Accepted Manuscript

Analytical Methods

Synthesis, characterization, and application of a Zn (II)-imprinted polymer graf-ted on graphene oxide/magnetic chitosan nanocomposite for selective extractionof zinc ions from different food samples

Elahe Kazemi, Shayessteh Dadfarnia, Ali Mohammad Haji Shabani, MansourehRanjbar

PII: S0308-8146(17)31035-XDOI: http://dx.doi.org/10.1016/j.foodchem.2017.06.053Reference: FOCH 21275

To appear in: Food Chemistry

Received Date: 6 March 2017Revised Date: 3 June 2017Accepted Date: 7 June 2017

Please cite this article as: Kazemi, E., Dadfarnia, S., Haji Shabani, A.M., Ranjbar, M., Synthesis, characterization,and application of a Zn (II)-imprinted polymer grafted on graphene oxide/magnetic chitosan nanocomposite forselective extraction of zinc ions from different food samples, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.06.053

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Synthesis, characterization, and application of a Zn (II)-imprinted polymer grafted on

graphene oxide/magnetic chitosan nanocomposite for selective extraction of zinc ions from

different food samples

Elahe Kazemi, Shayessteh Dadfarnia٭, Ali Mohammad Haji Shabani, Mansoureh Ranjbar

Department of Chemistry, Yazd University, Safaieh, 89195-741, Yazd, Iran

* Corresponding author. Tel: +983531232667; fax: +983538210644

E-mail addresses: sdadfarnia@yazd.ac.ir (S. Dadfarnia), hajishabani@yahoo.com (A. M. Haji

Shabani), baran_6079@yahoo.com (E. Kazemi)

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Abstract

A novel Zn(II) imprinted polymer was synthesized via a co-precipitation method using

graphene oxide/magnetic chitosan nanocomposite as supporting material. The synthesized

imprinted polymer was characterized by Fourier transform infrared spectrometry (FTIR) and

scanning electron microscopy (SEM) and applied as a sorbent for selective magnetic solid phase

extraction of zinc followed by its determination by flame atomic absorption spectrometry. The

kinetic and isothermal adsorption experiments were carried out and all parameters affecting the

extraction process was optimized. Under the optimal experimental conditions, the developed

procedure exhibits a linear dynamic range of 0.5–5.0 µg L-1 with a detection limit of 0.09 µg L-1

and quantification limit of 0.3 µg L-1. The maximum sorption capacity of the sorbent was found

to be 71.4 mg g-1. The developed procedure was successfully applied to the selective extraction

and determination of zinc in various samples including well water, drinking water, black tea,

rice, and milk.

Keywords: Magnetic solid phase extraction; Flame atomic absorption spectroscopy; Ion

imprinted polymer; Graphene oxide; Chitosan; Zinc

Running title: Synthesis & analytical application of novel Zn(II)-imprinted polymer

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1. Introduction

Zinc is one of the most important essential trace elements and considered to be a vital

micronutrient as it has shown a wide range of biochemical functions in all living organisms. Zinc

deficiency in the human body causes several disorders including growth retardation, diarrhea,

eye and skin lesions, immunity depression and malfunction of wound healing (Scherz &

Kirchhoff, 2006). On the other hand, an excessive intake of zinc can be toxic and harmful and

lead to various chronic and acute adverse effects (Zhao, Han, Zhang, & Wang, 2007).

Accordingly, trace amount determination of zinc in different matrices is of great importance.

Various analytical techniques including inductively coupled plasma optical emission

spectrometry (Feist & Mikula, 2014), voltammetry (Behnia, Asgari, & Feizbakhsh, 2015),

spectrophotometry (Pourreza & Naghdi, 2014; Ribas, Tóth, & Rangel, 2017), and induced

plasma-atomic emission spectrometry (Ozbek & Akman, 2016) have been employed for the

determination of zinc. Furthermore, based on our literature survey flame atomic absorption

spectrometry is the most frequently used technique for this purpose (Behbahani, Salarian,

Bagheri, Tabani, Omidi, & Fakhari, 2014; Lemos, Bezerra, & Amorim, 2008; Roushani, Abbasi,

Khani, & Sahraei, 2015; Shakerian, Dadfarnia, & Haji Shabani, 2012; Shamsipur, Rajabi,

Pourmortazavi, & Roushani, 2014).

