen reported till now. Because of the matrix interferences andthe very low concentration of DPG in environmental samples, aseparation/pre-concentration process is required. At present, solid-phase extraction (SPE) is the mostly often used method for thepretreatment of samples. The problem is that the selectivity of theconventional SPE sorbents is not high. Although the subsequentlyreported immunoadsorbents provide high selectivity, the instabil-ity, high cost and low mechanical intensity make their applications

limited. It is thus important to prepare novel SPE sorbents with highselectivity and stability for analysis and potential environmentalapplications [7].

Molecular imprinting is known as a technique for the prepa-ration of polymers with a predetermined selectivity for the target

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olecule [8–10]. Molecularly imprinted polymer can be synthe-ized by the complexation of a target molecule with functionalonomers through non-covalent or covalent bonds followed by a

olymerization step in the presence of a cross-linker. The templateolecules are then removed to produce a polymer with molecular

ecognition sites, which are able to selectively rebind the tem-late and analytes with similar structures [11]. It is important forhe imprinted polymer that non-specific interactions should be

inimized since other compounds cannot be involved in thesenteractions with the functional monomer. This is also related tohe nature of the shape arrangement of the complex and the char-cter of the actual cavities (pores) or “binding pockets” themselves12]. During the last years, molecularly imprinted polymers haveeen utilized in capillary electrophoresis [13,14], liquid chromatog-aphy [15–19] and used as catalysts and biosensors [20–23] dueo their high selectivity to the target molecules and their struc-ural analogs. In addition, molecularly imprinted polymer has thedvantages of the simplicity of preparation and being stable at highemperatures or in organic solvents, acids or bases [24]. Therefore,

olecularly imprinted polymer holds promise in the developmentf highly selective SPE methods for the determination of trace ana-

ytes. Furthermore, applications of molecularly imprinted polymern SPE [25–28] demonstrated that these polymers have great poten-ial for the complex sample clean-up, especially when the analytend impurities have similar properties. This promoted the applica-ions of molecularly imprinted polymer in environmental analysisn the presence of various contaminants with similar structures.herefore, using molecularly imprinted polymer as SPE sorbent toeparate and enrich DPG from environmental samples will be con-idered in the present work. To date, only one case was reportedor the utilization of compounds containing the guanidine group asemplate to synthesize molecularly imprinted polymer [29]. UsingPG as template to synthesize molecularly imprinted polymer hasot yet been reported.

In this work, we prepared a molecularly imprinted polymer, forhe first time, using DPG as the template and methacrylic acid ashe functional monomer by bulk polymerization. The static bindingapacity and selectivity of the polymer for DPG were evaluated inoth acetonitrile and aqueous buffer. Then the solid-phase extrac-ion procedure was performed, and the results indicated that underptimal conditions, the imprinted polymer can be applied success-ully to separate DPG from its structural analogs in both acetonitrilend aqueous buffer.

. Experimental

.1. Materials and equipments

Methacrylic acid (MAA) and ethylene dimethacrylate (EDMA)ere purchased from Acros Company (NJ, USA). Azobisisobuty-

onitrile (AIBN), potassium dihydrogen phosphate and dipotassiumydrogen phosphate were purchased from Beijing Chemicaleagent Company (Beijing, China). DPG was obtained from Shang-ai Dunhuang Chemical Factory (Shanghai, China). Methanol andcetic acid glacial were obtained from Tianjin No. 3 Chemicaleagent Factory (Tianjin, China), and acetonitrile and chloroformere products from Tianjin kemiou Chemical Reagent Limited Com-any (Tianjin, China). All chemicals were of analytical reagent gradexcept for AIBN, which was of chemical purity grade. MAA andDMA were distilled before use in order to remove the polymer-zation inhibitor. AIBN was recrystallized from methanol and then

ried under reduced pressure before use. Deionized water was usedhroughout the experiments.

A T6 new century UV–vis spectrophotometer (Shanghai, China)as used for the determination of absorbance at a given wave-

ength. A pHS-3C digital pH meter (Hangzhou, China) was used

Acta 639 (2009) 42–50 43

for the pH measurements. IR spectra (4000–800 cm−1) in KBrwere recorded using a Perkin-Elmer 983 infrared spectrophotome-ter (Norwalk, USA). The SEM micrographs of the sorbents wereobtained at 20.0 kV on a JSM-5610LV scanning electron microscopy(JEOL, Japan). The specific surface area of particles in dry state wasdetermined by a 3H-2000 Brunauer–Emmett–Teller (BET) appara-tus (Beijing, China).

2.2. Spectrophotometric analysis of the interaction between DPGand the functional monomer

A series of solutions was prepared with a fixed concentrationof DPG (0.1 mmol L−1) but varied amounts of MAA (0, 0.2, 0.4, 0.6,0.8 mmol L−1) in acetonitrile. After standing for 1 h, the change inabsorbance was determined with corresponding solutions withoutDPG as reference.

