Journal of Food Composition and Analysis 34 (2014) 81–89

Original Research Article

Synthesis, characterization and analytical application ofZn(II)-imprinted polymer as an efficient solid-phase extractiontechnique for trace determination of zinc ions in food samples

Mohammad Behbahani a, Mani Salarian b, Akbar Bagheri a,*, Hadi Tabani a,Fariborz Omidi c, Alireza Fakhari a

a Department of Chemistry, Shahid Beheshti University, G.C., Tehran 1983963113, Iranb Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, United Statesc Department of Occupational Health, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

A R T I C L E I N F O

Article history:

Received 23 August 2012

Received in revised form 27 August 2013

Accepted 27 October 2013

Keywords:

Ion imprinted polymer

Copolymerization

Zinc

Solid-phase extraction

Preconcentration

Food analysis

Food Composition

Flame atomic absorption spectrometry

Food safety

Metal contamination levels in food

A B S T R A C T

Zinc imprinted polymer (Zn(II)-IIP) as a solid-phase extraction technique was applied for

preconcentration and determination of zinc in environmental and food samples by FAAS. Zn(II)-IIP

was synthesized by copolymerization of 2-vinylpyridine as the functional monomer, ethylene glycol

dimethacrylate (EGDMA) as the cross-linker, 2,2-azobisisobutyronitrile as the initiator, Zn(NO3)2 as the

template ion, diphenylcarbazone as the ligand, and methanol/dichloromethane (50:50) as the solvent.

The synthesized polymer particles were characterized by thermogravimetry analysis (TGA), differential

thermal analysis (DTA), scanning electron microscopy (SEM), Fourier transform infrared spectrometry

(FT-IR), and X-ray diffraction (XRD). The optimization process was carried out using the central

composite design (CCD). With this design, the optimum conditions for the adsorption step were 6.6,

30 mg, and 23 min, for pH, mass of polymer and adsorption time respectively, and for desorption step the

optimal conditions were 14 mL, 12% (v/v), and 23 min, for volume of eluent, concentration of eluent, and

desorption time, respectively. Under the optimized conditions, the detection limit of the proposed

method was found to be 0.15 mg L�1, while the relative standard deviation (RSD) for 10 replicate

measurements was calculated to be 2.8%. Therefore the introduced selective solid phase extraction

method can be used as a reliable technique for selective and sensitive trace detection of zinc in different

food matrixes, and the reliability of the proposed solid phase extraction technique was established by the

analysis of standard reference materials.

� 2013 Elsevier Inc. All rights reserved.

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

jo u rn al ho m epag e: ww w.els evier . c om / lo cat e/ j fc a

1. Introduction

Some trace heavy metals are considered to be among the vitalmicronutrients, and they have a wide range of biochemicalfunctions in all living organisms (Ferreira et al., 2007; Saracogluet al., 2007; Uluozlu et al., 2007). Zinc is one of the significantingredients of proteins involved in intermediary metabolism,hormone secretion pathway, and immune system, and itsenzymes participate in synthesis and decomposition of proteins,lipids, carbohydrates, and nucleic acids (Mcdowell, 1992;Solomons and Rosenberg, 1992). Zinc is mobile and easily takenby plants in the environment (Ferreira et al., 2007; Zhu et al.,2007). Zinc deficiency might lead to several disorders such as

* Corresponding author. Tel.: +98 21 29903259; fax: +98 21 22403041.

E-mail address: me-bagheri@sbu.ac.ir (A. Bagheri).

0889-1575/$ – see front matter � 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.jfca.2013.10.003

growth retardation, diarrhea, immunity depression, eye and skinlesions, malfunction of wound healing, and other skin diseases(Scherz and Kirchhoff, 2006). In order to observe the symptomsand trace element absorption, a biological sample such as bloodis often analyzed. Deficiency in trace elements includingdeviation of zinc from its normal level in blood is used for earlydiagnosis of certain diseases (Kruusma et al., 2004). Moreover,metabolic disorders of zinc are thought to be associated with anumber of neurological diseases such as Alzheimer, Parkinson,Epilepsy, and Hypoxia-ischemia (Flinn et al., 2005). Althoughtrace metals like zinc are vital for humans, the excess utilizationcan be harmful and toxic, and thus it should be used according tophysiological needs.

Due to the importance of analysis of zinc ions in trace levels,separation and enrichment of trace zinc in complex matrix shouldbe considered, and the purification of processed liquids containingheavy metal ions requires meticulous attention.

M. Behbahani et al. / Journal of Food Composition and Analysis 34 (2014) 81–8982

Modern instrumental methods including spectrometry, induc-tively coupled plasma mass spectrometry (ICP-MS), inductivelycoupled plasma atomic emission spectrometry (ICP-AES), atomicabsorption spectrometry (AAS). Dadfarnia and McLeod (1994), DosSantos et al. (2005) and Sreenivasa Rao et al. (2002) have been usedfor determination of traces of metal ions in various fields. However,direct determination of metal ions at trace levels by flame atomicabsorption spectrometry (FAAS) is limited not only due toinsufficient sensitivity, but also to matrix interference. Underthese circumstances, in order to determine trace levels of Zn aseparation and enrichment step may be beneficial prior to theirdeterminations. A wide range of sorbents has been applied to metalion preconcentration by solid phase extraction technique andthese include: silica gel, resins, alumina oxide, cellulose, activatedcarbon, chitosan, metal-organic frameworks, modified conductivepolymers, grapheme and titanium oxides (Bagheri et al., 2012a;Behbahani et al., 2013a,b,c,d,e; Camel, 2003; Lemos et al., 2008;Meesri et al., 2007; Poole, 2003; Pyrzynska and Wierzbicki, 2004;Stafiej and Pyrzynska, 2008; Tabakci and Yilmaz, 2008; Tabaniet al., 2013). The most popular polymer resin – styrene-divinylbenzene (S-DVB) demonstrates good sorption capacity,high surface area, and good chemical and physical stability.However, owing to its insufficient selectivity, structure modifica-tions are often made on the sorbent. Many examples of chemicaland physical immobilization of a complexing agent onto a polymermatrix may be found in literature, both for commercially availableresins (Chelex, Amberlit) (Bezerra et al., 2007; Ferreira et al., 2000;Lemos et al., 2006, 2007a) and new materials (Lemos et al., 2007b;Walas et al., 2008). Recently, the idea of molecular/ion imprintinghas been introduced as a method of polymer structure modifica-tion which resulted in obtaining new selective sorbents (Behba-hani et al., 2012, 2013c; Ebrahimzadeh et al., 2013; Prasada Raoet al., 2006). Selectivity of IIPs is strongly influenced by selectivityof ligand, size of metal ion, coordination number, and its geometrywhich makes them very applicable for elimination of matrixinterferences. Several approaches have been developed for IIPssynthesis, e.g. cross-linking of bi-functional monomers, surfaceimprinting, and complex trapping or chemical immobilization of aligand in the entire volume of a polymer (Kato et al., 2003;Sellergren, 2001).