However, the trace level determination of metal ions in complex matrices commonly

necessitate a step of sample preparation before instrumental analysis. The application of

imprinted polymers (IIPs) as cost-effective and robust smart materials with the peculiar

recognition properties in trace or ultra-trace analysis provide significant breakthroughs in

separation or preconcentration chemistry. Bulk and precipitation polymerization are the most

commonly utilized method for the preparation of imprinted polymers. However, some major

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drawbacks have been reported in the resultant polymers. For example, heterogeneous distribution

of the binding sites inside their structure leads to poor site accessibility of the target and therefore

undesirable sorption/desorption kinetics as well as slow mass transfer (Su, Li, Li, Liu, Lei, Tan,

et al., 2015). It has proven that controlling the position of the binding sites on the material’s

surfaces via surface imprinting can efficiently resolve these problems. This methodology is

based on the synthesis of the imprinted polymer on the surface of a supporting material (Deng,

Qi, Deng, Zhang, & Zhao, 2008). Among various supporting materials, Fe3O4 nanoparticles

individually (Kazemi, Haji Shabani, & Dadfarnia, 2015) or incorporated into other nano or micro

materials (Qiu, Luo, Sun, Lu, Fan, & Li, 2012) has enjoyed wide range of application as it

provides large external surface area to volume ratio as well as facilitating the separation of the

sorbent via external magnetic field.

Graphene oxide (GO) with possessing extremely large surface area, extraordinary

physical and mechanical stability and especially plentiful presence of hydroxyl, epoxide, and

carboxylic functional groups which enables its surface chemical modification has also attracted

great attention in preparation of imprinted polymers (Ning, Peng, Li, Chen, & Xiong, 2014; Qiu,

Luo, Sun, Lu, Fan, & Li, 2012). There are some other reports using chitosan in the preparation of

imprinted polymers. Chitosan is a biocompatible and biodegradable hydrophilic polymer with

abundant hydroxyl and amino functional groups which is known to be one of the most abundant

natural amino-polysaccharide (Chang, Zhang, Ying, Li, Lv, & Ouyang, 2010; Wang, Wang, Wu,

Li, Zhu, Zhu, et al., 2014). In recent years, the research interest has turned into integrating

materials with different properties in order to provide multi imprinting site, enhanced surface

area, and sorption capacity. In this regards, the integration of graphene oxide and chitosan which

occurred through the special interaction between the epoxy groups of GO and primary amine

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groups of chitosan as well as the H-bonding between them have been utilized in preparation of

imprinted polymers (Deng, Xu, & Kuang, 2014; Han, Yan, Chen, & Li, 2011). However, based

on our literature review there is only two works including our previous study dealing with the

triplicate combination of chitosan, magnetic nanoparticles and graphene oxide in preparation of

imprinted polymers (Barati, Kazemi, Dadfarnia, & Haji Shabani, 2017; Duan, Li, Wang, Wang,

Li, & Luo, 2015).

Up to now, several kinds of Zn(II)-imprinted polymers have been synthesized and

applied for selective extraction of zinc from different samples which all of them have been

prepared using the traditional methods (Behbahani, Salarian, Bagheri, Tabani, Omidi, & Fakhari,

2014; Behnia, Asgari, & Feizbakhsh, 2015; Roushani, Abbasi, Khani, & Sahraei, 2015;

Shakerian, Dadfarnia, & Shabani, 2012; Shamsipur, Rajabi, Pourmortazavi, & Roushani, 2014;

Zhao, Han, Zhang, & Wang, 2007). The aim of the present study was to prepare a sorbent

exhibiting high selectivity, high sorption capacity and significantly fast mass transfer for zinc

ions with simple and ease of separation. For this purpose, a novel zinc imprinted polymer on

supporting material exploiting the unique characteristic of graphene oxide, chitosan and

magnetic nanoparticles was synthesized and its performance for selective separation,

preconcentration, and extraction of Zn2+ ions from different matrices was evaluated. The

structure and binding properties of the synthesized polymer were thoroughly studied and all the

important parameters influencing the extraction of zinc ions were investigated and optimized.

Finally, the performance of the synthesized polymer and feasibility of the developed methods for

the selective extraction and determination of zinc from water and different food samples was

evaluated.

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2. Experimental

2.1. Reagents and materials

Ethylene glycol dimethacrylate (EGDMA), acrylic acid (AA), styrene, glutaraldehyde (25

wt.%), zincon (C20H16N4O6S), ferric chloride (FeCl3.6H2O), ferrous chloride (FeCl2.4H2O),

graphite powder, ethylenediaminetetraacetic acid (EDTA), histidine (C6H9N3O2) and all solvents

used in this study were of analytical reagent grade and purchased from Merck Company

(Darmstadt, Germany). Chitosan with an average molecular weight of 350 kDa and deacetylation

degree of >75% was obtained from Sigma-Aldrich (Missouri, USA). 2, 2' -

Azobisisobutyronitrile (AIBN) was prepared from ACROS Company (New Jersey, USA). All

solutions throughout the study were prepared using double distilled water. A stock solution of

zinc at 1000 mg L-1 was prepared by dissolving an appropriate amount of Zn(NO3)2.6H2O in

double distilled water. Working solutions were prepared through serial dilutions of the stock

solution with doubly distilled water.

2.2. Instrumentation

An Analytik Jena novAA 300 atomic absorption spectrometer (model 330, Jena,

Germany) equipped with a Zn-hollow cathode lamp (HCL) and an air-acetylene flame atomizer

was used for determination of zinc ions. The operation conditions were adjusted according to the

manufacturer’s recommendation as follows: HCL wavelength: 213.9 nm, Lamp current: 4.0 mA,

spectral bandwidth: 0.5 nm. The pH measurements were carried out using a digital pH meter,

Metrohm model 827 (Herisau, Switzerland), equipped with a combined glass calomel electrode.