2.3. Preparation of the diphenylguanidine-imprinted polymer(MIP)

For the preparation of the diphenylguanidine-imprinted poly-mer, the template (DPG, 0.11 g, 0.50 mmol) was dissolved in thechloroform (CHCl3, 5.00 mL) in a 25 mL thick-walled glass tube. Thefunctional monomer (MAA, 0.17 mL, 2.00 mmol) was then addedand the mixture was allowed to stand for 1 h to ensure completecomplex formation of the template with MAA. Then the cross-linking monomer (EDMA, 1.89 mL, 10.00 mmol) and the initiator(AIBN, 0.03 g, 0.18 mmol) were added. The above solution wasdegassed under sonication with nitrogen for 10 min to eliminateoxygen, and then the glass tube was sealed. The polymerizationreaction was allowed to proceed at 58 ◦C for 24 h in a water bath.The resultant rigid polymer was grounded in a mortar and passedthrough 96 and 178 �m sieves. The obtained particles were soxhletextracted with a mixture of methanol/acetic acid (9:1, v/v) for 72 hto remove the template untill the absorbance peak disappeared at240 nm. Then the particles were washed with methanol to removethe residual acetic acid and dried to constant weight under vacuumat 60 ◦C. For comparison purpose, non-imprinted polymer (NIP) wasprepared by using the same recipe, except for the addition of thetemplate, and the same procedure.

2.4. Static binding experiments

2.4.1. Binding characteristics of DPG on the polymers and theselectivity of MIP in aqueous buffer and in acetonitrile

20.0 mg of the polymer particles was mixed with 5.0 mL ofsolvent (acetonitrile or phosphate buffer) containing DPG orits structural analogs at a known concentration. The mixtureswere incubated at room temperature for a fixed period of time.After incubation, concentration of these compounds in the super-natant solution was determined by measuring their maximum UVabsorption using a UV–vis spectrophotometer. The amount of thecompounds bound to the polymers, Q (�mol g−1) was calculatedby subtracting the amount of the unbound compounds from thatof the added compounds to the mixture. This method was used tostudy the binding capacity, binding kinetics and selectivity of thepolymers in acetonitrile, as well as the effects of pH, buffer con-centration and ionic strength on these binding characteristics inaqueous buffer.

2.4.2. Specific binding of DPG on the polymers in

acetonitrile–water solutions

DPG/acetonitrile/water solutions were prepared by fixing theDPG concentration at 2.5 mmol L−1 and changing the volume ratioof acetonitrile/water (100:0; 90:10; 70:30; 50:50; 30:70; 10:90.).20.0 mg of the polymer particles was mixed with 5.0 mL of the

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PG/acetonitrile/water solutions. Such mixtures were incubated atoom temperature for 24 h. Absorbance of the supernatants waseasured at their �max with spectrophotometer. Then the amount

f the compounds bound to the polymers can be calculated fromhe amount of the unbound and the added compounds.

.5. Column experiments

For the column experiment, 50.0 mg of MIP or NIP waslurred in methanol, and then poured into a glass columnØ3 mm × 17 mm). The column was treated successively with ace-onitrile and phosphate buffer (0.067 mol L−1, pH 4.5). Then DPGr its structural analogs, dissolved in acetonitrile or phosphateuffer (0.067 mol L−1, pH 4.5), was passed through the column atcertain flow rate. The compounds retained on the column wereashed with proper washing solvent and eluted with the solu-

ion of methanol/water/acetic acid (79:20:1, v/v/v) at a flow rate of.13 mL min−1. The eluates were analyzed by spectrophotometry.

. Results and discussion

.1. Interaction between DPG and the functional monomer

The principle of molecular imprinting lies in the preservation ofhe prepolymerized host/guest structure into a polymer matrix. Sot is significant that the template and the monomer can form sta-le complexes through hydrogen bonding, ionic bonding or other

nteraction forces in the prepolymerization mixture. The study ofnteraction between template and functional monomer will beelpful to understand molecular recognition mechanism. Since theross-linker and initiator would be much less important for thenteraction and DPG has strong absorption in UV–vis range, spec-rophotometric method was employed to elucidate the possiblenteraction between DPG and the functional monomer at different

olar ratios of DPG to MAA in acetonitrile. As shown in Fig. 1, thebsorption spectra showed a blue shift of the absorption band andhe absorbance value decreased with the increase of the amountsf MAA. This indicated that strong interactions were producedetween DPG and MAA. It is probably that C NH of the DPG

nteracts with MAA through ionic bonding or hydrogen bonding,nd the NH of the DPG also interacts with the COOH of theAA through hydrogen bonding [29]. Meanwhile, the absorption

pectrum of DPG in acetonitrile is very sensitive to the presence ofmall amounts of MAA. However, when the concentration of MAA

ig. 1. Absorption spectra of DPG in the presence of MAA in acetonitrile. Concen-ration of DPG, 0.1 mmol L−1; concentrations of MAA, 1: 0.0, 2: 0.2, 3: 0.4, 4: 0.6, 5:.8 mmol L−1; l = 1 cm; room temperature.

Acta 639 (2009) 42–50

was more than 0.4 mmol L−1 (about four times of DPG concentra-tion), the change in absorption spectra is not significant. Existenceof excess MAA will lead to the increase of the non-specific adsorp-tion. So in our experiment, the molar ratio of 1:4 for DPG and MAAwas used.

3.2. Characteristics of the IR spectra, SEM image and BET

The result of IR spectra determination showed that the IR spectraof MIP were similar to that of NIP, indicating that the template hadbeen removed completely. O H stretching vibration was reflectedat 3442 cm−1, stretching vibration of CH2 or CH3 was locatedat around 2959 cm−1, and the band at 1731 cm−1 was assigned tothe absorption band for COOH. The observed features at around1261 and 1158 cm−1 indicated the C O C stretching vibrations. Thebands at 1457 and 1637 cm−1suggest the CH3 bending vibrationand the C C vibration, respectively.