Most of the practical works involving IIP optimization usestrategies based on systematic studies of one variable at a time(OVAT). Such an approach does not allow selecting variables thathave the most influence on the response, and it does not considerpossible interdependence between variables. On the other hand, amultivariate method allows simultaneous changes of all chosenfactors affecting the responses of experiment and avoids highconsumption of reagents and materials, and time-consumingexperiments (Lundstedt et al., 1998). The experimental designmethodology enables the simultaneous study of several control-ling factors and the development of mathematical models thatpermit assessment of the relevance and statistical significance ofthe factors being studied. They also facilitate the evaluation ofinteraction effects among factors, and the optimum operationconditions are attained by using quadratic response surfaceexperimental designs (Tarley et al., 2009).

In the present study, Zn(II)-IIP followed by FAAS was applied fortrace monitoring of Zn(II) in different real samples. Zn(II)-IIP wassynthesized by co-polymerization of 2-vinylpyridine (2-VP),ethylene glycol dimethacrylate (EGDMA), 2,2-azobisisobutyroni-trile (AIBN), Zn(NO3)2 as the template ion, and diphenylcarbazoneas the ligand. Central composite design (CCD) was used to optimizethe influence of factors in adsorption and desorption steps,separately. Finally, the proposed method was successfully appliedfor extraction and determination of Zn(II) ions in river water,vegetables, red meat, and milk samples.

2. Materials and methods

2.1. Apparatus

AA-680 Shimadzu (Kyoto, Japan) flame atomic absorptionspectrometer with a deuterium background corrector was used fordetermination of Zn(II) ions. A zinc hollow cathode lamp (HCL) wasused as the light source operated at 7 mA. The wavelength was setat 213.9 nm resonance line and the spectral band width at 0.5 nm.All measurements were carried out in an air/acetylene flame. Adigital pH meter, WTW Metrohm 827 Ion analyzer (Herisau,Switzerland), equipped with a combined glass calomel electrodewas used for the pH adjustments at 25 � 1 8C temperature.Heidolph heater stirrer model MR 3001 (Schwabach, Germany)was used for heating and stirring of the solutions. IR spectra wererecorded on a Bruker IFS-66 FT-IR Spectrophotometer (Billerica, MA,USA). High angle X-ray diffraction (XRD) patterns were obtained on aPhilips-PW 17C diffractometer with Cu Ka radiation. SEM wasperformed by gently distributing the powder sample on the stainlesssteel stubs, using SEM (Philips, XL30, Almelo, the Netherlands)instrument. The thermal properties of samples were determined,using a BAHR-Thermoanalyse GmbH (Hullhorst, Germany) employ-ing, heating and cooling rates of 10 8C min�1 and using a condenser asthe coolant. The samples were weighed as a thin film and carefullypacked into a clean aluminum pan (11.5–12.5 mg), and sealed bycrimping an aluminum lid on the pan (Shimadzu universal crimper).An Al2O3 empty pan sealed with a cover pan was used as a referencesample. A scanning range of 10–800 8C was used for samples at10 8C min�1 in nitrogen gas. The Brunauer–Emmett–Teller (BET) ofthe prepared ion imprinted polymer was analyzed by nitrogenadsorption in a nitrogen adsorption apparatus (BEL, BELSORP-18 Plus,Tokyo, Japan). The BET surface area (SBET) was determined using theadsorption data in the relative pressure (P/P0) range of 0–1 by amultipoint BET method. The samples were degassed at 150 8C prior tothe nitrogen adsorption measurements.

2.2. Material and reagents

2-Vinyl pyridine and diphenylcarbazone of high purity werepurchased from Merck (Darmstadt, Germany). Ethylene glycoldimethacrylate (EGDMA) was obtained from Fluka (Buchs,Switzerland). 2,20-Azobisisobutyronitrile (AIBN) was obtainedfrom Acros Organics (Fair Lawn, NJ, USA). NaOH, HCl, HNO3, aceticacid, dichloromethane, and methanol were purchased from Merck(Darmstadt, Germany). All the other reagents used were ofanalytical grade and purchased from Merck (Darmstadt, Germany).Ultrapure water was prepared using a Milli-Q system fromMillipore (Bedford, MA, USA). Ore polymetallic gold Zidarovo-PMZRZ 206 BG 326 (Bulgaria) and Tomato leaves NIST 1573 (NIST,Gaithersburg, MD, USA) were purchased and used as a referencematerial.

2.3. Preparation of standard solutions

The two reference materials (Ore polymetallic gold Zidarovo-PMZRZ 206 BG 326 and Tomato leaves NIST 1573) were digestedwith 6 mL of HCl (37%, v/v) and 2 mL of HNO3 (65%, v/v) in amicrowave digestion system. Digestion was carried out for 2 minat 250 W, 2 min at 0 W, 6 min at 250 W, 5 min at 400 W, 8 min at550 W, and then venting for 8 min. The residue from digestion wasthen diluted with ultrapure water (Bagheri et al., 2012b).