The magnetic phase separation was carried out by means of a strong magnet (1.2 T, 10 cm × 5

cm × 2 cm).

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2.3. Synthesis of the imprinted sorbent

In the first step for the synthesis of magnetic ion imprinted polymer, Fe3O4 nanoparticles,

and graphene oxide were separately synthesized via co-precipitation method (Tahmasebi &

Yamini, 2012) and a modified Hummers method (Kazemi, Dadfarnia, & Haji Shabani, 2015),

respectively. Next, to prepare the magnetic composite of graphene oxide/chitosan (GO/Chm), an

accurately weighted amount of pure chitosan (2.0 g) was dissolved in 100.0 mL of acetic acid

solution (2.0% v/v), and mixed by sonication for 30 min. 0.75 g of Fe3O4 nanoparticles was

added to the mixture and stirred for 2 h. Thereafter, 30.0 mL of glutaraldehyde (25.0 wt.% in

water) and 1.5 g of graphene oxide was added to the mixture and the pH was adjusted to 9.0–

10.0 by the addition of ammonia solution. The mixture was stirred at 80 ºC. After 1 h, the

precipitate of GO/Chm nanocomposites was collected using an external magnet and dried under

vacuum at 60 ºC (Travlou, Kyzas, Lazaridis, & Deliyanni, 2013).

The prepared magnetic nanocomposite (GO/Chm) was then modified with acrylic acid.

For this purpose, 1.0 g of the nanocomposite was added to 100.0 mL of ethanol under sonication.

10.0 mL of acrylic acid was added and the mixture was stirred at the rate of 300 rpm for 2h. At

the end of this process, the precipitate of the acrylic acid modified GO/Chm nanocomposite was

collected by the aid of an external magnet and dried under vacuum at 50 ºC (Liu, Han, Guan,

Wang, Liu, & Zhang, 2011).

The imprinted polymer supported on the magnetic nanocomposite of graphene

oxide/chitosan (IIP-GO/Chm) was synthesized through coprecipitation polymerization. For this

purpose, 1.0 mmol zincon was dissolved in 60.0 mL mixture solution of ethanol/acetonitrile (1:2

v/v). Then, 1.0 mmol Zn(NO3)2.6H2O was added and the mixture was stirred for 1h to allow

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completion of the dark blue complex formation between zinc and zincon. 1.0 g of the previously

prepared nanocomposite was added to the mixture and mixed by mechanical stirring for another

hour. Then, 32.0 mmol of EGDMA and 8.0 mmol styrene was added and stirred for a few second

to achieve a homogenized solution. 50.0 mg of AIBN were added, and the mixture was

transferred to an ice bath (0 ºC) and purged with nitrogen for 15 min. The reaction flask was

sealed and thermally polymerized in a water bath at 60 ºC for 24 h. The obtained magnetic

polymer was collected using an external magnet, washed with ethanol and distilled water, and

dried at 60 ºC under vacuum. At the final step, the template ion was removed by repetitive

extraction with EDTA solution (0.005 mol L-1, pH = 5.5) until no analyte was detected by flame

atomic absorption spectrometry. The product which was turned from blue to purple due to

leaching was collected, dried and stored for further use. Non-imprinted polymer (NIP) was

synthesized using the same procedure, without the presence of zinc ions. Fig. S1 shows the

schematic representation of the synthesis process.

2.4. Real sample preparation

The water samples including drinking water and well water were filtered through a 0.45

mm pore filter. The amount of zinc in 100.0 mL of these samples was determined using the

developed procedure.

The rice sample, purchased from a local supermarket, was cleaned, rinsed with double

distilled water, finely grounded and oven-dried at 60 ºC. 100.0 mg of the pre-treated sample was

transferred to 100.0 mL beaker. 10.0 mL of concentrated nitric acid (65% w/w) was added and

the mixture was heated to dryness. The residue was cooled to the ambient temperature, then 3.0

mL of H2O2 (30%, w/w) was added and again heated to dryness. The residue was then dissolved

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in 10.0 mL of double distilled water and filtered. After adjustment of pH using 2.5 mL borate

buffer (pH= 8.5), the filtrate was diluted to 100.0 mL with double distilled water and treated

according to the developed procedure (Abbasi-Tarighat, Shahbazi, & Niknam, 2013).

The black tea sample was prepared from a local supermarket. The tea sample was rinsed

with cold distilled water, oven-dried at 60 ºC and grounded. 10.0 mL of concentrated nitric acid

(65% w/w) was added to 100.0 mg of the sample and the mixture was heated to dryness. The

resultant residue was dissolved in 10.0 mL of double distilled water and filtered. After

adjustment of pH using 2.5 mL borate buffer (pH = 8.5), the filtrate was diluted to 100.0 mL

with distilled water and treated according to developed procedure (Dadfarnia, Haji Shabani,

Shirani Bidabadi, & Jafari, 2010).