A comparison of scanning electron microscopy observations forMIP and NIP showed that NIP has a rather smooth surface com-pared with that of MIP. This may be explained by the fact that theaddition and removal of template affected the outer surfaces of themolecularly imprinted polymer.

The specific surface area of the particles in dry state was deter-mined by a 3H-2000 Brunauer–Emmett–Teller (BET) apparatus(Beijing, China). 1.0 g of the particle was placed in a sample holderand degassed in a N2-gas stream at 150 ◦C for 1 h. Adsorption anddesorption of the gas were performed at 100 and 60 ◦C, respec-tively. Values obtained from desorption step were used for thespecific surface area calculation. The result indicated that the spe-cific surface areas of MIP and NIP particles were 172 and 149 m2 g−1,respectively. This was consistent with the conclusion obtained fromscanning electron microscopy.

3.3. Static binding studies

3.3.1. Binding kinetics curve of DPG on the MIP in acetonitrileBinding kinetics experiment was performed by adding 5 mL

(3.0 mmol L−1) of DPG in acetonitrile to the MIP and then deter-mining the concentration of unbound DPG as a function of time. Asshown in Fig. 2, the amounts of DPG bound on the MIP increased

rapidly with the time during the first 3 h, then increased slowly.After 9 h, the amounts of the bound DPG did not change any longer,indicating that binding equilibrium was reached. It is reasonableto assume that the flat pores in the polymer surface are benefitto the rapid binding, whereas the deep pores are benefit to the

Fig. 2. Binding kinetical curve of DPG on the MIP in acetonitrile. Polymer, 20.0 mg;V = 5 mL; initial DPG concentration, 3.0 mmol L−1; room temperature.

imica Acta 639 (2009) 42–50 45

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low binding. Here, the pseudo-first order kinetic model (1) andhe pseudo-second order kinetic model (2):

n(Qe − Qt) = ln Qe − kat (1)

t

Qt= t

Qe+ 1

kbQ 2e

(2)

ere used to describe the binding process [30]. In these equations,a and kb are the rate constants for the first order sorption (t−1) andhe second order sorption (�mol g−1 s−1), respectively. Qt stands forhe binding capacity (�mol g−1) at any given time t, and Qe indicateshe equilibrium binding capacity (�mol g−1).

The pseudo-first order kinetic model is the most widely usedate equation to describe the sorption of a solute from liquid solu-ion. In this model, reaction on the surface, i.e., the transitionrom free to adsorbed states is the step controlling the rate of thedsorption process, and the solute molecules react with one kindf adsorption site. When the pseudo-first order kinetic model waspplied to our experimental data, a good linear relationship with aorrelation coefficient of 0.9878 was observed. Although effectiven many cases, the pseudo-first order equation has been found to bensatisfactory in providing a concrete mechanism for the adsorp-ion process in an equally good number of cases [31,32]. In suchases, the experimental results differ in two important aspects: (i)a(Qe − Qt) does not represent the number of available sites, and (ii)n Qe is not equal to the intercept of the plot of ln (Qe − Qt) against t.n our case, the experimental ln Qe values did not match the valuesbtained from the plot of ln(Qe − Qt) versus t. So the binding pro-ess does not fit strictly to a pseudo-first order kinetic model. Theseudo-second order model based on Eq. (2) was therefore applied,hich considered the formation of chemisorptive bond between

he adsorbate and the adsorbent as the rate-limiting step. In thisodel, the solute molecules react with two kinds of adsorption site.hen the pseudo-first order kinetic model was used, the date of t/Qt

ersus t yielded a linear plot with correlation coefficient of 0.9989,ndicating that the binding process can be described by the pseudo-econd order kinetics, and the chemisorption is the rate-controllingtep [32].

.3.2. Binding isotherms of DPG on the MIP and Scatchardnalysis in acetonitrile

The binding capacity of DPG on the MIP is an importantarameter for determining how much molecularly imprinted poly-er is required to quantitatively bind a specific amount of DPG

rom solution. So it would be very important to investigate thePG binding capacities on the MIP. For this purpose, the binding

sotherms were determined in the initial DPG concentrations rangef 1.0–6.5 mmol L−1. It was found that in the studied concentrationange, the amounts of DPG bound on the MIP increased with thencrease of the initial concentration of DPG. Compared with NIP,he higher affinity of MIP is ascribed to its specific binding for DPG,hich is related to the selectivity/specificity of MIP. This suggests

hat there existed specific rebinding sites for DPG in the MIP. Inhe high concentration range, the binding capacities incline to betable.

Analysis of the binding date can be performed by using Langmuirodel (shown in Fig. 3). The Langmuir isotherm is a function that

escribes a relationship between the equilibrium concentration ofhe bound (Ceq) and the free (Q) guests in homogeneous system withwo different coefficients according to the following equation:

QmaxCeq

=B + Ceq

(3)

here Q stands for the binding capacity (�mol g−1), Ceq equilib-ium concentration of DPG (mmol L−1), Qmax the maximum bindingapacity (�mol g−1), and B a constant. In order to examine our equi-

Fig. 3. Langmuir isotherms for the binding of DPG on the MIP and NIP in acetonitrile(n = 3). Polymer, 20.0 mg; V = 5 mL; adsorption time, 24 h; room temperature.

librium binding date more directly, Eq. (3) was changed into theform of Eq. (4):

Ceq

Q= Ceq

Qmax+ B

Qmax(4)

which indicates a linear relationship between Ceq/Q and Ceq. Fromthe slope and intercept of the linear plot, the maximum bindingcapacity of DPG on both MIP and NIP was calculated to be 474and 389 �mol g−1, respectively. Although the chemical composi-tion of MIP and NIP was similar, their spatial structure was differentgreatly. There existed cavities whose shape and position of func-tional groups matched DPG in MIP but not in NIP, the differencein their Qmax values was based on the selective binding of thesecavities.