Standard Stock solutions (1000 mg L�1) of Pb(II), Ni(II), Mn(II),Fe(II), Ca(II), Zn(II), Cu(II), Cd(II), Al(III), and Ag(I) from their nitratesalts purchased from Merck (Darmstadt, Germany, with puritygreater than 99.97%) were prepared in 2% (v/v) HNO3. The usedstandard solutions were prepared by appropriate dilution of the

Fig. 1. Schematic illustration of imprinting process for the preparation of structure

zinc imprinted polymer by bulk polymerization technique.

M. Behbahani et al. / Journal of Food Composition and Analysis 34 (2014) 81–89 83

stock solution with double distillated water. All of these solutionswere stored in ambient temperature.

2.4. Real sample pretreatment

2.4.1. Vegetables (Parsley, Basil)

Two types of vegetables were chosen for analysis. These wereparsley and basil which were collected from the local supermarket.Parsley and basil samples were air-dried followed by drying at105 8C for 2 h and then powdered. 0.5 g of basil sample and 0.3 g ofparsley sample were taken in Teflon flasks, and 5.0 mL ofconcentrated nitric acid and 5.0 mL of 30% (v/v) hydrofluoric acidwere added. After standing for 8 h, the samples were digested in amicrowave system for 10 min at 106 8C. The clear solutionsobtained were transferred to a 400 mL beaker; and diluted to300 mL, then adjusted to the pH of 6.6.

2.4.2. Red meat

The meat samples were brought to the laboratory in plasticbags, cleaned and washed with distilled water, before cooking,they were cut in pieces with a stainless steel knife, and dried in anoven at 65 8C for 48 h discontinuously until constant weight wasobtained. The dried samples were then powdered. Accurately, 1 gof dried samples were weighted and put in polyethylene vials, anddigested with a mixture of concentrated H2SO4 and HNO3 (1:3, v/v). A small amount of 30% H2O2 was added to eliminate nitrogendioxides. The digestion process was continued until the solutionbecame clear. The samples were transferred into 500 mL beakerand diluted to 300 mL with distilled water.

2.4.3. Milk

Fifteen mL of Low fat cow’s milk sample was first defatted bycentrifugation at 3000 rpm for 10 min at 4 8C. Then 1 mol L�1 HClsolution was added to the sample of the resulting defatted milk toadjust the pH to 4.6. After incubation for 30 min at roomtemperature, the sample was again centrifuged at 4000 rpm for30 min at 4 8C. Then the clear solution from the top was separatedand transferred into a volumetric flask.

2.4.4. River water

Two water samples were collected from two rivers (Amirie andAbbarik) in Northern city of Tehran, Iran. First, both samples werefiltered and then 300 mL of Abbarik water sample and 300 mL ofAmirie water sample were transferred into 500 mL beakers. Afterthat the pH of both samples were adjusted to 6.6.

2.5. Synthesis of ion-imprinted polymeric particles

The Zn(II)-imprinted particles were prepared by bulk polymer-ization technique. In the first step, 4 mmol of 2-VP (functionalmonomer) and 1 mmol of diphenylcarbazone (ligand) weredissolved in 25 mL of methanol/dichloromethane (50:50) in a100 mL glass flask. Subsequently, as the second step, 0.5 mmol ofZn(NO3)2 as an imprinted metal ion (template) was added slowlyto a glass flask and the resulted mixture was stirred for 5 h at roomtemperature. In the third step, 25 mmol of EGDMA and 0.3 mmol ofAIBN were added as cross-linker and initiator. The oxygen of thesample solution was removed by bubbling nitrogen through thesample for 10 min. Polymerization was performed in an oil bath at65 8C for 24 h in the presence of nitrogen under magnetic stirring at400 rpm. The prepared polymer was washed several times with1:4 (v/v) methanol/water to remove the unreacted materials andthen with HCl 12% (v/v) for leaching the imprinted metal ions untilthe washing solution was free from Zn(II) ions. Finally, it waswashed with double distilled water until it reached a neutral pH.The resulting fine powder was dried under vacuum in a desiccator

before sorption and desorption studies. In the same way, the nonimprinted polymer (NIP) was also prepared without ions of zinc(Fig. 1 provides the images of synthesized IIP after each step).

2.6. IIP extraction procedure

Batch experiments were used for adsorption and desorptionstudies. The dry imprinted polymer (30 mg) was immersed insolution containing unknown concentration of zinc ions. Thesolution was pH-adjusted to 6.6, stirred for 23 min, then thesolution was filtered, and ultrapure water was used for washingthe zinc ions on the surface of the polymer. The solid polymer wassuspended in 25 mL of HCl 12% (v/v) as the eluent and stirred for23 min. The resulting solution was subsequently filtered andconcentration of zinc ions was determined by FAAS.

3. Results and discussion

3.1. Characterization of IIP

The resulting imprinted nanoparticles were characterized by IRspectroscopy, scanning electron microscopy (SEM), thermogravi-metric analysis (TGA), Differential thermal analysis (DTA), andXRD. The IR spectra of unleached and leached ion imprintedpolymer (IIP) were recorded by using KBr pellet method. In the IRspectra, the absorptions due to C55O (1728 cm�1), C–O(1262 cm�1), C–N (1153 cm�1), C55N (1639 cm�1), N–H(3443 cm�1) and C–H bands (756, 1458 and 2958 cm�1) wereobserved. The N–H and C55N bands at 3443 and 1639 cm�1 inunleached IIP were shifted to 3444.5 and 1647 cm�1 in leached IIP.This amount of reduction in band frequencies indicates that the

Fig. 2. TG and DTA curve of leached (a) and unleached (b) Zn(II)-imprinted polymer.