The milk sample was prepared as follows: 10.0 mL of concentrated nitric acid followed

by 2.0 mL of 30% (w/w) hydrogen peroxide were added to 0.15 g of the accurately weighed

dried milk sample. The mixture was heated at 100 ºC for 15 minutes, and allowed to cool at

room temperature. Afterward, the mixture was filtered through a 0.45 mm pore filter, the pH was

adjusted to 8.5 by the addition of 2.5 mL of borate buffer, transferred to a 100.0 mL volumetric

flask and the volume was adjusted with distilled water (Machado, Bergmann, & Pistón, 2016).

The resulting solution was analyzed according to the given general procedure.

2.5. General procedure

The extraction of zinc ions using the prepared sorbent was carried out by the batch

experiments as follows: 20.0 mg of the synthesized magnetic ion imprinted polymer was added

to 100.0 mL of sample/standard solution containing no more than 0.5 µg zinc and the pH was

adjusted to 8.5 by the addition of 2.5 mL of borate buffer. The mixture was stirred mechanically

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for 10 minutes to complete the extraction process. Thereupon, the sorbent was held by an

external magnet and the supernatant was decanted. 0.5 mL of EDTA solution (0.005 mol L-1, pH

= 5.5) was then added to the sorbent and the mixture was sonicated for 5 minutes to desorb the

retained zinc ions. The desorbing solution was separated by the aid of an external magnet and

introduced to atomic absorption spectrometry for quantification.

2.6. Adsorption studies

To study the binding and adsorption properties of the synthesized ion imprinted polymer,

the batch mode sorption experiments were carried out as follow: 20.0 mg of the prepared sorbent

was equilibrated with 100.0 mL of the zinc solutions with a different initial concentration within

the range of 50.0–300.0 mg L-1 at the optimized conditions. At certain time intervals up to 6h,

the concentration of zinc remaining in the solution was determined by flame atomic absorption

spectrometry. The amount of zinc retained on the sorbent (qt) at the time of t was calculated

using a mass balance relationship as follow:

�� =��� − ���V

W

Where Co is the initial concentration of zinc and Ct (mg L-1) represent the remaining

concentration of zinc in the solution at time t. V represent the volume of solution (L) and W is

the sorbents mass (g). The results of this experiments are depicted in Fig. S2. As it can be seen,

the retained amount of Zn2+ ion per unit mass of the polymer was increased almost linearly with

the increase in the initial concentration of the zinc ions and gradually reached a plateau

indicating the saturation of the active binding sites on the synthesized IIP-GO/Chm.

Furthermore, the adsorption equilibrium in solutions with different initial concentrations of zinc

was achieved after about 2.5 h, representing the significantly high rate of mass transfer.

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3. Results and discussion

3.1. Sorbent characterization

3.1.1. FTIR analysis

The correct synthesis of Fe3O4 nanoparticles, graphene oxide, the magnetic composite of

graphene oxide/chitosan and modified magnetic nanocomposite by zinc-imprinted polymer was

investigated using the FTIR spectroscopy (Fig. 1). The spectrum of pure Fe3O4 nanoparticles

(Fig. 1a) shows the characteristic peak at 540 cm−1 corresponding to the Fe-O stretching mode of

the Fe3O4 lattice. The characteristic absorption peaks of graphene oxide corresponding to C=O

stretching of the carboxyl group at 1740 cm-1, C=C stretching vibration of the remaining sp2

character at 1620 cm-1, C-O-C stretching of the epoxy at 1248 cm-1 and C-O-H stretching of the

hydroxyl group at 1051 cm-1 can be seen in Fig. 1b. The spectrum of magnetic composite of

graphene oxide/chitosan (Fig. 1c) exhibits the characteristic peaks of Fe3O4 nanoparticles at 600

cm-1 and the C=O stretching corresponding to carboxyl group of graphene oxide is downshifted

to 1712 cm-1 which is the exhibition of hydrogen bonding between the carboxyl group in the

graphene oxide and NH2 in chitosan. The appearance of carboxyl peak at 1400 cm-1 indicates the

formation of (-COO- +H3N-R). Furthermore, the appearance of two characteristic peaks at 1633

and 1566 cm-1 which can be ascribed to the C–O stretching vibration of –NHCO– and the N–H

bending of NH2 confirms the successful preparation of magnetic nanocomposite of chitosan

grafted on the graphene oxide.

The spectra of zincon and zinc-zincon complex (Fig. 1d and 1e) show the main

characteristic absorption peaks of zincon at 1042 cm-1 corresponding to S=O stretching vibration,

at 1107 cm-1 corresponding to C-N stretching vibration, at 1200 cm-1 corresponding to C-O or O-

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H bending vibration of benzene ring, at 1471 cm-1 corresponding to stretching vibration of

benzene ring, at 1580 cm-1 corresponding to N=N stretching vibration and at 1700 cm-1 attributed

to C=O stretching. These peaks are all observed in the spectrum of the complex (Fig. 1e) with a

slight shift. The peak at 1603 cm-1 is attributed to the bending vibration of N-H which is

disappeared in the spectrum of complex due to interaction with zinc.