Langmuir model can provide us the maximum binding capacityof DPG on the MIP, but it cannot illuminate the binding characteris-tic of the MIP to DPG in detail. In order to validate the Qmax values ofthe MIP and further understand its binding characteristic, Scatchardmodel:

Q

Ceq= Qmax − Q

Kd(5)

was used. In the above equation, Kd stands for the equilibrium dis-sociation constant (mmol L−1). The relationship between Q/Ceq andQ, namely, the Scatchard plot, was shown in Fig. 4. It is appar-ent that the plot in Fig. 4(a) contains two distinct linear sections.This suggests that there exist two types of binding site in MIP,which provides a strong support for the results obtained from bind-ing kinetics study. From the slope and intercept of the Scatchardplot, Kd and Qmax for the higher-affinity binding sites have beencalculated to be 0.088 ± 0.013 mmol L−1 and 373 ± 16 �mol g−1,whereas Kd and Qmax for the lower affinity binding sites were0.310 ± 0.019 mmol L−1 and 488 ± 5 �mol g−1, respectively. The twokinds of binding sites may be produced in the following way: DPGformed two kinds of complexes with MAA in the process of poly-merization, and they were fixed in the MIP matrix. After removalof the template, two types of binding sites with distinct affin-ity existed in the MIP. Fig. 4(b) shows that the Scatchard plot ofNIP was a single straight line, suggesting that there existed one

kind of binding site in the NIP. The Kd and Qmax values calculatedfor NIP were 0.287 ± 0.019 mmol L−1 and 391 ± 6 �mol g−1, respec-tively. The latter value (391 �mol g−1) was in excellent agreementwith that obtained from Langmuir model (389 �mol g−1).

46 J. Fan et al. / Analytica Chimica Acta 639 (2009) 42–50

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Table 1Binding selectivity of MIP and NIP for DPG and other compounds in acetonitrile(n = 3, t0.05,2 = 4.30).a.

Compound QMIP (�mol g−1) QNIP (�mol g−1)

DPG 371.9 ± 7.8 306.7 ± 6.7Diphenylcarbazide 50.9 ± 2.8 46.8 ± 3.0Diphenylthiourea 31.5 ± 2.0 46.7 ± 3.01,4-Diphenylsemicarbazide 24.2 ± 1.8 27.6 ± 1.6

with the increase of water content, leading to the increased bind-

ig. 4. Scatchard plots for the binding of DPG on the MIP and NIP in acetonitrile: (a)IP and (b) NIP. Polymer, 20.0 mg; V = 5 mL; initial DPG concentration, 2.5 mmol L−1;

dsorption time, 24 h; room temperature.

.3.3. Selectivity evaluation of the MIP in acetonitrileSelectivity test of the MIP was carried out by using a series of

tructural analogs 1,4-diphenylsemicarbazide, diphenylcarbazidend diphenylthiourea (pKa < 2) whose structures were shown inig. 5. Binding of these compounds on the MIP and NIP was inves-igated by the equilibrium binding experiments. The results wereisted in Table 1. It is obvious that MIP exhibited much higher bind-ng affinity for DPG (pKa = 10.12) than for the structural analogs.

he binding capacity of NIP for DPG was lower than that of MIP,ut their binding capacities for the structural analogs were closeo each other. These results suggest that the imprinting methodreated a micro-environment based on the shape and position of

Fig. 5. Structures of the compounds used

a The conditions for the measurements were as follows: polymer, 20.0 mg; initialconcentration of the compounds, 2.5 mmol L−1; V = 5.0 mL; adsorption time, 24 h;room temperature.

the functional groups which recognized the template molecule.The selectivity test results also give some insights into the possi-ble molecular recognition mechanism. There were one C NH andtwo NH groups, which can interact with the COOH within themicrocavities of the MIP through ionic bonding or hydrogen bond-ing. Among the tested structural analogs, diphenylthiourea is themost similar to DPG in molecular size and structure, but its bind-ing capacity was still very low. This is possibly due to the fact thatunlike DPG, no C NH group exist in diphenylthiourea. Size of theother structural analogs is larger than DPG, so it is not easy forthese compounds to go into and out the microcavities. Meanwhile,there is no C NH group in these structural analogs, which is abasic function group and has a significant effect on the formationof the complex between DPG and MAA. From these results, it canbe concluded that both non-covalent interactions and the size ofthe target compounds were very important in the binding process.Although the chemical composition of MIP and NIP is the same, thelatter polymer can only bind the test compounds by non-specificadsorption because no proper cavities and recognition sites wereformed in this polymer.