M. Behbahani et al. / Journal of Food Composition and Analysis 34 (2014) 81–8984

zinc ions have been coordinated with non-bonding electron pairsof nitrogen in N–H and C55N groups in diphenylcarbazone, and as aresult, it demonstrates the presence of zinc ions in unleached IIPstructure. Moreover, the presence of C55N and N–H bands in the IRspectra of these materials indicated that diphenylcarbazone hadbeen sufficiently immobilized in the polymer matrix. Thermalstability of the leached and unleached polymer particles wasevaluated by thermo gravimetric analysis. Fig. 2 shows that TGAand DTA plots for (a) unleached and (b) leached ion imprintedpolymer. In DTA plots of zinc imprinted polymer, exothermic peakswere observed at 352.2, 494.9, and 567.3 (for unleached) and239.6, 343.2, 478.4 and 565.8 8C (for leached). As it is shown in TGplots for ion imprinted particles, weight loss for Zn(II)-IIP wasabout 97%, and this amount of reduction in weight was related tothe presence of Zn(II) ions in polymer bead. While decrease inweight for leached imprinted polymer up to 100%, is due toabsence of zinc ions in polymer. These observations indicate thatthe formation of Zn(II)-imprinted polymer and elution of Zn(II)ions from the polymer was performed successfully. The morphol-ogy of the Zn(II)-IIP was assessed by SEM, and the patterns areshown in Fig. 3. The XRD patterns of the leached (a) and unleached(b) IIP particles are shown in Fig. 4. The XRD patterns of the leached(a) and unleached (b) IIP particles indicated different patterns. Inunleached IIP, peaks related to zinc can be seen while these peaks

were absent in the curve of leached IIP. In order to further examinethe porous structure of the synthesized imprinted polymer, N2

adsorption-desorption isotherms were employed. The surfacearea, pore volume and mean pore size of the sorbent determined byBET method were about 7.3 m2 g�1, 0.15 cm3 g�1 and 8.23 nm,respectively.

3.2. Optimization strategy

The Zn(II) ions were extracted from 25 mL sample solutions.First, the extraction conditions were optimized for water, and thenthe conditions were applied to environmental samples. In alloptimization experiments, the concentration of Zn(II) ions was1 mg L�1.

First, CCD was used to optimize the conditions of the adsorptionstep, and then the influence of desorption solvent on extractionefficiency was evaluated by one variable at a time. Finally, in the laststep, CCD was used to determine the optimum conditions ofdesorption step. According to preliminary studies, in the adsorptionstep three variables (pH, extraction time, and amount of polymer)and in desorption step three variables (elution volume, elution time,and concentration of eluent) were studied. The levels of the factorsare shown in Table 1. The experimental design matrix and dataanalysis were carried out by Stat Graphics Plus Package, version 5.1.

Fig. 3. SEM images of Zn(II) imprinted polymer.

M. Behbahani et al. / Journal of Food Composition and Analysis 34 (2014) 81–89 85

3.2.1. Adsorption step

CCD was employed to optimize three factors (pH, extractiontime, and amount of polymer). The examined levels of the factorsare given in Table 1. This design permitted the responses to bemodeled by a second-order polynomial fit, which can be expressedas the following equation:

YðxÞ ¼ b0 þX3

i¼1

biXi þX3

i¼1

biX2i þ

X3

i¼1

X3

j¼1

bi jXiX j

where Y(x) is the response function (percentage of retention), b0 isan intercept, bi and bij are constant regression coefficients of the

Fig. 4. XRD patterns of leached (a) and un

model, and Xi and Xj (i = 1,3; j = 1,3 and i 6¼ j) represent the codedlevel of an independent variable.

The number of experimental points (N) is defined by theexpression below:

N ¼ 2 f þ 2 f þ C;

where f is the number of factors, and C is the number of centerpoints. This design consists of a factorial design (2f) augmentedwith (2f) star points, and central points (C) (Kamarei et al., 2010).The star points are located at + and � from the center of theexperimental domain. An axial distance, a was selected with thevalue of 1.682 in order to establish the rotatability condition of thecentral composite design. In this study, f and C were both set at 3and 4, respectively, which meant that 18 experiments had to bedone.

The experimental data showed a good accordance with thesecond order polynomial equation. The appropriateness of the fitof the quadratic polynomials is expressed by coefficient ofdetermination, R2. According to Joglekar and May (Loi et al.,2010), R2 should be at least 0.80 for a good fit of a model. In thisstudy, the R2 was more than 0.94 which means that the obtainedequation is appropriate for correlating the experimental results.Also, this result can be interpreted with the analysis of variance(ANOVA) by F-ratio and P-value which are presented in Table 3. Ifthe P-value of a factor or interaction between factors is higher than0.05, it means that it does not have dominant effect, while a factorwith the P-value of lower than 0.05 would be significant. In orderto estimate the adequacy of the proposed equation, the residualsum of squares has been split into two parts consist of pureexperimental error and inadequacy of model (lack of fit). The lackof fit test is designed to determine whether the selected model isadequate to describe the observed data, or whether a morecomplicated model should be used. The test is performed bycomparing the variability of the current model residuals to thevariability between observations at replicate settings of thefactors. Since the P-value for lack of fit in the ANOVA table is higherthan 0.05, the model appears to be adequate for the observed dataat 95.0% confidence level.

The ANOVA results produced the Pareto chart of main andinteraction effects are shown in Fig. 5. The standard effect wasestimated for computing the t-statistic for each effect. The vertical

leached (b) Zn(II) imprinted polymer.

Table 1Experimental variables, levels and star points of the CCD.