The IR spectrum of the imprinted polymer before and after leaching is demonstrated in

Fig. 1f and 1g. As it can be seen, these spectra exhibit similar features of absorption peaks at

1154 cm-1 corresponding to S=O stretching vibration of zincon, 1296 cm-1 corresponding to

stretching vibration of phenolic group in zincon and 1724 cm-1 attributed to stretching vibration

of C=O in the polymer backbone. The peak observed at 1638 cm-1 is attributed to the amide

group in the supporting material which is slightly shifted due to interaction with acrylic acid in

the functionalization process. Accordingly, the successful synthesis of ion imprinted polymer

and incorporation of zincon in its structure as well as the stability of polymer in all leaching

process can be concluded.

3.1.2. SEM analysis

The morphology of the Fe3O4 nanoparticles, graphene oxide, the magnetic

nanocomposite of graphene oxide-chitosan and IIP coated GO/Chm was characterized by

scanning electron microscopy. Figure 2a shows the synthesized spherical nanoparticles of Fe3O4

and Fig.2b. shows the typical flake-like shape of graphene oxide. The SEM of the magnetic

nanocomposite of GO/Chm (Fig. 2c) shows the successful aggregation of magnetic nanoparticles

and chitosan on the surface of graphene oxide. Fig. 2d illustrates the highly porous three-

dimensional structure as well as the spherical shape of IIP-GO/Chm. This evidence confirms the

successful synthesis of IIP coated GO/Chm.

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3.1.3. Adsorption Isotherms

The adsorption isotherms provide the interactive behavior between the solute and the

adsorbent. Thus, the analysis of isotherm data is important in estimating the adsorption capacity

as well as describing the affinity and adsorbent surface properties. In this study, Langmuir

(Langmuir, 1916), Freundlich (Surikumaran, Mohamad, & Sarih, 2014) and Temkin (Barati,

Kazemi, Dadfarnia, & Haji Shabani, 2017) models were used to assess the binding properties of

the synthesized IIPs-GO/Chm. The studied models expressed by the following equations:

Langmuir: �� �

= � � ��� + � �

����

Freundlich: �� �� = �� �� + �� �� ��

Temkin: �� = � �� �� + � �� ��

� = ���

Where qe (mg g-1) and Ce (mg L-1) are the amount of zinc adsorbed at equilibrium and the

equilibrium concentration of zinc in the solution, respectively. qm and b are the Langmuir

constant corresponding to the maximum monolayer capacity (mg g-1), and the sorption energy,

respectively. The Langmuir model assumes that the sorbent has a homogeneous surface with

similar and energetically equivalent binding sites which provide monolayer adsorption. The

Freundlich model is commonly utilized to describe multilayer adsorption onto heterogeneous

surfaces. Kf (mg g-1) and n are the Freundlich constants, and 1/n represent the surface

heterogeneity or exchange intensity. A value of 1/n smaller than 1.0 accounts for a favorable

adsorption.

The Temkin model illustrates the homogeneous distribution of binding energies up to

some maximum and postulates the linear decrease of the heat of the sorption of all molecules in

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the layer with the surface coverage due to the interactions between the solute molecules and the

adsorbent. KT is the equilibrium binding constant (mol-1) ascribed to the maximum binding

energy, and the constants A and b represent the heat of sorption and the heat of adsorption

respectively.

The obtained diagrams for Langmuir, Freundlich and Temkin models are illustrated in

Fig. S3 and their corresponding parameters including values of R2 are listed in Table S1. The

applicability of the studied models was judged by evaluating the correlation coefficients, R2

values. As shown in Table S1, the R2 value of the Langmuir isotherm was greater and more

proximate to unity (0.999) than that of the other models. Therefore, the experimental data of zinc

adsorption onto IIP-GO/Chm fitted well to the Langmuir isotherm model indicating the

homogeneous solid surface of the synthesized ion imprinted polymer and the regular monolayer

adsorption of zinc molecules (Barati, Kazemi, Dadfarnia, & Haji Shabani, 2017). Furthermore,

based on the data summarized in Table S1, the maximum predictable sorption capacity of the

adsorbent for zinc ions was found to be 71.4 mg g-1.