3.3.4. Effect of the content of water in acetonitrile on the bindingcapacity

In order to provide more information for recognition mecha-nism, we decided to examine the binding characteristic of MIPby changing water content in acetonitrile. As shown in Fig. 6, theamounts of DPG bound to MIP and NIP decreased rapidly withthe water content changing from 0% to 10%. The possible reasonis that ionic bonding and hydrogen bonding were dominant at themoment, and the addition of water can interfere with these inter-actions due to its high polarity and strong ability to form hydrogenbonding [33]. Above 10% water, the DPG binding capacities on theMIP and NIP increased with the increase of water content, butspecific binding decreased gradually in the process. This can beexplained by the fact that the hydrophobic interactions increased

ing capacities of DPG. However, hydrophobic interaction does nothave selectivity, which decreased the specific binding. These resultssuggest that ionic bonding and hydrogen bonding interactions aredominant for the selectivity of MIP for DPG.

in the selectivity test in acetonitrile.

J. Fan et al. / Analytica Chimica Acta 639 (2009) 42–50 47

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ig. 6. Effect of water content in acetonitrile on the binding capacity of DPG onhe polymers (n = 3). Polymer, 20.0 mg; initial concentration of DPG, 2.5 mmol L−1;= 5 mL; adsorption time, 24 h; room temperature.

.3.5. Effect of pH on the binding capacity in phosphate bufferThe effect of pH on the binding capacities was studied in phos-

hate buffer from pH 2.3 to 8.1. It can be seen from Fig. 7 that in thetudied pH range, the binding capacity of MIP for DPG increasedrom 21.57 to 400.6 �mol g−1, compared with that of NIP increasedrom 19.42 to 375.2 �mol g−1. Generally, it is expected that numberf the ionized COOH was increased with increasing values of pH34], and as a result, the ionic bonding interaction between DPGnd the polymers was enhanced. In addition, DPG binding capacityn the MIP was still higher than that of NIP in the pH range studied,uggesting that MIP was capable of binding DPG more strongly thanIP even in the aqueous solutions. The specific binding was found

o increase as the value of pH changed from 2.3 to 4.5, and was notensitive to the pH values from 4.5 to 8.1. Therefore, pH 4.5–8.1 washosen for the further experiments. It should be indicated that theoor solubility of DPG in alkaline solution limits our study for theinding capacity of the polymers in solutions with pH > 8.3.

.3.6. Effect of the buffer concentration on the binding capacity inhosphate buffer

Fig. 8 illustrates the effect of phosphate buffer concentration onhe DPG binding capacities of MIP and NIP in concentration range

ig. 7. Effect of pH on the binding capacity of DPG on the polymers (n = 3). Polymer,0.0 mg; initial concentration of DPG, 2.5 mmol L−1; V = 5 mL; adsorption time, 24 h;oom temperature; buffer, phosphate buffer (0.067 mol L−1).

Fig. 8. Effect of buffer concentration on the binding capacity of DPG on the polymers.Polymer, 20.0 mg; initial concentration of DPG, 2.5 mmol L−1; V = 5 mL; adsorptiontime, 24 h; room temperature; buffer, phosphate buffer (pH 7.4).

from 0.025 to 0.20 mol L−1 when pH was fixed at 7.4. It can be seenthat binding capacities of both MIP and NIP decreased with increas-ing buffer concentration at concentrations lower than 0.10 mol L−1.On the other hand, the binding capacities did not vary any more atconcentrations above 0.10 mol L−1. Binding should be due mostlyto ionic interaction where the buffer ions can more effectively actas competitors. MIP and NIP were, however, affected equally, indi-cating that the specific part of the total binding was constant. In allsubsequent experiments, a buffer concentration of 0.067 mol L−1

was used in order to have sufficient buffer capacity in the incubationmixtures.

3.3.7. Effect of ionic strength on the binding capacity inphosphate buffer

The effect of NaCl concentrations on the binding capacities ofthe polymers was also investigated. As shown in Fig. 9, high ionicstrength weakens the binding of DPG on the polymers when moreNaCl was added up to the concentration of 0.12 mmol L−1. The

decrease in the binding capacities of DPG on the polymers may becaused by the following factors: (i) the increase of ionic strengthin the solution would lead to the decrease of ionic bonding inter-action as ionic bonding and hydrophobic interactions occur at the

Fig. 9. Effect of ionic strength on the binding capacity of DPG on the polymers (n = 3).Polymer, 20.0 mg; initial concentration of DPG, 2.5 mmol L−1; V = 5 mL; adsorptiontime, 24 h; room temperature; buffer, phosphate buffer (0.067 mol L−1, pH 7.4).

4 imica Acta 639 (2009) 42–50

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Table 2Recovery of DPG from the MIP and NIP columns using phosphate buffer/methanolsolution as a washing solvent.a.

Recovery (%)

MIP NIP

Breakthrough (1 mL) 0 0First washing (5 mL) 0 0Second washing (5 mL) 5.8 34.0Third washing (5 mL) 13.5 28.2Fourth washing (5 mL) 14.8 13.0Elution (5 mL) 68.6 28.9Total recovery (%) 102.7 104.1

8 J. Fan et al. / Analytica Ch

ame time [35]; (ii) the counter ions of salt interact with DPG viaharge–charge interactions and mask the binding sites; (iii) theecrease in the binding capacity as the ionic strength increasesan be attributed to the repulsive electrostatic forces between theolymers and DPG molecules. As NaCl concentrations were variedrom 0.12 to 0.20 mol L−1, the binding capacity increased first andhen kept constant. At the moment, we could not give a reasonablexplanation for such a phenomenon. At NaCl concentrations below.20 mol L−1, the decrease in binding capacities was equal for bothIP and NIP. This indicated that only non-specific part of the total

inding was affected below this concentration.