Factors Level Star points (a = 1.682)

Lower Central Upper �a +a

Extraction step A: pH 3 5.5 8 1.296 9.764

B: Extraction time (min) 10 20 30 3.182 36.818

C: Amount of polymer 15 27.5 40 6.478 48.522

Desorption step A: Elution volume (mL) 5 10 15 1.591 18.409

B: Elution time (min) 10 20 30 3.182 36.818

C: Concentration eluent 5 10 15 1.591 18.409

M. Behbahani et al. / Journal of Food Composition and Analysis 34 (2014) 81–8986

line on the plot indicates the statistically significant effects. The bargoing beyond the line corresponds to the effects that arestatistically significant at 95% confidence level (StatGraphics Plus5.1). Furthermore, the positive or negative sign (corresponding to acolored or colorless response) can be enhanced or reduced,respectively, when passing from the lowest to the highest levelset for the specific factor. According to Fig. 5, the pH has a largeinfluence on the adsorption of Zn(II) ions on IIP this effect waspositive upon the extraction. The adsorption of Zn(II) increases asthe pH increases. In acidic solution, adsorption is very low. Thisobservation is due to protonation of the active sites of the polymer.As the pH increases, the protonation of ligand decreases and thecondition becomes more favorable for complex formation andsorption of Zn(II) ions to the imprinted sorbent.

The Response Surface Methodology (RSM) (Fig. 6a) was appliedto analyze simultaneous effects of time and pH variables on theresponse and the two-dimensional contour plot (Fig. 6b) thatdisplayed the interaction between independent variables. Theadsorption of Zn(II) ions increased along with the increase in pHbut the time factor has no significant effect on the extraction ofZn(II) ions. However, in pH above 7.0 the amount of adsorption wasdecreased due to precipitation of Zn(II) ions in basic conditions.

The design permitted the response to be modeled by a second-order polynomial fit, which can be expressed as the followingequation:

Y ¼ �108:094 þ 45:295A þ 2:03523B þ 2:38499C � 3:37321A2

� 0:016AC � 0:0481912B2 þ 0:006BC � 0:0398933C2

According to the overall results of the optimization study, thefollowing experimental conditions were chosen: pH, 6.6; extrac-tion time, 23 min; amount of polymer, 30 mg.

Table 2Tolerance limit of various ions on the determination of zinc.

Foreign ion Tolerable concentration

ratio X/Zn

Zinc imprinted polymer

Ra (%) � Sb

(coexisting ions)

Ra (%) � Sb

(template ion)

K+ 10,000 7.0 � 1.0 99.0 � 2.0

Na+ 10,000 6.0 � 2.0 99.0 � 2.0

Ag+ 1000 8.0 � 1.0 98.0 � 1.0

Ca2+ 1000 8.0 � 2.0 98.0 � 1.0

Al3+ 900 7.0 � 2.0 96.0 � 2.0

Cu2+ 800 6.0 � 2.0 98.0 � 1.0

Fe2+ 800 7.0 � 2.0 97.0 � 1.0

Cd2+ 700 8.0 � 1.0 97.0 � 2.0

Pb2+ 600 6.0 � 1.0 95.0 � 2.0

Mn2+ 800 5.0 � 2.0 97.0 � 1.0

Ni2+ 800 6.0 � 1.0 96.0 � 2.0

Cr3+ 700 5.0 � 2.0 97.0 � 1.0

Conditions: sample pH: 6.6, sample volume: 100 mL, 0.01 mg of zinc ion, Sorption

time: 23 min; 14 mL of HCl 12% (v/v); and desorption time: 23 min. X: concentration

of diverse ions.a Recovery.b Standard deviation (N = 3).

3.2.2. Selection of desorption solvent

In this work three solvents (HCl, HNO3 and CH3COOH) wereused as desorption solvent. Other factors were kept constantduring the optimization (pH, 6.6; extraction time, 23 min; amountof polymer 30 mg; elution volume, 14 mL; elution time, 23 min;concentration of eluent, 12% (v/v). Based on measurements, thebest quantitative recovery was obtained with HCl as the eluentsolvent.

3.2.3. Desorption step

Three factors were studied in desorption step using experi-mental design: elution volume (mL), elution time (min), andconcentration of eluent (v/v, %). In these conditions, a responsesurface design could be done without previously performing ascreening design. The CCD was chosen because it requires the leastnumber of experiments (20, including six central points). Thelevels of the factors are listed in Table 1. The adsorption conditionswere the same for all the experiments fixed at proposed optimumlevels for Zn(II) ions in the previous design.

The experimental data showed a good correlation with thesecond-order polynomial equation. The coefficients of determina-tion, R2, were higher than 0.92, which they were statisticallyacceptable at P below 0.05 levels. Since the P-value for lack of fit inthe ANOVA table is higher than 0.05, the model appears to beadequate for the observed data at 95.0% confidence level (Table 4).

The data obtained were evaluated by ANOVA. The effectsdetermined by this analysis are shown using Pareto chart in Fig. 7(AA, BB and CC are the quadratic effects of the elution volume,extraction time and concentration of eluent, respectively. AB, ACand BC are the interaction effects between elution volume andextraction time; elution volume and concentration of eluent, andextraction time and concentration of eluent, respectively).

Based on CCD, all of the factors were significant. As Fig. 7 shows,elution volume has the greatest influence on the extractionrecovery and a positive effect upon the extraction efficiency.

The RSM (Fig. 8a) was applied to analyze simultaneous effects ofelution time and elution volume variables on the responses. Thetwo-dimensional contour plot (Fig. 8b) displays the interactionbetween independent variables. The extraction efficiency of Zn(II)

Table 3Results of ANOVA for the fitted model (adsorption).

Source Sum of squares df Mean square F-Ratio P-Value

A 5126.18 1 5126.18 396.87 0.0003

B 101.475 1 101.475 7.86 0.0677

C 105.984 1 105.984 8.21 0.0643

AA 5622.23 1 5622.23 435.27 0.0002

AB 0.0 1 0.0 0.0 1.0000

AC 2.0 1 2.0 0.15 0.7203

BB 293.762 1 293.762 22.74 0.0175

BC 4.5 1 4.5 0.35 0.5965

CC 491.475 1 491.475 38.05 0.0086

Lack-of-fit 547.037 5 109.407 8.47 0.0544

Pure error 38.75 3 12.9167

Table 4Results of ANOVA for the fitted model (desorption).