3.1.4. Adsorption kinetics study

To investigate the mechanism of zinc adsorption onto the surface of the IIP-GO/Chm,

four kinetic models including the pseudo-first-order, pseudo-second order (Barati, Kazemi,

Dadfarnia, & Haji Shabani, 2017), intraparticle diffusion (Cheung, Szeto, & McKay, 2007) and

Elovich (Özacar & Şengil, 2005) were used to explore the experimental data. These models are

expressed by the following equations:

Pseudo-first-order: log��� − ��� = log�� − !"�#.%�%

Pseudo-second order: � &= �

!' �'+ � �

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Intraparticle diffusion:�� = �() *"' + +

Elovich:�� = �, ln�./� +

�, ln *

qe and qt which are the same in all equations represent the amounts of zinc ion (mg g-1) sorbed at

the equilibrium time and time t, respectively. k1 (min-1) and k2 (g mg-1 min-1) are the pseudo-first

order and the pseudo-second order rate constant of sorption, respectively. The Kid and I are the

intraparticle diffusion rate constant (mg g-1 min-1/2) and the intercept determined from the plot of

qt versus t1/2, respectively. In Elovich equation α is the initial sorption rate constant (mg g-1 min-

1) and β represent the amount of the surface coverage and the activation energy of chemisorption

(g mg-1).

Fig. S4, 5, 6 and 7 shows the experimental data fitted with pseudo-first-order, pseudo-

second order, Elovich, and intraparticle diffusion, respectively. As it can be concluded from data

summarized in Table S2, the correlation coefficient (R2) values of the pseudo-second-order

model are higher than that’s of other models and closer to unity. Therefore, the pseudo-second

order equation which suggests the chemisorption of solute onto the sorbent was more suitable to

describe the adsorption of zinc ions into the polymer.

3.2. Optimization of extraction conditions

In order to have the most desirable extraction performance and achieve satisfactory

results, all parameters affecting the extraction efficiency of zinc using the synthesized ion

imprinted polymer was investigated and optimized. All optimization experiments were

performed in triplicates and the results were averaged.

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3.2.1. Effect of pH

The sample solution pH exerts a profound influence on the overall performance of the

SPE procedure, as it can affect the sorptive uptake of the target analyte by changing the surface

chemistry of both sorbent and analyte. The driving force for adsorption of zinc ions onto the

synthesized ion imprinted polymer is the ability of the zinc in the formation of a stable chelate

with N-containing groups existing in zincon. By considering the structure of zincon (Fig. 3a) and

its pKa values (Fig. 3b) it is expected that the best complex formation between zinc and zincon

take place at alkaline medium (Säbel, Neureuther, & Siemann, 2010). With this perception, the

influence of the sample solution pH on the extraction of zinc ions was investigated in the pH

range of 4.0–11.0. The pH of the sample solution was adjusted using nitric acid or ammonia

solution as well as phosphate buffer (6.0–8.0) and borate buffer (8.0–10.0). The results

represented in Fig. 4a showed that as expected the maximum extraction recovery is achieved in

the pH range of 7.0–9.0. The decrease in extraction recoveries at lower pH can be explained by

protonation of the ligand which leads to incomplete complexation and retention of the analyte by

the sorbent, whereas at higher pH, zinc ions are prone to the precipitation as hydroxide.

Accordingly, pH of 8.5 was selected as the optimum pH.

3.2.2. Optimization of desorption condition

In order to assure the achievement of satisfactory recovery of retained zinc ions, the

effect of type of desorbing solution on the extraction performance was investigated. For this

purpose, 1.0 mL of different potential desorbing solution including EDTA (0.05 mol L-1),

histidine (0.05 mol L-1), HNO3 (1.0 mol L-1), H2SO4 (1.0 mol L-1) and CH3COOH were

examined. As it can be concluded from the results represented in Fig. 4b, EDTA (0.05 mol L-1)

17

provided the most effective desorption of the retained zinc ions which can be explained by the

competition of the EDTA with zincon in the complex formation with zinc. As the complexation

and consequently the desorption capability of EDTA toward zinc may be affected by the pH, a

series of EDTA solutions with pHs varying in the range of 2.0–10 was used for desorption of

zinc. The results implied that the maximum extraction recovery was obtained in the pH range of

4.5–10.0. Furthermore, the effect of concentration of EDTA in the range of 5 × 10-4 – 0.05 mol

L-1 was investigated and 0.005 mol L-1 was chosen as the optimum concentration. The minimum

volume of desorption solution required to efficiently desorb the analyte was also investigated by

varying its volume in the range of 0.3 to 1.5 mL. The quantitative results were achieved by 0.5

mL of EDTA. Accordingly, 0.5 mL EDTA solution with a concentration of 0.005 mol L-1 and

pH of 5.5 was selected for the desorption stage.

3.2.3. Effect of time

The adsorption process of the target analyte must be done for enough time to achieve

equilibrium. The optimum time for adsorption of zinc defined as the time between the addition of

the sorbent to the sample solution and its separation for the sample was determined by

application of different time intervals from 1 to 30 minutes. Based on the results (Fig. 4c), 10

minutes was selected as the optimum adsorption time. The effect of desorption time was also

investigated in the range of 3-20 minutes and 5 minutes was found to be sufficient for

quantitative desorption of analyte.

3.2.4. Effect of amount of sorbent, sample volume and ionic strength

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The dependency of extraction efficiency to the amount of sorbent was studied by

changing the amount of IIP-GO/Chm from 5.0 to 30.0 mg. The results showed that 20.0 mg of

the prepared sorbent was adequate to achieve satisfactory results. Consequently, 20.0 mg of IIP-

GO/Chm was utilized in the subsequent studies.