.4. Solid-phase extraction of DPG and its structural analogs

.4.1. Effect of flow rate on the binding of DPGIn the column experiment, 50.0 mg of MIP was mixed with

ethanol and then packed in a glass column. The column was equi-ibrated with acetonitrile and phosphate buffer (0.067 mol L−1, pH.5). Then 5 mL of phosphate buffer (0.067 mol L−1, pH 4.5) contain-

ng 250 �g of DPG was passed through the column. DPG elutionrom the column was carried out with 10 mL of MeOH/H2O/HAc79:20:1, v/v/v). The flow rate of DPG solution through the packedolumn was a very important parameter affecting the binding ofPG. Hence, the influence of flow rate on the DPG binding was

tudied in the range of 0.04–1.25 mL min−1. As shown in Fig. 10,00% of DPG was bound by MIP when the flow rate was less than.25 mL min−1. However, as the flow rate was up to 1.25 mL min−1,he binding of DPG was only 87.7%. The possible reason is that theast flow rate makes DPG do not have sufficient time to be boundy MIP in the column. Therefore, we choose 0.25 mL min−1 as theest flow rate in our experiment.

.4.2. Selection of the elution solventIt was known that elution solvent had great influence on the

ecovery of DPG. In order to optimize the elution procedure, sev-ral elution solvents such as methanol, methanol/water/acetic acid79:20:4, v/v/v) and methanol/water/acetic acid (79:20:1, v/v/v)ere used to obtain the maximum recovery of DPG. For this pur-ose, 5 mL of phosphate buffer (0.067 mol L−1, pH 4.5) containing

50 �g of DPG was passed through the column. DPG elution fromhe column was carried out with 10 mL of elution solvent at the flowate of 0.13 mL min−1. The results indicated that methanol could note used as elution solvent because the buffer substances retainedn the column could not be completely dissolved in methanol. It

ig. 10. The effect of flow rate on the binding of DPG on MIP. Polymer, 50.0 mg; CDPG,0 mg L−1; V = 5 mL; room temperature.

a The conditions for the measurements were as follows: loading solvent, phos-phate buffer (0.067 mol L−1, pH 4.5); CDPG, 50 mg L−1; V = 1 mL; washing solvent,phosphate buffer (0.067 mol L−1, pH 4.5)/methanol (90:10, v/v); elution solvent,methanol/water/acetic acid (79:20:1, v/v/v).

is found that the recoveries of DPG showed an elution efficiencyof approximately 100% when methanol/water/acetic acid (79:20:4,v/v/v) and methanol/water/acetic acid (79:20:1, v/v/v) were usedas elution solvents. Consequently, methanol/water/acetic acid(79:20:1, v/v/v) was employed in our further work, and a flow rateof 0.13 mL min−1 was selected for elution solvent.

3.4.3. Selection of the washing solventIt was necessary to choose the appropriate washing solvent in

order to minimize the non-specific interaction between the ana-lytes and the imprinted polymer. In the washing process, DPGnon-specifically bound to the polymer will be eluted, whereas partof the DPG specifically bound remains trapped in the polymer dueto the specific interactions. In the NIP, quantitative elution of thetemplate was expected in order to eliminate the disturbance of thenon-specific interactions.

When phosphate buffer was used as loading solvent, phosphatebuffer (0.067 mol L−1, pH 4.5)/methanol was used as the wash-ing solvent. To investigate the washing effect of different volumeratios of phosphate buffer (0.067 mol L−1, pH 4.5)/methanol (100:0;90:10; 80:20, v/v), 5 mL of DPG in phosphate buffer (50 mg L−1)was loaded into the column. Then, the column was washed withthe washing solvents mentioned above. The results indicated thatfor these solvents, no significant difference was observed in wash-ing effect. It was found that if phosphate buffer/methanol solution(100:0, v/v) was used as washing solvent, more volumes of thesolvent were needed. Thus, phosphate buffer/methanol solution

(90:10, v/v) was chosen for further studies. In order to verifythe reliability of this washing solvent, 1 mL (50 mg L−1) and 5 mL(50 mg L−1) of DPG in phosphate buffer were, respectively, loadedinto the column, and the column was then washed with thephosphate buffer/methanol solution (90:10, v/v) and eluted with

Table 3Recoveries of DPG and moroxydine hydrochloride (MXHCl) from the MIP and NIPusing phosphate buffer as a loading solvent.a.

Recovery (%)

MIP NIP

DPG MXHCl DPG MXHCl

Breakthrough (5 mL) 0 83.0 0 88.5First washing 5 mL) 15.7 21.2 35.6 17.8Second washing (5 mL) 28.9 0 39.8 0Third washing (5 mL) 21.3 0 14.8 0Fourth washing (5 mL) 12.3 0 4.9 0Elution (5 mL) 25.8 0 13.7 0Total recovery (%) 104.0 104.2 108.8 106.3

a The conditions for the measurements were as follows: loading solvent, phos-phate buffer (0.067 mol L−1, pH 4.5); concentration, 50 mg L−1; washing solvent,phosphate buffer (0.067 mol L−1, pH 4.5)/methanol (90:10, v/v); elution solvent,methanol/water/acetic acid (79:20:1, v/v/v).

J. Fan et al. / Analytica Chimica Acta 639 (2009) 42–50 49

Table 4Recoveries of DPG, diphenylthiourea (DPT), diphenylcarbazide (DPC), and 1,4-diphenylsemicarbazide (DPSC) from the MIP and NIP using acetonitrile as loading solvent.a.