Source Sum of Squares df Mean square F-Ratio P-Value

A 1684.66 1 1684.66 78.48 0.0003

B 630.25 1 630.25 29.36 0.0029

C 874.314 1 874.314 40.73 0.0014

AA 454.146 1 454.146 21.16 0.0058

AB 60.5 1 60.5 2.82 0.1540

AC 50.0 1 50.0 2.33 0.1875

BB 513.151 1 513.151 23.90 0.0045

BC 8.0 1 8.0 0.37 0.5682

CC 748.045 1 748.045 34.85 0.0020

Lack-of-fit 267.118 5 53.4236 2.49 0.1699

Pure error 107.333 5 21.4667

Fig. 5. Pareto chart of the main effects in the CCD. AA, BB and CC are the quadratic

effects of the pH, extraction time and amount of polymer, respectively. AB, AC and

BC are the interaction effects between pH and extraction time; pH and amount of

polymer and extraction time and amount of polymer, respectively.

Fig. 7. Pareto chart of the main effects in the CCD for desorption step.

M. Behbahani et al. / Journal of Food Composition and Analysis 34 (2014) 81–89 87

ions increased along with the increase in elution time and elutionvolume.

The design permitted the responses to be modeled by a second-order polynomial fit, which can be expressed as the followingequation:

R ¼ �67:7162 þ 8:81223A þ 3:81619B þ 8:76395C � 0:224546A2

� 0:055AB � 0:1AC � 0:0596722B2 � 0:02BC � 0:288185C2

According to the overall results of the optimization study, thefollowing experimental conditions were chosen: elution volume,14 mL; elution time, 23 min; concentration of eluent, 12% (v/v).

3.3. Effect of sample volume

In the analysis of real samples, the sample volume is one of theimportant parameters affecting the preconcentration factor.Therefore, the effect of sample volume on quantitative adsorptionof Zn(II) ion was investigated. For this purpose, 30 mg of thepolymer was suspended in different sample volumes (25, 50, 100,250, 500, 750 1000, 1100, 1250, and 1500 mL) containing 0.1 mg ofZn(II) ions. All solutions were extracted under the optimumcondition by the proposed method. The results demonstrated that

Fig. 6. (a) RSM and (b) contour plot obtained by plotting of pH vs. extraction time

using the CCD.

the dilution effect was not significant for sample volumes up to1100 mL for Zn(II) ions.

3.4. Sorption capacity

The sorption capacity which is defined as maximum amount ofmetal ions sorbed per gram of the polymer is an important factor forevaluation of the synthesized IIP. To evaluate this factor, 500 mL of asolution containing 25 mg of zinc ion was applied to the extractionprocedure and the sorption capacity was calculated. Based on threereplicate measurements, the sorption capacities of the ion imprintedpolymer and non imprinted polymer were calculated to be68.6 � 0.07 mg g�1 and 8 � 0.09 mg g�1, respectively.

Fig. 8. (a) RSM and (b) contour plot obtained by plotting of elution volume vs.

elution time using the CCD.

Table 5Results of zinc ion analysis in different real samples.

Sample COriginal (N = 3) (mg kg�1) Cadded (mg kg�1) Cexpected (mg kg�1) Cfound (N = 3) (mg kg�1) Recovery (%) RSD (%)

Parsley 24.2 10.0 34.2 33.7 98.5 3.5

Basil 25.8 10.0 35.8 34.9 97.5 3.4

Red meat 80.4 10.0 90.4 90.4 100 3.4

Milk 512.5 10.0 522.5 521.2 99.8 3.1

Amirie river water 54.4 10.0 64.4 63.7 98.9 2.4

Abbarik river water 110.2 10.0 120.2 119.5 99.4 3.5

M. Behbahani et al. / Journal of Food Composition and Analysis 34 (2014) 81–8988

3.5. Effect of potentially interfering ions

In order To investigate the effect of various cations existed innatural samples; elements that are known as alkaline, alkalineearth, and transition metals were added to 100 mL of solutioncontaining 0.01 mg zinc ions. The degree of tolerance for somealkaline, alkaline earth, and transition metal ions is presented inTable 2. From the tolerance data, it can be found that the foreignions have no significant effects on preconcentration of zinc ions atpH 6.6.

3.6. Statistical and calibration parameters

Under optimum conditions that have been described, the IIP-SPE showed a linear calibration curve within concentrationranging from 0.5 to 120 mg L�1. The least square equation atabove dynamic linear range was as follows:

A ¼ 0:21CZnðmg L�1Þ þ 0:08 ðR2 ¼ 0:98Þ

The limits of detection, defined as CLOD = 3Sb/m, where Sb is thestandard deviation of 8 replicate blank signals and m is the slope ofthe linear section of calibration curve after preconcentration, for asample volume of 750 mL, was found to be 0.15 mg L�1 for zincions. The relative standard deviation (RSD) for six separate batchexperiments with 30 mg of sorbent for determination of 1 mg Zn(II)in 100 mL of water was 2.8%.

3.7. Real samples and certified reference material analysis

To evaluate the capability of this method for analysis of realsamples with different matrices containing various amounts ofdiverse ions, the method was used for the extraction of zinc ionsfrom different samples. As shown in Table 5, a good correlation wasobtained between the added and measured amounts of analyte.

Ore polymetallic gold zidarovo-PMZrZ (206 BG 326) andTomato leaves (NIST 1573, Gaithersburg, MD, USA) Certifiedreference materials were used for the validation of the proposedmethod. As Table 6 shows, a good correlation was obtainedbetween the certified amounts and the amounts found by thecurrent method. Therefore, Zn(II)-IIP particle can be used as areliable solid-phase for extraction and determination of zinc infood and environmental samples.

Table 6Validation of the proposed method by comparing the data obtained by this method

and data for certified reference materials.