To investigate the breakthrough volume of the sample solution, 0.5 µg of zinc was

extracted from different volumes of sample solutions (25.0–200.0 mL). The results revealed that

up to 100.0 mL of the sample solution, the extraction process was quantitative. Accordingly, in

order to achieve a higher preconcentration factor, 100.0 mL of the sample solution was selected

for the extraction of analyte in the following experiments.

The influence of the ionic strength on the extraction efficiency of zinc ions was

investigated by conducting the extraction process in the presence of different amount of sodium

chloride (0-1.5 mol L-1). It was observed (Fig. 4d) that the presence of salt up to 1.0 mol L-1 did

not interfere with the extraction process. However, the extraction efficiency decreased at higher

concentration of salt. This can be related to the competition of Na+ ions at high concentration

with zinc for the sorption sites or the hindrance of mass transfer due to the increase in the

viscosity of the sample in the presence of high amount of salt. Accordingly, the following

experiments were performed without the addition of salt.

3.3. Selectivity and reusability of the sorbent

The selectivity of the synthesized IIP-GO/Chm toward zinc ions was investigated by

considering competitive extraction of zinc in the presence of other ions including some transition

metals such as Cu2+, Co2+, Ni2+ and Cd2+ which are capable of forming a complex with zincon.

The tolerance limit was taken as the maximum concentration of the ions which caused an error

19

not exceeding ±5%. The results summarized in Table S3 showed that the examined ions at the

given mole ratio did not interfere in the extraction and determination of zinc ions. Accordingly,

the developed procedure using IIP-GO/Chm exhibits high selectivity toward zinc.

In order to evaluate the renewability and reusability of the synthesized sorbent, a set of

experiments were performed by using the same sorbent repeatedly. For this purpose, the

synthesized IIP-GO/Chm was subjected to several adsorption and desorption cycles, under the

same experimental conditions. Based on the obtained results (Fig. S8a), the IIP-GO/Chm was

stable and could be regenerate without any significant loss of analytical performance up to 9

adsorption/desorption cycle. Furthermore, the analytical performance of IIP-GO/Chm was

compared with NIP-GO/Chm by application of these polymers for the extraction of zinc ions

under the similar condition. As it can be concluded from Fig. S8b, the application of IIP-

GO/Chm result in much higher extraction efficiency indicating the higher affinity of the

synthesized IIP-GO/Chm toward zinc ions due to imprinting effect.

3.4. Analytical performance

The developed procedure was validated by determination of quality parameters including

linear dynamic range, coefficient of determination (R2), limit of detection, preconcentration

factor, and precision. Under the optimal concentration, the method showed a good linear

correlation of analytical signal to the zinc concentration within the range of 0.5–5.0 µg L-1 with a

regression equation of A (signal) = 0.019 CZn (µg L-1) + 0.028 and correlation coefficient of R2 =

0.999. The limit of detection of 0.09 µg L-1 and limit of quantification of 0.3 µg L-1 were

obtained based on 3Sb/m and 10Sb/m (Sb is the standard deviation of the blank and m is the

slope of the calibration graph), respectively. Five replicate measurement of zinc solutions at the

20

concentration of 2.0 µg L-1 gave a relative standard deviation of 2.7%. The enrichment factor,

ascribed to the ratio of the maximum volume of the sample solution (100.0 mL) to the final

volume of the extract (0.5 mL) was 200.

A comparison of the analytical performance of the current procedure with other literature

is provided in Table 1. It is obvious that the developed procedure achieved better or comparable

precision, higher preconcentration factor and with one exception (Lemos, Bezerra, & Amorim,

2008) the lower detection limit in comparison with other previously reported methods.

Furthermore, it is noteworthy that the capacity of the synthesized ion imprinted polymer based

on graphene oxide/magnetic chitosan is significantly higher than most of the other zinc imprinted

polymers which confirm the superiority of the polymer synthesized by surface imprinting

methodology in comparison with traditional methods.

3.5. Application

To verify the feasibility of the method, the developed procedure was applied to the

determination of zinc ions in real samples including well water, drinking water, black tea, rice,

and milk. The accuracy of the procedure was assessed through the recovery experiments from

samples spiked with the known amount of zinc as well as a comparison of the results with data

obtained from independent analysis using electrothermal atomic absorption spectrometry

(ETAAS). The results summarized in Tables 2 demonstrate that the recoveries of the spiked

sample are in a satisfactory range of 96.0-106.0 and at 95% confidence levels there is no

significant differences between the results of the current method and ETAAS analysis. These

results signify the capability and suitability of the developed procedure for the determination of

zinc in a wide variety of matrices.