Recovery (%)

MIP NIP

DPG DPT DPC DPSC DPG DPT DPC DPSC

Breakthrough (5 mL) 3.6 90.3 84.8 82.5 3.0 91.8 89.8 89.3First washing (5 mL) 7.9 14.1 24.0 19.3 95.5 13.5 17.2 17.5Elution (10 mL) 98.0 0 0 0 9.9 0 0 0Total recovery (%) 109.5 104.4 108.8 101.8 108.4 105.3 107.0 106.8

a The conditions for the measurements were as follows: loading solvent, acetonitrile; concentration, 50 �g mL−1; washing solvent, 0.5% acetic acid/acetonitrile; elutionsolvent, methanol/water/acetic acid (79:20:1, v/v/v).

Table 5The recoveries of DPG in different water samples (n = 3).

Sample Before column pre-concentration After column pre-concentration Amount of polymer (mg) Added DPG (�g) Recovery (%) R.S.D (%)

Tap water – – 50.0 25.0 105.2 1.4RL

msNwsww

cdtwlritmwt

3

atptTssds1twatos8Dto

iver water – –ake water – –

ethanol/water/acetic acid (79:20:1, v/v/v). The recoveries werehown in Tables 2 and 3. It was obvious that DPG bound on theIP was washed off from the column in the second times washing,hereas DPG bound on MIP was washed off more slowly due to the

trong specific interaction between MIP and DPG. After four timesashing, most of the DPG bound by the non-specific binding wasashed off.

The structural analogs of DPG such as 1,4-diphenylsemi-arbazide, diphenylcarbazide and diphenylthiourea cannot beissolved in aqueous solution. We investigated, therefore, the selec-ivity of MIP for these compounds in acetonitrile. Towards this end,e optimized the washing solvent when acetonitrile was used as

oading solvent. It was found that 0.5% acetic acid/acetonitrile was aight washing solvent. For example, when 5 mL (50 mg L−1) of DPGn acetonitrile was loaded into the column, and the column washen washed once with 0.5% acetic acid/acetonitrile and eluted with

ethanol/water/acetic acid (79:20:1, v/v/v), only 7.9% of DPG wasashed off from the MIP, while 95.5% of DPG was washed off from

he NIP.

.4.4. Separation of DPG from its structural analogs by the MIPTo assess the possibility to separate DPG from its structural

nalogs using the MIP column, each of the 5 mL (50 mg L−1) of solu-ions prepared by dissolution of DPG or its structural analogs inhosphate buffer (0.067 mol L−1, pH 4.5) or acetonitrile was passedhrough the MIP and NIP columns at the flow rate of 0.25 mL min−1.hen the columns were washed and eluted by the selected washingolvent and the elution solvent. Considering the poor and differentolubility of the structural analogs, separation of DPG from moroxy-ine hydrochloride was investigated in phosphate buffer, whereaseparation of DPG from diphenylthiourea, diphenylcarbazide and,4-diphenylsemicarbazide was studied in acetonitrile. The separa-ion results were given in Tables 3 and 4. It was clear that no matterhat loading solvent was used, binding ratios of all the structural

nalogs were less than 18% on the MIP, indicating that serious break-hrough was observed in the loading step. In contrast, binding ratiosf DPG was more than 95% in this step. After the first washing, all the

tructural analogs were washed off from the column, but more than4% of DPG was still bound on the MIP. These results suggest thatPG can be separated completely from its structural analogs using

he washing procedure described. Meanwhile, the high selectivityf MIP for DPG was further verified.

50.0 25.0 103.6 2.450.0 25.0 102.6 2.5

3.5. Determination of DPG in spiked water samples

Water samples used in the present work include lake water fromnational natural conservation area for birds and wetland of Yubei,river water from Yellow river, and tap water from the campus ofHenan Normal University. The samples were filtered to eliminateany solid impurity. The DPG in the water samples was determined.However, no DPG in the waters could be detected because of itslow concentration. Thus, 25.0 mL of each water sample was passedthrough the column used above. 5.0 mL of eluent was collectedand the concentration of DPG in the eluent was determined. Then25.0 mL of each water sample spiked with DPG at a concentrationlevel of 1.0 mg L−1 was passed through the column. 5.0 mL of eluentwas collected and the concentration of DPG in the eluent was deter-mined. The results were listed in Table 5. As can be seen, recoveriesranging between 102.6% and 105.2% were obtained for tap, lake andriver waters. This confirmed the practical analytic application of theMIP prepared in the present work.

4. Conclusions

In this work, a diphenylguaindine-imprinted polymer wasprepared by molecular imprinting technique using MAA as func-tional monomer. The static adsorption properties and solid-phaseextraction performance of this polymer were studied. From theexperimental results described in the above sections, conclusionscan be drawn as follows: (i) MIP has the more developed surface anda higher specific surface area than NIP; (ii) the imprinted polymerexhibited relatively high binding capability for DPG both in ace-tonitrile and in aqueous solution, and two kinds of bonding siteswas found in the MIP; (iii) it was suggested that hydrogen bondingand ionic bonding between the binding sites and the DPG playedan important role in molecular recognition in acetonitrile, whereasionic bonding and hydrophobic interactions are dominant in aque-ous solution; (iv) the selectivity of imprinted polymer was higherin acetonitrile than in aqueous solution, which was ascribed to thehigher non-specific interaction in aqueous solution; (v) the result of

solid-phase extraction confirmed that careful choice of the solventscan disrupt non-specific binding, allowing a selective extraction ofDPG by the molecular imprinted polymer; (vi) the prepared molec-ularly imprinted polymer can be used for the pre-concentration andselective separation of DPG from environmental water samples.