Sample Element Concentration

(mg kg�1)

Relative

error (%)

Certified Found

Ore polymetallic gold Zn 4.06 3.94 �2.95

Tomato leaves (NIST 1573) Zn 62.0 61.3 �1.1

4. Conclusion

The ion imprinted polymer method is a useful technique for thepreparation of sorbent for preconcentration of metal ions, such aszinc from aqueous solutions. The prepared ion-imprinted polymerparticles show an increased selectivity toward Zn(II) ion over arange of competing metal ions with the same charge and similarionic radius. The Zn(II)-imprinted particles can be used repeatedlywith no significant decrease in their binding affinities. Due torelatively high preconcentration factor, trace metal ions at ng mL�1

levels in high-volume samples can be determined and separated byion imprinted polymers.

Acknowledgement

We gratefully acknowledge financial support from the ResearchCouncil of Shahid Beheshti University.

References

Bagheri, A., Behbahani, M., Amini, M.M., Sadeghi, O., Taghizade, M., Baghayi, L.,Salarian, M., 2012a. Simultaneous separation and determination of traceamounts of Cd(II) and Cu(II) in environmental samples using novel diphenyl-carbazide modified nanoporous silica. Talanta 89, 455–461.

Bagheri, A., Taghizadeh, M., Behbahani, M., Asgharinezhad, A.A., Salarian, M.,Dehghani, A., Ebrahimzadeh, H., Amini, M.M., 2012b. Synthesis and characteri-zation of magnetic metal-organic framework (MOF) as a novel sorbent, and itsoptimization by experimental design methodology for determination of palla-dium in environmental samples. Talanta 99, 132–139.

Behbahani, M., Babapour, M., Amini, M.M., Sadeghi, O., Bagheri, A., Salarian, M.,Rafiee, B., 2013a. Separation/enrichment of copper and silver using titaniumdioxide nanoparticles coated with poly-thiophene and their analysis by flameatomic absorption spectrophotometry. American Journal of Analytical Chemis-try 4, 90–98.

Behbahani, M., Bagheri, A., Amini, M.M., Sadeghi, O., Salarian, M., Najafi, F., Taghi-zadeh, M., 2013b. Application of multiwalled carbon nanotubes modified bydiphenylcarbazide for selective solid phase extraction of ultra-traces Cd(II) inwater samples and food products. Food Chemistry 141, 48–53.

Behbahani, M., Bagheri, A., Taghizadeh, M., Salarian, M., Sadeghi, O., Adlnasab, L.,Jalali, K., 2013c. Synthesis and characterisation of nano structure lead(II) ion-imprinted polymer as a new sorbent for selective extraction and preconcen-tration of ultra trace amounts of lead ions from vegetables, rice, and fishsamples. Food Chemistry 138, 2050–2056.

Behbahani, M., Najafi, M., Amini, M.M., Sadeghi, O., Bagheri, A., Salarian, M., 2013d.Dithizone-modified nanoporous fructose as a novel sorbent for solid-phaseextraction of ultra-trace levels of heavy metals. Microchimica Acta 180, 911–920.

Behbahani, M., Salarian, M., Amini, M.M., Sadeghi, O., Bagheri, A., Bagheri, S., 2013e.Application of a new functionalized nanoporous silica for simultaneous traceseparation and determination of Cd(II), Cu(II), Ni(II), and Pb(II) in food andagricultural products. Food Analytical Methods 6, 1320–1329.

Behbahani, M., Taghizadeh, M., Bagheri, A., Hosseini, H., Salarian, M., Tootoonchi, A.,2012. A nanostructured ion-imprinted polymer for the selective extraction andpreconcentration of ultra-trace quantities of nickel ions. Microchimica Acta178, 429–437.

Bezerra, M.A., dos Santos, W.N.L., Lemos, V.A., Korn, A., das Gracas, M., Ferreira,S.L.C., 2007. On-line system for preconcentration and determination of metalsin vegetables by inductively coupled plasma optical emission spectrometry.Journal of Hazardous Materials 148, 334–339.

Camel, V., 2003. Solid phase extraction of trace elements. Spectrochimica Acta PartB 58, 1177–1233.

Dadfarnia, S., McLeod, C.W., 1994. On-line trace enrichment and determination ofuranium in waters by flow injection inductively coupled plasma mass spec-trometry. Applied Spectroscopy 48, 1331–1336.

Dos Santos, W.N.L., Da Silva, E.G.P., Fernandes, M.S., Araujo, R.G.O., Costa, A.C.S.,Vale, M.G.R., Ferreira, S.L.C., 2005. Determination of copper in powdered

M. Behbahani et al. / Journal of Food Composition and Analysis 34 (2014) 81–89 89

chocolate samples by slurry-sampling flame atomic absorption spectrometry.Analytical Bioanalytical Chemistry 382, 1099–1102.

Ebrahimzadeh, H., Behbahani, M., Yamini, Y., Adlnasab, L., Asgharinezhad, A.A.,2013. Optimization of Cu(II)-ion imprinted nanoparticles for trace monitoringof copper in water and fish samples using a Box–Behnken design. Reactive andFunctional Polymers 73, 23–29.

Ferreira, H.S., Dos Santos, W.N.L., Fiuza, R.P., Nobrega, J.A., Ferreira, S.L.C., 2007.Determination of zinc and copper in human hair by slurry sampling employingsequential multi-element flame atomic absorption spectrometry. Microchemi-cal Journal 87, 128–131.

Ferreira, S.L.C., Lemos, V.A., Moreira, B.C., Costa, A.C.S., Santelli, R.E., 2000. An on-linecontinuous flow system for copper enrichment and determination by flameatomic absorption spectroscopy. Analytical Chimica Acta 403, 259–264.

Flinn, J.M., Hunter, D., Linkous, D.H., Lanzirotti, A., Smith, L.N., Brightwell, J., Jones,B.F., 2005. Enhanced zinc consumption causes memory deficits and increasedbrain levels of zinc. Physiology and Behavior 83, 793–803.

Kamarei, F., Ebrahimzadeh, H., Yamini, Y., 2010. Optimization of solvent barmicroextraction combined with gas chromatography for the analysis of ali-phatic amines in water samples. Journal of Hazardous Materials 178, 747–752.