21

4. Conclusion

The present study reports the synthesis and analytical application of a novel Zn(II)

imprinted polymer grafted on graphene oxide/magnetic chitosan. The synthesized polymer was

well characterized using FTIR and SEM. The Langmuir isotherm model fitted well with

experimental data demonstrating the homogeneous solid surface of the synthesized ion imprinted

polymer and the regular monolayer adsorption of zinc molecules. The adsorption kinetic studies

showed that the adsorption process followed a pseudo-second-order kinetic model indicating

chemisorption of target analyte onto the sorbent. The synthesized ion imprinted polymer

exhibited high selectivity, high sorption capacity and significantly fast mass transfer which can

be attributed to the extremely large surface area and multi imprinting sites of the magnetic

chitosan/graphene oxide. The magnetic field sensitivity enables the simple, rapid and efficient

separation of the sorbent thereby shortening the separation and improving the efficiency of the

extraction process. In summary, the specific recognition capability, high adsorption capacity,

high preconcentration factor, low detection limit and good precision and accuracy signifies that

the developed procedure is a promising methodology for the selective and accurate determination

of zinc in a wide variety of real samples.

Conflict of interest

The authors declare no conflict of interest.

22

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Figure captions

Fig. 1. FT-IR spectra of magnetic nanoparticles (a) graphene oxide (b) graphene oxide/magnetic

chitosan nanocomposite (c) zincon (d) zinc-zincon complex (e) Zn(II) imprinted polymer grafted

on graphene oxide/magnetic chitosan before (f) and after (g) leaching.

Fig. 2. SEM images of magnetic nanoparticles (a) graphene oxide (b) graphene oxide/magnetic

chitosan nanocomposite (c) Zn(II) imprinted polymer grafted on graphene oxide/magnetic

chitosan (d).

Fig. 3. The structure of zincon and complex formation between zinc and zincon (a). The pKa

values and ionization process of zincon (b).

Fig. 4.Fig. 4.Fig. 4.Fig. 4. The effect of pH (a) type of desorbing solution (b) sorption time (c) and ionic strength of

the sample solution (d) on the extraction efficiency of zinc. Conditions: sample volume, 100.0

mL; zinc concentration, 2.0 µg L-1; amount of sorbent, 20.0 mg; desorbing solution volume, 0.5

mL; desorption time, 5 min.

28

- The first synthesis of Zn(II) ion imprinted polymer grafted on GO/Chm.

- Development of a novel MSPE/FAAS method for the determination of Zinc in food samples.

- Achievement of high selectivity, high sorption capacity & fast mass transfer.

- The capability of detection of zinc ions with a low detection limit of 0.09 µg L-1.

29

30

31

Fig. 1

Fig. 2

32

Fig. 3

33

Fig. 4

34

Table 1

Comparison of analytical characteristics of the present method and some previously reported methods for the extraction and determination of zinc.

Ref

RSD

(%)

PF

LOD

(µg L-1

)

Sorbent capacity

(mg g-1

)

Matrix

Analytical

system

Sorbent

(Shakerian et al., 2012) 3.4 118 0.8 2.73 Water, cereals FAAS IIP

(Roushani et al., 2015) 3.0 100 1.0 22.11 Milk, Rice, tea, water FAAS IIP

(Shamsipur et al., 2014) 2.7 120 0.33 0.13 Water, Juice, syrup FAAS IIP

(Behbahani et al., 2014) 2.8 78 0.15 68.6 Vegetable, Meat, Milk, Water FAAS IIP

(Feist et al., 2014) 1.3 80 1.5 2.40 Fruits ICP-OES Activated carbon

(Lemos et al., 2008) 3.4 62 0.077 - Standard reference materials FAAS

Functionalized resin

This work 2.7 200 0.09 71.4 Milk, Rice, tea, water FAAS IIP -GO/Chm

LOD; Limit of detection, PF; Preconcentration factor, RSD; Relative standard deviation

35

Table 2

Determination of zinc in different water and food samples.

Sample Zinc Recovery

(%) ET-AAS

Experimental

t٭ Added Found

Well water 0 N.D - N.D -

(µg L-1) 1.0 1.05 ± 0.05 105.0 - -

2.0 1.95 ± 0.13 97.5 - -

Drinking water 0 1.00 ± 0.01 - 0.97 ± 0.04 1.26

(µg L-1) 1.0 2.06 ± 0.07 106.0 - -

2.0 2.95 ± 0.09 97.5 - -

Black tea 0 2.00 ± 0.02 - 2.05 ± 0.08 1.05

(µg g-1) 1.0 2.96 ± 0.08 96.0 - -

2.0 3.94 ± 0.14 97.0 - -

Rice 0 1.01 ± 0.02 - 1.09 ± 0.05 2.56

(µg g-1) 1.0 1.98 ± 0.05 97.0 - -

2.0 2.96 ± 0.12 97.5 - -

Milk 0 1.16 ± 0.05 - 1.22 ± 0.03 1.78

(µg g-1) 1.0 2.12 ± 0.07 96.0 - -

2.0 3.18 ± 0.08 101.0 - -

The t for degree of freedom of 4 at 95% confidence level is 2.78, N.D: not detected ٭