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cknowledgments

This work was supported financially by the Science and Tech-ology Department of Henan Province (Grant nos. 072102320007nd 082300423202).

eferences

[1] M.A. Bempong, E.V. Hall, J. Toxicol. Environ. Health 11 (1983) 869.[2] Y. Yasuda, T. Tanimura, J. Environ. Pathol. Toxicol. 4 (1980) 451.[3] http://www.jetoc.or.jp/HP SIDS/pdffiles/102-06-7.pdf, SIDS Initial Assessment

Profile, SIAM 14, March 26–28, 2002.[4] http://www.epa.gov/HPV/pubs/summaries/13dphnlg/c14886tp.pdf, SIDS Ini-

tial Assessment Profile, 201-14886A, 2004.[5] National Toxicology Program (NTP), NTP Technical Report on Toxicity Studies

of 1,3-Diphenylguanidine, Research Triangle Park, NC, 1995, No. 42.[6] M.X. Lin, M. Li, A. Rustum, J. Pharm. Biomed. Anal. 45 (2007) 747.[7] M.L. Hu, M. Jiang, P. Wang, S.R. Mei, Y.F. Lin, X.Z. Hu, Y. Shi, B. Lu, K. Dai, Anal.

Bioanal. Chem. 387 (2007) 1007.[8] C. Alexander, H.S. Andersson, L.I. Andersson, R.J. Ansell, N. Kirsch, I.A. Nicholls,

J.O. Mahony, M.J. Whitcombe, J. Mol. Recognit. 19 (2006) 106.[9] A.J. Hall, M. Emgenbroich, B. Sellergren, Top. Curr. Chem. 249 (2005) 317.

10] D.A. Spivak, Adv. Drug Deliv. Rev. 57 (2005) 1779.11] F. Chapuis, V. Pichon, F. Lanza, S. Sellergren, M.C. Hennion, J. Chromatogr. A 999

(2003) 23.12] S.A. Piletsky, T.L. Panasyuk, E.V. Piletskaya, I.A. Nicholls, M.J. Ulbricht, J. Membr.

Sci. 802 (1998) 263.13] V.T. Remcho, Z. Tan, J. Anal. Chem. 71 (1999) 248A.

[[[

[

Acta 639 (2009) 42–50

14] C. Cacho, L. Schweitz, E. Turiel, C. Perez-Conde, J. Chromatogr. A 1179 (2008)216.

15] M. Kempe, K. Mosbach, J. Chromatogr. A 664 (1994) 276.16] L. Fischer, R. Muller, B. Ekberg, K. Mosbach, J. Am. Chem. Soc. 113 (1991) 9358.

[17] J. Matsui, I.A. Nicholls, T. Takeuchi, Tetrahedron: Assymetry 7 (1996) 1357.[18] F.G. Tamayo, M.M. Titirici, A. Martin-Esteban, B. Sellergren, Anal. Chim. Acta 542

(2005) 38.19] Y.Q. Xia, T.Y. Guo, M.D. Song, B.H. Zhang, B.L. Zhang, React. Funct. Polym. 66

(2006) 1734.20] J.T. Huang, S.H. Zheng, J.Q. Zhang, Polymer 45 (2004) 4349.21] R. Thoelen, R. Vansweevelt, J. Duchateau, F. Horemans, Biosens. Bioelectron. 23

(2008) 913.22] E. Mazzotta, R.A. Picca, C. Malitesta, S.A. Piletsky, E.V. Piletska, Biosens. Bioelec-

tron. 23 (2008) 1152.23] J. Wang, Z.Y. Chen, M.P. Zhao, Y.Z. Li, Chin. Chem. Lett. 18 (2007) 981.24] H.R. Park, S.H. Chough, Y.H. Yun, S.D. Yoon, J. Polym. Environ. 13 (2005) 81.25] X.M. Jiang, W. Tian, C.D. Zhao, H.X. Zhang, M.C. Liu, Talanta 72 (2007) 119.26] Y.Q. Lv, Z.X. Lin, W. Feng, X. Zhou, T.W. Tan, Biochem. Eng. J. 36 (2007) 221.27] M.L. Mena, P. Martinez-Ruiz, A.J. Reviejo, J.M. Pingarron, Anal. Chim. Acta 451

(2002) 297.28] D.M. Hana, G.Z. Fang, X.P. Yan, J. Chromatogr. A 1100 (2005) 131.29] P.C.L. Edward, Y.F. Sherry, Microchem. J. 75 (2003) 159.30] W. Rudzinski, W. Plazinski, J. Phys. Chem. B 110 (2006) 16514.

32] Y.S. Ho, G. McKay, Trans. Ichem. E 77B (1999) 165.33] J. Zhou, X.W. He, Anal. Chim. Acta 381 (1999) 85.34] J.J. Ou, J. Dong, T.J. Tian, J.W. Hu, M.L. Ye, H.F. Zou, J. Biochem. Biophys. Methods

70 (2007) 71.35] M. Odabasi, R. Say, A. Denizli, Mater. Sci. Eng. C 27 (2007) 90.