Kato, K., Uchida, E., Kang, E.T., Uyamaa, Y., Ikada, Y., 2003. Polymer surface with graftchains. Progress in Polymer Science 28, 209–259.

Kruusma, J., Banks, C.E., Nei, L., Compton, R.G., 2004. Electroanalytical detection ofzinc in whole blood. Analytical Chimica Acta 510, 85–90.

Lemos, V.A., Teixeira, L.S.G., Bazerra, M.D., Costa, A.C.S., Castro, J.T., Cardoso, L.A.M.,de Jesus, D.S., Santos, E.S., Baliza, P.X., Santosi, L.N., 2008. New materials forsolid-phase extraction of trace elements. Applied Spectroscopy Reviews 43,303–334.

Lemos, V.A., Goncalves de Silva, D., Lago de Carvalho, A., de Andrade Santana, D., dosSantos Novaes, G., Sousa dos Passos, A., 2006. Synthesis of amberlite XAD-2-PCresin for preconcentration and determination of trace elements in food samplesby flame atomic absorption spectrometry. Microchemical Journal 84, 14–21.

Lemos, V.A., Santos, E.S., Gama, E.M., 2007a. A comparative study of two sorbents forcopper in a flow injection preconcentration system. Separation and PurificationTechnology 56, 212–219.

Lemos, V.A., Santos, E.S., Gama, E.M., 2007b. Synthesis of a new ion imprintedpolymer material for separation and preconcentration of traces of uranyl ions.Reactive and Functional Polymers 67, 966–976.

Loi, C.C., Boo, H.C., Mohamed, A.S., Ariffin, A.A., 2010. Application of headspace solid-phase microextraction and gas chromatography for the analysis of furfural incrude palm oil. Journal of the American Oil Chemists’ Society 87, 607–613.

Lundstedt, T., Seifert, E., Abramo, L., Thelin, B., Nystrom, A., Pettersen, J., Bergman, R.,1998. Experimental design and optimization. Chemometrics and IntelligentLaboratory Systems 42, 3–40.

Mcdowell, L.R., 1992. Minerals in Animal and 459 Human Nutrition. AcademicPress, New York, USA.

Meesri, S., Praphairaksit, N., Imyim, A., 2007. Extraction and preconcentration oftoxic metal ions from aqueous solution using benzothiazole-based chelatingresins. Microchemical Journal 87, 47–55.

Poole, C.F., 2003. New trends in solid-phase extraction. Trends in AnalyticalChemistry 22, 362–373.

Prasada Rao, T., Kala, R., Daniel, S., 2006. Metal ion-imprinted polymers – novelmaterials for selective recognition of inorganics. Analytical Chimica Acta 578,105–116.

Pyrzynska, K., Wierzbicki, T., 2004. Preconcentration and separation procedures forthe spectrochemical determination of platinum and palladium. MichrochimicaActa 147, 59–64.

Saracoglu, S., Saygi, K.O., Uluozlu, O.D., Tuzen, M., Soylak, M., 2007. Determinationof trace element contents of baby foods from Turkey. Food Chemistry 105,280–285.

Scherz, H., Kirchhoff, E., 2006. Trace elements in foods: zinc contents of raw foods –a comparison of data originating from different geographical regions of theworld. Journal of Food Composition and Analysis 19, 420–433.

Sellergren, B., 2001. Techniques and Instrumentation in Analytical chemistry,Molecularly Imprinted Polymers, vol. 23. Elsevier, Amsterdam.

Solomons, N.W., Rosenberg, I.H., 1992. Absorption and Malabsorption of MineralNutrients. Alan R Liss, New York, USA.

Sreenivasa Rao, K., Balaji, T., Prasada Rao, T., Babu, Y., Naidu, G.R.K., 2002. Determi-nation of iron, cobalt, nickel, manganese, zinc, copper, cadmium and lead inhuman hair by inductively coupled plasma-atomic emission spectrometry.Spectrochimica Acta B 57, 1333–1338.

Stafiej, A., Pyrzynska, K., 2008. Solid phase extraction of metal ions using carbonnanotubes. Microchemica Journal 89, 29–33.

.Tabakci, M., Yilmaz, M., 2008. Sorption characteristics of Cu(II) ions onto silica gel-

immobilized calixarene polymer in aqueous solutions: batch and columnstudies. Journal of Hazardous Materials 151, 331–338.

Tabani, H., Fakhari, A.R., Shahsavani, A., Behbahani, M., Salarian, M., Bagheri, A.,Nojavan, S., 2013. Combination of graphene oxide-based solid phase extractionand electro membrane extraction for the preconcentration of chlorophenoxyacid herbicides in environmental samples. Journal of Chromatography A 1300,227–235.

Tarley, C.R.T., Silveira, G., Santos, W.N.L., Matos, G.D., Silva, E.G.P., Bezerra, M.A.,Miro, M., Ferreira, S.L.C., 2009. Chemometric tools in electroanalytical chemis-try: methods for optimization based on factorial design and response surfacemethodology. Microchemical Journal 92, 58–67.

Uluozlu, O.D., Tuzen, M., Mendil, D., Soylak, M., 2007. Trace metal content in ninespecies of fish from the Black and Aegean Seas, Turkey. Food Chemistry 104,835–840.

Walas, S., Tobiasz, A., Gawin, M., Trzewik, B., Strojny, M., Mrowiec, H., 2008.Application of a metal ion imprinted polymer based on salen–Cu complex toflow injection preconcentration and FAAS determination of copper. Talanta 76,96–101.

Zhu, X.B., Chang, X.J., Cui, Y.M., Zou, X.J., Yang, D., Hu, Z., 2007. Solid-phaseextraction of trace Cu(II), Fe(III) and Zn(II) with silica gel modified withcurcumin from biological and natural water samples by ICP-OES. Microchemi-cal Journal 86, 189–194.